Enzymatic asymmetric synthesis of the silodosin amine intermediate

Article abstract of DOI:10.1016/j.tetasy.2013.12.012

Various enantiocomplementary ω-transaminases (ωTAs) were investigated in kinetic resolution and asymmetric reductive amination reactions to prepare silodosin amine. Whilst the enzymatic kinetic resolution gave moderate to good results with respect to the yield and enantioselectivity, the asymmetric reductive amination proved to be superior. The best results were obtained with the ωTA originating from (R)-Arthrobacter sp. which afforded the desired bioactive (R)-enantiomer in enantiomerically pure form (ee >97%) at excellent conversion (conv. >97%) under mild and benign reaction conditions.

Full text of DOI:10.1016/j.tetasy.2013.12.012

Tetrahedron: Asymmetry 25 (2014) 284–288
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enzymatic asymmetric synthesis of the silodosin amine intermediate
Robert C. Simon a, Johann H. Sattler c, Judith E. Farnberger a, Christine S. Fuchs a,
Nina Richter a, Ferdinand Zepeck b, Wolfgang Kroutil a,c,
⇑
a ACIB GmbH, Heinrichstraße 28, A-8010 Graz, Austria
b Sandoz GmbH, Biocatalysis Lab, Biochemiestraße 10, A-6250 Kundl/Tirol, Austria
c University of Graz, Institute of Chemistry, Organic and Bioorganic Chemistry, Heinrichstraße 28, A-8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 November 2013
Accepted 18 December 2013
Various enantiocomplementary
asymmetric reductive amination reactions to prepare silodosin amine. Whilst the enzymatic kinetic res-
olution gave moderate to good results with respect to the yield and enantioselectivity, the asymmetric
x-transaminases (xTAs) were investigated in kinetic resolution and
reductive amination proved to be superior. The best results were obtained with the
xTA originating from
(R)-Arthrobacter sp. which afforded the desired bioactive (R)-enantiomer in enantiomerically pure form
(ee >97%) at excellent conversion (conv. >97%) under mild and benign reaction conditions.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
BzO
HO
H2N
O
CN
F3C
O
O
Silodosin (UrorecÒ, RapafloÒ) is a single enantiomer pro-drug
with an (R)-configuration which was first disclosed in 1995 as a
therapeutic agent for the treatment of dysuria and urinary distur-
N
N
X
HN
(
)
R
bance.1 It acts as a selective
a1A-adrenergic receptor (AR) antago-
rac-1: X = NH2
silodosin
nist and is currently used for the treatment of disurya associated
with benign prostatic hyperplasia (BPH). This chronical progressive
disease affects more than 90% of male patients over 85 years.2 The
first marketing approval was granted in Japan in 2006, two years
later in the US and another two years later in the EU. So far, only
a few chemical strategies for its synthesis have been published in
the literature,3 most of which are in the form of patents.4 Common
approaches to access enantiomerically pure silodosin involve two
key-intermediates (Scheme 1). For example, the racemic amine
rac-1 was resolved via diastereomeric crystallisation employing
2:
X = O
Scheme 1. Silodosin and two possible key-intermediates.
general,7
xTAs work under mild reaction conditions and show
broad substrate specificity to afford the products with high to
excellent regio- and stereoselectivity.8 Recent successful applica-
tions of
xTAs include the chemoenzymatic synthesis of chiral
key intermediates for pharmaceuticals9 such as Suvorexant (a
potent dual orexin inhibitor),10 MK-7246 (a chemo-attractant
receptor antagonist expressed on Th2 cells),11a MK6096 (an orexin
receptor antagonist in clinical trials for the treatment of insom-
nia),11b sitagliptine11cand (S)-rivastigmine.12 Natural products such
as dihydropinidine13 and isosolenopsin14 have also been
(+)-L
-tartaric acid1 or (+)-mandelic acid after coupling with the phe-
nolic moiety.3a,4b An alternative route to access the desired amine
(R)-1 involves diastereoselective reduction of the imine obtained
by condensation of ketone 2 with a chiral amine [e.g., (R)-phenyl
ethylamine or (R)-phenyl glycinol].5 However, the resolution is
hampered a priori by a theoretical overall yield of only 50%;6 the
imine reduction also requires additional reaction and purification
steps. Both methods involve multiple crystallisation steps in order
to achieve a sufficient level of enantiomeric purity, thus making
them rather uneconomical in terms of overall efficiency.
synthesised by employing an
xTA catalysed asymmetric reductive
amination as the key step. Consequently, the two silodosin
key-intermediates amine rac-1 and ketone 2 are of interest with
respect to
amine (R)-1.
xTAs and the preparation of enantiomerically pure
In contrast to these exclusively chemical methods,
x-transam-
2. Results and discussion
inases ( TAs) have become a promising biocatalytical alternative
x
to obtain enantiomerically pure amines. Like all biocatalysts in
2.1. Kinetic resolution
Initially, racemic amine rac-1 (3.6 g Lꢀ1, 10 mM) was incubated
⇑
Corresponding author. Tel.: +43 316 380 53 50.
E-mail address: wolfgang.kroutil@uni-graz.at (W. Kroutil).
with various (R)- and (S)-selective
xTAs in a buffer, whereby
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.12.012

R. C. Simon et al. / Tetrahedron: Asymmetry 25 (2014) 284–288
285
10 vol % DMSO were added to improve substrate-solubility. The
biotransformations were shaken for 24 h at 30 °C. The results indi-
cated that rac-1 was transformed by various enzymes (Table 1). For
(entry 5), which afforded the desired (R)-isomer with excellent
conversion (P97%) and with excellent stereoselectivity (ee
P97%), as already indicated in the KR (Table 1, entry 7). Also the
Table 1
The kinetic resolution of amine rac-1 using various
xTAs
CN
R
CN
CN
R
R
N
N
N
ωTA, PLP
NH2
NH2
*
O
+
pyruvate
alanine
rac-1
(R)- or (S)-1
ketone 2
R = (CH2)3OBz
Entry
x
-Transaminase (origin)
Conv.a (%)
eeb (%)
E-valuec
1
2
3
4
5
6
7
8
9
Chromobacterium violaceum
Bacillus megaterium
Pseudomonas putida I
Pseudomonas putida II
Arthrobacter citreus
Pseudomonas fluorescens
(R)-Arthrobacter sp.
Aspergillus terreus
70
40
35
35
32
30
48
42
23
44 (R)
68 (R)
62 (R)
44 (R)
36 (R)
44 (R)
96 (S)
83 (S)
36 (S)
2
>200
27
15
11
>100
>200
39
Hyphomonas neptunium
15
a
Reactions were performed in triplicate. Conversion determined by HPLC on a reverse phase column; Conditions: 3.6 g Lꢀ1 substrate rac-1 (10 mM), 10 vol % DMSO, KPi
buffer (100 mM, pH 7.0), 1.0 mM PLP, 10.0 mM sodium pyruvate, 24 h, 30 °C.
b
Determined via HPLC on a chiral phase after derivatisation to the corresponding trifluoroacetamide.
c
Calculated: E = [(ln(1 ꢀ c) ꢁ (1 ꢀ eeS)]/[(ln(1 ꢀ c)ꢁ(1 + eeS)].16
instance, Chromobacterium violaceum15a (entry 1) readily deami-
Table 2
Asymmetric reductive amination of ketone 2 using various
xTAs
nated the amine although with low enantioselectivity (E = 2),
which is also indicated by the conversion exceeding 50%; in an
ideal kinetic resolution, the conversion would stop at 50%. Low
enantioselectivity (E) was also observed for biotransformation
CN
CN
R
R
N
N
ωTA, PLP
O
NH2
*
using the
robacter citreus15c (entry 5). However, the
terium15d (entry 2) and Pseudomonas fluorescens15e (entry 6) gave
high enantioselectivities with E >100. Whilst the first six TAs
transformed the (S)-enantiomer leaving the (R)-enantiomer
behind, the
TA from Arthrobacter sp.15f (entry 7) transformed
x
TA from Pseudomonas putida15b (entries 3–4) and Arth-
x
TAs from Bacillus mega-
2
1
(R)- or (S)-
pyruvate
(recycling/removal)
alanine
R = (CH2)3OBz
x
Entry
x
-Transaminase (origin)
Conv.a (%)
eeb (%)
x
1
2
3
4
5
6
Chromobacterium violaceum
Bacillus megaterium
Arthrobacter citreus
Pseudomonas fluorescens
(R)-Arthrobacter sp.
Aspergillus terreus
22
25
16
84
>97
8
88 (S)
95 (S)
89 (S)
>97 (S)
>97 (R)
>97 (R)
the (R)-enantiomer leading to an enriched remaining (S)-enantio-
mer at a conversion close to 50% and excellent enantioselectivity
(E >200). This indicated a strong preference of the enzyme for
the desired (R)-enantiomer. Consequently, the enzymes were
tested for the reductive amination of the corresponding ketone 2,
since this is the preferred reaction regarding the theoretical overall
yield.
a
Reactions were performed in duplicate, conversion determined by HPLC on a
reverse phase column. Conditions: 3.6 g Lꢀ1 substrate 2 (10 mM),
D- or L-alanine
(500 mM), 10 vol % DMSO, KPi buffer (100 mM, pH 7.0), 1.0 mM PLP, 1.0 mM NAD+,
11 U FDH, 12 U AlaDH, 24 h, 30 °C.
2.2. Asymmetric reductive amination
b
Determined via HPLC on a chiral phase after derivatisation to the corresponding
trifluoroacetamide.
Employing
xTAs for the asymmetric reductive amination
allowed us to theoretically convert prochiral ketones in quantitative
yield but suffers from an unfavorable equilibrium when alanine is
used as the amine donor.8 Various biocatalytic strategies were
employed to shift the equilibrium by removing the formed
co-product pyruvate by a second enzyme (e.g., via lactate dehydro-
genase,17 pyruvate decarboxylase,18 or recycling/removing via
alanine dehydrogenase19).
x
TA from A. terreus15f furnished the amine (R)-1 with high
enantiomeric purity (ee P97%) although with low conversion
(8%) thus rendering it inappropriate for further transformations.
Overall, the asymmetric reductive amination revealed two enan-
tio-complementary enzymes, which were able to provide the
enantiomerically pure amine (R)- and (S)-1 in high conversions,
Consequently, different enantiocomplementary
x
TAs were as-
that is, the xTA from Arthrobacter sp. and P. fluorescens.
sayed using the alanine-dehydrogenase (AlaDH) system. From
the enzymes showing an (S)-preference (Table 2, entries 1–4),
2.3. Reaction engineering
the best results were obtained with the
xTA from P. fluorescens
(entry 4): the (S)-enantiomer was obtained with high conversion
(84%) and excellent stereoselectivity of ee P97% thus confirming
the high enantioselectivity observed in the kinetic resolution. Bet-
ter results were obtained with the enzyme from Arthrobacter sp.
Since ketone 2 is almost insoluble in water and nonpolar aprotic
organic solvents (e.g., toluene, EtOAc, CH2Cl2, TBME and Et2O), we
investigated polar and water miscible co-solvents (DMSO, DMF,
THF and MeCN) to increase the bioavailability and concentration

286
R. C. Simon et al. / Tetrahedron: Asymmetry 25 (2014) 284–288
of the substrate. Consequently the conversion was studied in the
presence of an organic co-solvent employing the TA from Arthro-
niques under dry N2 atmosphere in oven-dried (120 °C) glassware.
1H and 13C NMR spectra were recorded at 20 °C on a 300 Bruker
NMR unit; chemical shifts are given in ppm relative to Me4Si
(1H: Me4Si = 0.0 ppm) or relative to the resonance of the solvent
(1H: CDCl3 = 7.26 ppm; 13C: CDCl3 = 77.0 ppm). Biocatalytic small
scale reactions were shaken on a thermo shaker EppendorfÒ
Comfort. Preparative scale reactions were performed in an Infors
Unitron shaker.
x
bacter sp. All reactions were conducted at various substrate con-
centrations (5–25 mM ketone 2) at 30 °C for 24 h. Subsequent
analysis revealed that the addition of the co-solvents THF and
MeCN led to an almost complete loss of enzymatic activity. Inde-
pendent of the substrate concentration, the conversions did not
exceed 20% (Fig. 1).
4.2. Enzymes and microorganisms
100
5 mM
Formate dehydrogenase (FDH) from Candida boidinii (2.2 U/mg)
and b-NAD free acid were purchased from Codexis Inc. Lyophilised
10 mM
15 mM
80
20 mM
Escherichia coli cells containing overexpressed
x
TA were prepared
-alanine dehy-
25 mM
as previously reported.15f,20 Purified recombinant
drogenase (AlaDH) was prepared as described.15f
L
60
40
20
0
4.3. Biotransformations (analytical scale)
4.3.1. Kinetic resolution
Lyophilised cells of E. coli containing the corresponding overex-
pressed -transaminase (20 mg) were rehydrated in a potassium
DMSO
DMF
THF
MeCN
x
phosphate buffer (pH 7.0, 100 mM) containing PLP (1.0 mM) and
sodium pyruvate (10 mM) at room temperature for 30 min. The
substrate was then added (3.6 mg dissolved in 100 lL DMSO)
and the reaction was carried out at 30 °C in an eppendorf shaker
(700 rpm) for 24 h (1.0 mL total volume).
Figure 1. Reductive amination of ketone 2 as a function of organic co-solvent
(10 vol %) and substrate concentration (5–25 mM) using the (R)-selective -TA
x
from Arthrobacter sp. Conditions:
D-alanine (500 mM), 10 vol % co-solvent, KPi
buffer (100 mM, pH 7.0), 1.0 mM PLP, 1.0 mM NAD+, 11 U FDH, 12 U Ala-DH, 24 h,
30 °C.
4.3.2. Reductive amination
In contrast, DMSO and DMF were better tolerated as co-
solvents: in the case of DMSO a decrease in the conversion was
observed between 10 and 15 mM substrate concentration, whilst
the conversion remained >90% when using DMF with up to
15 mM substrate concentration (5.4 g Lꢀ1). The enantiomeric pur-
ity remained excellent (ee P97%) independent of the solvent used.
Using these conditions (10 vol % DMF), the transformation was
successfully performed on a preparative scale (56 mg ketone 1,
15 mM). The desired bioactive amine (R)-1 was isolated in 93%
yield (ee P97%) ready for coupling towards the pharmaceutical
drug silodosin.
Lyophilised cells of E. coli containing the corresponding overex-
pressed
xTA (20 mg) were rehydrated in a potassium phosphate
buffer (pH 7.0, 100 mM) containing PLP (1.0 mM), NAD+
(1.0 mM), ammonium formate (150 mM), FDH (11 U), AlaDH (12
U) and
D
- and
L
-alanine (500 mM), respectively, at 21 °C for
30 min. The substrate was added (3.6 mg dissolved in 100
DMSO) and the reaction was run at 30 °C in an eppendorf orbital
shaker (700 rpm) for 24 h (1.0 mL total volume).
l
L
4.4. Analytical methods
3. Conclusion
4.4.1. Determination of conversion
After the indicated reaction time, aqueous saturated Na2CO3
Various (R)- and (S)-selective
x-transaminases were investi-
solution was added (100
The water was evaporated under reduced pressure via SPEEDVAC
and the residue resuspended with MeCN (500 L). After filtration
through a plug of Celite 545, additional MeCN was added (500 L)
and the process repeated. HPLC conditions: column = Luna RP-
C18 (Phenomenex); length = 25 cm, internal diameter = 4.6 mm;
isocratic flow with 1.0 mL/min using MeCN/H2O 65:35 as eluent
(+0.1 vol % TFA); Rt 1 = 2.51 min, Rt 2 = 9.68 min.
lL) and the mixture shaken vigorously.
gated in the kinetic resolution (KR) and asymmetric reductive
amination of two silodosin precursors to prepare an enantiomeri-
cally pure amine intermediate. In case of the KR via deamination,
the enzyme originating from B. megaterium afforded the bioactive
(R)-enantiomer without optimisation with reasonable conversion
and high enantioselectivity (E >100). Superior results were ob-
l
l
tained in the asymmetric reductive amination: the
xTA from Arth-
robacter sp. furnished the desired enantiomer (R)-1 with excellent
conversion (P97%) in enantiomerically pure form (ee P97%) at a
substrate concentration of 3.6 g Lꢀ1. A subsequent solubility study
allowed us to increase the concentration to 5.4 g Lꢀ1 without
affecting the stereochemical outcome using DMF as the co-solvent.
4.4.2. Determination of the enantiomeric excess (ee) after
derivatisation to the corresponding TFA-acetamide
A sample of MeCN-solution was withdrawn (500
centrated using a rotary evaporator. The residue was dissolved in
CH2Cl2 (200 L) and trifluoroacetic acid (TFAA) (3 equiv) was
lL) and con-
l
added. After shaking for 15 min at 30 °C, the sample was concen-
trated again. The sample was then ready for measurements after
re-uptake in an organic solvent (n-heptane/2-PrOH 50:50) and
filtration through a plug of cotton. HPLC conditions: column =
OD-H (Chiracel), length = 25 cm, internal diameter = 4.6 mm, iso-
cratic flow with 1.5 mL/min; eluent: n-heptane/2-PrOH 95:5. Rt
[(R)-1-TFA-amide] = 19.35 min, Rt [(S)-1-TFA-amide] = 22.85 min
(Fig. 2).
4. Experimental
4.1. General
All starting materials were obtained from commercial suppliers
and used as received with the exception of amine rac-1 and ketone
2; both compounds were generously provided by Sandoz GmbH.
Chemical reactions were carried out with standard Schlenk tech-

R. C. Simon et al. / Tetrahedron: Asymmetry 25 (2014) 284–288
287
(54 mg, 0.15 mmol) dissolved in 1.0 mL DMF was added and the
reaction was shaken for 26 h at 30 °C. Aqueous saturated Na2CO3
solution was then added (1.00 mL) and the mixture was concen-
trated to dryness under reduced pressure. The residue was resus-
pended with MeCN (3 ꢁ 5 mL) and filtered though a plug of
Celite to afford the enantiomerically pure amine (R)-1 in 93% yield.
Spectroscopic data matched the authentic sample provided by San-
mAu
40000
30000
20000
10000
doz. 1H NMR (300 MHz, CD3CN): dH [ppm] = 0.97 (d,3J3 ,2 = 6.3 Hz,
0
0
3
3 H, 30-H), 2.10 (tt, J2 ,3 = 6.2 Hz,3J2 ,1 = 7.1 Hz, 2 H, 200-H), 2.32
00 00
00 00
0
(dd,3J1 a,2 = 7.5 Hz,2J1 a,1 b = 13.3 Hz, 1 H, 10-Ha), 2.43 (dd, J1 b,2
=
3
0
0
0
0
0
0
racemic
5.8 Hz,2J1 b,1 a = 13.4 Hz, 1 H, 10-Hb), 2.92 (t, J3,2 = 8.8 Hz, 2 H,
3
0
0
(R)-enantiomer
3-H), 2.93 (mc, 1 H, 20-H), 3.57 (t,3J2,3 = 8.7 Hz, 2 H, 2-H), 3.72
(t,3J1 ,2 = 7.2 Hz, 2 H, 100-H), 4.41 (t, J3 ,2 = 6.2 Hz, 2 H, 300-H), 6.94
(brs, 1 H, arom.-Hind), 7.01 (brs, 1 H, arom.-Hind), 7.46 (mc, 2 H,
arom.-H), 7.66 (mc, 1 H, arom.-H), 8.02 (mc, 2 H, arom.-H). 13C
NMR (75 MHz, CDCl3): dC [ppm] = 23.8 (C-30), 27.5 (C-200), 27.9
(C-3), 45.9 (C-10), 46.1 (C-100), 49.3 (C-20), 53.9 (C-2), 63.6 (C-300),
88.2 (C-7), 120.4 (CN), 129.4 (arom.-CH), 130.0 (arom.-Cipso),
130.3 (arom.-CH), 130.9 (arom.-CHind), 131.3 (arom.-Cipso), 132.1
(arom.-CHind), 134.0 (arom.-CH), 134.1 (arom.-Cipso), 152.4
(arom.-Cipso), 167.2 (COO).
3
00 00
00 00
(S)-enantiomer
18 19 20 21 22 23 24 25 26 27
retention time (min)
Figure 2. Overlay of the chromatograms of the analysis of TFA-acetamide of 1 on a
chiral phase.
4.4.3. Synthesis of the reference compound for the chiral
analysis: 3-(7-cyano-5-(2-(2,2,2-trifluoroacetamido)propyl)
indolin-1-yl)propyl benzoate
Acknowledgments
This work has been supported by the Austrian BMWFJ, BMVIT,
SFG, Standortagentur Tirol and ZIT through the Austrian
FFG-COMET-Funding Program. Financial support by NAWI Graz is
acknowledged. We thank Barbara Grischek for her ongoing support
in all projects.
O
O
CN
O
1''
1
N
HN
CF3
2
1'
3'
References
2'
3
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(1.7 mg, 14 mol). The reaction was quenched after 12 h by the
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Indoline
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l
l
l
addition of aqueous saturated NaHCO3 and then extracted three
times with CH2Cl2. The combined organic layers were dried over
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EtOAc = 80/20) to afford the product as a slightly yellow oil in
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61% yield (77 mg, 168 l
mol). RF [(PE/EtOAc) 60/40] = 0.41.1H
NMR (300 MHz, CDCl3): dH [ppm] = 1.20 (d,3J3 ,2 = 6.6 Hz, 3 H,
0
0
30-H), 2.17 (tt,3J2 ,1 = 6.5 Hz,3J2 ,3 = 7.1 Hz,
2
H, 200-H), 2.61
00 00
00 00
3
(dd,3J1 a,2 = 7.4 Hz,2J1 a,1 b = 13.8 Hz, 1 H, 10-Ha), 2.72 (dd, J1 b,2
=
0
0
0
0
0
0
3
5.9 Hz,2J1 b,1 a = 13.8 Hz, 1 H, 10-Hb), 2.97 (t, J3,2 = 8.7 Hz, 2 H,
0
0
3-Hind), 3.61 (t,3J2,3 = 8.7 Hz, 2 H, 2-Hind), 3.76 (t,3J3 ,2 = 7.2 Hz, 2
00 00
3
H, 300-H), 4.15 (mc, 1 H, 20-H), 4.47 (t, J1 ,2 = 6.3 Hz, 2 H, 100-H),
00 00
6.11 (d,3JNH,2 = 7.9 Hz, 1 H, NH), 6.93 (mc, 2 H, arom-Hind), 7.44
0
(mc, 2 H, arom.-H), 7.55 (mc, 1 H, arom.-H), 8.06 (mc, 2 H,
arom.-H). 13C NMR (75 MHz, CDCl3): dC [ppm] = 19.5 (C-30), 27.3
(C-200), 27.5 (C-3), 40.9 (C-10), 45.3 (C-100), 47.5 (C-20), 53.5 (C-2),
62.7 (C-300), 88.0 (C-7), 119.4 (CF3), 125.5 (CN), 128.8 (arom.-CH),
129.5 (arom.-CHind), 129.9 (arom.-CH), 130.3 (arom.-Cipso), 132.0
(arom.-CHind), 133.2 (arom.-Cipso), 133.3 (arom.-Cipso), 152.2
(COCF3), 166.8 (COO). 19F NMR (285 MHz, CDCl3): dF [ppm] =
ꢀ76.00 (s, 3 F, CF3).
8. Selected reviews (a) Kroutil, W.; Fischereder, E.-M.; Fuchs, C. S.; Lechner, H.;
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Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol. 2010, 28,
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4.5. Biotransformation (preparative scale)
Lyophilised cells of E. coli containing overexpressed
xTA from
(R)-Arthrobacter sp. (225 mg) were rehydrated in a KPi buffer
(9 mL, pH 7.0, 100 mM) containing PLP (1.00 mM), NAD+
(1.00 mM), ammonium formate (150 mM), FDH (11 U), Ala-DH
(12 U) and
D
-alanine (500 mM) at 22 °C for 30 min. Ketone 2

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