Chemoenzymatic Synthesis of Sertraline

Article abstract of DOI:10.1002/ejoc.201901810

A chemoenzymatic approach has been developed for the preparation of sertraline, an established anti-depressant drug. Ketoreductases (KREDs) were employed to yield a key chiral precursor. The bioreduction of the racemic tetralone exhibited excellent enantioselectivity (>99 % ee) and diastereomeric ratio (99:1) at 29 % conversion (the maximum theoretical yield is 50 %) after 7 hours. The resulting (S,S)-alcohol was efficiently oxidized to an enantiopure (S)-ketone, an immediate precursor of sertraline, by using sodium hypochlorite as oxidant and 2-azaadamantane N-oxyl (AZADO) as organocatalyst. Alternative routes aiming at the direct biocatalytic amination using imine reductases and transaminases were unsuccessful.

Full text of DOI:10.1002/ejoc.201901810

A Journal of
Accepted Article
Title: Chemoenzymatic Synthesis of Sertraline
Authors: Lisa Marx, Nicolas Rios-Lombardia, Philipp Süss, Matthias
Höhne, Francisco Moris, Javier Gonzales-Sabin, and Per
Berglund
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201901810
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10.1002/ejoc.201901810
European Journal of Organic Chemistry
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Chemoenzymatic Synthesis of Sertraline
Lisa Marx,a,b,c Nicolás Ríos-Lombardía,b Philipp Süss,c Matthias Höhne,d Francisco Morís,b
Javier González-Sabínb* and Per Berglunda*
N-methylimine precursor.[5] Having both these precedents and
Abstract: A chemoenzymatic approach has been developed for
the preparation of sertraline, an established anti-depressant
drug. Ketoreductases (KREDs) were employed to yield a key
chiral precursor. The bioreduction of the racemic tetralone
exhibited excellent enantioselectivity (>99% ee) and
diastereomeric ratio (99:1) at 29% conversion (the maximum
the current biocatalytic toolbox in mind, we envisaged alternative
chemoenzymatic approaches from the commercially available
racemic tetralone precursor 2 (Scheme 1). Given the structural
features of 1, the inherent enantiopreference of an enzyme
ideally should set both stereocenters of the molecule at once.
For this, we devised an approach starting from 2 based on
ketoreductases (KREDs), according to Scheme 1.
theoretical yield is 50%) after
7
hours. The resulting
(S,S)-alcohol was efficiently oxidized to an enantiopure
(S)-ketone, an immediate precursor of sertraline, by using
sodium hypochlorite as oxidant and 2-azaadamantane N-oxyl
(AZADO) as organocatalyst. Alternative routes aiming at the
direct biocatatalytic amination using imine reductases and
transaminases were unsuccessful.
Results and Discussion
Asymmetric bioreduction of rac-2 catalyzed by KREDs was
explored to yield the chiral alcohol 4 intermediate, followed by
re-oxidation and selective amination to yield (S,S)-1 (Scheme
1).[6,7] Ideally a KRED would be found that displays 4S-selectivity
of 2. This however means that inherently the maximum
theoretical yield for the target (S,S)-4, or alternatively (R,S)-4 is
50%, as it is assumed that no spontaneous racemization of 2
takes place. Accordingly, two different outcomes would be
Introduction
(S,S)-sertraline (1), (1S,4S)-4-(3,4-dichlorophenyl)-N-methyl-
1,2,3,4-tetrahydronaphthalen-1-amine,
is
the
active
anticipated for our synthetic purposes: On the one hand,
a
pharmaceutical ingredient (API) in Zoloft® (Pfizer), a drug for the
treatment of depression and anxiety.[1] The implemented
diastereo- and enantioselective bioreduction in which only one
enantiomer of the ketone reacts (50% conversion), namely (S)-2,
leading to a 1:1 mixture of the target alcohol (S,S)-4 or (R,S)-4
and remaining ketone (R)-2 (Scheme 2) or,
industrial process of
1 relies on a resolution of the racemic
tetralone precursor followed by diastereoselective reductive
amination.[2] With its two stereocenters, 1 is a challenging target,
and in addition, the rather low yield of the established
methodology, triggered us to seek new synthetic routes. The
excellent properties displayed by enzymes in terms of selectivity
and reactivity under mild reaction conditions make biocatalysis
an attractive alternative for the production of enantiopure
building blocks and pharmaceuticals.[3] Interestingly, the plethora
[a]
L. Marx, Prof. P. Berglund,
Department of Industrial Biotechnology,
KTH Royal Institute of Technology,
SE-106 91 Stockholm (Sweden)
Phone: +46 87907037
of reported syntheses of
1 includes one chemoenzymatic
approach based on a stereoselective enzyme- and ruthenium-
catalyzed dynamic kinetic asymmetric transformation (DYKAT)
employing Candida antarctica lipase B.[4] Very recently, Pfizer
unveiled an imine reductase (IRED) capable of producing
sertraline by diastereoselective reduction of the enantiopure (S)-
email: perbe@kth.se
www.kth.se/profile/perbe
[b]
L. Marx, Dr. N. Ríos-Lombardía, Dr. F. Morís, Dr. Javier González-
Sabín
EntreChem, S.L., E-33011 Oviedo (Spain)
Phone: +34 985 259 021
email: jgsabin@entrechem.com
https://entrechem.com/en/index.html
[c]
[d]
L. Marx, Philipp Süss
Enzymicals AG, DE-17489 Greifswald (Germany)
Prof. Matthias Höhne
Institute of Biochemistry
University of Greifswald, DE-17487 Greifswald (Germany)
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
1
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10.1002/ejoc.201901810
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2a) NaOCl,
AZADO
O
OH
HN
O
3i) MeNH2, EtOH
3ii) NaBH4
chemical step
1) KREDs
enzymatic step
2b) Laccase,
TEMPO or
AZADO
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
(S,S)-4
(S)-2
(S,S)-1
rac-2
cis/trans 99:1,
>99% ee
>99% ee
>99% ee
Scheme 1. Synthesis of the API Sertraline (S,S-1). The first step 1) is a KRED–catalysed selective bioreduction to the alcohol precursor (S,S)-4. The second step
can either be achieved chemically 2a) by NaOCl/AZADO or enzymatically 2b) by Trametes versicolor laccase/AZADO. Step 3) comprises of a direct amination
employing methylamine followed by reduction with NaBH4.
on the other hand, an enantio- but non-diastereoselective
process in which all the ketone is converted (100% conversion),
leading to a diastereoisomeric mixture of alcohols (S,S)-4 and
(S,R)-4. Taking the further purification into account, the first
scenario would be preferable due to the different polarity of
ketone and alcohol. The initial screening for the bioreduction of
rac-2 was performed using NADPH-dependent KREDs from the
Codex® KRED Screening Kit (standard conditions: 30 ºC and pH
7.5) with propan-2-ol for cofactor recycling and as co-solvent.
Upon these conditions and after 24 h, half of the enzymes in the
kit were very active and reached conversions higher than 90%
(Supporting Information). Regarding the enantioselectivity, some
KREDs displayed excellent selectivity towards the target cis-
enantiomer (S,S)-4 with ee up to 99%. However, none of the
enzymes displayed diastereoselectivity and a 1:1 cis:trans ratio
of 4 was obtained in all cases (Scheme 2). Further optimization
was aimed at finding conditions in which KREDs are also
diastereoselective (Table 1). For this, five KREDs from the initial
screening emerged as promising candidates, namely P1-B02,
P1-B05, P1-B10, P1-B12 and P1-A12 (Table 1, entry 6-10), with
regards to de and ee after pH optimization (pH 5-10; full results
in Supporting Information). As deduced from Table 1, the overall
conversion decreased at pH 10 (9.0 for P1-A12) for all KREDs
(entries 6-10). More interestingly, P1-B10, P1-B12 and P1-A12
additionally exhibited
a remarkable increase in selectivity
towards one of the desired isomers - (S,S)-4 - (entries 8-10) with
a cis:trans ratio up to 9:1. Accordingly, the specific conversion
towards (S,S)-4 turned out to be higher despite a lower overall
conversion for those variants. Likewise, a temperature screen
was conducted with the pH being kept at pH 7.5. At 50 ºC the
overall conversion decreased although the diastereo- and
enantioselectivity of the process was improved (entries 11-13).
In particular, P1-B10 and P1-B12 led to (S,S)-4 with cis:trans
ratios of 10:1 and 16:1, respectively, with enantiomeric excess of
the cis isomers of >99 % (entries 11-12).
Scheme 2. Bioreduction of racemic tetralone (rac-2) by NADPH-dependent KREDs. The four possible diastereomers are shown with the target alcohol isomers
(S,S)-4 (continuous) and (R,S)-4 (dashed) framed. The co-factor NADPH is self-regenerated by the KREDs using 2-propanol as the sacrificial co-substrate.
2
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Table 1. Optimisation of the KRED bioreduction of rac-2 to (S,S)-4.[a]
Entry
KRED
[2] (mM)
pH
T (°C)
conversion (%)[b]
cis:trans[b]
ee (%)[b]
cis
trans
1
2
P1-B02
P1-B05
P1-B10
P1-B12
P1-A12
P1-B02
P1-B05
P1-B10
P1-B12
P1-A12
P1-B10
P1-B12
P1-A12
P1-B10
P1-B12
P1-B12
P1-B12
20
20
20
20
20
7
7.5
7.5
7.5
7.5
7.5
10
10
10
10
9
30
30
30
30
30
30
30
30
30
30
50
50
50
50
50
50
50
92
40
80
74
32
87
27
29
41
26
29
39
15
43
34
34
29[d]
1:1
1:1
85 (S,S)
99 (S,S)
93 (S,S)
89 (S,S)
96 (S,S)
80 (S,S)
99 (S,S)
95 (S,S)
96 (S,S)
98 (S,S)
>99 (S,S)
>99 (S,S)
95 (S,S)
98 (S,S)
96 (S,S)
99 (S,S)
>99 (S,S) [d]
80 (S,R)
87 (S,R)
85 (S,R)
65 (S,R)
88 (S,R)
69 (S,R)
79 (S,R)
44 (S,R)
43 (S,R)
87 (S,R)
66 (S,R)[c]
54 (S,R) [c]
95 (S,R)
57 (R,S)
52 (R,S)
18 (S,R)
n.d.
3
1:1
4
1:1
5
1:1
6
1:1
7
7
1:1
8
7
9:1
9
7
9:1
10
11
12
13
14
15
16
17
7
3:1
7
7.5
7.5
7.5
7.5
7.5
9
10:1
16:1
1:4.5
9:1
7
7
55
55
55
7
4:1
16:1
99:1[d]
10
[a] Reaction conditions: 2 (variable concentration), KRED (100% w/w), KPi buffer 125 mM pH 7.5 (1.25 mM MgSO4, 1 mM NADPH), DMSO (5% v/v), i-PrOH (15% v/v), 250 rpm. [b] conversion, cis:trans ratio and ee
were determined simultaneously by chiral HPLC analysis at 24 h reaction time. [c]due to low concentrations high standard deviations in this value. [d] Conversion and stereoselectivity determined at 7 h reaction time.
3
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10.1002/ejoc.201901810
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Next, to get more insight into the process, we tested higher Experimental Section
substrate concentrations. A higher loading of rac-2 such as
55 mM produced a slight decrease of diastereomeric ratio and
ee (entries 14-16). Finally, the synergistic effect of higher
temperature and pH, namely 50 ºC and pH 10, was evaluated
for KRED-P1-B12 at 7 mM and 55 mM (entries 16-17). At the
lower concentration and after 7 h only cis-(S,S)-4 was formed
in >99% ee and 29% conversion (entry 17).
General Remarks
A
kit of KREDs was purchased from Codexis. All other commercial
chemicals and enzymes were purchased from Sigma Aldrich. For the
biotransformations, commercially available solvents were used. Thin-
layer chromatography was performed on precoated TLC plates of Merck
silica gel 60F254, using potassium permanganate as developing
reagent. For column chromatography, Merck silica gel 60 (particle size,
After separating the resulting enantiopure alcohol (S,S)-4 from
the remaining R-ketone by flash chromatography, next step
consists of re-oxidizing this alcohol to the ketone (S)-2. Two
catalytic methods proved to be effective. The enzymatic
oxidation employing the laccase from Trametes versicolor with
either TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) or
AZADO (2-azaadamantane-N-oxyl) as mediator reached full
conversion in 16 h at room temperature.[8],[9] Alternatively, the
combination of sodium hypochlorite (NaOCl) as oxidant and
AZADO as organocatalyst (0.1 mol-%) in a biphasic medium
(buffer:trifluorotoluene) oxidized quantitatively (S,S)-4 to ketone
(S)-2 in 1 h at room temperature (95% yield).[10] In the last step
the methylamine group is selectively added in quantitative yield
following a reported synthetic procedure consisting of the imine
formation followed by Raney-Ni-catalyzed hydrogenation,[11] the
target compound Sertraline, (S,S)-1 being obtained in 16%
overall yield and >99% ee.
40
- 63 µm) was used. 1H NMR and proton-decoupled 13C NMR
spectra were obtained using a 400 MHz NMR spectrometer using the δ
scale (ppm) for chemical shifts; calibration was made on the CDCl3
(13C; 76.95 ppm) or the residual CHCl3 (1H; 7.26 ppm) signals. HPLC
analyses to determine degree of conversions were carried out in an
Agilent RR1200 HPLC system, using a reversed phase column (Zorbax
Eclipse XDB-C18, RR, 18μm, 4.6 x 50 mm, Agilent). HPLC analyses to
determine ee were performed on
a Hewlett Packard 1100 LC liquid
chromatograph, using normal phase columns (Daicel Chiralcel OD-H
and Daicel Chiralpak OJ-H).
Bioreduction of 2 employing KREDs
We have also explored alternative routes aiming at the direct
biocatatalytic amination of rac-2 using imine reductases and
transaminases but these routes were unsuccessful (Supporting
Information).
Initial screening: In
a 1.5 mL Eppendorf tube, KRED (1 mg), rac-2
(20 mM), DMSO (50 µL or as indicated) and i-PrOH (200 µL or as
indicated) were added to 900 µL of 100 mM KH2PO4 buffer (1.25 mM
MgSO4, 1 mM NADP+) pH 7.0 or 900 µL of 250 mM KH2PO4 buffer
(2 mM MgSO4, 1.1 mM NADP+, 80 mM D-glucose, 10 U/mL glucose
dehydrogenase) pH 7.0 in the case of KRED-101, KRED-119, KRED-
130, KRED-NADH-101 and KRED-NADH-110 (or as specified in the SI).
The reaction was shaken at 250 rpm and 30 ºC for 24 h. To determine
the conversion, 10 µL of the mixture was diluted with 90 µL of Milli-Q
water and analyzed by achiral reverse phase HPLC with previous
centrifuging and filtering of the sample. Then the mixture was extracted
with ethyl acetate (3x500 μL), and the ee and de were measured by
HPLC in chiral normal phase.
Conclusions
A chemoenzymatic route towards sertraline has been explored
from a racemic ketone precursor providing an alternative route
to the existing processes. In
a KRED-based approach, the
stereoselectivity could be optimized to 99:1 cis:trans ratio and
>99% ee of the desired alcohol stereoisomer (S,S)-4, which
was obtained at 29% conversion. The improvement could be
achieved solely by condition optimization without resorting to
protein engineering methods. Further organo-catalytic oxidation
with a NaOCl/AZADO system provided nearly quantitatively the
corresponding enantiopure ketone (S)-2 within 1 h at 0.1 mol-%
mediator loading. Finally, reaction with methylamine and
selective chemical hydrogenation gave the final product.
Overall, this new three-step chemo-enzymatic route yielded the
final product (S,S)-1 in 16% overall yield from the racemic
ketone.
Oxidation of (S,S)-4 to (S)-2
Chemical oxidation
Borttaget: ¶
To a solution of (S,S)-4 (20 mg, 0.08 mmol) and AZADO (0.1 mol-%) in
PhCF3 (150 μL), a 0.40 M solution of NaOCl (pH = 8.9; 1.0-1.4 equiv)
was added. The mixture was vigorously stirred at room temperature
during 1 h. Once the starting material disappeared (TLC control), the
reaction mixture was extracted with ethyl acetate (3 ´ 600 μL). 1H-NMR
analysis of the crude product showed the corresponding ketone (S)-2 in
high purity.
4
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10.1002/ejoc.201901810
European Journal of Organic Chemistry
FULL PAPER
[1]
a) B. K. Koe, A. Weissman, W. M. Welch, R. G. Browne, J.
Enzymatic oxidation
Pharmacol. Exp. Ther. 1983, 226, 686–700; b) W. M. Welch, A. R. Kraska, R.
Sarges, B. K. Koe, J. Med. Chem. 1984, 27, 1508–1515.
To a solution of (S,S)-4 (15 mg, 0.05 mmol) in citrate buffer pH 5.5 (900
μL) and DMSO (100 μL) was added TEMPO or AZADO (10 mol-%) and
the mixture was stirred vigorously in an open-to-air vial at 20 ºC for 16 h.
The reaction mixture was extracted with ethyl acetate (3 ´ 600 μL). 1H-
NMR analysis of the crude product showed the corresponding ketone
(S)-2 in high purity.
[2]
a) W. M. Welch, C. A. Harbert, B. K. Koe, A. R. Kraska,
Derivatives of Cis-4-Phenyl-1,2,3,4-Tetrahydro-1-
Antidepressant
Naphthalenamine, 1985, US4536518A; b) M. T. William, G. Quallich, J.
Chem. Ind. 1990, 10, 315-319; c) G. J. Quallich, R. C. Friedmann, M. T.
Williams, J. Org. Chem. 1990, 55, 4971–497.
[3]
a) S. Pedragosa-Moreau, A. Le Flohic, V. Thienpondt, F. Lefoulon, A.-
M. Petit, N. Ríos-Lombardía, F. Morís, J. González-Sabín, Adv. Synth. Catal.
2017, 359, 485–493; b) L. Marx, N. Ríos-Lombardía, J. F. Farnberger, W.
Kroutil, A. I. Benítez-Mateos, F. López-Gallego, F. Morís, J. González-Sabín,
P. Berglund, Adv. Synth. Catal. 2018, 360, 2157-2165; c) J. Albarrán-Velo, D.
González-Martínez, V. Gotor-Fernández, Biocatal. Biotransform. 2018, 36,
102-130.
Sertraline [(S,S)-1]
(S)-2 (30 mg, 0.10 mmol) was dissolved in 3.0 mL of a methylamine
solution (2.0 M in methanol) and the mixture refluxed during 6 h. After
this time, evaporation to dryness gave access to the corresponding N-
methylimine. Without further purification, this intermediate was
dissolved in methanol (2.0 mL) and reduced by conventional treatment
with NaBH4 (10 mg, 0.15 mmol). After purification by flash
chromatography, (S,S)-1 was obtained in excellent yield (28 mg, 92%).
[4]
P. Krumlinde, K. Bogár, J. E. Bäckvall, Chem. Eur. J. 2010, 16, 4031–
4036.
[5]
D. L. Hughes, Org. Process Res. Dev. 2018, 22, 1063–1080.
[6]
M. J. Sorgedrager, F. van Rantwijk, G. W. Huisman, R. A. Sheldon,
Adv. Synth. Catal. 2008, 350, 2322–2328.
[7] Y.-C. Thai, A. Szekrenyi, Y. Qi, G. W. Black, S. J. Charnock, W.-D.
Fessner, Bioorg. Med. Chem. 2018, 26, 1320–1326.
[8] L. Martínez-Montero, V. Gotor, V. Gotor-Fernández, I. Lavandera,
Green Chem. 2017, 19, 474–480.
[9] For an alternative enzymatic oxidation procedure see: T. Eggert, D.
Acknowledgements
Bakonyi, W. Hummel, J. Biotechnol. 2014, 191, 11-21.
LM, PS, FM, JGS and PB acknowledge funding from the
European Union’s Horizon 2020 MSCA ITN-EID program
[10] a) E. Liardo, N. Ríos-Lombardía, F. Morís, F. Rebolledo, J. González-
Sabín, ACS Catal. 2017, 7, 4768–4774; b) E. Liardo, N. Ríos-Lombardía, F.
Morís, J. González-Sabín, F. Rebolledo, Eur. J. Org. Chem. 2018, 23, 3031–
3035.
under
grant
agreement
No
634200
(Project
BIOCASCADES). We acknowledge Prof. Dr. Sebastian
Günther and Dr. Merdivan (University of Greifswald) for
providing access to HPLC.
[11] For alternative procedures, see: a) G. Wang, C. Zheng, G. Zhao,
Tetrahedron: Asymm. 2006, 17, 2074-2081; b) D. Talwar, N. P. Salguero, C.
M. Robertson, J. Xiao, Chem. Eur. J. 2014, 20, 245–252; c) C. Wang, A.
Pettman, J. Basca, J. Xiao, Angew. Chem. Int. Ed. 2010, 49, 7548–7552.
Keywords: Ketoreductases, AZADO, Asymmetric synthesis,
Sertraline, Biocatalysis
5
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FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Lisa Marx,a,b,c Nicolás Ríos-Lombardía,b
Philipp Süss,c Matthias Höhne,d
Francisco Morís,b Javier González-
Sabín,b* and Per Berglunda*
A
chemoenzymatic approach has
chemical
step
been developed for the preparation of
sertraline. Ketoreductases (KREDs)
were employed to yield a key chiral
precursor. The bioreduction of the
racemic tetralone exhibited excellent
enantioselectivity (>99% ee) and
diastereomeric ratio (99:1) at 29%
conversion.
O
OH
HN
O
KREDs
Cl
Cl
Cl Page No. – Page No.
Cl
enzymatic
step
Cl
Cl
Cl
Cl
Sertraline
Chemoenzymatic Synthesis of
Sertraline
6
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