Biocatalytic approaches to a key building block for the anti-thrombotic agent ticagrelor

Article abstract of DOI:10.1039/c6ob01382a

Three complementary biocatalytic routes were examined for the synthesis of the cyclopropyl amine (1R,2S)-2, which is a key building block for the anti-thrombotic agent ticagrelor 1. By employing either a ketoreductase, amidase or lipase biocatalyst, the k

Full text of DOI:10.1039/c6ob01382a

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DOI: 10.1039/C6OB01382A
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Biocatalytic Approaches to a Key Building Block for the Anti-
Thrombotic Agent Ticagrelor
Katharina G. Hugentoblera,b, Humera Sharifa, Marcello Rasparinic,d, Rachel S. Heatha and Nicholas
Received 00th January 20xx,
Accepted 00th January 20xx
J. Turnera*
DOI: 10.1039/x0xx00000x
Three complementary biocatalytic routes were examined for the synthesis of the cyclopropyl amine
(1R, 2S)-2, which is a key building block for the anti-thrombotic agent ticagrelor 1. By employing
either a ketoreductase, amidase or lipase biocatalyst, the key building blocks for synthesis of the
amine 2 were obtained in 99.9, 94.6 and 48.8 % e.e., respectively.
www.rsc.org/
The first approach envisaged was to explore asymmetric
reduction of ketone 3 to either (R)- or (S)-4 using a
ketoreductase (KRED) (Scheme 1[a]). Actavis recently
Introduction
reported9 enantioselective reduction of ketone 3 to alcohol
(R)-4 using CBS-borane. Subsequent cyclopropyl ring
formation via a Mitsunobu type reaction yielded the
nitrocyclopropane (1R,2S)-5 which was then reduced to the
desired amine 2 with an e.e. = 99% (Scheme 2). The (S)-
alcohol has also been converted to the target amine (1R,2S)-2
via initial bromination to give the bromide 6 followed by
cyclopropane formation with DBU and reduction of the nitro
group.
During the past two decades, the pharmaceutical industry
has sought to address environmental challenges associated
with manufacturing by embracing the principles of green
chemistry1. One of these guidelines is directed towards
reducing the waste2 while another addresses the use of
reagents from sustainable sources to render a process more
environmentally sustainable. In this context biocatalysis
represents an attractive option in that enzymes offer the
possibility of highly selective (enantio-, regio-, chemo-)
transformations based upon sustainable catalysts with high
catalytic rates of turnover. Ticagrelor 1 is one of the most
potent drugs on the market for the treatment of acute
coronary syndrome. The cyclopropyl amine 2 represents a
challenging target, in particular to explore and ultimately
compare alternative biocatalytic routes for its synthesis.
Previously reported routes to the amine 2 include the use of
chiral auxiliaries (e.g. ruthenium based chiral ligands3,
Oppolzer’s chiral camphorlactam4,5,or menthol6,7) as well as
kinetic resolution of the corresponding racemate by
formation of diastereomeric salts4,6. We opted to employ a
biocatalytic retrosynthetic approach8 in order to develop and
evaluate potential routes towards the target compound
(Scheme 1).
Scheme 1: Biocatalytic approaches towards the target
cyclopropylamine 2.
Results and Discussion
a.
School of Chemistry, University of Manchester, Manchester Institute of
Biotechnology, 131 Princess Street, Manchester M1 7DN, UK.
Institute of Pharmaceutical Chemistry, University of Freiburg,
Albertstraße 25, 79104 Freiburg, Germany
Chemessentia Srl., Via G. Bovio 6 - 28100 Novara, Italy
Janssen Pharmaceutica API small molecule development,
Turnhoutseweg 30, B-2340 Beerse, Belgium
We initially screened a number of ketoreductases from the
Codexis Codex® KRED Screening Kit as well as the RasADH
from Ralstonia sp.10,11 for activity towards 3. We were able to
identify biocatalysts for generation of both the (R)- and (S)-
alcohol 4 with good to excellent e.e. (Table 1). KRED-130
b.
c.
d.
e.
† Electronic Supplementary Information (ESI) available: experimental details and
compound characterisation. See DOI: 10.1039/x0xx00000x
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(Codexis) proved to be the best candidate (entry 1) yielding has previously been reported to undergo a sVtieewreAortsicpleeOcnilfinice
6,13
DOI: 10.1039/C6OB01382A
the (S)-enantiomer with an enantiomeric excess of 99.9% and Curtius rearrangement to yield (1R, 2S)-2
. The
100% conversion after 48h. The use of 10% DMSO enhanced microorganism Rhodococcus rhodochrous has previously
the solubility of the substrate and although alternative been shown to hydrolyse cyclopropyl-containing
systems for NADPH co-factor recycling were examined, the carboxamides with high enantioselectivity14–16 and thus we
use of glucose/glucose dehydrogenase (GDH) proved to be prepared racemic amide 7 and tested it as a substrate using
optimal (data not shown). RasADH was also found to yield whole cells of R. rhodochrous IFO 15564. In agreement with
(S)-4 although with lower e.e. Using KRED 110 (Codexis), the previous studies the amidase was found to exhibit (S)-
(R)-alcohol 4 can also be prepared but with low conversion selectivity towards 7 yielding (1S, 2S)-acid 8.
(23%) and modest e.e. (71.6%). Although the
enantioselectivities and conversions obtained with the KRED
were good, the catalyst loading in this approach suggested
further optimisation would be required. In contrast to a
theoretical 100% yield in an asymmetric approach such as [a],
hydrolase-mediated approaches are limited to a 50% yield
maximum. However we weighted catalyst loading against
theoretical yields and shifted our attention to approaches [b]
and [c] (Scheme 1).
In order to optimise conversion, the effect of different cell
densities (OD600), co-solvents and temperature were
examined (see table 2). Amide 7 dissolved readily in methanol
and ethanol, but partially precipitated after coming into
contact with the aqueous reaction medium. The precipitate
disappeared over the course of reaction as the amide was
hydrolysed by the amidase. In the absence of co-solvent, the
reaction medium formed a suspension between the cell
suspension and the hydrophobic amide. Under these
conditions, it is likely that only small amounts of substrate
will have dissolved, thereby creating a substrate/catalyst ratio
similar to that attained in a reaction performed at high cell
density.
Scheme 2: Ketoreductase mediated preparation of (R)- and
(S)-alcohol 4.
Table 2: Hydrolysis of amide rac-7.
E
OD
T
t
Entry 600 [°C] [h]
ees
eep conv.
(Config.)
1
2
1.5
1
26
26
26
26
26
4
23
23
23
20
20
26
97.8 43.2
96.8 60.2
69.4
61.7
10 (S)
16 (S)
17 (S)
3
0.5
98.0 60.0 62.0
4
5a
0.25
0.25
0.25
0.25
92.5 86.6
11.8 88.7
32.0 94.9
51.6
11.7
46 (S)
19 (S)
6
7b
52.8 (S)
16.7 (S)
25.2
48.8
4
16.7 95.3 14.9
a no co-solvent, b Methanol (10% v/v) was used as a co-solvent.
The data in Table 2 shows an inverse relationship between
the enantioselectivity of the hydrolysis and the cell loading.
A possible explanation for this phenomenon is related to the
uptake of the substrate. Resting cells are employed in these
experiments, therefore the observed amidase activity has to
be due to an intracellular enzyme (extracellular enzymes are
removed through several washing steps). Although we were
able to attain both amide and carboxylic acid in high
enantiomeric purity and with a conversion close to the
theoretical 50% 8 (table2 entry 4) via this approach, the
comparably lower yields obtained prevented further scale-up
of this method.
Table 1: KRED mediated reduction of ketone 3.
Entry
1
Enzyme
T [C] t [h]
conv.
100
ee (Config.)
KRED-
130
30
48
99.9 (S)
2
3
KRED-110
RasADH
30
30
48
48
23
34
71.6 (R)
91.6 (S)
As a third approach, lipase mediated hydrolysis of the
corresponding racemic ethyl ester 9 (Scheme 1, [c]) was
investigated, with the goal of identifying either an (R)- or (S)-
selective biocatalyst for the generation of the corresponding
enantiomerically pure recovered ester or carboxylic acid
product (Scheme 3). In either case the target would be the
For subsequent approaches to amine 2, hydrolase mediated
kinetic resolutions of suitable precursors12 were explored to
access the acid 8 (Scheme 1 [b] and [c]). The (1R, 2R)-acid 8
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carboxylic acid (1R, 2R)-8 which as before could be converted
to the target amine 2 via a Curtius rearrangement.
Experimental
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DOI: 10.1039/C6OB01382A
Ketoreductases Conditions for the KRED-130 (Codexis):
Reactions were performed in 100 mM potassium phosphate
buffer (pH 7) with 5 g/L ketone 3 in 10% DMSO, 5g/L
ketoreductase, 1 g/L GDH, 1 g/L NADPH, 1.25 molar
Scheme 3: lipase mediated hydrolysis of precursor ester 9.
equivalents of glucose. The reaction was kept stirring
(magnetic agitation 500 rpm) at 30ºC for 48 h. Conditions for
the RasADH (W. Kroutil, I. Lavandera): The lyophilised enzyme
was rehydrated in buffer for 30 min prior to the addition of the
substrate. RasADH screens were performed in 50 mM Tris HCl
buffer pH 7.5 with 8 g/L ketone 3, 37 g/L ketoreductase, 0.7
g/L NADPH, 1 g/L, 2 molar equivalents of glucose. The reaction
was kept stirring (magnetic agitation 500 rpm) at 30ºC for 48
h. Workup: All reactions were stopped in all cases by
extraction of the aqueous phase with two volumes of MTBE.
The organic phases were combined and dried over MgSO4 and
the crude was analysed via HPLC and NMR.
Hydrolases Amidase-mediated hydrolysis of compound 7: A
suspension of arrested cells in a 0.1 M phosphate buffer at pH
7.0 was used for all biotransformations performed. The
bacterial solutions employed showed an OD650 of 1.5, 1.0, 0.5
and 0.25, respectively. Amide rac-7 was added to the bacterial
solution in 10% (w/v) ethanol to obtain a final concentration of
0.1 % (w/v). The reactions were shaken for 22h (orbital shaker,
temperatures as indicated) and were then stopped by
centrifugation and decantation of the supernatant. The
resulting aqueous solution was basified with NaHCO3 and
extracted with dichloromethane by shaking and centrifugation.
Subsequently the resulting aqueous phase was acidified (1 N
HCl) and again extracted with AcOEt. The organic phases were
dried over MgSO4 and the solvent evaporated in a rotary
evaporator. The compounds were dried under vacuum and
could be isolated in pure state with yields of ≥ 90%. Lipase-
mediated ester hydrolysis of compound rac-9: 1ml Screens
were performed in MTBE in a 2 mL Eppendorf tube. The
substrate ester rac-9 was added to the solvent (1 mL) as a 0.5%
(w/v; 5 mg) solution. The enzyme was added to give a 5% (w/v;
50 mg enzyme suspension) solution. The reactions were left to
shake in an orbital incubator at 30°C for 48h. Medium scale
reactions were performed in phosphate buffer 0.1M, pH 7.0 (5
mL) and 10% co-solvent in a 10 mL flask. The substrate ester
rac-9 was added as a 1% (w/v; 50 mg) solution to the solvent.
The enzyme was added to give a 2% (w/v; 100 mg enzyme
suspension) solution. The reactions were stirred at room
temperature (18°C) for 48h.
A number of lipases and esterases from different commercial
sources (Almac, Johnson Matthey) were screened, most of
which showed no activity and/or enantioselectivity towards
9. However, the lipase from Thermomyces lanuginosus (TlL)
showed good activity on an initial small scale screen (5 mg
ester rac-9, 5 wt% lipase in 1 mL MTBE, 48h at 30°C orbital
shaking) and yielded the desired (1R, 2R)-ester 9 and (1S, 2S)-
acid 8 with ees = 14.7 and eep = 99.9 (conv. = 12.8%, E > 200).
Based upon these initial results the reaction was
subsequently scaled up and optimised with respect to co-
solvents (Table 3).
Table 3: use of co-solvents in TlL mediated hydrolysis of rac-
9.
E
Entry
co-solvent
ees
eep
conv.
(Config.)
1
2
3
4
AcOEt
MTBE
0
0
0
0
3.1
76.5
96.9
99.1
3.9
8 (S)
Cyclohexane 46.3
buffer 18.4
32.3
15.6
101 (S)
> 100 (S)
Lipase activity is known to be enhanced by interfacial
activation17 and hence the reactions were performed in 10%
water-immiscible co-solvents18. In ethyl acetate no hydrolysis
of ester 9 was observed. Addition of 10% (v/v) methyl tert-
butyl ether (MTBE) had a negative influence on the reaction
compared to a biotransformation performed only in buffer
(cf. table 3, entry 2 and 4). Finally, addition of cyclohexane
seemed to have a beneficial influence on the conversion of
the biotransformation. The degree of conversion was double
compared to the conversion obtained in a buffered system
without co-solvent (cf. table 3, entry 4).
Conclusion
Three complementary biocatalytic approaches have been
examined towards the key intermediate cyclopropylamine 2
used in the synthesis of Ticagrelor. While the KRED-
mediated approach afforded alcohol-4 with good to excellent
enantioselectivities, the catalyst loading was somewhat high.
The hydrolase-mediated approaches closely met the
theoretical 50% yield. The amidase from R. rhodochrous
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yielded the target amide (1R, 2R)-7 in acceptable
enantiomeric purity, although the comparably lower yields
obtained prevented further scale-up of this method. The
enantioselectivities and conversions obtained in the lipase-
mediated approach show high potential for further process
optimisation and thus represent a key step in applying
enzyme technologies for the synthesis of the anti-thrombotic
agent Ticagrelor.
75, 6614–24.
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Acknowledgements
The authors thank Dr. Iván Lavandera for a sample of the
RasADH and Dr. Nicholas Weise for fruitful discussion and
suggestions. KGH thanks CHEMO for funding. NJT thanks the
Royal Society for a Wolfson Research Merit Award.
Notes and references
‡ ees and eep were determined by chiral HPLC, conversion was
푒푒푠
determined as c =
, and Enantioselectivity E determined
푒푒푠+ 푒푒푝
19
from ees and eep
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