Assessing biocatalysis for the synthesis of optically active tetrahydropyrazolo[1,5-α]pyrimidines (THPPs) as novel therapeutic agents

Article abstract of DOI:10.1016/j.molcatb.2013.11.012

Enantiomerically pure THPPs are useful pharmacological agents in relevant therapeutic areas such as tuberculosis, osteoporosis and hepatitis C. Typically resolved by preparative chiral HPLC, in this work the catalytic approach - via hydrolase-catalyzed ki

Full text of DOI:10.1016/j.molcatb.2013.11.012

Journal of Molecular Catalysis B: Enzymatic 100 (2014) 1–6
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Assessing biocatalysis for the synthesis of optically active
tetrahydropyrazolo[1,5-␣]pyrimidines (THPPs) as novel
therapeutic agents
Elena Fernández-Álvaroa,∗, Jorge Esquiviasa, María Pérez-Sánchezb,
Pablo Domínguez de Maríab, Modesto J. Remuin˜án-Blancoa
a Tres Cantos Medicines Development Campus (TCMDC), Diseases of the Developing World, GlaxoSmithKline, c/Severo Ochoa, 2.
Tres Cantos, 28760 Madrid, Spain
b Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringer Weg 1., 52074 Aachen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Enantiomerically pure THPPs are useful pharmacological agents in relevant therapeutic areas such as
tuberculosis, osteoporosis and hepatitis C. Typically resolved by preparative chiral HPLC, in this work the
catalytic approach – via hydrolase-catalyzed kinetic resolution – has been demonstrated for the first time.
After biocatalytic successful proof-of-concept, future opportunities for protein engineering and process
set-up appear.
Received 20 September 2013
Received in revised form
11 November 2013
Accepted 12 November 2013
Available online 27 November 2013
© 2013 Elsevier B.V. All rights reserved.
Keywords:
CAL-B
Enantioselective biotransformations
Tetrahydropyrazolo[1,5-␣]pyrimidines
Therapeutic agents
1. Introduction
than the (S)-enantiomer [1a], in the case of hepatitis C inhibitors
(5S,7R)-isomers are the active form [2] and as TB inhibitors the
The 2,4-disubstituted tetrahydropyrazolo[1,5-␣]pyrimidine
(THPP, Fig. 1) is a chemical scaffold with increasingly impor-
tant biological and pharmacological activities. A broad number
of THPP-derivatives have found use as modulators of calcium-
sensing receptors (CaSR) with potential to deliver agents to
treat osteoporosis (CaSR antagonists) [1], or to prevent and
treat viral infections and diseases, especially those caused by
hepatitis C virus [2]. Remarkably, in addition to these uses,
hits from these chemical series active against Mycobacterium
tuberculosis have been recently identified in several phenotypic
screenings [3], and medicinal chemistry approaches have yielded
promising structures with in vivo anti-tubercular properties
[4].
(5R,7S)-THPPs are at least two orders of magnitude more active
than the (5S,7R)-compounds [4]. Furthermore, the regulations of
enantiopurity applied by Food and Drug Administration (FDA) for
novel pharmaceuticals [5] force to the development of optically
active THPPs for its further therapeutic use and commercializa-
tion. Herein, preparative chiral HPLC has been always industrially
used for the separation of these enantiomers, regardless of the
final medical application (Scheme 1). Albeit effective, such chro-
matographic techniques may involve tedious and time-consuming
processes, while contributing significantly to solvent consump-
tion and waste formation. Actually, solvents account for 80% of
material use and waste production in the manufacture of active
pharmaceutical ingredients (APIs) [6]. Conclusively, due to the
emerging importance of THPPs as therapeutic agents, the set-up
of more environmentally friendly and practically oriented strate-
gies in the production of optically active THPPs would be highly
desirable.
In the chemical synthesis of THPP series, hydrogenation of the
pyrimidine ring leads to a racemic mixture of cis-enantiomers
(Scheme 1). Whereas in the case of THPPs as CaSR antagonists the
(R)-enantiomer displays 10-fold higher pharmacological activity
To our knowledge, asymmetric catalysis – in its broadest sense
– has never been assessed for the chirality generation/resolution
in THPPs. Among the catalytic strategies for asymmetric synthesis,
biotransformations have emerged in the last decades as a powerful
∗
Corresponding author. Tel.: +34 650 42 16 87.
E-mail address: elena.a.fernandez-alvaro@gsk.com (E. Fernández-Álvaro).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.11.012

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E. Fernández-Álvaro et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 1–6
R3
N
N
R2
R4
N
CF3
R1
N
THPP
N
N
N
N
H
N
H
NH
O
NH
O
CaSR antagonists
(osteoporosis)
OCH3
(Anti-tuberculars)
CF3
O
N
N
HN
N
H
O
S
Antivirals
(for hepatitis C)
Fig. 1. General structure of tetrahydropyrazolo[1,5-␣]pyrimidine (THPP), with several concrete examples of pharmaceuticals [1–4].
and economically attractive option for the provision of optically
active drugs and building blocks, under mild and environmen-
tally friendly conditions [7]. Typically, enzymes are introduced
as the core step for the chirality generation/resolution within a
multi-step synthetic protocol. A recent relevant example is the
industrial implementation of a transaminase-catalyzed process for
the sitagliptin synthesis. In this case, a combination of a cosolvent
(DMSO) to solubilize the substrate in the aqueous solution, together
with an engineered enzyme variant, led to great results both in
activity and enantiomeric excess [8a,b]. Likewise, that example
shows the tremendous potential that biocatalysis may have for
pharmaceuticals, starting from an inefficient wild-type enzyme,
and ending up with a robust industrial process, once reaction set-
up and protein engineering are judiciously aligned [7a,8]. Although
some outstanding examples of enzymes being used at industrial
scale have been described [8], the number of applications of bio-
catalysis to industrially interesting substrates remains to be low for
its envisaged potential.
2. Experimental
2.1. Materials
All chemicals were purchased from Sigma–Aldrich. Enzymes
were purchased from Sigma–Aldrich, Enzagen (Cambridge, United
Kingdom) or kindly donated by Novozymes (Bagsvaerd, Denmark)
and c-LEcta (Lepzig, Germany).
2.2. Analytics
Achiral UPLC–MS analysis was performed using an Acquity BEH-
C18 1.7 ␮m 2.1 × 50 mm column (Waters), in a Waters Acquity
UPLC (Waters) coupled to an API 2000 Q-Trap (Applied Biosystems).
Mobile phase ACN, 0.1% formic acid. Chiral HPLC analyses were per-
formed in a Chiralpak IC column 4.6 × 250 mm (Chiral Technologies
Europe Cedex, France) in a HPLC 1200 from Agilent (Boeblingen,
Germany), using different mixtures of hexane, ethyl acetate and
TFA as mobile phases.
Within a lead optimization program of THPPs as antitubercular
agents [3a,4a,c], we considered the relevance of applying biocatal-
ysis for the synthesis of optically active THPPs. Following these
considerations, in this work the biocatalytic kinetic resolution of
optically active THPPs is reported for the first time. To this end,
hydrolases were assessed to perform the kinetic resolution of the
formed racemic esters in aqueous solutions.
2.3. Deep Eutectic Solvent (DES) synthesis
Choline chloride and hydrogen-bond-donors (HBD) were mixed
in different equimolar ratios and gently stirred and heated up to
80 ◦C, until the DES was formed (30–90 min). The water content of
Scheme 1. Synthesis for the provision of optically active THPPs with therapeutic uses.

E. Fernández-Álvaro et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 1–6
3
Fig. 2. Lipase-catalyzed hydrolysis of THPP-ester 1a using CAL-B under screening conditions. Conditions: 2.5 ␮M substrate, phosphate buffer saline 10 mM (pH 7.4), 30 ◦C,
1000 rpm.
the DESs was determined by Karl Fischer titration at 20 ◦C, and was
typically found between 0.5 and 1.0% (w/w).
90:10:0.1 as mobile phase for compounds 1a–1b, and 95:5:0.1
for compounds 2a–2b and 3a–3b. Conversion and E values were
then calculated from the enantiomeric excesses from substrate and
product following equations reported in the literature [9].
2.4. Activity screening
The hydrolytic activity of a battery of 50 enzyme prepara-
tions against substrate 1a was tested under screening conditions:
300 ␮L of a 2.5 ␮M solution of 1a in PBS (phosphate buffer saline,
10 mM, pH 7.4). Reactions were stirred at 30 ◦C and 1000 rpm in a
Thermomixer for 48 h. After this time, 100 ␮L sample were taken
and mixed with 150 ␮L precipitating agent (methanol:aceonitile,
3:1, 1 ␮M Resorufin as internal standard) and 50 ␮L 0.1% formic
acid in 96-well microtiter plates. Plates were centrifuged at
3700 rpm and 10 ◦C for 15 min and reactions were analyzed by
UPLC–MS.
2.7. General procedure for the synthesis of
tetrahydropyrazolopyrimidine derivatives 1a–3a
Ethyl esters of THPPs 1a–3a were synthesized as previously
described [1]. For the synthesis of compound 1a, a mixture of ethyl
3-aminopyrazole-4-carboxylate (0.635 g, 4.090 mmol) and 1-(4-
ethyl-phenyl)-4,4,4-trifluoro-butane-1,3-dione (1 g, 4.090 mmol)
in AcOH (10 mL) was heated under reflux for 6 h. After cooling to
room temperature the reaction mixture was poured onto ice (60 g).
The solid formed was filtered off, triturated with hexane and dried
to afford a pale yellow solid (950 mg, 2.61 mmol, 64%). This interme-
diate was resuspended (480 mg, 1.321 mmol) in dry MeOH (11 mL)
and sodium borohydride (175 mg, 4.620 mmol) was added in 4 por-
tions at room temperature. The reaction was stirred for 5 h and
followed by LC–MS. When completion of the reaction was observed,
the reaction was quenched with saturated citric acid solution,
concentrated in vacuo and extracted with EtOAc (3 × 10 mL). The
organic solution was washed with aqueous NaHCO3 solution, water
and brine, dried over anhydrous MgSO4 and concentrated to afford
1a as a pale yellow solid (452 mg, 1.23 mmol, 93%). 1H NMR
(400 MHz, CDCl3) ı ppm: 7.74 (s, 1H), 7.36 (d, J = 8.3 Hz, 2H), 7.25 (d,
J = 8.3 Hz, 2H), 6.12 (bs, 1H), 4.87–4.81 (m, 1H), 4.55 (dd, J = 2.5 and
11.6 Hz, 1H), 4.28–4.22 (m, 2H), 2.68 (q, J = 7.8 Hz, 2H), 2.57–2.51
(m, 1H), 2.41–2.32 (m, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.26 (t, J = 7.6 Hz,
3H). [ES+MS] m/z 368 (M+H)+.
2.5. Enzymatic hydrolysis in analytic scale
To a reaction mixture of 2.5 ␮M 1a in 800 ␮L PBS (phosphate
buffer saline, 10 mM, pH 7.4), 20 ␮g enzyme preparation was
added (PLE, ERL or CAL-B). Reactions were incubated at 30 ◦C and
1000 rpm in a Thermomixer (Fischer, Germany). At periodic time
points 100 ␮L samples were withdrawn and mixed with 150 ␮L
precipitating agent (methanol:aceonitile, 3:1, 1 ␮M Resorufin as
internal standard) and 50 ␮L 0.1% formic acid in 96-well microtiter
plates. Plates were centrifugated at 3700 rpm and 10 ◦C for 15 min
and reactions were analyzed by UPLC–MS.
2.6. General procedure for CAL-B-catalyzed kinetic resolutions
2–4 mg substrate and the correspondent amount of cosolvent
(200 ␮L DMSO in standard reactions, other cosolvents and amounts
in optimization experiments) was added and stirred at 30 ◦C for
5–10 min. Sodium phosphate buffer (100 mM, pH 7.4) was added
to form a 5 mM solution of substrate and mixed for 20–30 min. The
reaction was started with addition of 400 ␮L liquid enzyme prepa-
ration (equivalent to 2 mg protein). Aliquots of 500 ␮L were taken
after 24 and 48 h, acidified with 20 ␮L HCl 1 N, and extracted three
times with 500 ␮L ethyl acetate. The organic phases were collected
and evaporated, the mixture was re-suspended in 100 ␮L methanol
and substrate/product concentration and optical purity was ana-
lyzed by chiral HPLC-UV (254 nm) using hexane:ethanol:TFA
Compound 2a was prepared by an analogous method using
ethyl 3-aminopyrazole-4-carboxylate (399 mg, 2.57 mmol)
and
4,4,4-trifluoro-1-(4-methylthiazol-2-yl)butane-1,3-dione
(554 mg, 2.336 mmol) as starting reactants. Compound 2a (345 mg,
0.957 mmol, 70%) was obtained as a pale yellow solid. 1H NMR
(400 MHz, DMSO-d6) ı ppm: 7.65 (s, 1H), 7.53 (d, J = 3.0 Hz, 1H),
7.19 (d, J = 1.0 Hz, 1H), 5.32–5.27 (m, 1H), 5.04–5.00 (m, 1H),
4.24–4.15 (m, 2H), 2.72–2.66 (m, 1H), 2.60–2.55 (m, 1H), 2.34 (s,
3H), 1.26 (t, J = 7.1 Hz, 3H). [ES+MS] m/z 361 (M+H)+.
Compound 3a was prepared by an analogous method
using ethyl 3-aminopyrazole-4-carboxylate (398 mg, 2.56 mmol)
and 1-(1-ethyl-1H-pyrazol-4-yl)-4,4,4-trifluorobutane-1,3-dione

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E. Fernández-Álvaro et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 1–6
Fig. 3. Screening of cosolvents for the hydrolysis of THPPs. Reaction conditions: 5 mM 1a, sodium phosphate buffer 100 mM (pH 7.4), 10% co-solvent, 30 ◦C, magnetic stirring.
Conversion was measured after 18 h.
(600 mg, 2.56 mmol) as starting reactants. Compound 3a (575 mg,
1.609 mmol, 74%) was obtained as a pale yellow solid. 1H NMR
(400 MHz, CDCl3) ı ppm: 7.72 (s, 1H), 7.55 (s, 1H), 7.50 (s, 1H),
6.04 (bs, 1H), 4.84–4.78 (m, 1H), 4.60 (dd, J = 2.5 and 11.6 Hz,
1H), 4.28–4.23 (m, 2H), 4.18 (q, J = 7.3 Hz, 2H), 2.59–2.54 (m, 1H),
2.42–2.33 (m, 1H), 1.51 (t, J = 7.3 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H).
[ES+MS] m/z 358 (M+H)+.
3. Results and discussion
The application of hydrolases for kinetic resolution purposes
may start from esters – to perform enantioselective hydrolysis
in aqueous media – or conversely from carboxylic acids, to carry
out (trans)esterifications in non-aqueous solutions [10]. Given the
envisaged synthetic approach to obtain the substrates, whereby
Table 1
CAL-B-catalyzed hydrolysis of different THPP-esters. Reaction conditions: 5 mM substrate concentration, sodium phosphate buffer 100 mM (pH 7.4), 10% DMSO as co-solvent,
30 ◦C, magnetic stirring.
.
Substrate/no.
eep (%)
ees (%)
Conversion (%)
E-valuea
Abs. config-
uration
(product)b
96
59
38
83
5R,7S-1b
95
98
51
>100
5R,7S-2b
93
96
51
>100
5R,7S-3b
a
Conversion and E values were then calculated from the enantiomeric excesses (ee) from substrate and product following equations reported in the literature.
Absolute configuration was determined by VCD [4a]. Reaction time was 24 h.
b

E. Fernández-Álvaro et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 1–6
5
esters were the obtained products (Scheme 1), hydrolytic reactions
4. Conclusion
using lipases and esterases in aqueous buffer were set to probe
biocatalysis. From a screening of 50 commercially available lipases
and esterases (detailed list provided in Supplementary Informa-
tion) using substrate 1a, only three active hits were found, pig and
rabbit liver esterase (PLE and ERL), and Candida antarctica lipase B
(CAL-B). The low hit rate of active enzymes is likely attributed to
the bulky substrate employed. The use of PLE and ERL – mixture
of different isoenzymes displaying different enantioselectivities –
virtually led to full conversions of the ester (100%) with poor or neg-
ligible enantiomeric excess [11]. Conversely, CAL-B afforded 50%
conversion, and the reaction stopped at that point, suggesting a
high enantioselectivity for the THPP used as prototypical substrate
(Fig. 2).
Encouraged by the results achieved with CAL-B under the
screening conditions reported (Fig. 2), that enzyme was selected for
a more in-depth assessment of biocatalysis in THPPs. To this end,
different THPP analogues based on structure–activity relationships
(SAR) were selected, by changing the nature of the phenyl ring,
aiming at improving physico-chemical and developability features.
Conversions and enantioselectivities were measured, and results
are summarized in Table 1.
This work has successfully explored for the first time
the use of biocatalysis for the kinetic resolution of different
tetrahydropyrazolo[1,5-␣]pyrimidines (THPPs), structures that are
having an increasingly importance as therapeutic agents (e.g. as
agents to treat osteoporosis, as antivirals and as anti-tuberculars)
whose enantiomeric centres are located far away from the
carbonyl moiety. After screening a number of commercially avail-
able hydrolases, CAL-B was selected as the biocatalyst to undertake
enantioselective hydrolysis using DMSO as cosolvent, with
excellent enantiomeric excesses and conversions. Moreover, envis-
aging a further real use of the process, different easy-to-handle
and (preferentially) bio-based cosolvents were tested. Herein, tert-
butanol and aqueous solutions of different deep-eutectic-solvents
(DES) appear promising for this specific case. Looking beyond, to
convert this preliminary proof-of-concept into a robust industrial
process, considerable improvements must be made, especially in
delivering genetically improved enzyme variants that may perform
this challenging transformation in reduced reaction times [8]. Being
the first example of asymmetric catalysis in THPP synthesis – and
given its foreseeable future importance for many therapeutic areas
– it may be expected that improved (bio)catalytic performances
will be disclosed in the coming years.
The use of CAL-B led to the formation of the desired optically
active drugs under very mild hydrolytic conditions (24 h, 30 ◦C).
In all cases moderate-to-full conversions (for a kinetic resolution)
were achieved, with high enantiomeric excesses in all cases. Albeit
Acknowledgements
the overall productivities achieved are in the range of ∼1 g L−1 d−1
,
still far from industrially sound conditions [12], the enzymatic reso-
lution of this highly valuable scaffold with both asymmetric centres
far from the catalytic centre has been achieved, which is a remark-
able and underexplored fact for biocatalysis.
GSK thanks Fundación Universidad Empresa and GATB for finan-
tial support. Dr. Peter Sutton, Dr. Radka Snajdrova, Dr. Santiago
Ferrer, Ángel Santos and Juan Miguel Siles are kindly acknowl-
edged for fruitful discussions and technical support. PDdM and MPS
thank financial support from DFG training group 1166 “BioNoCo”
(“Biocatalysis in Non-conventional Media”).
The addition of a cosolvent (10% DMSO) to the buffer enabled
the dissolution of both substrates and products. Yet, envisag-
ing future real process set-up conditions, the use of DMSO often
leads to problems during work-up procedure, with the genera-
tion of considerable amounts of wastewater and consequent yield
losses and environmental challenges [13]. Aiming at providing
more practical solutions combining ecology for pharmaceutical
industries, in recent years the use of bio-based cosolvents, such
as 2-methyltetrahydrofuran (2-MeTHF) [13b,14], or tert-butanol
[15], have been suggested for enzymatic reactions. In addition, the
use of ionic liquids and deep-eutectic-solvents (DES) as cosolvents
have been put forth as well for several enzymatic catalytic systems
by different research groups [16]. To further assess the enzymatic
kinetic resolution of THPPs and aiming to improve substrate solu-
bility, a broad screening of co-solvents was conducted (Fig. 3).
Our screening revealed that apart from DMSO other cosolvents
enabling a more straightforward work-up (e.g. distillation) and/or
a potentially biogenic origin (2-MeTHF, tert-butanol and different
DES) could also be satisfactorily used in the lipase-catalyzed kinetic
resolution of THPPs. 2-MeTHF did not dissolve the substrate in the
conditions applied, what may explain the lower enzymatic activ-
ity observed, compared to other more successful lipase-catalyzed
processes reported in the literature for 2-MeTHF [14,17]. Remark-
ably the use of DES as cosolvents (10% in buffer) led to promising
results in hydrolytic reactions, with comparable outcomes to those
of DMSO (Fig. 3). Albeit DES did not fully dissolve the substrate satis-
factorily at those conditions, and furthermore, it would be expected
that the DES structure would be disaggregated under aqueous
solutions, it appears that some beneficial effect may be found
for hydrolytic enzymatic reactions. Analogous improved results
for DES as cosolvents have also been reported for other enzymes
(epoxide hydrolases, cellulases) [18], as well as for proteases in
DES-containing water solutions [19]. Overall, the use of more envi-
ronmentally friendly and easy-to-handle cosolvents (compared to
DMSO) results compatible with the kinetic resolution of THPPs.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.11.012.
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