Dual Mechanoenzymatic Kinetic Resolution of (±)-Ketorolac

Article abstract of DOI:10.1002/cctc.201902292

Recently, biocatalysis mediated by mechanical energy has become a convenient strategy to obtain highly valuable products following the principles of green chemistry. In particular, mechanoenzymatic techniques have allowed the isolation of chiral drugs wit

Full text of DOI:10.1002/cctc.201902292

Accepted Article
Title: Dual Mechanoenzymatic Kinetic Resolution of (±)-Ketorolac
Authors: Mario Pérez-Venegas, Agustín Mario Rodríguez-Treviño, and
Eusebio Juaristi
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201902292
Link to VoR: http://dx.doi.org/10.1002/cctc.201902292
A Journal of
www.chemcatchem.org

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
Dual Mechanoenzymatic Kinetic Resolution of (±)-Ketorolac
Mario Pérez-Venegas,[a] Agustín Mario Rodríguez-Treviño,[a] and Eusebio Juaristi*[a],[b]
Abstract: Recently, biocatalysis mediated by mechanical energy has
become a convenient strategy to obtain highly valuable products
following the principles of green chemistry. In particular,
mechanoenzymatic techniques have allowed the isolation of chiral
drugs with high enantiomeric purity. In this regard, being aware of the
preponderant anti-inflammatory activity of Ketorolac’s (S)-isomer
relative to its (R)-enantiomer, we developed a mechanoenzymatic
kinetic resolution protocol using Candida antarctica Lipase B to yield
both enantiomers with high enantiopurity. The significant
enantiopreference of CALB for the (R)-isomer of Ketorolac allowed
the isolation of the active (S)-enantiomer, either by means of an
enantioselective esterification of racemic Ketorolac or via
enantioselective hydrolysis of suitable ester derivatives. Both
techniques proceeded in short reaction times, with high enantiomeric
excess (> 83%), remarkable enantiodiscrimination (E >> 500) and
rather good conversion (46%). Furthermore, (R)-Ketorolac, an
effective drug for ovarian cancer management, was obtained in
essentially enantiopure form (ee > 99%).
products,[12] including several pharmaceutically relevant
molecules.[13,14] These extraordinary developments have
promoted the study of the underlying consequences of
mechanical activation on the catalytic activity of different enzymes,
demonstrating that milling has
a
negligible detrimental
repercussion in the enzymatic activity of protected -
glucosidases.[15] In fact, even a positive effect that increases the
enzymatic activity in one order of magnitude, without loss of
stereoselectivity, was observed when an immobilized lipase was
submitted to exhaustive milling.[16]
Figure 1. Mirror images of Ketorolac enantiomers.
On the other hand, (R/S)-Ketorolac (Figure 1) is a potent
nonsteroidal anti-inflammatory drug (NAID) with strong analgesic,
anti-inflammatory and antipyretic activity.[17] Recent reports have
evidenced the preponderant anti-inflammatory activity of the (S)-
enantiomer of Ketorolac with respect to the (R)-isomer.[17]
Nevertheless, as it is the case with several cyclooxygenase
inhibitors such as Etodolac and Ibuprofen, Ketorolac is
commercialized as a racemic mixture.[18, 19]
Several strategies for the preparation of enantiopure (S)-
Ketorolac have been described, including enantioselective
synthesis via a highly efficient procedure that unfortunately makes
use of potential toxic reagents.[20] Furthermore, the recently
reported chromatographic separation of diastereomeric
derivatives constitutes a noteworthy separation strategy but
requires the use of a large amount of solvent.[21] Furthermore,
biocatalytic methods employing proteases or lipases for kinetic
resolution procedures of racemic Ketorolac have been
disclosed.[22]
Introduction
The use of enzymes in academy and industry, especially in the
pharmaceutical and fine chemistry field,[1] has been expanded
from the use of proteases and lipases[2] to the application of
transaminases[3] and ketoreductases,[4] as well as complex
proteins such as the cytochrome P450s.[5] Moreover, recent
developments in directed enzyme evolution and protein
immobilization on inert materials have permitted to overcome
several limitations in the use of enzymes in organic synthesis.
Furthermore, these novel procedures have actually enhanced
numerous enzymatic properties such as the regio- and
enantioselectivity of a biocatalytic process, as well as the
enzymatic conversion rate and recycling.[6] Thereby, boosted
biocatalytic features have allowed the combined use of
biocatalysts with different energy sources such as microwaves,[7]
ultrasound waves (sonochemistry)[8] and mechanical energy,[9]
leading to a new and fertile field in biocatalysis that genuinely fulfill
most of the twelve green chemistry principles.[10,11]
Currently, biocatalysis mediated by mechanical force has become
a powerful strategy in asymmetric organic synthesis avoiding the
use of bulk amounts of solvents to obtain a wide range of
[a]
B. Sc. M. Pérez-Venegas, B. Sc. A. M. Rodríguez-Treviño and Prof.
Dr. E. Juaristi
Department of Chemistry
Centro de Investigación y de Estudios Avanzados,
Av. IPN 2508, 07360 Ciudad de México, Mexico
E-mail: juaristi@relaq.mx; ejuarist@cinvestav.mx
Prof. Dr. E. Juaristi
El Colegio Nacional
Luis Gonzáles Obregón 23, Centro Histórico, 06020 Ciudad de
México, Mexico.
Scheme 1. Dual enzymatic kinetic resolution of (±)-Ketorolac by means of (a)
enantioselective esterification and (b) enantioselective hydrolysis of ester
derivatives.
[b]
E-mail: juaristi@relaq.mx or ejuarist@cinvestav.mx
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
In this regard and considering current advances in
mechanoenzymatic techniques, and in line with our search for
more sustainable protocols for the synthesis of biologically active
chiral compounds using CALB in a minimal amount of solvent and
employing a highly efficient source of energy such as mechanical
milling,[13] we decided to develop mechanoenzymatic strategies
for the isolation of both enantiomers of Ketorolac.
results were obtained when methanol is used as esterification
agent in the enantioselective kinetic resolution, affording
enantioenriched recovered (S)-Ketorolac (ee = 58%) with a high
conversion (c = 37%, of the theoretical 50%) and an extraordinary
enantiodiscrimination (E = ln[1−c(1+%eep)]/ln[1−c(1−%eep)]) >
300. The effect of the alcohol chain length corroborates previous
reports in which short-chain alcohols (one and two carbons)
exhibit the best results in asymmetric esterification of racemic
mandelic acid using CALB.[24]
In the present work, it is shown that the enzymatic deracemization
of (±)-Ketorolac using lipases can be conveniently conducted
through two alternative synthetic routes (Scheme 1).
e e (S)-1 (% )
In the first approach, enzymatic enantioselective esterification of
the racemic carboxylic acid (Scheme 1, a), results in the
formation of the (R)-Ketorolac alkyl ester and the concomitant
isolation of unreacted (S)-Ketorolac. The second strategy
involves the enantioselective hydrolysis of a racemic mixture of a
Ketorolac alkyl ester (Scheme 1, b) yielding the (R)-isomer of the
free drug and the ester of the complementary (S)-enantiomer.
Among the lipases used in the kinetic resolution of (±)-Ketorolac,
Candida antarctica Lipase B (CALB, commercially available as
Novozym 435) proved to be rather efficient in the stereoselective
preparation of both enantiomers of Ketorolac.
1 0 0
e e (R )-2 (% )
c
(% )
(1 0 -1
)
8 0
6 0
4 0
2 0
0
E
l
l
l
o
l
l
o
o
o
o
n
n
n
a
n
n
a
a
a
a
t
u
h
p
x
e
h
t
t
o
E
e
r
B
-
H
-
P
M
-
n
n
n
E s te r ify in g a g e n t
Figure 2. Mechanoenzymatic resolution of rac-1 using immobilized CALB (30
mg, 300 IU) and different esterification agents and agate milling-jar and ball.
Results and Discussion
The mechanoenzymatic resolution of (±)-Ketorolac was initially
achieved via the enantioselective enzymatic esterification of its
carboxylic acid function. For this, a screening of potential
esterification agents was carried out, employing the previously
reported conditions for the esterification of Ketorolac in
solution[22c] and using short-chain aliphatic alcohols (one to four
and six carbon chains). CALB was selected for the present
resolution of (±)-Ketorolac. For the mechanochemical procedure
0.1 mmol of racemic Ketorolac, 1.0 equivalent of the
corresponding alcohol, a small amount of solvent (CH3CN, 0.2
mL) and 30 mg of immobilized CALB (that corresponds to 300 IU,
that is 80 IU of CALB/mL of CH3CN) were charged in an agate
reactor with one agate ball, the milling jar was placed in an
automatized mixer mill (Retsch, MM200) and the content was
milled at 25 Hz of frequency by 90 minutes (Scheme 2, see also
Experimental Section).
It is worth pointing out that the material of which milling-jars and
balls are made plays a key role for the effectiveness of the
mechanochemical methods.[25] Consequently, we decided to
investigate the relative efficiency of stainless-steel and Teflon® in
the mechanoenzymatic resolution of interest (see Supporting
Information [SI], Table S2). When a Teflon® (PTFE) milling-jar
and ball were used, the enantiomeric excess of the (S)-
enantiomer decreased considerably from ee = 58% when using
an agate reactor to ee = 17%. In contrast, when stainless-steel
accessories were used, the enantiomeric excess of (S)-1
increased (ee = 75%) although the enantiopurity of the ester
diminished slightly (ee = 98%). Considering the metal impurities
generated during the milling process when metal components are
employed and taking into account the fluctuating results when
harder materials are used,[9,13,16] we decided to use agate
components throughout this study.
Minimization of solvent use is a determinant factor for the
fulfillment of the green chemistry principles. In this regard, and in
contrast with previous reports of enzymatic resolution of racemic
Ketorolac in which the use of solvent is excessive,
mechanoenzymatic reactions can be conducted under the liquid-
assisted grinding (LAG) technique,[26] which employs minimal
amounts of solvent to carry out chemical transformations in a ball
mill. Aiming to increase the enantiomeric excess of the (S)-
enantiomer of Ketorolac in the present mechanoenzymatic
approach several LAG additives (considering that methanol is
being used here as a reagent) were evaluated (Table 1).
Scheme 2. Mechanoenzymatic resolution of (±)-Ketorolac through
enantioselective esterification. CH3CN: acetonitrile. The three balls on the arrow
represent the mechanochemical method.[23]
By comparison with the use of acetonitrile, solvents such as 2-
methyl-2-butanol (2M2B) or dioxane, that can be anticipated to
prevail throughout the mechanoenzymatic process, did not
increase significantly the enantiomeric excess of the (S)-
enantiomer of Ketorolac (Table 1, entries 1 and 2). Nevertheless,
when methyl t-butyl ether (MTBE) a low boiling solvent with
As it is shown in Figure 2, CALB has an extraordinary preference
for the (R)-isomer of Ketorolac reaching high values of
enantiomeric excess (ee > 99%) of the corresponding esters,
independently of the esterification agent. Nevertheless, best
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
moderate polarity was used, the enantiopurity of the active (S)
form of the drug increased from ee = 58% to ee = 72% with a
As indicated in Figure 3, the enzymatic kinetics for the resolution
of rac-1 under conventional heat (45°C) and stirring conditions
(Figure 3, a) the reaction was complete after 60 minutes of milling,
affording an excellent ee value for (R)-2 (ee > 99%), but a
moderate enantiopurity for the recovered (S)-enantiomer of
Ketorolac (ee = 69%). It is also appreciated that the enantiomeric
excess of (S)-1 begins to decrease after it reaches a value for ee
= 62% at 120 minutes of reaction (E > 300). The loss in
enantiopurity when thermal energy is used could be associated to
a well-known reversible (hydrolytic) reaction that hydrolyzes the
preformed ester, decreasing the amount of pure (R)-2.
concomitant
improvement
in
conversion
and
enantiodiscrimination, suggesting that the moderate polarity and
moderate boiling point of MTBE makes more efficient the reaction
of CALB with the corresponding substrates.[27]
Table 1. Effect of LAG additive in the mechanoenzymatic resolution of rac-1.
entry
LAG[a]
ee(S)-1
(%)[b]
ee(R)-2
(%)[b]
c (%)[c]
E (%)[d]
1
2
3
4
5
2M2B
Dioxane
CH3CN
DIPE
57
57
58
89
72
> 99
98
37
37
37
57
42
> 300
> 100
> 300
15
> 99
67
MTBE
> 99
> 400
[a] Reaction conditions: rac-1 (0.10 mmol), methanol (1.00 mmol), CALB (30
mg), LAG additive (0.2 mL), agate milling-jar and ball. [b] Determined by
HPLC with chiral stationary phase. [c] Calculated from c = eeS/(eeS+eeR). [d]
E = ln[1 - c(1 + eeR)]/ln[1 - c(1 - eeR)]. 2M2B: 2-methyl-2-butanol. CH3CN:
acetonitrile. DIPE: diisopropyl ether. MTBE: methyl t-butyl ether.
Biocatalyst and esterification agent loads were evaluated in the
kinetic resolution using MTBE as LAG additive (see SI, Tables S3
and S4). A decrease in enzymatic load resulted in a deficient
resolution of racemic Ketorolac, reaching low values of ee for
recovered (S)-1. On the other hand, an increase in the amount of
CALB also rises the enantiopurity of the active drug (see SI, Table
S3), but with a small decrease in ee for the ester derivative (ee
(R)-2 = 98%). In this context, the number of equivalents of
esterification agent has a significant influence on the efficiency of
the mechanoenzymatic deracemization of (±)-Ketorolac,
obtaining the best results when 12.5 equivalent of methanol were
employed (see SI, Table S4).
Figure 3. Enzyme kinetics for the kinetic resolution of racemic Ketorolac using
methanol as an esterification agent with a) conventional heat (45°C) and stirring
conditions and b) mediated by mechanical activation.
Contrary to the process carried out under conventional conditions,
the mechanoenzymatic reaction proceeds without loss on
enantiopurity of the recovered (S)-enantiomer of Ketorolac
(Figure 3, b). After 90 minutes of continuous milling unreacted
(S)-1 exhibited ee (S)-1 = 83%, whereas the product consisted of
essentially enantiopure ester (ee (R)-2 > 99%). Furthermore, the
enzymatic reaction proceeded with high values of conversion and
enantiodiscrimination (c = 46%, E >> 500). As it has been
established recently,[16] the low energy that is involved in the
mechanochemical process, and the corresponding increased
enzymatic accessibility lead to a more efficient process. Thus,
significant benefits from the use of mechanical force in the
biocatalytic transformation can be appreciated. The enantiomeric
excess of the (S)-enantiomer of Ketorolac is higher at 60 minutes
The optimal conditions for the CALB enzymatic resolution of
racemic Ketorolac by means of enantioselective esterification and
mediated by mechanical activation are shown in Scheme 3.
of reaction (ee (S)-1,
process in solution (ee (S)-1,
= 79%), to be compared with the
60min
Scheme 3. Optimal conditions for the mechanoenzymatic resolution of rac-1.
Immobilized CALB: 30 mg (that corresponds to 300 IU), MTBE: 0.2 mL.
= 69%). Furthermore, the
60min
solvent usage was substantially reduced, from 5 mL in the
conventional reaction to 200 µL in the mechanochemical protocol.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
In this regard, when the enzymatic kinetic resolution is carried out
under mechanical activation it is possible to obtain the free active
form of Ketorolac, the (S)-enantiomer, with a high enantiomeric
excess (ee = 83%) and the ester form of the (R)-isomer in an
enantiopure form (ee > 99%), in a very short time, and with
exceptional enantiodiscrimination (E >> 500).
As it can be appreciated in Figure 4, the enantiomeric excess of
the non-converted isomer increases together with an increase of
the size of the ester chain, until it reaches the highest value (ee =
45%) with Ketorolac n-propyl ester (rac-4). Nevertheless, longer
aliphatic chains appear to be detrimental for an adequate
biocatalytic process (Figure 4), possibly due to unfavourable
interactions between the hydrocarbon chain and the active site of
the enzyme.[28]
As it was mentioned above, in this work the kinetic resolution of
Ketorolac could be carried out through two parallel strategies that
consist in either, a) the esterification of the racemic carboxylic acid
of Ketorolac or b) the hydrolysis of an ester derivative (Scheme
4).
6 0
4 5
3 0
1 5
0
r
r
r
r
r
r
e
e
e
e
e
t
t
t
s
t
t
s
s
s
s
e
l
e
e
e
l
e
l
l
l
y
h
y
y
t
u
y
x
y
p
h
t
t
E
o
e
e
B
H
P
M
Scheme 4. Representative mechanoenzymatic resolution of racemic Ketorolac
methyl ester through enantioselective hydrolysis. The three balls represent the
mechanochemical method.[23]
Figure 4. Dependency of the enantiomeric excess of the unreacted racemic
Ketorolac alkyl ester with respect to the aliphatic chain length in the
mechanoenzymatic hydrolytic resolution.
Previously, the enzymatic hydrolysis of Ketorolac derived alkyl
esters was explored by Sih, Kim, and coworkers using proteases
and lipases.[22a,b] Nevertheless, the amount of solvent used in the
resolution is much higher than that used with the
mechanoenzymatic strategies. Additionally, some values of
enantiomeric excess are low (ee values as low as 4% were
reported).[22a] In this regard and considering the extraordinary
results achieved in the mechanoenzymatic resolution by means
of enantioselective esterification (vida supra), we decided to
explore a hydrolytic strategy for the isolation of the (S)-enantiomer
of Ketorolac.
In the event, a family of racemic Ketorolac esters were
synthesized and used as substrate in the mechanoenzymatic
hydrolytic protocol, following the best conditions obtained for the
resolution of Ketorolac through esterification (Scheme 5). CALB
shows a remarkable enantioselectivity for the hydrolysis of the
(R)-isomer of racemic ketorolac esters. As it can be anticipated,
the (R)-enantiomer is also the more reactive in the
mechanoenzymatic resolution by means of esterification (vide
supra). Nevertheless, the enantiodiscrimination in the hydrolytic
process seems to be dependent to some extent on the size of the
ester’s aliphatic chain.
As it was the case in the resolution of racemic Ketorolac by means
of enantioselective esterification, seeking here to increase the
enantiopurity of the less reactive (S)-enantiomer, several
parameters such as the nature of the LAG additive, the material
of the milling-jar and balls, and the enzymatic load were evaluated
in the resolution process (see SI, Tables S6, S7 and S8 for
details). In the event, tert-amyl alcohol (2M2B) a recently
introduced green solvent,[29] proved rather effective in the kinetic
resolution of n-propyl ester rac-4, yielding enantiopure free (R)-
Ketorolac (ee (R)-1 > 99%) and a highly enantioenriched
recovered (S)-Ketorolac n-propyl ester (ee (S)-4 = 75%). Agate
components and 30 mg of CALB were used in this
mechanoenzymatic protocol (see SI, Table S8 for details).
Furthermore, it was observed that the presence of water is
required for the enantioselective enzymatic hydrolytic process to
take place. In principle, half equivalent of water should be
sufficient for the hydrolytic deracemization process; however,
optimal conditions were reached when 6 equivalents of water
were employed in the mechanoenzymatic resolution (Table 2,
entry 5), yielding a highly enantioenriched unreacted (S)-
Ketorolac n-propyl ester (ee (S)-4 = 85%).
The presence of increased amounts of water (e.g. 10 equivalents,
Table 2, entry 6), was unsatisfactory since no increase in the
enantiopurity of the (S)-enantiomer was observed. This negative
effect in the biocatalytic process may be due to induced hindrance
of the active site of CALB as indicated in previous reports by
Peters et al.[30]
In order to compare the effectiveness of the kinetic resolution of
racemic Ketorolac ester under conventional heating and stirring
conditions, vis-a-vis mechanical activation, enzymatic kinetics
were carried out using the optimal conditions previously
established (Cf. Table 2, entry 5). As shown in Figure 5, kinetic
resolution via enantioselective hydrolysis of Ketorolac’s esters
Scheme 5. Mechanoenzymatic resolution of racemic Ketorolac esters through
enantioselective hydrolysis with CALB employing 1 equivalent of water, MTBE
as LAG additive, and agate components. The three balls represent the
mechanochemical method.[23]
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
under conventional heating and stirring conditions proceeds
smoothly being complete after 90 minutes of reaction, with
excellent enantiomeric excess for the free form of (R)-Ketorolac
(ee (R)-1 > 99%) and the recovered (S)-Ketorolac n-propyl ester
(ee (S)-4 = 86%), in addition to a very high conversion (c = 46%)
and excellent enantiodiscrimination (E >> 500).
potential drug in the management of ovarian cancer.[31] (R)-
Ketorolac has also proved efficient as a noncompetitive inhibitor
of GTPases,[33] a not visible effect when diverse NSAIDs were
used.[34]
Table 2. Effect of water equivalents in the mechanoenzymatic resolution of rac-
4
entry
Water
(eq.)[a]
ee(S)-4
(%)[b]
ee(R)-1
(%)[b]
c (%)[c]
E (%)[d]
1
2
3
4
5
6
0.5
1
71
75
77
80
85
86
> 99
> 99
> 99
> 99
> 99
> 99
42
43
44
45
46
46
> 400
> 400
> 400
> 400
> 500
> 500
1.5
3
6
10
[a] Reaction conditions: rac-4 (0.08 mmol), water, CALB (30 mg), 2-methyl-
2-butanol (2M2B) (0.2 mL), agate milling-jar and ball. [b] Determined by
HPLC with chiral stationary phase. [c] Calculated from c = eeS/(eeS+eeR). [d]
E = ln[1 - c(1 + eeR)]/ln[1 - c(1 - eeR)].
Figure 5. Enzyme kinetics for the kinetic resolution of racemic Ketorolac n-
propyl ester using 2M2B as LAG and 6 equivalents of water under a)
conventional heating (45°C) and stirring conditions, and b) mediated by
mechanical activation.
Surprisingly, the mechanoenzymatic resolution proceeds ca. 50%
faster than the process under traditional thermal conditions,
reaching the highest values for the resolution (ee (R)-1 > 99%, ee
(S)-4 = 85%, c = 46%, E >> 500) after 45 minutes of milling.
Enzyme kinetics determined for the hydrolytic protocol clearly
Enzyme kinetics for the mechanoenzymatic resolution of rac-1 via
enantioselective esterification (“EE”) and via enantioselective
hydrolysis (“EH”) of rac-4, can be used for the suitable isolation of
both enantiomers of Ketorolac. Nevertheless, in order to obtain a
more quantitative measure of these enantioselective reactions, it
was decided to evaluate the difference in Gibbs free energy
(∆∆퐺‡ ) in each enzymatic reaction.
demonstrate
the
viability
and
superiority
of
the
mechanoenzymatic strategy over the process in solution, allowing
for the isolation of both enantiomers of Ketorolac with excellent
enantiopurity (ee > 85%), in a very short time (45 minutes),
employing a minimal amount of a green solvent.
In the present work, both enantioselective esterification of
racemic Ketorolac and enantioselective hydrolysis of derived
aliphatic esters lead to the full resolution of the less active (less
anti-inflammatory activity) (R)-enantiomer with outstanding
results (ee (R)-isomer > 99%) in a short period of time. It is worth
pointing out that recent studies have showed that, even when the
(R)-isomer of Ketorolac has a limited anti-inflammatory activity, it
can be successfully employed as a noncompetitive inhibitor of
Rac1 (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell
division control protein 42) GTPases, that are regulatory enzymes
of several functions related to tumor development such as
progression, metastasis and chemo-resistance (Figure 6).[31]
Furthermore, the expression of these GTPases, specifically the
Rac1, could be used as a prognostic factor of ovarian cancer
patients.[32] Furthermore, (R)-Ketorolac has been used as a
Figure 6. Dual activity of ketorolac enantiomers as an anti-inflammatory drug
(left) and as an inhibitor of tumoral regulatory enzymes (right).
Thus, under the experimentally established optimal conditions
both resolution strategies were carried out in solution, using
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
mixture of racemic Ketorolac (0.1 mmol), CALB (30 mg), esterification
alcohol (12.5 eq.) and CH3CN (0.2 mL) was placed in an agate milling-jar
(12 mm of diameter, 4.6 mL capacity) together with the corresponding
agate ball (6 mm of diameter, 480 mg of weight). Alternatively, stainless-
steel milling-jar (15 mm of diameter, 4.6 mL of capacity) with the
corresponding stainless-steel ball (8 mm of diameter, 1.5 g of weight) or
Teflon® milling-jar (10 mm of diameter, 6.5 mL of capacity) together with
the corresponding Teflon® ball (8 mm of diameter, 1.2 g of weight). The
resulting mixture was milled for 90 minutes at 25 Hz of frequency. The
milling-jar content was extracted with acetone, centrifuged at 3800 rpm
and concentrated for HPLC analysis. When methanol was used as
esterification agent, the crude product was purified using either hexane
and ethyl acetate (95:5) to isolate the ketorolac ester and CH2Cl2 and
methanol (1:1) to isolate the Ketorolac in free form.
different temperatures within the stable range of CALB (30-60°C).
Application of Eyring’s transition state theory (see SI, Table
S9),[35] enabled estimation of Gibbs free energy ∆∆퐺퐸‡퐸
=
1.44 푘푐푎푙 푚표푙−1 and ∆∆퐺퐸‡퐻 = 1.06 푘푐푎푙 푚표푙−1
,
for the
enantioselective resolution by means of enantioselective
esterification and enantioselective hydrolysis, respectively.
From these data, it could be anticipated that the kinetic resolution
mediated by esterification should give rise to
a more
enantioselective process as a result of the larger (by 0.38
kcal/mol) ∆∆퐺‡ ; experimentally however, (R)-Ketorolac was
formed with similar enantiopurity in both strategies.
Conclusions
General method for the mechanoenzymatic kinetic resolution of
racemic Ketorolac by means of enantioselective hydrolysis of alkyl
ester derivatives. A mixture of the corresponding racemic Ketorolac ester
(0.08 mmol), CALB (30 mg), water (6 eq.) and 2M2B (0.2 mL) was placed
in an agate milling-jar (12 mm of diameter, 4.6 mL capacity) with the
corresponding agate ball (6 mm of diameter, 480 mg of weight).
Alternatively, stainless-steel milling-jar (15 mm of diameter, 4.6 mL of
capacity) with the corresponding stainless-steel ball (8 mm of diameter,
1.5 g of weight) or Teflon® milling-jar (10 mm of diameter, 6.5 mL of
capacity) with the corresponding Teflon® ball (8 mm of diameter, 1.2 g of
weight). The resulting mixture was milled for 90 minutes at 25 Hz of
frequency. The milling-jar content was extracted with acetone, centrifuged
at 3800 rpm and concentrated for HPLC analysis. When the racemic
Ketorolac methyl ester was used, the crude product was purified using
hexane and ethyl acetate (95:5) to isolate the unreacted Ketorolac ester
and CH2Cl2 and methanol (1:1) to isolate the produced enantioenriched
Ketorolac in free form.
The anti-inflammatory (S)-enantiomer of Ketorolac was
conveniently isolated (ee > 83%) by means of two alternative
mechanoenzymatic strategies that involve either the kinetic
resolution of a racemic mixture of free Ketorolac or the
enantioselective hydrolysis of racemic Ketorolac alkyl esters.
Both strategies proceeded with an equally high conversion (c =
46%) and with remarkable enantiodiscrimination (E >> 500). The
resolution by means of enantioselective hydrolysis of (±)-
Ketorolac ester proceeds between 50% and 200% faster than
similar strategies reported in solution, evidencing the efficiency of
the mechanoenzymatic technique. Both enzymatic resolution
procedures conduct to the enantiopure form of (R)-Ketorolac (ee
> 99%), and highly enantioenriched (ee ca. 83%) (S)-enantiomer.
In summary, the activation of immobilized CALB by mechanical
force employing a minimal amount of solvent, constitutes a
promising green strategy in the pharmaceutical field. Indeed, by
increasing the effective concentration of reagents in the
enzymatic reaction a more efficient and more sustainable kinetic
resolution protocol is achieved.
Acknowledgments
We are indebted to fund SEP-CINVESTAV via grant 126. M. P.-
V. thanks CONACYT for doctoral scholarship 70766.
Keywords: Ketorolac • Mechanoenzymatic • CALB •
Sustainable • Deracemization
Experimental Section
1
General Information. H and 13C spectra were recorded on a BRUKER
[1]
(a) M. T. Reetz, J. Am. Chem. Soc. 2013, 135, 12480-12496. (b) R. A.
Sheldon, D. Brady, Chem. Commun. 2018, 54, 6088-6104. (c) P. N.
Devine, R. M. Howard, R. Kumar, M. P. Thompson, M. D. Truppo, N. J.
Turner, Nat. Rev. Chem. 2018, 2, 409-421.
DP300 (300MHz). High resolution mass spectra were recorded on a HPLC
1100 coupled to a MSD-TOF Agilent series HR-MSTOF model 1969 A.
Chromatograms were acquired in a Dionex HPLC Ultimate 3000 with a
UV/VIS detector, with a diode array, at 210 and 254 nm. High-Speed Ball-
Milling reactions were carried out in a Retsch, Mixer Mill (MM200).
Ketorolac was extracted from commercial tablets of ketorolac-
trometahmine. All reagents were purchased from Sigma-Aldrich (Merck)
and used as received. Immobilized CALB was purchased from Novozymes,
Novozym 435® (Immobilized on acrylic resin, IU/g > 10000).
[2]
(a) A. C. Lustosa de Melo Carvalho, T. De S. Fonseca, M. C. de Mattos,
M. Da. C. Ferreira de Oliveira, T. L. Gomes de Lemos, F. Molinari, D.
Romano, I. Serra, Int. J. Mol. Sci. 2015, 16, 29682-29716. (b) D. J.
Pollard, J. M. Woodley, Trends Biotechnol. 2006, 25, 66-73.
S. A. Kelly, S. Pohle, S. Wharry, S. Mix, C. C. R. Allen, T. S. Moody, B.
F. Gilmore, Chem. Rev. 2018, 118, 349-367.
[3]
[4]
F. Xu, B. Kosjek, F. L. Cabirol, H. Chen, R. Desmond, J. Park, A. P.
Gohel, S. J. Collier, D. J. Smith, Z. Liu, J. M. Janey, J. Y. L. Chung, O.
Alvizo, Angew. Chem. Int. Ed. 2018, 57, 6863-6867.
(a) V. B. Urlacher, S. Eiben, Trends Biotechnol. 2006, 24, 324-330. (b)
K. Schroer, M. Kittelmann, S. Lütz, Biotechnol. Bioeng. 2010, 106, 699-
706.
General method for the preparation of racemic esters of Ketorolac
esters (rac-2 to rac-6). To a benzene (5 mL) solution of racemic Ketorolac
(0.1 mmol) was added 3 equivalent of the corresponding esterification
alcohol and 0.1 equivalents of p-toluenesulfonic acid. The resulting mixture
was heated to 120°C for 20 minutes in a Monowave 50 apparatus. The
crude product was extracted with ethyl acetate (25 mL), dried with
anhydrous Na2SO4, concentrated and purified using a mixture of hexane
and ethyl acetate (9:1) to obtain the corresponding Ketorolac ester as a
racemic mixture.
[5]
[6]
(a) S. B. Rubin-Pitel, H. Zhao, Comb. Chem. High Throughput Screen.
2006, 9, 247-257. (b) R. A. Sheldon, Adv. Synth. Catal. 2007, 349, 1289-
1307.
[7]
[8]
D. D. Young, J. Nichols, R. M. Kelly, A. Deiters, J. Am. Chem. Soc. 2008,
130, 10048-10049.
I. Gonçalves, C. Silva, A. Cavaco-Paulo, Green Chem. 2015, 17, 1362-
1374.
General method for the mechanoenzymatic kinetic resolution of
racemic Ketorolac by means of enantioselective esterification. A
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
[9]
J. G. Hernández, M. Frings, C. Bolm, ChemCatChem 2016, 8, 1769-
1772.
[10] P. T. Anastas, J. C. Warner in Green Chemistry: Theory and Practice,
Vol. 1, Oxford University Press, Oxford, 1998.
[11] R. A. Sheldon, J. M. Woodley, Chem. Rev. 2018, 118, 801-838.
[12] (a) K. J. Ardila-Fierro, D. E. Crawford, A. Körner, S. L. James, C. Bolm,
J. G. Hernández, Green Chem. 2018, 20, 1262-1269. (b) F. Hammerer,
L. Loots, J.-L. Do, J. P. D. Therien, C. W. Nickels, T. Friščić, K. Auclair,
Angew. Chem. 2018, 130, 2651-2654. (c) J. P. D. Therien, F. Hammerer,
T. Friščić, K. Auclair, ChemSusChem 2019, 12, 3481-3490. For a
thorough review see: (d) C. G. Avila-Ortiz, M. Pérez-Venegas, J. Vargas-
Caporali, E. Juaristi, Tetrahedron Lett. 2019, 60, 1749-1757. (e) S.
Kaabel, T. Friščić, K. Auclair, ChemBioChem 2019, DOI:
10.1002/cbic.201900567.
[13] (a) M. Pérez-Venegas, G. Reyes-Rangel, A. Neri, J. Escalante, E.
Juaristi, Beilstein. J. Org. Chem. 2017, 13, 1728-1734. (b) M. Pérez-
Venegas, E. Juaristi, Tetrahedron 2018, 74, 6453-6458.
[14] J. G. Hernández, K. J. Ardila-Fierro, D. Crawford, S. L. James, C. Bolm,
Green Chem. 2017, 19, 2620-2625.
[15] F. Hammerer, S. Ostadjoo, T. Friščić, K. Auclair, ChemSusChem 2019,
DOI: 10.1002/cssc.201902752
[16] M. Pérez-Venegas, M. M. Tellez-Cruz, O. Solorza-Feria, A. López-
Munguía,
E.
Castillo,
E.
Juaristi,
ChemCatChem
2019,
DOI:10.1002/cctc.201901714.
[17] (a) A. M. Evans, Eur. J. Clin. Pharmacol. 1992, 42, 237-256. (b) P. J.
Hayball, J. Wrobel, J. G. Tamblyn, R. L. Nation, Br. J. Clin. Pharmac.
1994, 37, 75-78. (c) P. J. Hayball, Drugs 1996, 52, 47-58. (d) A. Handley,
P. Cervoni, J. E. McCray, J. R. McCullough, J. Clin. Pharmacol. 1998,
38, 25-35.
[18] J. C. Gillis, R. N. Brogden, Drugs 1997, 53, 139-188.
[19] K. I. Kaitin, P. A. DiCerbo, L. Lasagna, J. Clin. Pharmacol. 1991, 31, 116-
122.
[20] P. S. Baran, J. M. Richter, D. W. Lin, Angew. Chem. Int. Ed. 2005, 44,
609-612.
[21] P. Malik, R. Bhushan, New J. Chem. 2017, 41, 13681-13691.
[22] (a) G. Fuelling, C. J. Sih, J. Am. Chem. Soc. 1987, 109, 2845-2846. (b)
Y. H. Kim, C. S. Cheong, S. H. Lee, K. S. Kim, Tetrahedron: Asymmetry
2001, 12, 1865-1869. (c) S. D. Shinde, G. D. Yadav, Process Biochem.
2015, 50, 230-236.
[23] N. R. Rightmire, T. P. Hanusa, Dalton Trans. 2016, 45, 2352-2362.
[24] Y. Pan, K.-W. Tang, C.-Q. He, W. Yi, W. Zhu, Y.-N. Liu, Biotechnol. Appl.
Biochem. 2014, 61, 274-279.
[25] (a) K. C. McKissic, J. T. Caruso, R. G. Blair, J. Mack, Green Chem. 2014,
16, 1628-1632. (b) J. Andersen, J. Mack, Angew. Chem. Int. Ed. 2018,
57, 13062-13065.
[26] T. Friščić, A. V. Trask, W. Jones, W. D. S. Motherwell, Angew. Chem. Int.
Ed. 2006, 45, 7546-7550.
[27] S. D. Banik, M. Nordblad, J. M. Woodley, G. H. Peters, ACS Catal. 2016,
6, 6350-6361.
[28] M. N. Varma, G. Madras, Appl. Biochem. Biotechnol. 2008, 144, 213-223.
[29] C. M. Alder, J. D. Hayler, R. K. Henderson, A. M. Redman, L. Shukla, L.
E. Shuster, H. F. Sneddon, Green Chem. 2016, 18, 3879-3890.
[30] S. D. Banik, M. Nordblab, J. M. Woodley, G. H. J. Peters, Catalysts 2017,
7, 227-244.
[31] L. G. Hudson, L. S. Cook, M. M. Grimes, C. Y. Muller, S. F. Adams, A.
Wandinger-Ness, Cancers 2019, 11, 1049-1065.
[32] L. G. Hudson, J. M. Gillette, H. Kang, M. R. Rivera, A. Wandinger-Ness,
Cancers 2018, 10, 358-384.
[33] Y. Guo, S. R. Kenney, C. Y. Muller, S. Adams, T. Rutledge, E. Romero,
C. Murray-Krezan, R. Prekeris, L. A. Sklar, L. G. Hudson, A. Wandinger-
Ness, Mol. Cancer Ther. 2015, 14, 2215-2227.
[34] P. Forget, J. Vandenhende, M. Berliere, J.-P. Machiels, B. Nussbaum, C.
Legrand, M. D. Kock, Anesth. Analg. 2010, 110, 1630-1635.
[35] P. L. A. Overbeeke, J. Ottosson, K. Hult, J. A. Jongejan, J. A. Duine,
Biocatal. Biotransformation 1999, 17, 61-79.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201902292
ChemCatChem
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Both (R) and (S) enantiomers of
pharmaceutically relevant chiral drug
Ketorolac were isolated in high
enantiopurity (ee > 85%) through a
Mechanochemistry
M. Pérez-Venegas, A. M. Rodríguez-
Treviño, E. Juaristi*
dual
mechanoenzymatic kinetic
Page No. – Page No.
resolution strategy using Candida
Dual mechanoenzymatic kinetic
resolution of ketorolac
antarctica
mechanoenzymatic
proceed with
enantiodiscrimination (E
avoiding the use of bulk solvent.
Lipase
B.
These
reactions
extraordinary
500),
>
This article is protected by copyright. All rights reserved.