Enzymatic kinetic resolution of ketorolac

Article abstract of DOI:10.1016/S0957-4166(01)00332-9

Ketorolac 1 was resolved into each enantiomer by interesterification using lipase B from Candida antarctica. The acid reacted with various alcohols and the ester and acid were resolved up to >99% e.e. when reacted with octanol, which was the best result.

Full text of DOI:10.1016/S0957-4166(01)00332-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1865–1869
Enzymatic kinetic resolution of ketorolac
Young Hee Kim,a Chan Seong Cheong,a,* So Ha Leea and Kwan Soo Kimb
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, South Korea
bDepartment of Chemistry, Yonsei University, Seoul 120-749, South Korea
Received 11 June 2001; accepted 23 July 2001
Abstract—Ketorolac 1 was resolved into each enantiomer by interesterification using lipase B from Candida antarctica. The acid
reacted with various alcohols and the ester and acid were resolved up to >99% e.e. when reacted with octanol, which was the best
result. To increase reactivity and enantioselectivity, the experimental conditions were adjusted by varying temperature, solvent,
alcohols and reaction time. © 2001 Published by Elsevier Science Ltd.
1. Introduction
2. Results and discussion
Ketorolac 1 (Toradol™), [( )-5-benzoyl-1,2-dihydro-
3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid] is a non-
steroidal anti-inflammatory drug (NSAID) with
cyclooxygenase inhibitory activity.1 It contains one
stereogenic carbon center and is on the market as a
racemic mixture. It is known that in several NSAIDs
such as ibuprofen and naproxen one enantiomer differs
in properties from the other.2 In 1996, Mroszczak et al.3
reported that its active enantiomer is the (−)- or (S)-
form, while the (R)-form is inactive from the chiral
kinetics and dynamics of ketorolac. In spite of its
beneficial activity, it has some adverse effects such as
gastrointestinal bleeding, renal impairment and platelet
inhibition with altered haemostasis. One way to mini-
mize these effects is to prescribe at the lowest dosage
necessary.4 If the active enantiomer of the racemate
could be used, the dosage might be halved. Thus, it is
important to resolve such compounds. Unfortunately,
only a few results have been reported to date. One
method involves resolution using hydrolase biocata-
lysts.5 The results were excellent only when proteases
were used. Herein, we report for the first time the
resolutions of ketorolac via esterification using lipase B
from Candida antarctica.
To resolve 1 using hydrolases, either hydrolase-cata-
lyzed hydrolysis or esterification is suitable. At first,
ketorolac butyl ester 3 was hydrolyzed as shown in
Scheme 1. The results are shown in Table 1.
As reported previously,5 the protease, Streptomyces
griseus, hydrolyzed 3 in favor of pure (S)-acid, while C.
antarctica lipase B favored the (R)-acid. The others
resolved 3 enantioselectively. In this reaction, the faster
reacting enantiomer was the (R)-isomer except when
using S. griseus. Although the residual ester is quite
pure using S. griseus, C. antarctica lipase B and Mucor
meihei, the final step in the synthesis of ketorolac from
the ester will induce racemization. Thus, it is important
to obtain the acid in pure (S)-form. We therefore
examined esterification as another method.
In search of screening esterifications it was most proba-
ble that 1 reacted with an alcohol such as 1-butanol
using C. antarctica lipase B (Scheme 2). At first, we
investigated the effect of the various alcohols on the
enantioselectivity in the organic solvent. The results are
shown in Table 2.
In this esterification, the straight chain alkanols inter-
acted with acid 1 smoothly and enantioselectively (E>
200), but the relatively hindered alcohols did not. The
fast reacting enantiomers are (R)-form in both hydroly-
sis and esterification reactions. These results are concor-
dant with the previous reports.6–8 However, the
stereospecificity is superior to them and among the
various alcohols, the ethanol, 1-butanol, 1-octanol, and
1-tetradodecanol reactions are the best. Secondly, in
O
O
N
OH
N
OR
O
O
+
( )
+
Ketorolac,
-1
( )-2
-
-
* Corresponding author.
0957-4166/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0957-4166(01)00332-9

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Y. H. Kim et al. / Tetrahedron: Asymmetry 12 (2001) 1865–1869
O
N
OC4H9
O
O
Enzyme, pH 7 phosphate buffer,
0.05M NaOH, pH stat
(-)-(S)-3, ester
N
OC4H9
O
+
+
( )-3
O
-
N
OH
O
(+)-(R)-1, acid
Scheme 1. Hydrolysis of ketorolac butyl ester 3.
Table 1.
Hydrolase
Time (h)
Conversion (%)
Acid e.e. (%)
Ester e.e. (%)
C. rugosa lipase
C. antarctica lipase B
S. griseus
7
28
0.5
3
1.5
29
22
32
0.5
2.5
47
84
19
60
7
68
11
15
26
96
91 (R)
–*
100 (R)
–*
100 (S)
–*
64 (R)
–*
–*
46 (S)
–*
99 (S)
–*
98 (R)
–*
1 (S)
–*
92 (S)
A. saitoi
M. meihei lipase
71 (R)
–*
* These values are very low.
O
N
OH
O
O
Candida antarctica lipase B, ROH
(-)-(S)-1, acid
N
OH
methylene chloride, 34 oC
O
+
+
( )
-1
-
O
N
OR
O
(+)-(R)-2, ester
Scheme 2. Esterification of racemic ketorolac 1 with various alcohols using CAL-B.
order to increase the enantioselectivity of the (S)-acid,
the reaction conditions were monitored by changing
solvent and temperature. In lipase-catalyzed esterifica-
tions, the reaction solvent is very important for increas-
ing reactivity and stereoselectivity.9–11 We studied the
effect of organic solvents from non-polar to polar and
showed conversion and enantioselectivity in Fig. 1 and
Table 3.
hydrofuran, acetonitrile, dimethylformamide and 1,2-
dichloroethane, it was very low. In view of obtaining
the pure (S)-acid, we are interested in the solvents such
as dichloromethane and 1,2-dichloroethane because 1
was resolved in >99% e.e. {[h]2D2 −154 (c 1.05,
CH3OH)}.5 This phenomenon cannot be fully explained
at present, but might result from the formation of
complex interactions.11 Finally, in these chlorinated
solvents, we controlled the reaction temperature to
increase the reaction rate without the enzyme destabi-
lization. To do this, we used 1,2-dichloroethane because
of its high boiling point (83°C) and these results are
shown in Fig. 2 and Table 4.
As shown in Fig. 1, in this reaction, the conversion rate
might be independent of the solvent polarity. In n-hex-
ane, dichloromethane and diisopropyl ether, it had the
same trend until 50% conversion. However, in tetra-

Y. H. Kim et al. / Tetrahedron: Asymmetry 12 (2001) 1865–1869
1867
Table 2.
Alcohol
Reaction time (h)
Conversion (%)
Enantiomeric excess
E
(% e.e.s) (S)
(% e.e.p) (R)
Ethanol
1-Butanol
1-Hexanol
1-Octanol
43
42
48
52
49
49
25
25
116
49
43
53
59
58
59
58
2
1
5
56
52
90
86
93
87
86
47
80
25
76
99
98
97
96
97
98
86
71
89
97
\200
\200
183
167
187
\200
21
14
21
151
1-Decanol
1-Tetradecanol
2-Chloroethanol
Benzyl alcohol
iso-Propanol
iso-Butanol
Figure 1. Solvent effect on the conversion in esterification of 1.
Table 3.
Solvent
Reaction time (h)
Conversion (%)
Enantiomeric excess
E
(% e.e.s) (S)
(% e.e.p) (R)
Acetonitrile
52
5
44
69
3
58
50
55
14
37
1
93
87
\99
13
96
99
\99
\99
99
167
Diisopropyl ether
Dichloromethane
Tetrahydrofuran
n-Hexane
Dimethylformamide
1,2-Dichloroethane
\200
\200
\200
\200
–
22
3
69
–
–
50
\99
\99
\200
As anticipated, the reaction time required to reach around
50% conversion reduced with increasing reaction temper-
ature. At 60°C, the reaction time required to obtain the
pure (S)-isomer was one third of that observed at 34°C.
Thus, we can select any reaction condition at either 34
or 60°C depending on time or energy considerations.
esterification with various alcohols in organic solvents
using C. antarctica lipase B. Among them, the resolving
efficiency was very high in n-octanol. To check solvent
effects, the acid 1 reacted with n-octanol in chlorinated
solvents such as dichloromethane and 1,2-dichloro-
methane and each enantiomer was resolved in up to
99% e.e., respectively. To get pure (S)-acid, the tem-
perature was acceptable from 34 to 60°C. These re-
3. Conclusion
solving conditions using C. antarctica lipase
B
will provide a useful guide for the other secondary chiral
acids.
In summary, we resolved ketorolac [( )-5-benzoyl-1,2-
dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid], by

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Y. H. Kim et al. / Tetrahedron: Asymmetry 12 (2001) 1865–1869
Figure 2.
Table 4.
Temperature (°C)
Reaction time (h)
Conversion (%)
Enantiomeric excess
E
(% e.e.s) (S)
(% e.e.p) (R)
34
40
50
60
69
58
30
24
50
49
55
53
99
85
98
99
98
\99
98
\200
\200
\200
\200
\99
4. Experimental
4.2. 5-Benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-
carboxylic acid butyl ester
4.1. General
A solution of ketorolac12 (100 mg, 0.392 mmol) in
benzene (5 mL) was stirred at room temperature. 1-
Butanol (35.9 mL, 3.92 mmol, 10 equiv.) and p-toluene-
sulfonic acid (7 mg, 0.0392 mmol, 0.1 equiv.) were
added to the solution. The mixture was heated under
reflux for 3 h then quenched with water. The product
was extracted with ether, the organic layer was washed
with saturated aqueous NaHCO3 and brine. The extract
was dried with anhydrous MgSO4, filtered and the
filtrate concentrated to an oil. The crude product was
purified by column chromatography (n-hexane:ethyl
acetate=10:1) to yield the ester as an oil (108 mg,
89%); 1H NMR (300 MHz, CDCl3) l 0.96 (t, 3H,
J=7.4), 1.37–1.44 (m, 2H), 1.64–1.69 (m, 2H), 2.77–
2.94 (m, 2H), 4.05–4.20 (m, 2H), 4.47–4.58 (m, 2H),
6.11 (d, J=3.4, 1H), 6.83 (d, J=4.0), 7.43–7.54 (m,
3H), 7.82 (dd, J=6.9 Hz, J=1.7 Hz, 2H); 13C NMR
(75 MHz, CDCl3) l 14.071, 19.501, 31.003, 31.286,
43.069, 47.989, 65.751, 103.474, 125.358, 127.560,
128.530, 129.281, 131.756, 139.679, 142.945, 171.679,
185.359; IR (KBr) 2960, 1734, 1624, 1268, 1180 cm−1;
GC/MSD retention time (min) 13.76, m/z 51, 65, 77,
91, 105, 115, 123, 132, 140, 152, 167, 180, 191, 210
(100), 226, 234, 254, 268, 282, 294, 311(M+).
The organic solvents, methanol, n-hexane and 2-
propanol were all HPLC-grade and purchased from J.
T. Baker. Trifluoroacetic acid (TFA) was obtained
from Aldrich. Candida rugosa lipase, S. griseus and
Aspergillus saitoi were obtained from Sigma Co. Mucor
miehei was obtained from Fluka. Candida antarctica
lipase B was obtained from NOVOZYME. The IR
spectra were measured on a Perkin–Elmer 16FPC FT-
IR grating infrared spectrophotometer. 1H and 13C
NMR spectra were recorded in CDCl3, at 300.13 and
75.46 MHz, respectively, on a Bruker AVANCE-300
spectrometer; chemical shifts being expressed in ppm
with reference to Me4Si, coupling constants (J) in Hz.
Mass spectra were obtained on a Hewlett–Packard 5890
series II; a Hewlett–Packard 5972 series mass selective
detector GC/MS spectrometer equipped with HP-5
cross-linked 5% phenyl methyl silicon fused silica capil-
lary column (25 m×0.20 mm i.d.). The optical rotations
were determined with an AUTOPOL III (Rudolph
Research, Flanders, NJ) recording polarimeter in
MeOH. A YOUNG-LIN high performance liquid chro-
matograph (Korea), equipped with a YOUNG-LIN
M930 Pump; a YOUNG-LIN M720 absorbance detec-
tor were used. The computer programs used were
Autochro 2.0 plus for chromatographic analysis. A
chiralpak AD column (0.46 cmƒ×25 cm) packed with
an amylose derivative coated on silica gel was used
(Daicel Chemical Industries). TLC was carried out on
Merck glass plates precoated with silica gel 60F-254.
Column chromatography was performed by Merck 70–
230 or 230–400 mesh silica gel. Enzymatic hydrolysis
was carried out on 718 STAT Titrino (Metrohom,
Switzerland).
4.3. Enzymatic hydrolysis of butyl ester of ketorolac
The butyl ester of ketorolac (40 mg, 0.16 mmol) was
dissolved in phosphate buffer (7 mL, pH 7.0) and
enzyme (0.2–2 mass equiv.) was added to the solution.
A pH stat regulated the addition of aqueous 0.02 M
NaOH solution at room temperature to maintain the
pH at 7.0. A sample was taken for HPLC analysis

Y. H. Kim et al. / Tetrahedron: Asymmetry 12 (2001) 1865–1869
1869
(chiralpak AD, n-hexane:IPA:TFA=90:10:0.1 v/v%,
310 nm, 0.8 mL/min). The results are shown in Table 1.
this work was supported by a grant from the Ministry
of Science and Technology in Korea.
4.4. Esterifications of ketolorac with alcohols using C.
antarctica lipase B
References
Ketorolac (10 mg, 0.039 mmol) was dissolved in MeCN
(5 mL) and alcohol (0.39 mmol, 10 equiv.), ground
molecular sieves (4 A, 30 mg) were added to the
solution. CAL-B (10 mg, 1 mass equiv.) was added to
the solution, and then mixture was shaken in incubator
at 33.8°C. The mixture was taken periodically from the
reactor and filtered and evaporated. The slurry was
dissolved in 1 mL MeOH for HPLC analysis (chiralpak
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Acknowledgements
A generous donation of C. antarctica lipase B by Novo
Nordisk (Seoul, Korea) is gratefully acknowledged and