Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Article abstract of DOI:10.1002/chir.23141

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.

Full text of DOI:10.1002/chir.23141

Received: 1 July 2019
Revised: 4 October 2019
Accepted: 5 October 2019
DOI: 10.1002/chir.23141
R E G U L A R A R T I C L E
Investigation of the chiral recognition ability of human
carboxylesterase 1 using indomethacin esters
Masato Takahashi
| Daisuke Takani | Masami Haba | Masakiyo Hosokawa
Faculty of Phramacy, Chiba Institute of
Science, Chiba, Japan
Abstract
Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role
in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation
of the chiral recognition ability of hCES1 was attempted using indomethacin
esters in which various chiral alcohols were introduced. Indomethacin was
condensed with various chiral alcohols to synthesize indomethacin esters.
The synthesized esters were hydrolyzed with a human liver microsome
(HLM) solution and a human intestine microsome (HIM) solution. High
hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis
reaction in the HLM solution but not in the HIM solution, and these indometh-
acin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin
esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates
of the esters were calculated. The stereoselectivity confirmed in HLM solution
was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters
in which a phenyl group is bonded next to the ester, the Vmax value of the (R)
form was 10 times larger than that of the (S) form.
Correspondence
Masato Takahashi, Faculty of Phramacy,
Chiba Institute of Science, 15–8, Shiomi‐
cho, Choshi, Chiba 288–0025, Japan.
Email: matakahashi@cis.ac.jp
Funding information
JSPS KAKENHI, Grant/Award Numbers:
JP17K15519 and JP16K08378
KEYWORDS
carboxylesterase, chiral recognition, ester prodrug, indomethacin, substrate specificity1
1 | INTRODUCTION
the liver.8-10 It is known that hCES1 efficiently hydrolyzes
a compound with a small alkoxy group and a large acyl
Among drug‐metabolizing enzymes, carboxylesterase
(CES, EC 3.1.1.1) is known to play the most important
role in the hydrolysis metabolism of ester compounds.1-4
Since CESs efficiently hydrolyze not only ester com-
pounds but also amide and thioester compounds, it is
involved in the metabolism and detoxification of many
pharmaceuticals, natural products, foods, and environ-
mental chemicals.3-7 Human CES1 (hCES1) and human
CES2 (hCES2) are mainly expressed in the liver and intes-
tine, respectively. Therefore, hCES1 plays a major role in
hydrolytic metabolism in the liver and hCES2 plays a
major role in hydrolytic metabolism in the small intes-
tine. hCES1 has been attracting attention as an enzyme
responsible for metabolic activation of ester prodrugs in
group and that hCES2 efficiently hydrolyzes a compound
with a small acyl group and a large acyl group. It has
been reported that substrates metabolized by hCES1 and
by hCES2 are distinctly different.11-13 In addition to
hCES, research on mouse CES and rat CES is also being
conducted.3,14,15
It has been confirmed that hCES1 has a chiral recogni-
tion ability, and methylphenidate (chiral center on the
acyl group),16 flurbiprofen ester (chiral center on the acyl
group),17,18 and propranolol ester (chiral center on the
alkoxy group) have been reported to have different meta-
bolic rates by CES between the (R) form and (S) form.19-21
Recently, we have reported that there were 2.1‐fold and
10.2‐fold differences in the rate of hydrolysis in an hCES1
Chirality. 2019;1–8.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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TAKAHASHI ET AL.
solution of the enantiomers of an indomethacin ester
prodrug with a 2‐butyl group and those with a 1‐
phenylpropyl group, respectively.9 However, these exam-
ples provide little information and it has not been possi-
ble to clarify in detail what structural or electronic
features hCES1 recognizes. It is considered to be impor-
tant to understand the metabolism of ester‐type drugs
by examining the chiral recognition ability of hCES1. In
this study, ester compounds were synthesized using indo-
methacin as a substrate and various alcohols having an
asymmetric center. These compounds were hydrolyzed
in human liver microsome (HLM) solution, human intes-
tine microsome (HIM) solution, and an hCES1 solution,
and hydrolytic parameters were calculated. We aimed
to elucidate what kind of asymmetric center hCES1
recognizes.
nitrophenyl acetate assay). HIM was purchased from
Corning (MA, USA) and was prepared from human intes-
tine derived from seven donors (mixed gender, 24‐69 years
old, Caucasian and African American). The HIM was
diluted in the SET buffer (pH 7.4) to obtain HIM solution
(5 mg/mL, 1396 nmol/min/mg according to the 4‐
nitrophenyl acetate assay). hCES1 and hCES2 were pur-
chased from Corning (MA, USA). The hCES1 and hCES2
were expressed from hCES1 (or hCES2) cDNA using a
baculovirus expression system. Baculovirus‐infected
insert cells were used to prepare these microsomes. The
hCES1 and hCES2 were diluted in the SET buffer
(pH 7.4) to obtain hCES1 solution (5 mg/mL,
489 nmol/min/mg according to the 4‐nitrophenyl acetate
assay) and hCES2 solution (5 mg/mL, 797 nmol/min/mg
according to the 4‐nitrophenyl acetate assay). The recom-
binant AADAC was expressed in Sf9 cells using a BAC‐
TO‐BAC Expression System (Invitrogen, CA, USA). The
pFAST BAC/AADAC was transformed into DH10Bac
cells. The recombinant bacmid DNA containing AADAC
cDNA was transfected into Sf9 cells. The virus was har-
vested 72 hours later. The cells were centrifuged at
1700 × G for 10 minutes to separate cells and virus. Cells
were routinely infected with the virus and harvested
72 hours after infection. Cytosol of Sf9 cells expressing
AADAC and that of Sf9 cells infected with mock virus
were prepared by subjecting the cell lysate to centrifuga-
tion (105 000 × G for 60 minutes at 4°C). The AADAC
was diluted in the SET buffer (pH 7.4) to obtain AADAC
solution (5 mg/mL, 38 nmol/min/mg according to the 4‐
nitrophenyl acetate assay).
2 | MATERIALS AND METHODS
2.1 | General
Melting points (mp) were measured using a Micro mp
apparatus (Yanaco, Kyoto, Japan) and are uncorrected.
Infrared (IR) spectra were recorded on a FT‐720 (Horiba,
1
Kyoto, Japan). H‐ and 13C‐ NMR spectra were obtained
1
on an Ascend 400 (Bruker, MA, USA). H‐NMR spectra
were recorded in CDCl3 as a solvent on a 400‐MHz spec-
trometer using tetramethylsilane (TMS) (0.00 ppm) as an
internal standard. 13C‐NMR spectra were recorded in
CDCl3 as a solvent on a 100‐MHz spectrometer using
the central peak of CDCl3 (77.0 ppm) as an internal
standard.
2.2 | Synthetic protocols
2.1.1 | Preparation of samples
A mixture of indomethacin (1) (0.50 mmol), N,N‐
dicyclohexylcarbodiimide (DCC, 0.75 mmol), and N,N‐
dimethylaminopyridine (DMAP, 0.065 mmol) in
CH2Cl2/alcohol (50/1, 5.1 mL) was stirred at room tem-
perature under an argon atmosphere for 1 day. After
addition of 10% citric acid solution, the whole mixture
was stirred at the same temperature for 5 minutes. The
mixture was extracted with CH2Cl2 (20 mL), and the
organic solution was dried over MgSO4 and evaporated
under reduced pressure. The residue was purified by col-
umn chromatography on SiO2 (ethyl acetate/n–hexane)
to give an ester derivative 2.
Human liver derived from single donor (male, 51 years
old, Caucasian, HIV, HBV, and HCV virus‐free) was
obtained from the Human and Animal Bridging (HAB)
Research Organization (Japan), in partnership with the
National Disease Research Interchange (NDRI). The use
of human livers was approved by the Ethics Committee
of Chiba Institute of Sciences (No. 22‐1), Japan, based
on the Helsinki declaration. The liver was chilled and
homogenized in 100 mM ice‐cold phosphate‐buffered salt
(PBS) solutions. The homogenate was centrifuged at
9000 × G for 20 minutes at 4°C, and the supernatant
was ultra‐centrifuged at 105 000 × G for 60 minutes at
4°C. The microsomal fraction was suspended in 0.25 M
sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA),
and 100 mM tris (hydroxymethyl)aminomethane (Tris)
HCl buffer (SET buffer, pH 7.4) to obtain an HLM solu-
tion (5 mg/mL, 1396 nmol/min/mg according to the 4‐
2.2.1 | Indomethacin pentan‐2‐yl ester 2a
1
mp 66°C to 67°C; IR (KBr) 1732, 1685 cm−1; H NMR
(400 MHz) δ 0.86 (3H, t, J = 7.2 Hz), 1.21 (3H, d,
J = 6.4 Hz), 1.22 to 1.31 (2H, m), 1.40 to 1.48 (1H, m),

TAKAHASHI ET AL.
3
1.53 to 1.59 (1H, m), 2.38 (3H, s), 3.63 (2H, s), 3.84 (3H, s),
4.89 to 4.97 (1H, m), 6.67 (1H, dd, J = 8.8, 2.4 Hz), 6.88
(1H, d, J = 8.8 Hz), 6.97 (1H, d, J = 2.4 Hz), 7.47 (2H,
d, J = 8.8 Hz), 7.65 (2H, d, J = 8.8 Hz); 13C NMR
(100 MHz) δ 13.3, 13.8, 18.6, 20.0, 30.7, 38.1, 55.7, 71.6,
101.3, 111.7, 113.0, 114.9, 129.1, 130.7, 130.9, 131.1,
134.0, 135.8, 139.2, 156.1, 168.3, 170.5; HPLC retention
time tR = 22.7 minutes.
2.2.5 | Indomethacin 1‐phenylethan‐1‐yl
ester 2e
1
mp 88°C to 89°C; IR (KBr) 1732, 1668 cm−1; H NMR
(400 MHz) δ 1.53 (3H, d, J = 6.8 Hz) 2.17 (4H, s),
2.35 (3H, s), 3.68 (2H, s), 3.76 (3H, s), 5.89 (1H, q,
J = 6.4 Hz), 6.66 (1H, dd, J = 9.2, 2.4 Hz), 6.89 (1H,
d, J = 9.2 Hz), 6.91 (1H, d, J = 2.4 Hz), 7.28 to 7.32
(5H, m), 7.46 (2H, d, J = 8.4 Hz), 7.64 (2H, d,
J = 8.4 Hz); 13C NMR (100 MHz) δ 13.4, 22.3, 30.7,
55.6, 73.1, 101.2, 111.9, 112.7, 115.0, 126.0, 127.9,
128.5, 129.1, 130.6, 130.8, 131.2, 134.0, 135.8, 139.2,
141.4, 156.1, 168.3, 170.1; HPLC retention time
tR = 23.8 minutes.
2.2.2 | Indomethacin
1‐methoxypropan‐2‐yl ester 2b
1
mp 57°C to 58°C; IR (KBr) 1736, 1687 cm−1; H NMR
(400 MHz) δ 1.23 (3H, d, J = 6.4 Hz), 2.38 (3H, s), 3.32
(3H, s), 3.38 to 3.46 (2H, m), 3.67 (2H, s), 3.84 (3H, s),
5.07 to 5.13 (1H, m), 6.67 (1H, dd, J = 9.2, 2.4 Hz), 6.88
(1H, d, J = 8.8 Hz), 6.97 (1H, d, J = 2.4 Hz), 7.47 (2H,
d, J = 8.8 Hz), 7.66 (2H, d, J = 8.8 Hz); 13C NMR
(100 MHz) δ 13.4, 16.6, 30.6, 55.7, 59.1, 70.0, 75.0, 101.4,
111.7, 112.8, 114.9, 129.1, 130.7, 130.9, 131.2, 134.0,
135.9, 139.2, 156.1, 168.3, 170.4; HPLC retention time
tR = 16.9 minutes.
2.3 | Hydrolysis reaction (HLM, HIM)
The indomethacin ester 2 (25 mM) in dimethylsulfoxide
(DMSO) solution (1 μL, final concentration of 0.25 mM)
was warmed with 5 mg/mL HLM or HIM solution (5 μL,
final concentration of 0.25 mg/mL), 200 mM 4‐(2‐
hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES)
buffer (50 μL, final concentration of 100 mM), and puri-
fied water (44 μL) at 37°C for 30 minutes (HLM) or
1440 minutes (HIM). Immediately after addition of
0.3 mM butyl para‐hydroxybenzoate (BPHB) in CH3CN
(100 μL, 50 nmol), the whole mixture was cooled in an
ice bath for 10 minutes. The mixture was centrifuged at
21,600 × G for 15 minutes at 4°C to precipitate proteins,
and then the supernatant was filtered and analyzed by
high‐performance liquid chromatography (HPLC).
2.2.3 | Indomethacin but‐3‐yn‐2‐yl ester 2c
1
mp 46°C to 47°C; IR (KBr) 1741, 1685 cm−1; H NMR
(400 MHz) δ 1.51 (3H, d, J = 6.4 Hz), 2.38 (3H, s), 2.45
(1H, d, J = 2.4 Hz), 3.67 (2H, s), 3.84 (3H, s), 5.45 (1H,
dq, J = 6.8, 2.4 Hz), 6.67 (1H, dd, J = 8.8, 2.4 Hz), 6.88
(1H, d, J = 8.8 Hz), 6.97 (1H, d, J = 2.8 Hz), 7.47 (2H,
d, J = 8.8 Hz), 7.66 (2H, d, J = 8.8 Hz); 13C NMR
(100 MHz) δ 13.4, 21.3, 30.3, 55.7, 60.7, 73.1, 81.9, 101.2,
111.8, 112.2, 115.0, 129.1, 130.6, 130.8, 131.2, 133.9,
136.0, 139.3, 156.1, 168.3, 169.7; HPLC retention time
tR = 17.4 minutes.
2.4 | Hydrolysis reaction (hCES1, hCES2,
AADAC)
Indomethacin ester (0.3125‐10 mM) 2 in DMSO solution
(1 μL, final concentration of 0.003125‐0.10 mM) was
warmed with 5 mg/mL enzyme solution (5 μL, final con-
centration of 0.25 mg/mL), 200 mM HEPES buffer (50 μL,
final concentration of 100 mM), and purified water
(44 μL) at 37°C for 30 to 180 minutes. Immediately after
addition of 0.3 mM BPHB in CH3CN (100 μL, 50 nmol),
the whole mixture was cooled in an ice bath at
10 minutes. The mixture was centrifuged at 21 600 × G
for 15 minutes at 4°C to precipitate proteins, and then
the supernatant was filtered and analyzed by HPLC.
2.2.4 | Indomethacin
3‐chloro‐1‐phenylpropan‐1‐yl ester 2d
1
mp 123°C to 124°C; IR (KBr) 1731, 1671 cm−1; H NMR
(400 MHz) δ 2.12 to 2.21 (1H, m), 2.31 to 2.39 (4H, m),
3.32 to 3.47 (2H, m), 3.70 (2H, s), 3.77 (3H, s), 5.94 (1H,
dd, J = 8.8, 5.6 Hz), 6.67 (1H, dd, J = 8.8, 2.4 Hz), 6.87
to 6.90 (2H, m), 7.23 to 7.33 (5H, m), 7.47 (2H, d,
J = 8.8 Hz), 7.64 (2H, d, J = 8.8 Hz); 13C NMR
(100 MHz) δ 13.3, 30.6, 39.0, 40.5, 55.7, 73.9, 101.1,
112.0, 112.4, 115.0, 126.3, 128.3, 128.6, 129.1, 130.5,
130.8, 131.2, 133.9, 135.8, 139.2, 139.3, 156.1, 168.3,
169.8; HPLC retention time tR = 24.6 minutes.
2.5 | HPLC analysis
HPLC was performed on a column of Mightysil RP‐18 GP
150‐4.6 (5 μm) at 30°C with elution at 1.0 mL/min using

4
TAKAHASHI ET AL.
the gradient of MeOH/0.1% H3PO4 solution = 65/35 to
90/10 for 25 minutes and then returned to 65/35 for
10 minutes. Indomethacin was monitored at 254 nm.
Analytical HPLC was performed in BPHB as an internal
standard (tR = 7.3 minutes). The hydrolysis rate was cal-
culated from the detecting area ratio of indomethacin
(tR = 11.8 minutes) and BPHB.
3.2 | Hydrolysis reaction in HLM and
HIM solutions
The synthesized indomethacin esters were hydrolyzed in
HLM solution (Figure 1), and the ratios of the hydrolysis
rates were calculated (Table 2). The hydrolysis rates of 2b
and 2c were large, and 2d and 2e had large differences in
hydrolysis rates between the (R) form and (S) form. It is
3 | RESULTS AND DISCUSSION
3.1 | Synthesis of indomethacin esters
Indomethacin esters (2a‐2e) were synthesized in
moderate yield by condensing various alcohols with
indomethacin (1) (Table 1). There were some esters
with low yields due to the difficulty in separating the
ester and alcohol. The synthesized compounds were
1
structurally determined by H‐NMR, 13C‐NMR, and IR
FIGURE 1 Hydrolytic rates of indomethacin ester derivatives in
human liver microsome (HLM). Values are means SD (n = 3)
spectrum data.
TABLE 1 Synthesis of indomethacin esters (2a‐2e) with a chiral center
Entry
Product 2
Yield (%)
1
2
(R)‐2a
(S)‐2a
65
58
3
4
(R)‐2b
(S)‐2b
88
61
5
6
(R)‐2c
(S)‐2c
57
69
7
8
(R)‐2d
(S)‐2d
62
16
9
(R)‐2e
(S)‐2e
41
57
10

TAKAHASHI ET AL.
5
TABLE 2 Hydrolytic rate ratios of major/minor in HLM
TABLE 3 Hydrolytic rate ratios of major/minor in HIM
Compound R = Ratio (major/minor)
–CH (CH3)CH2CH2CH3 1.07 (S/R)
Compound R =
Ratio (Major/Minor)
2a
2b
2c
2d
2e
–CH (CH3)CH2CH2CH3 1.44 (S/R)
2a
2b
2c
2d
2e
–CH (CH3)CH2OCH3
–CH (CH3)C≡CH
–CH (Ph)CH2CH2Cl
–CH (Ph)CH3
1.01 (S/R)
3.93 (S/R)
11.1 (R/S)
10.3 (R/S)
–CH (CH3)CH2OCH3
–CH (CH3)C≡CH
–CH (Ph)CH2CH2Cl
–CH (Ph)CH3
1.02 (S/R)
1.16 (S/R)
1.52 (R/S)
4.99 (R/S)
known that various hydrolytic enzymes including hCES,
hCES2, arylacetoamide deacetylase (AADAC), butyryl-
cholinesterase (BCHE), paraoxonase 1 (PON1), and
PON3 are expressed in the human liver.22 AADAC
mainly hydrolyzes drugs such as flutamide and phenace-
tin with an arylacetamide structure,23-25 and BCHE
mainly hydrolyzes drugs such as bunbuterol and cocaine
with a nitrogen atom near the ester. PON is known to
efficiently hydrolyze organophosphates, cyclic esters,
and cyclic carbonates.26-30 Alkyl esters such as indometh-
acin esters are hydrolyzed by hCES1 or hCES2. Since
indomethacin esters have an acyl group with a large
indole skeleton, it is thought that hydrolysis proceeds by
hCES1. From the above reasons, it is thought that hCES1
acts as a metabolic enzyme of indomethacin esters in
HLM solution. The benzyl esters of indomethacin were
confirmed to have a 10‐fold difference in hydrolysis rate
between the (R) form and the (S) form, which is thought
to be due to the chiral recognition ability of hCES1.
Abbreviation: HLM, human liver microsome.
Next, the indomethacin esters were hydrolyzed in HIM
solution (Figure 2) and the ratios of the hydrolysis rates
were calculated (Table 3). The hydrolysis rate in HIM
solution was very small compared with the hydrolysis
reaction in HLM solution. Furthermore, the
enantioselectivity confirmed in the hydrolysis reaction
in HLM solution was not confirmed in HIM solution.
Various hydrolytic enzymes are also present in the HIM
solution; however, hCES1 is not expressed in the human
intestine.1,2 The indomethacin esters 2 are thought to be
hydrolyzed by hCES2 in HIM solution. Since the active
site of hCES2 is larger than that of CES1, it is thought
that steric differences cannot be recognized.31
Abbreviation: HIM, human intestine microsome.
3.3 | Hydrolysis reaction in hCES1
solution
There was no evidence that the ester compounds were
activated by hCES1 because these metabolic reactions
were performed in HLM that contained various enzymes.
We compared the hydrolyzing activities of several CESs
including hCES1, hCES2, and AADAC (Figure 3).32-35
The ester (R)‐2c which showed the highest activity
among the indomethacin esters was used as a substrate.
In the hydrolysis reaction of ester (R)‐2c, the hydrolysis
rate in hCES1 was more than 30 times higher than the
hydrolysis rate of hCES2 and AADAC. It was found that
indomethacin ester is likely to be specifically hydrolyzed
by hCES1.The synthesized indomethacin esters (2a‐2e)
were hydrolyzed in hCES1 solution to create
a
Michaelis‐Menten plot using GraphPad Prism 7 software
(Figure 4). Same as the hydrolysis reaction in HLM solu-
tion, the hydrolysis rates of esters 2b and 2c were rela-
tively high and the hydrolysis rates of the other
indomethacin esters were relatively low in hCES1 solu-
tion. Recently, we have shown that if the steric hindrance
of the carbon atom near the ester is large, the hydrolysis
rate by
FIGURE 2 Hydrolytic rates of indomethacin ester derivatives in
human intestine microsome (HIM solution). Values are
means SD (n = 3)
FIGURE 3 Hydrolytic rates of indomethacin ester derivatives in
solutions of hCES1, hCES2, and AADAC. Values are means SD
(n = 3)

6
TAKAHASHI ET AL.
FIGURE 4 Michaelis‐Menten plot of
indomethacin esters (2a‐2e) in hCES1
solution. Values are means SE (n = 3)
hCES1 is greatly reduced. We have also shown that an
Vmax values of ester 2b between the (S) form and the
(R) form whereas there was a 4.5 times difference in the
Vmax values between the (S) form and the (R) form.
Although the reactivity of the ester 2b is enhanced
because the methoxy group is bonded to the ester, it is
thought that the enantioselectivity is lowered since there
is no difference in steric size between the methoxymethyl
group and the methyl group at the chiral center. The Vmax
values in the benzyl type esters 2d and 2e were more 10
times larger in the (R) form than in the (S) form. In addi-
tion, it was found that the Km value of the (R) form hav-
ing a large Vmax value was lower than that of the (S) form.
Since esters having a large Vmax value are thought to have
high affinity to the enzyme, these esters are thought to
have decreased the Km value. In a comparison of esters
2d and 2e, the largest difference was observed in the ester
2d which substituted a chloroethyl group at the chiral
center. Since the electron density of the ester decreased
ester for which electron density is lowered is more reac-
tive, and hydrolysis proceeds even on a substrate having
a large steric hindrance.36 In the 1‐methoxypropan‐2‐yl
ester 2b, the electron density of the ester is lowered by
the methoxy group substituted to the ester. It can be seen
that the propyn‐1‐yl ester 2c has the sp hybrid orbital car-
bon atom near the ester. For the above reasons, it is con-
sidered that hCES1 easily reacts with the substrate and
that esters 2b and 2c have increased reactivity in the
hCES1 solution. The enantioselectivity was greatly differ-
ent between esters 2d and 2e. In both 2d and 2e, a phenyl
group is present at the 1‐position of the alkoxy group and
it is considered that the presence of the phenyl group
greatly affects the enantioselectivity. The hydrolysis
parameters of hCES1 were calculated (Table 4). It was
found that the Vmax values were large in both esters 2b
and 2c. However, there was almost no difference in the

TAKAHASHI ET AL.
7
TABLE 4 Kinetic parameters of indomethacin esters (2a‐2e) in
hCES1 solution
a chiral center of the benzylic position. The findings
obtained in this study are expected to be useful for
predicting the metabolism of chiral ester drugs.
Compound
Vmax (pmol/mg protein/min)
Km (μM)
(R)–2a
(S)–2a
(R)–2b
(S)–2b
(R)–2c
(S)–2c
(R)–2d
(S)–2d
(R)–2e
(S)–2e
95.9 4.01
136 5.92
1485 5.92
1417 94.0
512 17.1
2291 111
125 4.31
3.38 0.15
232 16.5
15.6 2.36
3.41 0.700
4.74 0.899
10.3 1.77
25.7 4.39
1.74 0.400
11.2 1.80
4.22 0.660
23.6 2.79
7.09 1.92
77.4 21.1
ACKNOWLEDGMENTS
We thank the HAB Research Organization (Japan) for
providing human livers. This work was supported by JSPS
KAKENHI grants JP16K08378 and JP17K15519.
ORCID
Masato Takahashi https://orcid.org/0000-0003-3233-6783
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was also higher in ester 2d. Furthermore, since the
chloroethyl group of 2d is sterically bulkier than the ethyl
group of 2e and hCES1 has a structure in which the enan-
tiomers can be easily distinguished from each other, it is
thought that a difference in reaction rate is generated
between the enantiomers. In fact, it can be seen that
the asymmetric carbon in methylphenidate and
flurbiprofen,16-18 which has been reported to have differ-
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4 | CONCLUSION
This study aimed to elucidate the chiral recognition abil-
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hydrolytic metabolism in the human liver. Chiral indo-
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chiral alcohols with indomethacin. The synthesized indo-
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HLM and HIM solutions. Although enantioselectivity
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How to cite this article: Takahashi M, Takani D,
Haba M, Hosokawa M. Investigation of the chiral
recognition ability of human carboxylesterase 1
using indomethacin esters. Chirality. 2019;1‐8.
https://doi.org/10.1002/chir.23141
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