Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1

Article abstract of DOI:10.1016/j.bmcl.2018.02.035

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the V_max value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.

Full text of DOI:10.1016/j.bmcl.2018.02.035

Accepted Manuscript
Chemical Synthesis of an Indomethacin Ester Prodrug and Its Metabolic Acti-
vation by Human Carboxylesterase 1
Masato Takahashi, Tomohiro Ogawa, Hiroshi Kashiwagi, Fumiya Fukushima,
Misaki Yoshitsugu, Masami Haba, Masakiyo Hosokawa
PII:
S0960-894X(18)30140-9
https://doi.org/10.1016/j.bmcl.2018.02.035
BMCL 25631
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 December 2017
16 February 2018
16 February 2018
Please cite this article as: Takahashi, M., Ogawa, T., Kashiwagi, H., Fukushima, F., Yoshitsugu, M., Haba, M.,
Hosokawa, M., Chemical Synthesis of an Indomethacin Ester Prodrug and Its Metabolic Activation by Human
Carboxylesterase 1, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.02.035
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Chemical Synthesis of an Indomethacin Ester Prodrug and Its Metabolic
Activation by Human Carboxylesterase 1
Masato Takahashi*, Tomohiro Ogawa, Hiroshi Kashiwagi, Fumiya Fukushima, Misaki Yoshitsugu,
Masami Haba, Masakiyo Hosokawa
Faculty of Pharmacy, Chiba Institute of Science, 15–8, Shiomi-cho, Choshi, Chiba 288–0025, Japan
 Corresponding author. Tel.: +81-479-30-4512; fax: +81-479-30-4683; e-mail: matakahashi@cis.ac.jp
Keywords: indomethacin; prodrug; carboxylesterase; thioester; chiral recognition
Abstract: It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient
activation of the prodrugs in the body. Although many prodrugs have been synthesized with
consideration of these chemical properties, there has been little study on the design of a structure
with consideration of biological properties such as substrate recognition ability of metabolic enzymes.
In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated
by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to
hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes
(HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates
of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the
hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in
HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the
Vmax value of n–butyl thioester with chemically high reactivity was significantly lower than that of
n–butyl ester.
1

Introduction
In recent years, many types of prodrug have been developed for improvement of bioavailability
and reduction of side effects. Since various enzymes including carboxylesterases (CESs) participate
in the metabolic activation of prodrugs, there has been much discussion about the properties of these
enzymes. By catalyzing the hydrolysis of various ester, amide and thioester derivatives, CESs are
involved in the metabolic inactivation or activation of many drugs, biological materials, natural
products, foods and environmental materials.1 CESs are categorized into five groups (CES1–CES5)
based on the amino acid sequence homology, and the CES1 or CES2 group is the major group of
CESs in mammals.2-4 Substrates with a small alcoxy group, such as methyl ester derivatives
(cocaine5 and methylphenidate)6 and ethyl ester derivatives (meperidine,7 temocapril,8 and
oseltamivir),9 are mainly catalyzed by the CES1 isozyme. Conversely, substrates with a small acyl
group, such as benzoate derivatives (cocaine),5 (1,4'-bipiperidin)-1'-ylate derivatives (CPT–11 with a
huge alkoxy group)10 and acetate derivatives (heroin),11 are mainly catalyzed by the CES2 isozyme.
The organ distribution of CESs has also been investigated, and it has been shown that CESs have
ubiquitous tissue distribution profiles.12,13 The CES1 family is mainly distributed in the liver and
lung, whereas the CES2 family is mainly distributed in the small intestine and kidney. Therefore, it
has been suggested that the substrates catalyzed by CES1 or CES2 are specifically metabolized in
each tissue. Previous experiments by our group showed that atorvastatin prodrugs are not hydrolyzed
in human intestine microsomes (HIM) or human CES2 (hCES2) solution but are hydrolyzed in
human liver microsomes (HLM) and human CES1 (hCES1) solution.14
Nonsteroidal anti–inflammatory drugs (NSAIDs) such as aspirin, diclofenac, ibuprofen and
indomethacin are one of the most widely prescribed group of drugs. However, long-term use of
NSAIDs might cause gastroduodenal mucosal injury.15 For decreasing the side effects of
indomethacin, prodrugs such as acemethacin16,17 and indomethacin farnecil18 have been developed.
Recently, results of studies on an oral prodrug of indomethacin in which a cilexetil, pivoxil19 or
glycosyl20 group was substituted have also been reported. Some studies have suggested that an
indomethacin–conjugated anticancer prodrug is effective for targeting cancer cells.21,22 Thus, many
2

researchers have attempted to synthesize indomethacin prodrugs. However, there has been little
study on the design and synthesis of an indomethacin prodrug activated by hCES1.
The purpose of the present study was to synthesize a new indomethacin ester prodrug activated by
hCES1 and to investigate the detailed substrate specificity of hCES1 for theoretical design of
prodrugs. First, the chemical synthesis of indomethacin ester and thioester prodrugs was carried out.
Then the synthesized prodrugs were subjected to hydrolysis reactions in HLM, HIM and hCES1
solutions, and the hydrolysis rates of these prodrugs were evaluated.
Indomethacin derivatives (2a–2g) were prepared according to the reported synthetic method
(Scheme 1).23 Treatment of indomethacin and alcohol compounds with dicyclohexylcarbodiimide
(DCC) in the presence of dimethylaminopyridine (DMAP) provided the n-butyl ester 2a,23,24
sec-butyl ester 2b, iso-butyl ester 2c and neo–pentyl ester 2e in moderate yields. Although the yields
were low, other esters such as tert-butyl ester 2d,25 1–phenylpropyl ester 2f and n–butyl thioester 2g
were synthesized by the same method. Substrates with a chiral center, such as sec-butyl ester 2b and
1-phenylpropyl ester 2f, were synthesized using their respective chiral alcohol compounds. The
structures of the synthesized compounds were confirmed by 1H NMR, 13C NMR, and IR analyses.
Scheme 1. Synthesis of indomethacin ester derivatives (2a-2f) and a thioester derivative 2g.
First, the synthesized prodrugs (2a–2g) were subjected to hydrolysis reaction in HLM solution,
and the hydrolysis rates were investigated (Figure 1). The results showed that n-butyl ester 2a had
the highest hydrolysis rate among all of the prodrugs (2a–2g). A comparison of the C4–alkyl ester
derivatives (2a–2d) showed that the hydrolysis rate greatly differs depending on the structure and
configuration of carbon atoms of the alkoxy group. Although neo-pentyl ester 2e has a bulky
quaternary carbon, the hydrolysis rate of 2e was not significantly reduced in comparison with that of
tert–butyl ester 2d. Therefore, it was thought that the hydrolysis rate in HLM solution is greatly
influenced by steric hindrance of the neighboring carbon of the ester. In the sec-butyl ester derivative
2b with a chiral center, the hydrolysis rate of the (S)–isomer was 2.1–times higher than that of the
(R)–isomer. Surprisingly, 1-phenylpropyl ester 2f, in which the hydrolysis rate of the (R)–isomer was
3

8.2–times higher than that of the (S)–isomer, has a reversed enantioselectivity from sec–butyl ester
2b. Even for substrates with the same n–butyl group, it was found that the hydrolysis rate of thioester
2g was 2.7–times lower than that of ester 2a. The results suggested that indomethacin ester
derivatives are more effectively hydrolyzed than are indomethacin thioester derivatives in HLM
solution.
Figure 1. Hydrolytic activities of indomethacin ester derivatives (2a–2f) and a thioester derivative
(2g) in HLM solution. Values are means ±S.D. (n = 3)
Next, prodrugs (2a–2g) were subjected to hydrolysis reaction in HIM solution, and the hydrolysis
rates were investigated (Figure 2). The data showed that the hydrolysis rates of C4–alkyl ester
prodrugs (2a-2d) in HIM solution are similar to those in HLM solution. In the sec-butyl ester
derivative 2b, the hydrolysis rate of the (S)–isomer was 1.9–times higher than that of the (R)–isomer.
Despite the high enantioselectivity in HLM solution, the hydrolysis rates of 1–phenylpropyl ester
isomers 2f were almost the same as those in HIM solution. The hydrolysis rate of n–butyl thioester
2g was slightly higher than that of n–butyl ester 2a. In terms of enantioselectivity or functional group
selectivity, different results were obtained for the hydrolytic rates of indomethacin prodrugs in HLM
and HIM solutions. On the whole, the hydrolytic rates of all of the prodrugs (2a–2g) in HIM solution
were significantly lower than those in HLM solution. The results therefore suggested that all of the
prodrugs (2a–2g) are efficiently hydrolyzed not in the human intestine but in the human liver.
4

Figure 2. Hydrolytic activities of indomethacin ester derivatives (2a–2f) and a thioester derivative
(2g) in HIM solution. Values are means ±S.D. (n = 3)
Finally, the prodrugs (2a–2g) were subjected to hydrolysis reaction in hCES1b solution, and the
kinetic parameters were determined (Table 1). hCES1b is also referred to as CES1A1, which is
mainly expressed in the human liver. The Km and Vmax values were calculated by the
Michaelis-Menten equation using nonlinear regression analysis with software (GraphPad Prism 7),
and CLint was calculated by Km and Vmax. The Vmax values for all of the prodrugs (2a–2g) show
patterns similar to those for the hydrolytic activities in HLM solution. The n-butyl ester 2a has the
highest Vmax value among all of the prodrugs (2a–2g). The Vmax value of neo-pentyl ester 2e with a
bulky quaternary carbon is slightly lower than that of tert-butyl ester 2d. Therefore, it is thought that
hCES1b strongly recognizes the neighboring carbon of the ester. In the sec-butyl ester 2b, the Vmax
value of the (S) –isomer is 2.1–times higher than that of the (R)–isomer. Furthermore, in the
1-phenylpropyl ester 2f, the Vmax value of the (R)–isomer is 10.2–times higher than that of the
(S)–isomer. Previous studies showed that CESs have a chiral recognition ability for some drugs, such
as propranolol esters,26,27 flurbiprofen esters26 and methyl phenidate. 6 These drugs have a functional
group, such as a phenyl, amino or fluoro group, that is substituted next to the chiral center. In this
study, it was found that hCES1 recognizes a simple alkyl ester such as sec-butyl ester. The Vmax and
Km values of n-butyl thioester 2g are lower than those of n-butyl ester 2a. The n-butyl thioester 2 has
the highest CLint value among all of the prodrugs (2a–2g).
5

Table 1. Kinetic parameters of indomethacin ester derivatives (2a-2f) and a thioester derivative (2g).
Values are means ±S.E. (n = 3) nd: not detected
Vmax
(nmo/mg protein/min)
1.512±0.041
0.049±0.003
0.103±0.009
1.221±0.082
nd
Km
CLint
compound
(mM)
(mL/mg protein/min)
4.4±0.4
10.1±2.1
48.5±10.3
13.7±2.9
nd
0.34
0.0048
0.0021
0.089
nd
2a
(R)–2b
(S)–2b
2c
2d
0.322±0.014
0.021±0.001
0.002±0.0001
0.800±0.016
4.6±0.7
2.9±0.4
6.5±1.1
2.1±0.3
0.069
0.007
0.0003
0.38
2e
(R)–2f
(S)–2f
2g
Thus, it was shown that the values of Vmax and Km differ depending on the structure of the ester.
This difference can be explained by the reaction mechanism of hydrolysis by CES. The hydrolysis
reaction by CES is a typical ping–pong–bi–bi reaction,1 which is considered to proceed similarly for
indomethacin hydrolysis reaction (Scheme 2). First, an acyl–enzyme complex 4 is formed by
nucleophilic addition–elimination in the ester 2 by the hydroxyl group of Ser203 activated by Glu336
and His450 in CES (step 1). Subsequently, an exchange reaction between alcohol (or thiol) and water
occurs (step 2), and finally indomethacin (1) is produced, causing a nucleophilic
addition–elimination reaction in the acyl–enzyme complex 4 by water (step 3). In this reaction, steps
1 and 2 affect Vmax and Km depending on the type of ester. In the case of esters 2b and 2f in which
the bulky alkoxy group is substituted, it can be assumed that the value of Vmax was small since it is
thought that it is difficult for nucleophilic attack of the hydroxyl group of Ser203 in step 1. Next,
although the chemical reactivity of thioester was high in aqueous solution,28 the Vmax value of
thioester 2g was smaller than that of ester 2a. This is because exchange reaction between thiol and
water in step 2 is difficult to proceed in solution with a high concentration. Although the hydrolysis
reaction proceeds easily in a dilute solution, the amount of thiol produced by the reaction also
increases a solution with a high concentration, presumably because the reverse reaction of step 1
proceeds. Since thiol has higher nucleophilic ability than that of alcohol, it is thought that the reverse
reaction of step 1 proceeds more easily in thiol than in alcohol. Also, in the usual hydrolysis reaction,
since carboxylic acid is formed in one step, the reverse reaction to the thioester does not occur. This
is thought to be the reason for the The Vmax and Km values of n-butyl thioester 2g are lower than
those of n-butyl ester 2a. Thioester type prodrugs with a low Km value may become prodrugs with a
high metabolic activation rate at a low concentration.
6

Scheme 2. Proposed mechanism for the hydrolysis reaction by CES.
In conclusion, this study showed that indomethacin ester prodrugs have the possibility of specific
metabolic activation in the liver since these prodrugs were highly hydrolyzed in HLM solution but
not in HIM solution. The results suggested that the hydrolytic activities of indomethacin prodrugs in
hCES1 solution greatly change depending on the steric hindrance on the neighboring carbon of the
ester. In addition, there were new findings for the chiral recognition ability of hCES1: Even in simple
alkyl esters such as 2b, there is a 2.1–times difference in hydrolytic activity between (R)–2b and
(S)–2b, and for a substrate with a phenyl group, such as 1-phenylpropyl ester 2f, there is 10.2-times
difference in hydrolytic activity between (R)–2f and (S)–2f. Although the Vmax value of a thioester is
lower than that of an ester, a thioester has a higher CLint value than that of an ester. The results
provide important information regarding the relationship between the structure of an ester prodrug
and the rate of metabolic activation. Further elucidation of the substrate specificity for CESs is
desired for theoretical prodrug design.
Acknowledgement
We thank the Human and Animal Bridging (HAB) Research Organization (Japan) for providing
human livers. This work was supported by JSPS KAKENHI Grant Number JP17K15519.
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