Metabolic activation of N-thiazol-2-yl benzamide as glucokinase activators: Impacts of glutathione trapping on covalent binding

Article abstract of DOI:10.1016/j.bmcl.2010.01.041

Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioactivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding, metabolites in liver microsomes and glutathione (GSH) trap assay.

Full text of DOI:10.1016/j.bmcl.2010.01.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1619–1622
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Metabolic activation of N-thiazol-2-yl benzamide as glucokinase activators:
Impacts of glutathione trapping on covalent binding
*
Tomoharu Iino , Noriaki Hashimoto, Takuro Hasegawa, Masato Chiba, Jun-ichi Eiki, Teruyuki Nishimura
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many
pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety
in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong
GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioac-
tivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding,
metabolites in liver microsomes and glutathione (GSH) trap assay.
Received 28 October 2009
Revised 29 December 2009
Accepted 13 January 2010
Available online 20 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Glucokinase activator
Covalent binding
GSH trapping
Radio labeled compound
GK, a member of the hexokinase family, is expressed in the liver
and in pancreatic b-cells.1 This enzyme catalyzes the key initial
step for glucose metabolism, that is, phosphorylation of glucose
to glucose 6-phosphate. In the liver, GK promotes glycogen synthe-
sis, whilst it enhances insulin secretion from pancreatic b-cells.2–4
Therefore, GKAs can be expected to function as an anti-hyperglyce-
mic drug, by both increasing glucose uptake in the liver and the
potentiation of insulin secretion from pancreatic b-cells.5–8 As a re-
sult of promising preclinical data, many pharmaceutical companies
have actively pursued this target aiming at the development of
GKAs.9–19 Of these, several companies including Roche, Astra-Zen-
eca, and OSI/Prosidion have advanced into clinical studies.10,12
Most of the GKAs reported previously possess the N-amin-
othiazol-2-yl amide moiety in their structures because the amino-
thiazole substructure interacts with Arg63 of GK and shows strong
GK activation.20 We have also developed and reported 2-aminothi-
azole-2-yl-containing benzamides as GKAs exemplified by com-
pounds 1–3 (Fig. 1).13–15 Compound 3, for instance, showed
potent GK activation and glucose lowering effects in diabetic mice
models.15 Thus, the GKA 3 was evaluated further to establish its
potential for biochemical activation and its metabolic profiles.
As a part of drug discovery and development, the metabolic pro-
files of new drug candidates must be characterized. Drug metabo-
lites are the products of enzymatic modifications such as oxidation
or conjugate formation. Intermediates in the metabolic reactions,
or the products themselves, may be reactive and give rise to cova-
lent protein binding. Evaluation of this is important since covalent
protein modification may lead to unwanted toxicities and idiosyn-
cratic reactions in human.21
In brief, the potential of drug candidates to cause covalent bind-
ing is first evaluated in vitro by incubation of a radiolabeled analog
in the presence of rat and human liver microsomes under oxidative
conditions. In both cases, formation of covalent adducts with pro-
tein is determined by successive washing of protein pellets using
either Brandel harvester technique or centrifugation-based meth-
ods.22–24 A target value of 50 pmol-equiv/mg protein at 1 h for
the in vitro assay was proposed by Evans et al., considering these
O
S
O
S
O
S
N
N
N
N
N
N
H
H
N
NH2
O
Compound 1
Compound 2
MeO2S
O
S
O
N
HO
N
H
O
* Corresponding author at present address: Banyu Clinical Development Institute,
Banyu Pharmaceutical Co. Ltd, 1-13-12 Kudan-kita, Chiyoda-ku 102-8667, Tokyo,
Japan. Tel.: +81 3 6272 2012; fax: +81 3 6238 9097.
MeO2S
Compound 3
E-mail address: tomoharu_iino@merck.com (T. Iino).
Figure 1. Structures of GK activators 1–3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.041

1620
T. Iino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1619–1622
Table 1
O
S
O
S
Covalent binding data of compound 3 in human and rat liver microsomes
O
O
N
HO
N
N
HO
H
N
Covalent bindinga
Human MS (HM)
715
10 + 9
H
H
NADPH
(a)
Rat MS (RM)
O
O
H
H
H
H
(+)
(À)
2
378 23
0 + 0
S
T
S
O
O
[3H]- 3
O
O
a
Compound 3
Values reported as pmol-equiv binding/mg protein at 1 h.
Scheme 1. Preparing of tritium labeled 3. Reagents and conditions: (a) (1) T2O–H2O
(5 Ci/mL), DBU, THF, specific activity: 102.2 mCi/mmol, purity: >99.4%.
This paper herein describes the in vitro covalent binding results
that were observed during the course of evaluating a GKA 3 for the
treatment of type-2 diabetes. Work was first performed to identify
what reactive species was responsible for protein labeling, using a
glutathione (GSH) as trapping agent.
values are approximately 10-fold over the background of the as-
says and represent 1/20th of binding obtained for known
hepatotoxins.21
O
S
O
S
O
S
O
O
OH
O
N
N
H
HO
O
N
H
HO
O
N
H
NH2
HO
O
N
H
O
HM, RM
RM
O
O
O
S
S
S
O
O
O
Metabolite 2
Metabolite 1
Figure 2. Major metabolites of 3 in human and rat microsomes. HM: Human microsomes, RM: Rat microsomes.
389
-18
99
407
306+2H
GSH adduct
Compound 3
363+2H
O
S
H
N
COOH
O
O
HO
N
N
O
S
H
697
641+2H
S
NH
O
HO
N
H
N
H
-18
O
O
O
COOH
NH
O
S
625
O
349
2
O
O
S
423+2H 465
349
+
MH+, Exact Mass: 449.0841 (C20H21N2O6S2
Observed Mass: 449.0818
)
O
+
MH+, Exact Mass: 772.1628 (C30H38N5O13S3
)
Observed Mass: 772.1625
J -1 3 9 1 1 2
G S H + _ + 7 7 2 + 9 3 2 + 6 5 6 # 8 7 7 -9 3 8
R T : 2 1 .8 7 - 2 3 .1 8 A V: 1 2 S B : 5 3
1 8 .3 1 -2 1 .1 3
3 .8 2 E 6 T :
5 0 .0 0 -1 0 0 0 .0 0 ]
R M
7 7 2 .0 MH+=772
1 0 0
9 0
8 0
7 0
6 0
5 0
4 0
3 0
2 0
1 0
Full Scan MS
,
1 7 .8 6 -2 1 .4 2 N L :
+
c
E S I F u ll
m s [
7 7 3 .0
7 7 4 .0
7 8 9 .9
3 8 6 .6
3 4 9 .1
7 9 0 .9
8 1 0 .0
3 4 0 .7
6 8 0 .1
7 2 6 .2
3 0 8 .0
8 7 2 .0
3 8 7 .7
4 9 6 .4
6 2 5 .4 6 7 1 .9
6 2 5 .0
7 4 6 .9
5 5 6 .7
3 6 8 .0
4 2 4 .0
0
5 3
J -1 3 9 1 1 2
G S H + _ + 7 7 2 + 9 3 2 + 6 5 6 # 8 8 2 -9 2 7
R T : 2 1 .8 9 - 2 2 .9 5 A V: 1 0 N L : 2 .0 2 E 6
R M
-147(Glu+18)
625
F :
+ c E S I F u ll m s 2 7 7 2 .0 0 @ 3 0 .0 0 [
4 5
4 0
3 5
3 0
2 5
2 0
1 5
1 0
5
2 1 0 .0 0 -9 0 0 .0 0 ]
MS/MS Scan parent ion772
-364
-307(GSH)
407
349
-129(Glu)
643
6 4 2 .8
4 0 6 .9
4 2 4 .9
465
4 6 4 .9
3 0 7 .9
6 9 6 .9
7 0 0
7 5 3 .9
7 3 8 .0
2 5 9 .8
2 5 0
3 3 1 .9
3 5 0
3 6 4 .9
6 2 4 .3
6 4 3 .9
6 5 0
6 0 7 .9
7 5 5 .0
0
3 0 0
4 0 0
4 5 0
5 0 0
5 5 0
/z
6 0 0
7 5 0
8 0 0
8 5 0
9 0 0
m
Figure 3. Chemical structure of GSH adduct of 3 in rat microsomes.

T. Iino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1619–1622
1621
O
S
O
S
O
S
O
O
OH
O
N
N
HO
N
HO
O
N
NH2
HO
O
N
H
H
H
H
O
HM, RM
RM
O
O
O
S
S
S
O
O
S
O
O
Metabolite 2
Metabolite 1
Oxidation
+ H2O
OH
+GSH
+ Protein
O
N
GSH adduct
Covalent binding
O
S
N
N
(Migration)
S
N
N
H
N
or
H
H
Proposed mechanism
Figure 4. Proposed mechanism leading to GSH adduct formation and covalent binding.
Further investigation of compound 3 was initiated to assess its
potential to cause in vitro protein covalent binding. Radio labeled
compound 3, tritiated at the methylsulfone moiety, was synthesized
as shown in Scheme 1.25 A base-catalyzed exchange reaction was
conducted with tritiated water and DBU to afford tritium labeled
compound 3 giving specific radioactivity of 102 mCi/mmol.26,27
Using this labeled compound, in vitro liver microsomal covalent
binding experiments were performed, as described previously.28 As
shown in Table 1, tritium labeled 3 led to a significant increase in
protein labeling in both rat and human liver microsomes in an
NADPH dependent manner. Due to the potential for extensive
covalent binding through bioactivation, further evaluation of this
compound was discontinued.
tive (191[113] and 173[268] pmol-equiv/mg protein in human[rat]
microsomes, respectively). It was suggested that introduction of
substituents on the thiazole ring would be effective to reduce the
CYP-catalyzed oxidation.
In conclusion, GKA 3 containing a 2-aminothiazole substructure
was shown to cause protein labeling in presence of rat and human
liver microsomes under oxidative conditions. Presence of GSH as a
trapping agent led to reduction of in vitro covalent binding and to
concomitant formation of a glutathione adduct to the thiazole ring,
suggesting the potential implication of a reactive thio-lactone ring.
Comparison studies using not only other aminothiazole deriva-
tives but the other heteroaromatics are underway.
In vitro incubations of 3 were repeated using unlabeled material
and analyzed by high performance liquid chromatography coupled
to mass spectrometric detectors (HPLC–MS/MS).29 The major
metabolite of 3 was identified to be the ring-opened metabolite
1 in both human and rat microsomes (Fig. 2). In rat microsomes,
the thiourea metabolite 2 was also observed; this is considered
to be a toxic metabolite.30–32 To investigate the mechanism of for-
mation of metabolites, GSH trapping assay was conducted.
Chemical trapping agents such as GSH, cyanide, semicarbazide
or methoxylamine have been used in the past to identify reactive
metabolites.21,23 The potential of GSH, a soft nucleophile, to trap
the reactive metabolite of 3 was assessed by repeating the rat
in vitro liver microsomal covalent binding assay in the presence
or absence of physiologically relevant concentrations of GSH
(5 mM). A fivefold decrease in protein labeling was observed in
the presence of GSH (from 272 to 50 pmol-equiv/mg protein at
1 h) and a concomitant formation of new metabolite showing a
+323 u mass shift relative to 3, characteristic for oxidative oxygen
atom (+16) and glutathione adduct (+307). The GSH adduct was
analyzed by HPLC–MS/MS and identified to be a ring-opening thi-
oester as shown in Figure 3.
As observed for 3, addition of millimolar quantities of GSH led
to decreases in the extent of in vitro covalent binding in parallel
with increased formation of the corresponding GSH adduct. A
mechanism implicating CYP-catalyzed oxidation and subsequent
migration of oxygen atom, leading to formation of the thio-lactone
ring, was postulated.31 This metabolic pathway would account for
either the covalent binding or the GSH adduct (Fig. 4).
In preliminary experiments, it was found that the levels of cova-
lent binding of substituted thiazolyl derivatives of compound 3
were lower than those of 3. 2–5-fold decreases in covalent protein
labeling were observed in testing 4-hydroxymethyl-1,3-thiazol-2-
yl amide derivative and 5-methyl-1,3-thiazol-2-yl amide deriva-
Acknowledgement
The authors are grateful to Shinnosuke Abe, Hisao Ochiai and
Atsushi Ose for the metabolical studies. We would also like to
thank Dr. Peter Meinke and Kimihiko Sato for critical reading of
the manuscript.
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