Reactive metabolite trapping studies on imidazo- and 2-methylimidazo[2,1-b] thiazole-based inverse agonists of the ghrelin receptor s

Article abstract of DOI:10.1124/dmd.113.051839

The current study examined the bioactivation potential of ghrelin receptor inverse agonists, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]- 2,7-diazaspiro[3.5]nonan-7-yl}-2-(imidazo[2,1-b]thiazol-6- yl)ethanone (1) and 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7- diazaspiro[3.5]nonan-7-yl}- 2-(2-methylimidazo[2,1-b]thiazol-6-yl) ethanone (2), containing a fused imidazo[2,1-b]thiazole motif in the core structure. Both compounds underwent oxidative metabolism in NADPH- and glutathione-supplemented human liver microsomes to yield glutathione conjugates, which was consistent with their bioactivation to reactive species. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in the thiol conjugates was on the thiazole ring within the bicycle. Two glutathione conjugates were discerned with the imidazo[2,1-b]thiazole derivative 1. One adduct was derived from the Michael addition of glutathione to a putative S-oxide metabolite of 1, whereas, the second adduct was formed via the reaction of a second glutathione molecule with the initial glutathione- S-oxide adduct. In the case of the 2-methylimidazo[2,1-b]thiazole analog 2, glutathione conjugation occurred via an oxidative desulfation mechanism, possibly involving thiazole ring epoxidation as the rate-limiting step. Additional insights into the mechanism were obtained via 18O exchange and trapping studies with potassium cyanide. The mechanistic insights into the bioactivation pathways of 1 and 2 allowed the deployment of a rational chemical intervention strategy that involved replacement of the thiazole ring with a 1,2,4-thiadiazole group to yield 2-[2-chloro-4-(2H-1,2,3-triazol- 2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7- yl)-2-(2-methylimidazo[2,1- b][1,3,4]thiadiazol-6-yl)ethanone (3). These structural changes not only abrogated the bioactivation liability but also retained the attractive pharmacological attributes of the prototype agents.

Full text of DOI:10.1124/dmd.113.051839

1521-009X/41/7/1375–1388$25.00
http://dx.doi.org/10.1124/dmd.113.051839
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 41:1375–1388, July 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Reactive Metabolite Trapping Studies on Imidazo- and
2-Methylimidazo[2,1-b]thiazole-Based Inverse Agonists
of the Ghrelin Receptor s
Amit S. Kalgutkar, Tim F. Ryder, Gregory S. Walker, Suvi T. M. Orr, Shawn Cabral,
Theunis C. Goosen, Kimberly Lapham, and Heather Eng
Pharmacokinetics, Dynamics and Metabolism–New Chemical Entities, Cambridge, Massachusetts (A.S.K.);
Pharmacokinetics, Dynamics and Metabolism–New Chemical Entities, Groton, Connecticut (T.F.R., G.S.W., T.C.G., K.L., H.E.);
Worldwide Medicinal Chemistry, La Jolla, California (S.T.M.O.); and Pfizer Inc., Groton, Connecticut (S.C.)
Received March 4, 2013; accepted April 22, 2013
ABSTRACT
The current study examined the bioactivation potential of ghrelin metabolite of 1, whereas, the second adduct was formed via the
receptor inverse agonists, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]- reaction of a second glutathione molecule with the initial glutathi-
2,7-diazaspiro[3.5]nonan-7-yl}-2-(imidazo[2,1-b]thiazol-6-
yl)ethanone (1) and 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7-
diazaspiro[3.5]nonan-7-yl}-2-(2-methylimidazo[2,1-b]thiazol-6-yl)
one-S-oxide adduct. In the case of the 2-methylimidazo[2,1-b]thiazole
analog 2, glutathione conjugation occurred via an oxidative
desulfation mechanism, possibly involving thiazole ring epoxidation
ethanone (2), containing a fused imidazo[2,1-b]thiazole motif in as the rate-limiting step. Additional insights into the mechanism were
the core structure. Both compounds underwent oxidative metabo- obtained via 18O exchange and trapping studies with potassium
lism in NADPH- and glutathione-supplemented human liver micro- cyanide. The mechanistic insights into the bioactivation pathways
somes to yield glutathione conjugates, which was consistent with of 1 and 2 allowed the deployment of a rational chemical interven-
their bioactivation to reactive species. Mass spectral fragmentation tion strategy that involved replacement of the thiazole ring with
and NMR analysis indicated that the site of attachment of the
a 1,2,4-thiadiazole group to yield 2-[2-chloro-4-(2H-1,2,3-triazol-
glutathionyl moiety in the thiol conjugates was on the thiazole ring 2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-
within the bicycle. Two glutathione conjugates were discerned with
b][1,3,4]thiadiazol-6-yl)ethanone (3). These structural changes not
the imidazo[2,1-b]thiazole derivative 1. One adduct was derived
only abrogated the bioactivation liability but also retained the
from the Michael addition of glutathione to a putative S-oxide attractive pharmacological attributes of the prototype agents.
Introduction
homeostasis and insulin sensitivity, while eliciting beneficial effects
on body weight (Serby et al., 2006; Esler et al., 2007; Rudolph et al.,
2007; Soares et al., 2008; Costantino and Barlocco, 2009).
A recent report from our laboratory disclosed a proprietary series of
spirocyclic piperidine-azetidine derivatives as potent and orally active
inverse agonists of GHSR-1a (Kung et al., 2012). Structure-activity
relationship studies revealed that analogs with distributed polarity in
the form of the fused imidazo[2,1-b]thiazole acetamide and phenyl
triazole functionalities (e.g., 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-
yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(imidazo[2,1-b]thiazol-
Ghrelin is a 28-amino acid hunger-stimulating peptide that is
produced in the stomach, pancreas, and hypothalamus (Kojima et al.,
1999; Inui et al., 2004). Ghrelin is a ligand for the growth hormone
secretagogue receptor type 1a, a G-protein-coupled receptor expressed
primarily in the pituitary gland, brain, and to a lesser extent in the
periphery. Through its action on growth hormone secretagogue re-
ceptor type 1a, ghrelin exerts a variety of metabolic functions includ-
ing stimulation of growth hormone release, stimulation of appetite and
weight gain, and suppression of insulin secretion in rodents and
humans (Nakazato et al., 2001; Wren et al., 2001; Dezaki et al., 2008; 6-yl)ethanone (1) and 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]
Cardona Cano et al., 2012; Heppner et al., 2012). Thus, antagonizing
growth hormone secretagogue receptor (GHSR)-1a with small mole-
cule antagonists or inverse agonists is anticipated to improve glucose
2,7-diazaspiro[3.5]nonan-7-yl}-2-(2-methylimidazo[2,1-b]thiazol-
6-yl)ethanone (2) depicted in Fig. 1) demonstrated significant improve-
ments in binding affinity (IC50 , 10 nM) to the ghrelin receptor, while
consistently displaying inverse agonism in a ghrelin functional assay.
In addition, these structural changes also led to an improved pharmaco-
kinetic profile (↓, clearance; ↑, oral bioavailability) in animals, which
dx.doi.org/10.1124/dmd.113.051839.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: CID, collision-induced dissociation; compound 1, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-
yl}-2-(imidazo[2,1-b]thiazol-6-yl)ethanone; compound 2, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(2-methy-
limidazo[2,1-b]thiazol-6-yl)ethanone; compound 3, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(2-methylimidazo[2,
1-b][1,3,4]thiadiazol-6-yl)ethanone; DMSO-d6, dimethyl sulfoxide-d6; GHSR, growth hormone secretagogue receptor; GSH, reduced glutathione; HLM,
human liver microsomes; HPLC, high-performance liquid chromatography; HSQC, multiplicity edited heteronuclear single quantum coherence; L-
766,112, 5-[4-(methylsulfonyl)phenyl]-6-phenylimidazo[2,1-b]thiazole; LC-MS/MS, liquid chromatography-tandem mass spectrometry; m/z, mass-to-
charge ratio; P450, cytochrome P450; TCI, triple resonance probe; TOCSY, total correlation spectroscopy, tR, retention time.
1375

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Kalgutkar et al.
Fig. 1. Structures of ghrelin receptor inverse agonists 1–3 from the spirocyclic piperidine-azetidine series and oxidative bioactivation of the imidazo[2,1-b]thiazole motif in
L-766,112 to an electrophilic S-oxide species. M.W., molecular weight.
warranted a further examination of relevant preclinical safety endpoints of 1, whereas the formation of the second adduct involved the attachment
as a prelude to candidate nomination.
of a second GSH molecule to the initial GSH-S-oxide adduct with
From a drug metabolism perspective, the presence of the imidazo[2,1- concomitant liberation of the oxidized thiazole sulfur. With respect to the
b]thiazole scaffold in 1 and 2 raised a significant cause for concern, corresponding 2-methylimidazo[2,1-b]thiazole analog 2, GSH conjuga-
given the possibility of a cytochrome P450 (P450)-mediated S-oxidation tion occurred via an oxidative desulfation mechanism, possibly involving
on the thiazole sulfur (within the imidazo[2,1-b]thiazole framework) to thiazole ring epoxidation as the rate-limiting step. Characterization of the
yield an electrophilic S-oxide metabolite capable of reacting with GSH conjugate structure and additional trapping studies with cyanide ion
proteins and/or the endogenous antioxidant glutathione (GSH). Indeed, proved to be useful in deciphering the overall bioactivation mechanism
the characterization of a GSH conjugate derived from Michael addition associated with 2. The in vitro metabolism information was used to design
to an S-oxide metabolite of the imidazo[2,1-b]thiazole derivative, the corresponding thiadiazole analog, 1-{2-[2-chloro-4-(2H-1,2,3-triazol-
5-[4-(methylsulfonyl)phenyl]-6-phenylimidazo[2,1-b]thiazole inhibi- 2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(2-methylimidazo[2,1-
tor (L-766,112) (Fig. 1) provides precedence for this bioactivation b][1,3,4]thiadiazol-6-yl)ethanone analog 3 (see Fig. 1) that would be
pathway in liver microsomes (Thérien et al., 1997; Trimble et al., 1997). devoid of reactive metabolite formation. Indeed, 3 was resistant
Furthermore, a successful medicinal chemistry strategy involving interna- to bioactivation in HLM (inferred from the lack of GSH conjugate
lization of the thiazole motif, resulting in the elimination of reactive formation) while retaining inverse agonist potency for the ghrelin
metabolite liability of L-766,112, while retaining the primary pharma- receptor and HLM stability noted with 1 and 2.
cology (selective inhibition of cyclooxygenase-2) has been presented in
subsequent work (Fig. 1) (Roy et al., 1997). Other than thiazole-S-
Materials and Methods
oxidation, an additional bioactivation pathway can arise through thiazole
ring scission (via the C4–C5 epoxidation → diol pathway) (Chatfield
and Hunter, 1973; Mizutani et al., 1993; Yabuki et al., 1997; Yoon
et al., 1998; Obach et al., 2008). The corresponding thioamide meta-
bolites, which are the by-products of thiazole ring cleavage, can undergo
additional S-oxidation to electrophilic sulfenic acid derivatives (Dansette
et al., 2005; Mansuy and Dansette, 2011) capable of oxidizing or
forming mixed disulfide adducts with proteins or GSH and eliciting
toxicity (Ziegler-Skylakakis et al., 1998; Hajovsky et al., 2012).
In this report, we summarize our findings on the in vitro bioactivation
of 1 and 2 in human liver microsomes (HLM) utilizing exogenously
added nucleophiles to trap reactive species. In the case of the imidazo[2,1-
b]thiazole derivative 1, two GSH adducts were formed. One adduct
was derived from the addition of the thiol to a putative S-oxide metabolite
Materials. NADPH, GSH, and potassium cyanide were obtained from
Sigma-Aldrich (St. Louis, MO). HLM were prepared from a mixed sex pool of
50 donors (provided by BD Biosciences, Woburn, MA). H218O (97%) and
dimethyl sulfoxide (DMSO)-d6 “100%” were obtained from Cambridge Isotope
Laboratories Inc. (Andover, MA). All other commercially available reagents
and solvents were of either analytical or high-performance liquid chromatog-
raphy (HPLC) grade. The preparation of title compounds 1–3 is provided in the
Supplemental Methods and Supplemental Fig. 1.
Metabolite Identification and Reactive Metabolite Trapping Studies in
HLM. Liver microsomal incubations were conducted at 37°C for 60 minutes in
a shaking water bath. The incubation volume was 1.0 ml and consisted of 0.1 M
potassium phosphate buffer (pH 7.4), MgCl2 (3.3 mM), HLM (protein con-
centration = 1.0 mg/ml, P450 concentration = 0.5 mM), test compound (10 mM),
and NADPH (1.3 mM). Additional microsomal incubations also included GSH

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1377
(10 mM), N-acetylcysteine (10 mM), and/or potassium cyanide (5 mM) to trap to dryness. In the case of M3-1, it was difficult to obtain a pure isolate because
reactive metabolites. Reactions were terminated by the addition of acetonitrile of poor chromatographic resolution between M3-1 and M2-1. Hence, a crude
(3.0 ml). The solutions were centrifuged (3000g, 15 minutes), and the super- isolate of M3-1 was used in NMR characterization. The residues were
natants were transferred to clean 15-ml glass centrifuge tubes and concentrated to evacuated in a drybox for ~2 hours prior to reconstitution in DMSO-d6 for
dryness. The residue was reconstituted with 200 ml 10% acetonitrile in water and NMR analysis.
analyzed for metabolite formation using liquid chromatography-tandem mass
spectrometry (LC-MS/MS).
NMR Analysis of Metabolites. NMR spectra for 1 and M3-1 were recorded
on a Bruker Avance 600 MHz instrument (Bruker BioSpin Corporation,
Billerica, MA) controlled by TOPSPIN V3.0 and equipped with a 1.7 mm cryo-
triple resonance probe (TCI). NMR spectra for 2 and M5-2 were recorded on
a Bruker Avance 600 MHz instrument (Bruker BioSpin Corporation) controlled
by TOPSPIN V2.1 and equipped with a 5 mm cryo-TCI probe. Synthetic
samples and isolated materials were dissolved in 0.15 ml (5 mm TCI probe) or
0.05 ml (1.7 mm TCI probe) of DMSO-d6. All spectra were referenced using
residual DMSO-d6 (d = 2.49 ppm relative to tetramethylsilane, d = 0.00 for 1H
and d = 39.5 ppm relative to tetramethylsilane, d = 0.00 for 13C). One-
dimensional spectra were typically recorded using a sweep width of 8000 Hz
and a total recycle time of approximately 7 seconds. The resulting time-
averaged free induction decays were transformed using an exponential line
broadening of 1.0 Hz to enhance signal-to-noise ratio. The two-dimensional
data [correlation spectroscopy, total correlation spectroscopy (TOCSY), multi-
plicity edited heteronuclear single quantum coherence (HSQC), and hetero-
nuclear multiple bond correlation] were recorded using the standard pulse
sequences provided by Bruker. A 1 K Â 128 data matrix was acquired using
a minimum of 4 scans and 16 dummy scans. The data were zero-filled to a size
of 1 K Â 1 K. A mixing time of 80 milliseconds was used in the TOCSY
experiments.
18O Incorporation Studies in HLM. For H218O studies, 15-ml glass
centrifuge tubes containing 1.0 ml of 0.1 M phosphate buffer (pH 7.4) were
concentrated to dryness using an evaporative centrifuge. H218O (840 ml, 97%)
was added to the dried centrifuge containing phosphate buffer. Potassium
phosphate (pH 7.4) buffered H218O was used to create 1.0-ml incubations
containing 2 (10 mM) and 3.3 mM MgCl2 (20 ml) in HLM (protein
concentration = 1.0 mg/ml, P450 concentration = 0.5 mM). The isotopic
enrichment of H218O was ~89.7%. Identical incubations were also performed in
the presence of GSH (10 mM). Samples were placed in a shaking water bath set
to 37°C. After 60 minutes, the reactions were quenched with 3.0 ml of ace-
tonitrile and centrifuged at 3000g for 5 minutes. The supernatants were trans-
ferred to clean 15-ml centrifuge tubes and concentrated to dryness. The samples
were analyzed for 18O incorporation by LC-MS/MS.
LC-MS/MS Methodology for Metabolite Identification Studies. The
HPLC system consisted of an Acella quaternary solvent delivery pump, an
Accela autoinjector, and a Surveyor PDA Plus photodiode array detector (Thermo
Electron Corporation, Waltham, MA). Chromatography was performed on
a Phenomenex Hydro RP column (4 mm 4.6 mm  150 mm; Phenomenex,
Torrance, CA). The mobile phase was composed of 5 mM ammonium formate
buffer (pH = 3.0) (solvent A) and acetonitrile (solvent B). The flow rate was 1.0
ml/min. The LC gradient started at 10% B for 5 minutes, was ramped linearly to
50% B over 35 minutes, ramped linearly to 90% B over 10 minutes, returned to
the initial condition over 1.0 minute, and allowed to equilibrate for 4.0 minutes.
Post-column flow passed through the PDA detector to provide ultraviolet (l =
254 nm) detection prior to being split to the mass spectrometer such that mobile
phase was introduced into the electrospray source at a rate of 100 ml/min. The LC
system was interfaced to a Thermo Orbitrap mass spectrometer operating in
positive ion electrospray mode. Xcalibur software, version 2.0, was used to
control the HPLC/mass spectrometry system. Full scan data were collected at
15,000 resolution. Data dependent product ion scans of the two most intense ions
found in the full scan were obtained at 15,000 resolution. The dynamic exclusion
function was used with a 1.0-minute exclusion duration after three successive
product ion scans with an early exclusion if the precursor ion falls below a signal-
to-noise ratio of 20.
Biosynthesis of GSH Conjugates. GSH conjugates M3-1 and M5-2
detected in the course of reactive metabolite trapping studies with 1 and 2 were
biosynthesized from human liver microsomal incubations. Six incubations of
1 or 2 were carried out in 50-ml polypropylene centrifuge tubes at 37°C for
60 minutes in a shaking water bath. The incubation volume was 10 ml and
consisted of 0.1 M potassium phosphate buffer (pH 7.4), MgCl2 (3.3 mM),
HLM (protein concentration = 1.0 mg/ml), test compound (60 mM), GSH
(10 mM), and NADPH (1.3 mM). Reactions were terminated by the addition of
acetonitrile (30 ml). The solutions were centrifuged (3000g, 15 minutes), and
the supernatants were transferred to clean 50-ml polypropylene centrifuge tubes
and concentrated to dryness. The residue in each tube was reconstituted with
400 ml 10% acetonitrile in water. The reconstituted samples were combined in
a 2-ml HPLC vial and were placed onto an HPLC semipreparative system in
eight 3-ml injections. This system consisted of a Shimadzu SiL-HTC auto-
sampler, two LC-20AD solvent pumps, an SPD-M20A diode array detector,
and a FRC-10 A fraction collector (Shimadzu USA, Columbia, MD). Separa-
tion was performed on a Zorbax RX C8, 9.4 Â 250 mm, 5-mM semipreparative
HPLC column (Phenomenex). The mobile phase was composed of 0.1% formic
acid (solvent A) and acetonitrile (solvent B). The flow rate was 4.0 ml/min. The
LC gradient started at 10% B for 5 minutes, ramped linearly to 50% B over
35 minutes, ramped linearly to 90% B over 10 minutes, returned to the initial
condition over 1.0 minutes, and allowed to equilibrate for 4.0 minutes. HPLC
fractions were collected throughout the run at 1.0-minute intervals. Aliquots
(100 ml) of fractions at the retention time (tR) of the ultraviolet peak corre-
sponding to the GSH conjugate (M3-1 or M5-2) were analyzed by liquid
chromatography-mass spectrometry for verification. Fractions containing the
peak were combined into a single 15-ml glass centrifuge tube and concentrated
Results
In Vitro Metabolism of Imidazo[2,1-b]thiazole Derivative 1
in HLM. In HLM supplemented with NADPH and GSH, three
metabolites of 1 (denoted as M1-1, M2-1, and M3-1) were observed
(Fig. 2A). Collision-induced dissociation (CID) spectra of 1 and its
metabolites M1-1, M2-1, and M3-1 are shown in Figs. 3–5. In the case
of 1 (tR = 18.82 minutes; MH+ = 482.1524), the major fragment ions
present at mass-to-charge ratio (m/z) 318.1476, 301.1210, 262.1004,
192.0318, and 165.0112 were consistent with the structure (see CID
spectrum in Fig. 3A). Metabolite M1-1 eluted at tR = 17.70 minutes
and possessed MH+ = 516.1579, which is an addition of 34.0055 Da
to the molecular mass of 1 (Fig. 3B). The presence of the fragment
ions at m/z 318.1476 and 192.0318 in the CID spectrum of M1-1
indicated that the 2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl-2,7-
diazaspiro[3,5]-nonane] portion was unaltered. A proposed structure
of M1-1 that is consistent with the observed molecular weight and
mass spectrum is shown in Fig. 3B.
Metabolites M2-1 (tR = 17.08 minutes) and M3-1 (tR = 16.47
minutes) possessed MH+ at 1096.3200 and 805.2311 m/z, respec-
tively. The CID spectra of M2-1 and M3-1 are shown in Figs. 4 and 5,
respectively. M2-1 contained two molecules of GSH in its structure
as evident from the facile loss of two pyroglutamic acid components
(m/z 967.2753 and m/z 838.2335) in its CID spectrum. Furthermore,
the presence of the fragment ion at m/z 318.1468 implied that the 2-[2-
chloro-4-(2H-1,2,3-triazol-2-yl)benzyl-2,7-diazaspiro[3,5]-nonane] por-
tion in M2-1 was unchanged. A structure of M2-1 that is compatible
with the observed molecular weight and fragmentation pattern is shown
in Fig. 4, and a mechanistic hypothesis that rationalizes its formation is
provided in Fig. 15 in the Discussion.
The molecular mass of M3-1 (MH+ = 805.2312) suggested that the
metabolite was derived from the addition of GSH to an oxidized
metabolite of 1. The fragment ions at m/z 730.1973 and m/z 676.1872
in the CID spectrum of M3-1 were derived from the characteristic
losses of the glycine (75 Da) and glutamic acid components (129 Da) of
GSH (Baillie and Davis, 1993). The diagnostic fragment ions at m/z
488.0904, 359.0471, and 318.1476 indicated that the 2-[2-chloro-4-(2H-1,

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Kalgutkar et al.
Fig. 2. Extracted ion chromatograms of incubation
mixtures of 1 (A) and 2 (B) in NADPH- and GSH-
supplemented HLM conducted at 37°C for 60 minutes.
2,3-triazol-2-yl)benzyl-2,7-diazaspiro[3,5]-nonane] scaffold in 1 was un- C5 position of the thiazole ring with addition of GSH or through
modified. On the basis of the fragmentation pattern, we inferred that a P450-mediated S-oxidation of the thiazole sulfur followed by addition
oxidation and subsequent GSH conjugation had occurred on the of GSH to the S-oxide metabolite (structure depicted in Fig. 5), similar
imidazo[2,1-b]thiazole ring. There are two obvious pathways through to one discerned with the imidazo[2,1-b]-thiazole derivative L-766,112
which this can arise, either via a P450-mediated epoxidation at the C4- (Trimble et al., 1997). To distinguish between the two possibilities, a
Fig. 3. CID spectra of 1 (A) and M1-1 (B).

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1379
Fig. 4. CID spectrum of bis-GSH conjugate
M2-1. Structures of diagnostic fragment ions
(and corresponding theoretical exact masses)
that allow metabolite identification are depicted.
The assigned regiochemistry is arbitrary.
crude isolate of M3-1 was obtained from a large-scale HLM incubation Furthermore, there is also set of cross-peaks that indicate a pair of
1
of 1 in the presence of NADPH and GSH and subsequently charac- inequivalent methylene resonances with H shifts of d 3.56/4.65 ppm
terized by NMR spectroscopy.
and a correlation to a 13C at d 62.9 ppm in the HSQC data. If the
1
Observed in the H-13C HSQC spectrum of the M3-1 isolate (Fig. bioactivation of 1 is mediated via the epoxidation pathway, the
1
6B) is a methine cross-peak with a H chemical shift of d 6.05 ppm resulting molecule would have two adjacent methine protons with
that correlates to a 13C atom with a chemical shift of d 58.6 ppm. chemical shifts in the range of d 5.0–6.0 ppm with corresponding 13
C
Fig. 5. CID spectrum of GSH conjugate M3-1.
Structures of diagnostic fragment ions (and corre-
sponding theoretical exact masses) that allow an
insight into the structure are depicted.

1380
Kalgutkar et al.
shifts in the range d 80 ppm (for HCOH) and d 60 ppm (for HCSG). a fragment ion at m/z 334.1420 (addition of oxygen to m/z 318.1473)
Alternatively, if the bioactivation of 1 is mediated via the S-oxidation indicated that M4-1 was derived from a monohydroxylation on the
1
pathway, the resulting molecule would have one methine H adjacent spirocyclic-azetidine ring. In contrast, the CID spectrum of the major
1
to a methylene, again each with H chemical shifts in the range of d metabolite M5-1 (Fig. 8) contained fragment ions that suggested that
5.0–6.0 ppm. Unlike the GSH-epoxide addition product, the 13C the imidazo[2,1-b]thiazole motif was the site of oxidation. Further-
chemical shifts of the GSH-S-oxide conjugate would have 13C shifts more, the fragment ion at m/z 452.1401 (MH+-SO) implied that M5-1
for both carbons in the range of d 50–60 ppm. The chemical shifts was the S-oxide metabolite of 1. Addition of GSH to the NADPH-
1
observed in the H-13C HSQC spectrum of M3-1 are more consistent supplemented HLM incubation of 1 significantly diminished the levels
with the structure resulting from the S-oxidation pathway and are also of M5-1 (Fig. 7B), which is consistent with the adduction of M5-1 to
closely aligned with the NMR data reported for the GSH conjugate of GSH resulting in the formation of M3-1.
L-766,112 S-oxide (Trimble et al., 1997). Furthermore, the TOCSY
In Vitro Metabolism of 2-Methylmidazo[2,1-b]thiazole Deriva-
data set (Fig. 6A) for the M3-1 isolate contains cross-peaks between tive 2 in HLM. Figure 2B depicts an extracted ion chromatogram of
the d 6.05 ppm and the d 3.56/4.65 ppm resonances, indicating a direct an incubation mixture comprised of NADPH, GSH, and 2 conducted
coupling between all of these protons. On the basis of these data the at 37°C for 60 minutes. A total of five metabolites was observed in
M3-1 isolate is tentatively assigned as the thiazole-S-oxide with a NADPH-dependent fashion. The CID spectrum of 2 (tR = 19.47
a GSH molecule attached at the C3 position of the thiazole ring. This minutes, MH+ = 496.1680) and proposed structures of diagnostic
structure strongly infers that the route of formation of M3-1 is through fragment ions (m/z 318.1473, 276.1159, and 192.0317) is shown in
a biotransformation step involving S-oxidation.
Fig. 9A. Metabolites M1-2 (tR = 19.0 minutes) and M2-2 (tR = 21.46
The potential for S-oxide formation was then specifically examined minutes) possessed MH+ at 512.1630, suggesting that they were
in NADPH-supplemented HLM incubations of 1 (10 mM) in the isomers derived from a monohydroxylation in 2. The CID spectra of
absence and presence of GSH (10 mM) (Fig. 7, A and B, respec- both metabolites (Supplemental Figs. 3 and 4) revealed fragment ions
tively). Two monohydroxylated metabolites (MH+ = 498.1473) de- at m/z 494.1510 (loss of a water molecule) and m/z 334.1420 (addition
signated as M4-1 (minor) and M5-1 (major) were observed in HLM of oxygen to m/z 318.1473) indicative of monohydroxylation on the
incubations in the absence of GSH. The CID spectrum of the minor spirocyclic-azetidine ring. Postulated structures of M1-2 and M2-2 are
metabolite M4-1 is shown in Supplemental Fig. 2. The detection of depicted in Supplemental Figs. 3 and 4, respectively. Considering that
Fig. 6. Abbreviated 1H-1H TOCSY (A) and 1H-13C HSQC spectrum (B) of M3-1. Red cross peaks in (B) indicate methine, whereas the blue cross peaks indicate the
methylene resonances.

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1381
Fig. 7. Extracted ion chromatograms of monohydroxylated
metabolites (MH+ = 498.1473) of 1 in NADPH-supplemented
HLM in the absence (A) and presence (B) of GSH.
M2-2 elutes after 2 under the reversed phase HPLC conditions, we corresponding diol metabolite derived from hydrolysis of an intermediate
speculate that M2-2 is an N-oxide metabolite of 2 by inference to thiazole ring epoxide. The fragment ions at m/z 318.1473, 213.0322, and
related tertiary basic amine drugs, such as loperamide, that undergo 192.0317 were consistent with the proposed structure (Fig. 9B).
N-oxidation (Kalgutkar and Nguyen, 2004).
Metabolite M4-2 (tR = 17.1 minutes) also possessed a MH+ at
Metabolite M3-2 (tR = 18.0 minutes) displayed a MH+ at 530.1735. 512.1630, consistent with a monohydroxylation in 2. In contrast with
The addition of 34.0055 Da to the MH+ of 2 indicated that M3-2 was the M1-2 and M2-2, metabolite M4-2 appeared to be derived from an
Fig. 8. CID spectrum of M5-1 derived from an NADPH-
supplemented HLM incubation of 1.

1382
Kalgutkar et al.
Fig. 9. CID spectra of 2 (A) and M3-2 (B).
oxidation on the 2-methylimidazo[2,1b]thiazole ring system as indi- 13C chemical shifts of the methyl of the imidazothiazole (d = 2.37 ppm
cated by the fragment ion m/z 318.1473 in the CID spectrum of M4-2. for 1H and d = 13.1 ppm for 2 and d = 2.15 ppm for 1H and d = 26.7
A proposed structure for M4-2 that is compatible with the observed ppm for M5-2) and the appearance of a new methylene resonance in
1
mass spectrum is shown in Supplemental Fig. 5.
the H spectrum of M5-2 at d = 4.93 ppm (Fig. 11, top panel). In the
Metabolite M5-2 (tR = 15.89 minutes) possessed a MH+ at 787.2747 HSQC data set this new 1H resonance correlates with a 13C resonance
and demonstrated a fragment ion at m/z 658.2308, which was con- at d = 55.1 ppm (unpublished data). Additionally, the H resonances
1
sistent with the loss of a glutamic acid component (129 Da) of GSH for GSH as well as those from the remaining 1-{2-[2-chloro-4-(2H-
(Fig. 10A). This observation suggested that M5-2 was a GSH conjugate 1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]non-7-yl}-2-(2-methyl-
of 2. The presence of the fragment ion at m/z 318.1474 implied that the imidazo[2,1-b][1,3]thiazol-6-yl)ethanone can be rationalized. The
site of attachment of GSH was on the 2-methylimidazo[2,1-b]thiazole critical data for the structural determination of M5-2 is the 1H-13C
ring system. Furthermore, the fragment ion at m/z 514.1776 was as- heteronuclear multiple bond correlation data (Fig. 12). In this data set
signed as a cleavage adjacent to the cysteinyl thioether moiety with there are correlations from both the new methylene resonance (d =
charge retention on the imidazole residue. The occurrence of the frag- 4.93 ppm) and the methyl resonance (d = 2.37 ppm) to a 13C with
ment ion at m/z 514.1776 is consistent with the presence of an aromatic a chemical shift of d = 201.7 ppm. A 13C chemical shift in the range
thioether motif in M5-2 (Baillie and Davis, 1993). A proposed structure of 190–220 ppm is unique and indicates a carbonyl with no adjacent
for M5-2 that is consistent with its mass spectrum is shown in Fig. 10A. hetero-atom, either a ketone or aldehyde. A structure that satisfies all
Replacement of GSH with N-acetylcysteine as a trapping agent in these data is shown in Fig. 10A, the biosynthetic pathway of which
NADPH-supplemented HLM incubations of 2 led to the formation of will be revealed in the Discussion.
M6-2 (tR = 18.2 minutes, MH+ = 643.2212), an analogous conjugate of
18O Incorporation Studies. The source of the carbonyl oxygen in
M5-2. The CID spectrum of M6-2 and interpretation of key fragment GSH conjugate M5-2 was investigated in NADPH- and GSH-
ions is depicted in Fig. 10B. As such, elucidation of the structure of supplemented HLM incubations of 2 conducted in H218O. As ex-
the GSH conjugate (M5-2) was largely aided by the isolation of the pected, the diol metabolite M3-2 incorporated 18O (MH+ = 532.1778)
thiol adduct from large-scale HLM incubations of 2 in the presence consistent with epoxide ring opening by H218O (Supplemental Fig. 6).
of NADPH and GSH, and subsequent characterization by NMR MS/MS analysis of the molecular ions of 16O- and 18O-incorporated
spectroscopy.
M3-2 (MH+ = 530.1725 and MH+ = 532.1778, respectively) clearly
NMR Characteristics of GSH Conjugate M5-2. The 1H spectrum indicated the 2-atomic mass unit shift in the parent ion and in the
M5-2 contains several critical differences from that of a similarly acylium ion fragment m/z 213.0322 (16O M3-2) to m/z 215.0369
acquired spectrum of 2. Most notably the change in both the H and (18O M3-2) (compare Fig. 13 with Fig. 9B). In contrast, 18O incorporation
1

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1383
Fig. 10. CID spectra of the GSH conjugate M5-2 (A) and N-acetylcysteine conjugate M6-2 (B).
was not observed in the GSH adduct M5-2. This observation suggested scaled to the corresponding intrinsic clearance values using the well-
that the source of the carbonyl oxygen in M5-2 is derived from molecular stirred model (Obach, 1999). Under these experimental conditions, the
oxygen.
half-lives of 1, 2, and 3 in HLM were 19.1, 28.6, and 38.8 minutes,
Trapping Studies with Potassium Cyanide. Attempts to trap the which translated into intrinsic clearance values (ml/min/kg) of 48, 32,
putative iminium ion intermediate involved in the formation of GSH and 24, respectively.
conjugate M5-2 (see Discussion and Fig. 16 on the proposed
mechanism for the formation of M5-2) were initiated in NADPH-
supplemented HLM incubations of 2 in the presence of potassium
Discussion
cyanide (5 mM). Figure 14 indicates the CID spectrum of an adduct
The otherwise attractive pharmacological and pharmacokinetic
M7-2 (tR = 18.4 minutes, MH+ = 539.1739) formed in these in- attributes of ghrelin inverse agonists 1 and 2 were offset by the
cubations in a NADPH- and cyanide-dependent fashion. The proposed presence of the imidazo[2,1-b]thiazole bicycle as part of the core
structure of the cyano adduct M7-2 and corresponding fragment ions structure, especially in light of previous studies by Trimble et al.
that led to the structural elucidation are also shown in Fig. 14. A mech- (1997) who demonstrated the microsomal S-oxidation of the thiazole
anism for the formation of M7-2 is shown in Fig. 16 (see Discussion). portion (within the bicycle) to an electrophilic sulfoxide metabolite
In Vitro Bioactivation of 2-Methylimidazo[2,1-b]thiadiazole that underwent a 1,4-Michael addition reaction with GSH. In
Derivative 3 in Human Liver Microsomes. LC-MS/MS analysis of addition, evidence linking thiazole ring bioactivation with preclinical
an incubation mixture of 3 in NADPH- and GSH-supplemented HLM
(or clinical) toxicity has been presented for several thiazole-based
did not reveal the presence of any GSH conjugates of 3, suggesting xenobiotics and drugs (Kalgutkar et al., 2005). For example, the clin-
that 3 is devoid of the bioactivation liability associated with 1 and 2. ical hepatotoxicity noted with the nonsteroidal anti-inflammatory
Comparison of the In Vitro Ghrelin Receptor Pharmacology
drug sudoxicam (Supplemental Fig. 6) has been attributed to a metabolic
and HLM Stability of Compounds 1–3. Potency against the ghrelin process involving thiazole ring scission to a toxic thiourea metabolite
receptor was determined in the previously described GHSR-1a binding (Obach et al., 2008). The structurally related anti-inflammatory agent
assay using [125I]ghrelin (Kung et al., 2012). As shown in Fig. 1, the (and marketed drug) meloxicam does not possess the hepatotoxic
IC50 values for 1, 2, and 3 were 8.0, 4.7, and 2.6 nM, respectively. The liability associated with sudoxicam. Although introduction of a methyl
microsomal stability of compounds 1–3 (final concentration = 1 mM) group at the C5 position of the thiazole ring in meloxicam is the only
was assessed by monitoring substrate consumption after incubation structural difference, the change dramatically alters the metabolic
with HLM in the presence of NADPH cofactor for 30 minutes at 37°C. profile such that oxidation of the C5 methyl group to the alcohol
Microsomal half-lives, reflecting depletion of test compounds, were metabolite (see Supplemental Fig. 7) constitutes the principal metabolic

1384
Kalgutkar et al.
Fig. 11. Full 1H spectrum of M5-2 and 2.
fate of meloxicam in humans with virtually no detectable thiazole ring (including bioactivation) of both 1 and 2 was blocked when the HLM
opening (Chesné et al., 1998; Obach et al., 2008). incubations were conducted in the presence of ketoconazole (a
Taking into consideration the above mentioned structure-toxicity selective CYP34 inhibitor), which implicated a role for CYP3A4 in the
relationship and the growing evidence linking immune-mediated drug metabolism of the two imidazo[2,1-b]thiazole derivatives. LC-MS/MS
toxicity with reactive metabolite formation (Park et al., 2011; Stepan characterization of the GSH conjugates suggested that the site of
et al., 2011; Sakatis et al., 2012; Thompson et al., 2012), we decided bioactivation in both compounds was on the imidazo[2,1-b]thiazole
to examine the bioactivation potential of 1 and 2 in HLM. Incubation bicycle. In the case of the imidazo[2,1-b]thiazole derivative 1, the
of 1 and 2 in NADPH- and GSH-supplemented HLM led to the formation of the diol metabolite M1-1 was consistent with a bioactivation
formation of reactive species that were trapped with GSH. Metabolism pathway involving thiazole ring oxidation by P450 enzyme(s) at the
Fig. 12. Abbreviated 1H-13C heteronuclear multiple bond correlation (HMBC) spectrum of M5-2 (A and B represent specific 1H correlations highlighted in the text).

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1385
Fig. 13. MS/MS data reflecting 18O incorporation in
the diol metabolite M3-2 generated in an NADPH-and
H218O-supplemented HLM incubation of 2.
C2–C3 position to yield the electrophilic epoxide intermediate 12, the crude M3-1 isolate indicated that the GSH adduct was derived
which is hydrolyzed to the diol M1-1 or can potentially react with from addition of the thiol nucleophile to the electrophilic S-oxide M5-
GSH to yield the sulfydryl conjugate 13 (Fig. 15, pathway A). An 1 and not from the ring opening of epoxide 12. The hypothesis
alternate pathway that leads to M3-1 involves P450-mediated is further strengthened by the observation that addition of GSH
oxidation on the thiazole sulfur to the S-oxide metabolite M5-1, to NADPH-supplemented HLM incubations of 1 significantly di-
followed by the Michael addition of GSH to yield adduct M3-1 (see minished the levels of M5-1 formed in the incubations. Reason(s) for
Fig. 15, pathway B) as outlined previously with the imidazo[2,1- the lack of formation of the GSH adduct 13 shown in Fig. 15, pathway
b]thiazole analog L-766,112 (Trimble et al., 1997). NMR analysis of A, are not apparent from this analysis. It is possible that hydrolytic ring
Fig. 14. CID spectrum of the cyanide conjugate
M7-2 derived from NADPH- and potassium cyanide-
supplemented HLM incubations of 2.

1386
Kalgutkar et al.
opening of 12 proceeds at a faster rate than the corresponding reaction leaving group (alkylsulfoxide) at the C5 position, which is poised for
with GSH.
nucleophilic displacement by GSH.
Apart from the mono-GSH adduct M3-1, we also noted the
In the case of 2, LC-MS/MS data on the GSH and N-acetylcysteinyl
formation of a novel bis-GSH conjugate M2-1 with two molecules of conjugates M5-2 and M6-2, respectively, were also consistent with thiazole
GSH attached to the imidazo[2,1-b]thiazole framework. A plausible ring bioactivation. From a structure-activity relationship standpoint,
mechanism outlining the formation of M2-1 is presented in Fig. 15 the C-2 methyl group on the imidazo[2,1-b]thiazole ring in 2 did not
(pathway C) and involves the initial addition of a GSH molecule to the prevent thiazole ring bioactivation to reactive metabolite(s), which
putative S-oxide metabolite of 1 (i.e., M5-1) to generate M3-1 contrasts the structure-toxicity relationship noted for sudoxicam and
followed by addition of a second molecule of GSH across the electron- meloxicam. From a mechanistic perspective, we speculate that the
deficient C5 position on the imidazole ring system in M3-1, which rate-limiting step in the formation of M5-2 and M6-2 proceeds via a
results in imidazo[2,1-b]thiazole-S-oxide ring scission. The outlined P450-mediated oxidation on the thiazole ring in 2 to generate epoxide
mechanism is certainly not beyond the realm of possibilities, consi- 14, which upon hydrolysis would yield the diol metabolite M3-2
dering the well-known chemical (and glutathione transferase-mediated) or undergo ring scission to an iminium species 15 (Fig. 16). A two-
displacement reaction of GSH with electron-deficient heteroaromatic electron reduction of the iminium bond in 15, possibly mediated by
rings (e.g., pyridine, pyrimidine, etc.) that contain alkylsulfoxide and/or GSH, would lead to the 2-mercaptoimidazole 16, which may be trapped
alkylsulfone as leaving groups (Clapp, 1956; Colucci and Buyske, by GSH or N-acetylcysteine via a developing ring-opened isothiocya-
1965; Conroy et al., 1984; Graham et al., 1989; Yang et al., 2012). A nate 17 to afford sulfydryl conjugates M5-2 and M6-2, respectively.
literature example that bears much commonality to the present situation Overall the mechanistic possibility is compatible with the results of the
is evident with the proton pump inhibitor pantoprazole wherein the 18O labeling studies, wherein lack of incorporation of 18O in M5-2
benzimidazole-2-sulfoxide motif undergoes nucleophilic attack by GSH indicates that the source of the carbonyl oxygen in M5-2 is from
at the electron-deficient C2 position on the benzimidazole ring (Zhong molecular oxygen (i.e., from a P450-mediated oxidation).
et al., 2005). Certainly, one can view M5-1 (in Fig. 15, pathway C) as
To examine whether iminium species 15 is indeed formed as an
an electron-deficient heterocycle (imidazole ring) with an excellent intermediate in the pathway leading to M5-2, 2 was incubated in HLM
Fig. 15. Proposed bioactivation pathways for imidazo[2,1-b]thiazole derivative 1.

Bioactivation of Imidazo[2,1-b]thiazole Derivatives
1387
in the presence of NADPH and excess potassium cyanide, which is
Overall, given this information, it appeared reasonable to attempt
typically used to trap electrophilic iminium ions generated via a two- to eliminate bioactivation liability in 1 and 2 via small structural
electron oxidation of amines (Rose and Castagnoli, 1983; Argoti et al., modifications of the thiazole ring that would preserve the physicochem-
2005). The accurate mass of the metabolite M7-2 detected in these ical properties (e.g., molecular weight, lipophilicity) largely responsible
incubations was consistent with the addition of cyanide across 15, for pharmacologic potency and HLM stability. Consequently, the
which provided additional support for our overall mechanistic hypo- 2-methylimidazo[2,1-b][1,3,4]thiadiazole analog 3 was synthesized
thesis. One can rationalize the formation of M7-2 to occur via the initial to prevent thiazole ring epoxidation and/or addition of GSH to an
addition of cyanide across the iminium bond in 15 to yield the cyano S-oxide metabolite. An identical medicinal chemistry strategy had
adduct 18, which upon cyclization would lead to M7-2 (see Fig. 16). been successfully used to abrogate thiazole ring bioactivation with
With reference to the step involving the reduction of the iminium bond nonpeptidyl thrombopoietin receptor agonists containing the
in 15 (to yield 2-mercaptoimidazole 16), we invoked the involvement 2-aminothiazole motif (Kalgutkar et al., 2007). As such, the absence
of GSH as a reducing agent, because two-electron reduction of imines of the GSH conjugate formation in NADPH- and GSH-supplemented
by GSH is a well precedented reaction. For example, N-acetyl-p- HLM incubations with 3 indicates that the thiadiazole derivative is
benzoquinone imine, the oxidation product of acetaminophen, can react devoid of the reactive metabolite liability associated with 1 and 2
with GSH in a 1,4-Michael fashion to yield a stable GSH adduct or and demonstrates the success of this medicinal chemistry tactic.
undergo a two-electron reduction to the parent drug in the presence of Compound 3 also retained the potent in vitro ghrelin inverse agonism
the thiol (Rosen et al., 1984; Potter and Hinson, 1986). In the present and HLM stability characteristics discerned with lead compounds 1 and
situation, we anticipate that the thioaminal conjugation product obtained 2. In summary, we demonstrated the human liver microsomal bio-
through addition of GSH across the imine double bond in 15 will be activation of the imidazo[2,1-b]thiazole functionality present as part of
highly unstable and spontaneously decompose back to 15. Clearly, the core structure in novel ghrelin inverse agonists. Characterization of
a more rigorous experimental analysis of these individual mechanistic the GSH adducts using LC-MS/MS and NMR techniques provided
steps will be needed to fully validate our hypothesis on the oxidative indirect information on the structures of the reactive species, thereby
metabolism/bioactivation of these unique bicyclo-heterocylic com- providing insight into the bioactivation mechanism. The information
pounds. Toward this end, the imidazo[2,1-b]thiazole intermediates gained from these studies was used to direct medicinal chemistry efforts
used in the synthesis of the title compounds 1 and 2 (see Supplemental in the design of a compound with the attractive pharmacology and
Methods) could provide much utility in testing certain mechanistic hy- disposition characteristics observed with the prototypes while avoiding
potheses (e.g., the use of deuterium exchange to distinguish S-oxidation potential safety concerns associated with the bioactivation of the
from mono-hydroxylation).
imidazo[2,1-b]thiazole functionality. Additional lead optimization
Fig. 16. Proposed bioactivation pathway for 2-methylimidazo[2,1-b]thiazole derivative 2.

1388
Kalgutkar et al.
Kung DW, Coffey SB, Jones RM, Cabral S, Jiao W, Fichtner M, Carpino PA, Rose CR, Hank
RF, and Lopaze MG, et al. (2012) Identification of spirocyclic piperazine-azetidine inverse
agonists of the ghrelin receptor. Bioorg Med Chem Lett 22:4281–4287.
efforts on 3, which culminated in the discovery of the clinical can-
didate, will be reported in due course.
Mansuy D and Dansette PM (2011) Sulfenic acids as reactive intermediates in xenobiotic me-
tabolism. Arch Biochem Biophys 507:174–185.
Authorship Contributions
Participated in research design: Kalgutkar, Goosen, Lapham, Ryder,
Walker, Eng.
Mizutani T, Yoshida K, and Kawazoe S (1993) Possible role of thioformamide as a proximate
toxicant in the nephrotoxicity of thiabendazole and related thiazoles in glutathione-depleted
mice: structure-toxicity and metabolic studies. Chem Res Toxicol 6:174–179.
Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, and Matsukura S (2001)
Conducted in vitro experiments: Ryder, Walker, Eng.
Contributed new reagents or analytic tools: Orr, Cabral.
Performed data analysis: Kalgutkar, Ryder, Walker, Orr, Eng, Goosen.
Wrote or contributed to the writing of the manuscript: Kalgutkar, Ryder,
Walker, Orr, Eng.
A role for ghrelin in the central regulation of feeding. Nature 409:194–198.
Obach RS (1999) Prediction of human clearance of twenty-nine drugs from hepatic microsomal
intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding
to microsomes. Drug Metab Dispos 27:1350–1359.
Obach RS, Kalgutkar AS, Ryder TF, and Walker GS (2008) In vitro metabolism and covalent
binding of enol-carboxamide derivatives and anti-inflammatory agents sudoxicam and meloxicam:
insights into the hepatotoxicity of sudoxicam. Chem Res Toxicol 21:1890–1899.
Park BK, Boobis A, Clarke S, Goldring CE, Jones D, Kenna JG, Lambert C, Laverty HG, Naisbitt
DJ, and Nelson S, et al. (2011) Managing the challenge of chemically reactive metabolites in
drug development. Nat Rev Drug Discov 10:292–306.
References
Potter DW and Hinson JA (1986) Reactions of N-acetyl-p-benzoquinone imine with reduced
glutathione, acetaminophen, and NADPH. Mol Pharmacol 30:33–41.
Argoti D, Liang L, Conteh A, Chen L, Bershas D, Yu CP, Vouros P, and Yang E (2005) Cyanide
trapping of iminium ion reactive intermediates followed by detection and structure identifi-
cation using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chem Res
Toxicol 18:1537–1544.
Baillie TA and Davis MR (1993) Mass spectrometry in the analysis of glutathione conjugates.
Biol Mass Spectrom 22:319–325.
Cardona Cano S, Merkestein M, Skibicka KP, Dickson SL, and Adan RA (2012) Role of ghrelin
in the pathophysiology of eating disorders: implications for pharmacotherapy. CNS Drugs 26:
281–296.
Rose J and Castagnoli N, Jr (1983) The metabolism of tertiary amines. Med Res Rev 3:73–88.
Rosen GM, Rauckman EJ, Ellington SP, Dahlin DC, Christie JL, and Nelson SD (1984) Re-
duction and glutathione conjugation reactions of N-acetyl-p-benzoquinone imine and two
dimethylated analogues. Mol Pharmacol 25:151–157.
Roy P, Leblanc Y, Ball RG, Brideau C, Chan CC, Chauret N, Cromlish W, Ethier D, Gauthier JY,
and Gordon R, et al. (1997) A new series of selective COX-2 inhibitors: 5,6-diarylthiazolo[3,2-
b][1,2,4]-triazoles. Bioorg Med Chem Lett 7:57–62.
Rudolph J, Esler WP, O’connor S, Coish PD, Wickens PL, Brands M, Bierer DE, Bloomquist BT,
Bondar G, and Chen L, et al. (2007) Quinazolinone derivatives as orally available ghrelin
receptor antagonists for the treatment of diabetes and obesity. J Med Chem 50:5202–5216.
Sakatis MZ, Reese MJ, Harrell AW, Taylor MA, Baines IA, Chen L, Bloomer JC, Yang EY,
Ellens HM, and Ambroso JL, et al. (2012) Preclinical strategy to reduce clinical hepatotoxicity
using in vitro bioactivation data for .200 compounds. Chem Res Toxicol 25:2067–2082.
Serby MD, Zhao H, Szczepankiewicz BG, Kosogof C, Xin Z, Liu B, Liu M, Nelson LT,
Kaszubska W, and Falls HD, et al. (2006) 2,4-Diaminopyrimidine derivatives as potent growth
hormone secretagogue receptor antagonists. J Med Chem 49:2568–2578.
Stepan AF, Walker DP, Bauman J, Price DA, Baillie TA, Kalgutkar AS, and Aleo MD (2011)
Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the
risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in
the top 200 drugs marketed in the United States. Chem Res Toxicol 24:1345–1410.
Soares JB, Roncon-Albuquerque R, Jr, and Leite-Moreira A (2008) Ghrelin and ghrelin receptor
inhibitors: agents in the treatment of obesity. Expert Opin Ther Targets 12:1177–1189.
Thérien M, Brideau C, Chan CC, Cromlish WA, Gauthier JY, Gordon R, Greig G, Kargman S,
Lau CK, and Leblanc Y, et al. (1997) Synthesis and biological evaluation of 5,6-diarylimidazo
[2.1-b]thiazole as selective COX-2 inhibitors. Bioorg Med Chem Lett 7:47–52.
Thompson RA, Isin EM, Li Y, Weidolf L, Page K, Wilson I, Swallow S, Middleton B, Stahl S,
and Foster AJ, et al. (2012) In vitro approach to assess the potential for risk of idiosyncratic
adverse reactions caused by candidate drugs. Chem Res Toxicol 25:1616–1632.
Trimble LA, Chauret N, Silva JM, Nicoll-Griffith DA, Li CS, and Yergey JA (1997) Charac-
terization of the in vitro oxidative metabolites of the COX-2 selective inhibitor L-766,112.
Bioorg Med Chem Lett 7:53–56.
Chatfield DH and Hunter WH (1973) The metabolism of acetamidothiazoles in the rat. 2-
Acetamido-, 2-acetamido-4-methyl- and 2-acetamido-4-phenyl-thiazole. Biochem J 134:
869–878.
Chesné C, Guyomard C, Guillouzo A, Schmid J, Ludwig E, and Sauter T (1998) Metabolism of
Meloxicam in human liver involves cytochromes P4502C9 and 3A4. Xenobiotica 28:1–13.
Clapp JW (1956) A new metabolic pathway for a sulfonamide group. J Biol Chem 233:207–214.
Colucci DF and Buyske DA (1965) The biotransformation of a sulfonamide to a mercaptan and to
a mercapturic acid and glucuronide conjugate. Biochem Pharmacol 14:457–466.
Conroy CW, Schwam H, and Maren TH (1984) The nonenzymatic displacement of the sulfamoyl
group from different classes of aromatic compounds by glutathione and cysteine. Drug Metab
Dispos 12:614–618.
Costantino L and Barlocco D (2009) Ghrelin receptor modulators and their therapeutic potential.
Future Med Chem 1:157–177.
Dansette PM, Bertho G, and Mansuy D (2005) First evidence that cytochrome P450 may catalyze
both S-oxidation and epoxidation of thiophene derivatives. Biochem Biophys Res Commun
338:450–455.
Dezaki K, Sone H, and Yada T (2008) Ghrelin is a physiological regulator of insulin release in
pancreatic islets and glucose homeostasis. Pharmacol Ther 118:239–249.
Esler WP, Rudolph J, Claus TH, Tang W, Barucci N, Brown SE, Bullock W, Daly M, Decarr L,
and Li Y, et al. (2007) Small-molecule ghrelin receptor antagonists improve glucose tolerance,
suppress appetite, and promote weight loss. Endocrinology 148:5175–5185.
Graham SL, Shepard KL, Anderson PS, Baldwin JJ, Best DB, Christy ME, Freedman MB,
Gautheron P, Habecker CN, and Hoffman JM, et al. (1989) Topically active carbonic anhy-
drase inhibitors. 2. Benzo[b]thiophenesulfonamide derivatives with ocular hypotensive activity.
J Med Chem 32:2548–2554.
Hajovsky H, Hu G, Koen Y, Sarma D, Cui W, Moore DS, Staudinger JL, and Hanzlik RP (2012)
Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes. Chem
Res Toxicol 25:1955–1963.
Heppner KM, Müller TD, Tong J, and Tschöp MH (2012) Ghrelin in the control of energy, lipid,
and glucose metabolism. Methods Enzymol 514:249–260.
Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA,
and Bloom SR (2001) Ghrelin enhances appetite and increases food intake in humans. J Clin
Endocrinol Metab 86:5992–5995.
Yang Y, Qiu F, Josephs JL, Humphreys WG, and Shu Y-Z (2012) Bioactivation of 2-(alkylthio)-
1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles. Chem Res Toxicol 25:2770–2779.
Yabuki M, Shimakura IM, Kanamura H, Iba K, and Nakatasuka O (1997) Metabolism of 4-[1-(2-
fluoro-4-biphenyl)-ethyl]-2-methylaminothiazole (SM-8849), in rats. Eur
Pharmacokinet 22:25–33.
J Drug Metab
Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, and Fujimiya M (2004)
Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ.
FASEB J 18:439–456.
Kalgutkar AS, Driscoll J, Zhao SX, Walker GS, Shepard RM, Soglia JR, Atherton J, Yu L, Mutlib
AE, and Munchhof MJ, et al. (2007) A rational chemical intervention strategy to circumvent
bioactivation liabilities associated with a nonpeptidyl thrombopoietin receptor agonist con-
taining a 2-amino-4-arylthiazole motif. Chem Res Toxicol 20:1954–1965.
Kalgutkar AS, Gardner I, Obach RS, Shaffer CL, Callegari E, Henne KR, Mutlib AE, Dalvie DK,
Lee JS, and Nakai Y, et al. (2005) A comprehensive listing of bioactivation pathways of
organic functional groups. Curr Drug Metab 6:161–225.
Yoon WH, Yoo JK, Lee JW, Shim CK, and Lee MG (1998) Species differences in pharmaco-
kinetics of a hepatoprotective agent, YH439, and its metabolites, M4, M5, and M7, after
intravenous and oral administration to rats, rabbits, and dogs. Drug Metab Dispos 26:152–163.
Ziegler-Skylakakis K, Nill S, Pan JF, and Andrae U (1998) S-oxygenation of thiourea results in
the formation of genotoxic products. Environ Mol Mutagen 31:362–373.
Zhong D, Xie Z, and Chen X (2005) Metabolism of pantoprazole involving conjugation with
glutathione in rats. J Pharm Pharmacol 57:341–349.
Kalgutkar AS and Nguyen HT (2004) Identification of an N-methyl-4-phenylpyridinium-like
metabolite of the antidiarrheal agent loperamide in human liver microsomes: underlying
reason(s) for the lack of neurotoxicity despite the bioactivation event. Drug Metab Dispos 32:
943–952.
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, and Kangawa K (1999) Ghrelin is
a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660.
Address correspondence to: Amit S. Kalgutkar, Pharmacokinetics, Dynamics,
and Metabolism-New Chemical Entities, Pfizer Worldwide Research and De-
velopment, Cambridge, MA 02139. E-mail: amit.kalgutkar@pfizer.com