Reactive Metabolite Trapping Studies on Imidazo- and 2...
Transcript of Reactive Metabolite Trapping Studies on Imidazo- and 2...
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Reactive Metabolite Trapping Studies on Imidazo- and 2-Methylimidazo[2,1-
b]thiazole-based Inverse Agonists of the Ghrelin Receptor
Amit S. Kalgutkar, Tim 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, Groton, CT (T.R.,
G.S.W., T.C.G., K.L., H.E.) and Cambridge MA (A.S.K.)
Worldwide Medicinal Chemistry, La Jolla, CA (S.T.M.O.) and Groton, CT (S.C.)
Pfizer Inc.
DMD Fast Forward. Published on April 22, 2013 as doi:10.1124/dmd.113.051839
Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: Bioactivation of imidazo[2,1-b]thiazole derivatives
Address correspondence to: Amit S. Kalgutkar, Pharmacokinetics, Dynamics, and
Metabolism-New Chemical Entities, Pfizer Worldwide Research and Development,
Cambridge, MA 02139. Phone: (617)-551-3336. E-mail: [email protected]
Text Pages (including references): 35
Tables: 0
Figures: 16
References: 45
Abstract: 221
Introduction: 697
Discussion: 1550
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Abbreviations used are: GHS-R1a, growth hormone secretagogue receptor type 1a; SAR,
structure-activity relationship; 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-methylimidazo[2,1-
b]thiazol-6-yl)ethanone; CYP, cytochrome P450; GSH, reduced glutathione; L-766,112, 5-(4-
(methylsulfonyl)phenyl)-6-phenylimidazo[2,1-b]thiazole; HLM, human liver microsomes; 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; NADPH, nicotinamide adenine
dinucleotide phosphate; DMSO-d6, dimethyl sulfoxide-d6; LC-MS/MS, liquid chromatography
tandem mass spectrometry; TCI, triple resonance probe; COSY, correlation spectroscopy;
TOCSCY, total correlation spectroscopy; HSQC, multiplicity edited heteronuclear single
quantum coherence; HMBC, heteronuclear multiple bond correlation; tR, retention time; CID,
collision-induced dissociation.
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Abstract
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
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abrogated the bioactivation liability but also retained the attractive pharmacological attributes of
the prototype agents.
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Introduction
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 (GHS-R1a), a G-protein-coupled receptor expressed
primarily in the pituitary gland, brain, and to a lesser extent in the periphery. Through its action
on GHS-R1a, ghrelin exerts a variety of metabolic functions including stimulation of growth
hormone release, stimulation of appetite and weight gain, and suppression of insulin secretion in
rodents and humans (Wren et al., 2001; Nakazato et al., 2001; Dezaki et al., 2008; Cardona Cano
et al., 2012; Heppner et al., 2012). Thus, antagonizing GHSR-1a with small molecule
antagonists or inverse agonists is anticipated to improve glucose homeostasis and insulin
sensitivity, while eliciting beneficial effects on body weight (Serby et al., 2006; Rudolph et al.,
2007; Esler et al., 2008; 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 (SAR) 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-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)
depicted in Figure 1) demonstrated significant improvements 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 pharmacokinetic profile (↓
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clearance, ↑oral bioavailability) in animals, which warranted a further examination of relevant
preclinical safety endpoints as a prelude to candidate nomination.
From a drug metabolism perspective, the presence of the imidazo[2,1-b]thiazole scaffold in 1
and 2 raised a significant cause for concern, given the possibility of a cytochrome P450 (CYP)-
mediated S-oxidation on the thiazole sulfur (within the imidazo[2,1-b]thiazole framework) to
yield an electrophilic S-oxide metabolite capable of reacting with proteins and/or the endogenous
antioxidant glutathione (GSH). Indeed, the characterization of a GSH conjugate derived from
Michael addition to an S-oxide metabolite of the imidazo[2,1-b]thiazole derivative, 5-(4-
(methylsulfonyl)phenyl)-6-phenylimidazo[2,1-b]thiazole inhibitor (L-766,112) (Figure 1)
provides precedence for this bioactivation pathway in liver microsomes (Thérien et al., 1997;
Trimble et al., 1997). Furthermore, a successful medicinal chemistry strategy involving
internalization of the thiazole motif, resulting in the elimination of reactive metabolite liability of
L-766,112, while retaining the primary pharmacology (selective inhibition of cyclooxygenase-2)
has been presented in subsequent work (Figure 1) (Roy et al., 1997). Besides thiazole-S-
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 metabolites,
which are the by-products of thiazole ring cleavage, can undergo further S-oxidation to
electrophilic sulfenic acid derivatives (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).
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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 of 1, whereas, the
formation of the second adduct involved the attachment of a second GSH molecule to the initial
GSH-S-oxide adduct with concomitant liberation of the oxidized thiazole sulfur. With respect to
the corresponding 2-methylimidazo[2,1-b]thiazole analog 2, GSH conjugation occurred via an
oxidative desulfation mechanism, possibly involving thiazole ring epoxidation as the rate-
limiting step. Characterization of the GSH conjugate structure and additional trapping studies
with cyanide ion proved to be useful in deciphering the overall bioactivation mechanism
associated with 2. The in vitro metabolism information was used to design the corresponding
thiadiazole analog, 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 analog 3 (see Figure 1) that would
be devoid of reactive metabolite formation. Indeed, 3 was resistant to bioactivation in HLM
(inferred from the lack of GSH conjugate formation), while retaining inverse agonist potency for
the ghrelin receptor and HLM stability noted with 1 and 2.
Materials and Methods
Materials. Reduced nicotinamide adenine dinucleotide phosphate (NADPH), GSH and
potassium cyanide were obtained from Sigma-Aldrich (St. Louis, MO). HLM were prepared
from a mixed gender 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
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of either analytical or HPLC grade. The preparation of title compounds 1–3 is provided in the
supplementary section of this paper (see Supplemental Methods and Supplemental Figure 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 concentration = 1.0 mg/ml, CYP concentration = 0.5 μM), test
compound (10 µM) and NADPH (1.3 mM). Additional microsomal incubations also included
GSH (10 mM), N-acetylcysteine (10 mM) and/or potassium cyanide (5 mM) to trap reactive
metabolites. Reactions were terminated by the addition of acetonitrile (3.0 ml). The solutions
were centrifuged (3000g, 15 min), and the supernatants were transferred to clean 15 ml glass
centrifuge tubes and concentrated to dryness. The residue was reconstituted with 200 μl 10%
acetonitrile in water and analyzed for metabolite formation using liquid chromatography tandem
mass spectrometry (LC-MS/MS).
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 μl, 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 μM), and 3.3 mM MgCl2 (20 μl), in HLM (protein concentration = 1.0 mg/ml, CYP
concentration = 0.5 μM). 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 acetonitrile and
centrifuged at 3000g for 5 minutes. The supernatants were transferred to clean 15 ml centrifuge
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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 Accela quaternary solvent delivery pump, an Acella autoinjector, and a Surveyor PDA Plus
photodiode array detector (Thermo Electron Corporation, Waltham, MA). Chromatography was
performed on a Phenomenex Hydro RP column (4 μm 4.6 mm x 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 minutes, and allowed to equilibrate
for 4.0 minutes. Post-column flow passed through the PDA detector to provide UV (λ= 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 μl/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/MS 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 3 successive product ion scans with an early exclusion if the precursor
ion falls below a signal to noise 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 via a scale up of the
human liver microsomal incubations. Six incubations of 1 or 2 were carried out in 50 ml
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polypropylene centrifuge tubes at 37 °C for 60 min 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 µM), GSH (10 mM) and
NADPH (1.3 mM). Reactions were terminated by the addition of acetonitrile (30 ml). The
solutions were centrifuged (3000g, 15 min), 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 μl 10% acetonitrile in water. The reconstituted samples were combined in
a 2 ml HPLC vial and were onto an HPLC semipreparative system in 8 3 μl injections. This
system consisted of a Shimadzu SiL-HTC autosampler, two LC-20AD solvent pumps, an SPD-
M20A diode array detector, and a FRC-10 A fraction collector (Shimadzu USA, Columbia,
MD). Separation was performed on a Zorbax RX C8, 9.4 x 250mm, 5 μM semipreparative
HPLC column. 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 μl) of
fractions at the retention time (tR) of the UV peak corresponding to the GSH conjugate (M3-1 or
M5-2) were analyzed by LC/MS for verification. Fractions containing the peak were combined
into a single 15 ml glass centrifuge tube and concentrated to dryness. In the case of M3-1, it was
difficult to obtain a pure isolate due to poor chromatographic resolution between M3-1 and M2-
1. Hence, a crude isolate of M3-1 was used in NMR characterization. The residues were
evacuated in a drybox for ~ 2 hours prior to reconstitution in DMSO-d6 for NMR analysis.
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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, Billerica, MA) 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
(δ = 2.49 ppm relative to tetramethylsilane, δ = 0.00 for 1H and δ = 39.5 ppm relative to
tetramethylsilane, δ = 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. The two-dimensional data (correlation spectroscopy [COSY],
total correlation spectroscopy [TOCSY], multiplicity edited heteronuclear single quantum
coherence [HSQC], and heteronuclear multiple bond correlation [HMBC]) were recorded using
the standard pulse sequences provided by Bruker. A 1K x 128 data matrix was acquired using a
minimum of four scans and 16 dummy scans. The data was zero-filled to a size of 1K x 1K. A
mixing time of 80 ms was used in the TOCSY experiments.
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 (Figure 2, panel A). Collision-induced dissociation (CID) spectra of 1 and its
metabolites M1-1, M2-1 and M3-1 are shown in Figures 3–5. In the case of 1 (tR = 18.82
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minutes; MH+ = 482.1524), the major fragment ions present at m/z 318.1476, 301.1210,
262.1004, 192.0318, and 165.0112 were consistent with the structure (see CID spectrum in
Figure 3, panel A). 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 weight of 1 (Figure 3, panel B).
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 Figure 3, panel B.
Metabolites M2-1 (tR = 17.08 minutes) and M3-1 (tR = 16.47 minutes) possessed MH+ at
1096.3200 m/z and 805.2311 m/z, respectively. The CID spectra of M2-1 and M3-1 are shown
in Figures 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) portion
in M2-1 was unchanged. A structure of M2-1 that is compatible with the observed molecular
weight and fragmentation pattern is shown in Figure 4 and a mechanistic hypothesis that
rationalizes its formation is provided in Figure 15 in the discussion section.
The molecular weight 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-
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4-(2H-1,2,3-triazol-2-yl)benzyl-2,7-diazaspiro[3,5]-nonane) scaffold in 1 was unmodified.
Based on the fragmentation pattern, we inferred that oxidation and subsequent GSH conjugation
had occurred on the imidazo[2,1-b]thiazole ring. There are two obvious pathways through which
this can arise, either via a CYP mediated epoxidation at the C4-C5 position of the thiazole ring
with addition of GSH or through a CYP mediated S-oxidation of the thiazole sulfur followed by
addition of GSH to the S-oxide metabolite (structure depicted in Figure 5), similar to one
discerned with the imidazo[2,1-b]-thiazole derivative L-766,112 (Trimble et al., 1996). In order
to distinguish between the two possibilities, a crude isolate of M3-1 was obtained from a large-
scale HLM incubation of 1 in the presence of NADPH and GSH, and subsequently characterized
by NMR spectroscopy.
Observed in the 1H-13C HSQC spectrum of the M3-1 isolate (Figure 6, panel B) is a methine
cross peak with a 1H chemical shift of δ 6.05 ppm that correlates to a 13C atom with a chemical
shift of δ 58.6 ppm. Furthermore, there is also set of cross peaks that indicate a pair of
inequivalent methylene resonances with 1H shifts of δ 3.56/4.65 ppm and a correlation to a 13C at
δ 62.9 ppm in the HSQC data. If the bioactivation of 1 is mediated via the epoxidation pathway
the resulting molecule would have two adjacent methine protons with chemical shifts in the
range of δ 5.0–6.0 ppm with corresponding 13C shifts in the range δ 80 ppm (for HCOH) and δ
60 ppm (for HCSG). Alternatively, if the bioactivation of 1 is mediated via the S-oxidation
pathway the resulting molecule would have one methine 1H adjacent to a methylene, again each
with 1H chemical shifts in the range of δ 5.0–6.0 ppm. Unlike the GSH-epoxide addition
product, the 13C chemical shifts of the GSH-S-oxide conjugate would have 13C shifts for both
carbons in the range of δ 50–60 ppm. The chemical shifts observed in the 1H-13C HSQC
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spectrum of M3-1 are more consistent with the structure resulting from the S-oxidation pathway,
and are also closely aligned with the NMR data reported for the GSH conjugate of L-766,112 S-
oxide (Trimble et al., 1996). Furthermore, the TOCSY data set (Figure 6, panel A) for the M3-1
isolate contains cross peaks between the δ 6.05 ppm and the δ 3.56/4.65 ppm resonances
indicating a direct coupling between all of these protons. Based on these data the M3-1 isolate is
tentatively assigned as the thiazole-S-oxide with a GSH molecule attached at the C3 position of
the thiazole ring. This structure strongly infers that the route of formation of M3-1 is through a
biotransformation step involving S-oxidation .
The potential for S-oxide formation was then specifically examined in NADPH-
supplemented HLM incubations of 1 (10 μM) in the absence and presence of GSH (10 mM)
(Figure 7, panels A and B, respectively). Two monohydroxylated metabolites (MH+ = 498.1473)
designated as M4-1 (minor) and M5-1 (major) were observed in HLM incubations in the absence
of GSH. The CID spectrum of the minor metabolite M4-1 is shown in Supplemental Figure 2.
The detection of a fragment ion at m/z 334.1420 (addition of oxygen to m/z 318.1473) indicated
that M4-1 was derived from a monohydroxylation on the spirocyclic-azetidine ring. In contrast,
the CID spectrum of the major metabolite M5-1 (Figure 8) contained fragment ions which
suggested that the imidazo[2,1-b]thiazole motif was the site of oxidation. Furthermore the
fragment ion at m/z 452.1401 (MH+ - SO) implied that M5-1 was the S-oxide metabolite of 1.
Addition of GSH to the NADPH-supplemented HLM incubation of 1 significantly diminished
the levels of M5-1 (Figure 7, panel B), which is consistent with the adduction of M5-1 to GSH
resulting in the formation of M3-1.
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In Vitro Metabolism of 2-Methylmidazo[2,1-b]thiazole Derivative 2 in HLM. Figure 2
(panel B) depicts an extracted ion chromatogram of an incubation mixture comprising of
NADPH, GSH and 2 conducted at 37 °C for 60 minutes. A total of five metabolites were
observed in a NADPH-dependent fashion. The CID spectrum of 2 (tR = 19.47 minutes, MH+ =
496.1680), and proposed structures of diagnostic fragment ions (m/z 318.1473, 276.1159 and
192.0317) is shown in Figure 9, panel A. Metabolites M1-2 (tR = 19.0 minutes) and M2-2 (tR =
21.46 minutes) possessed MH+ at 512.1630, suggesting that they were isomers derived from a
monohydroxylation in 2. The CID spectra of both metabolites (Supplemental Figures 3 and 4)
revealed fragment ions at m/z 494.1510 (loss of a water molecule) and m/z 334.1420 (addition of
oxygen to m/z 318.1473) indicative of monohydroxylation on the spirocyclic-azetidine ring.
Postulated structures of M1-2 and M2-2 are depicted in Supplemental Figures 3 and 4,
respectively. Considering that M2-2 elutes after 2 under the reversed phase HPLC conditions,
we speculate that M2-2 is an N-oxide metabolite of 2 by inference to related tertiary basic amine
drugs such as loperamide that undergo N-oxidation (Kalgutkar and Nguyen, 2004).
Metabolite M3-2 (tR = 18.0 minutes) displayed a MH+ at 530.1735. The addition of 34.0055
Da to the MH+ of 2 indicated that M3-2 was the corresponding diol metabolite, derived from
hydrolysis of an intermediate thiazole ring epoxide. The fragment ions at m/z 318.1473,
213.0322 and 192.0317 were consistent with the proposed structure (Figure 9, panel B).
Metabolite M4-2 (tR = 17.1 minutes) also possessed a MH+ at 512.1630, consistent with a
monohydroxylation in 2. In contrast with M1-2 and M2-2, metabolite M4-2 appeared to be
derived from an oxidation on the 2-methylimidazo[2,1b]thiazole ring system as indicated by the
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fragment ion m/z 318.1473 in the CID spectrum of M4-2. A proposed structure for M4-2 that is
compatible with the observed mass spectrum is shown in Supplemental Figure 5.
Metabolite M5-2 (tR = 15.89 minutes) possessed a MH+ at 787.2747, and demonstrated a
fragment ion at m/z 658.2308, which was consistent with the loss of a glutamic acid component
(129 Da) of GSH (Figure 10, panel A). This observation suggested that M5-2 was a GSH
conjugate of 2. The presence of the fragment ion at m/z 318.1474 implied that the site of
attachment of GSH was on the 2-methylimidazo[2,1-b]thiazole ring system. Furthermore, the
fragment ion at m/z 514.1776 was assigned as a cleavage adjacent to the cysteinyl thioether
moiety with charge retention on the imidazole residue. The occurrence of the fragment ion at
m/z 514.1776 is consistent with the presence of an aromatic thioether motif in M5-2 (Baillie and
Davis, 1993). A proposed structure for M5-2 that is consistent with its mass spectrum is shown
in Figure 10, panel A. Replacement of GSH with N-acetylcysteine as a trapping agent in
NADPH-supplemented HLM incubations of 2 led to the formation of M6-2 (tR = 18.2 minutes,
MH+ = 643.2212), an analogous conjugate of M5-2. The CID spectrum of M6-2 and
interpretation of key fragment ions is depicted in Figure 10, panel B. As such, elucidation of the
structure of the GSH conjugate (M5-2) was largely aided by the isolation of the thiol adduct from
large-scale HLM incubations of 2 in the presence of NADPH and GSH, and subsequent
characterization by NMR spectroscopy.
NMR Characteristics of GSH Conjugate M5-2. The 1H spectrum M5-2 contains several
critical differences from that of a similarly acquired spectrum of 2. Most notably the change in
both the 1H and 13C chemical shifts of the methyl of the imidazothiazole (δ = 2.37 ppm for 1H
and δ = 13.1 ppm for 2 and δ = 2.15 ppm for 1H and δ = 26.7 ppm for M5-2), and the appearance
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of a new methylene resonance in the 1H spectrum of M5-2 at δ = 4.93 ppm (Figure 11, top
panel). In the HSQC data set this new 1H resonance correlates with a 13C resonance at δ = 55.1
ppm (data not shown). Additionally, the 1H resonances for GSH as well as those from the
remaining 1-{2-[2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]non-7-yl}-2-(2-
methylimidazo[2,1-b][1,3]thiazol-6-yl)ethanone can be rationalized. The critical data for the
structural determination of M5-2 is the 1H-13C HMBC data (Figure 12). In this data set there are
correlations from both the new methylene resonance (δ = 4.93 ppm) and the methyl resonance (δ
= 2.37 ppm) to a 13C with a chemical shift of δ = 201.7 ppm. A 13C chemical shift in the range of
190-220 ppm is unique and indicates a carbonyl with no adjacent hetero-atom, either a ketone or
aldehyde. A structure that satisfies all these data is shown in Figure 10 (panel A), the
biosynthetic pathway of which will be revealed in the discussion section.
18O Incorporation Studies. The source of the carbonyl oxygen in GSH conjugate M5-2 was
investigated in NADPH- and GSH-supplemented HLM incubations of 2, conducted in H218O.
As expected, the diol metabolite M3-2 incorporated 18O (MH+ = 532.1778), consistent with
epoxide ring opening by H218O (Supplemental Figure 6). MS/MS analysis of the molecular ions
of 16O- and 18O-incorporated M3-2 (MH+ = 530.1725 and MH+ = 532.1778, respectively) clearly
indicated the 2-atomic mass unit shift in the parent ion and in the acylium ion fragment m/z
213.0322 (16O M3-2) to m/z 215.0369 (18O M3-2) (compare Figure 13 with Figure 9, panel B).
In contrast, 18O incorporation was not observed in the GSH adduct M5-2. This observation
suggested that the source of the carbonyl oxygen in M5-2 is derived from molecular oxygen.
Trapping Studies with Potassium Cyanide. Attempts to trap the putative iminium ion
intermediate involved in the formation of GSH conjugate M5-2 (see discussion section and
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Figure 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 cyanide (5 mM). Figure 14
indicates the CID spectrum of an adduct M7-2 (tR = 18.4 minutes, MH+ = 539.1739) formed in
these incubations in a NADPH- and cyanide-dependent fashion. The proposed structure of the
cyano adduct M7-2 and corresponding fragment ions that led to the structural elucidation are also
shown in Figure 14. A mechanism for the formation of M7-2 is shown in Figure 16 (discussion
section).
In Vitro Bioactivation of 2-Methylimidazo[2,1-b]thiadiazole Derivative 3 in Human Liver
Microsomes. LC-MS/MS analysis of an incubation mixture of 3 in NADPH- and GSH-
supplemented HLM did not reveal the presence of any GSH conjugates of 3, suggesting that 3 is
devoid of the bioactivation liability associated with 1 and 2.
Comparison of the In Vitro Ghrelin Receptor Pharmacology and HLM Stability of
Compounds 1–3. Potency against the ghrelin receptor was determined in the previously
described GHSR-1a binding assay using [125I]-ghrelin (Kung et al., 2012). As shown in Figure
1, the IC50 values for 1, 2 and 3 were 8.0 nM, 4.7 nM, and 2.6 nM, respectively. The microsomal
stability of compounds 1–3 (final concentration = 1 μM) was assessed by monitoring substrate
consumption after incubation with HLM in the presence of NADPH co-factor for 30 min at 37
°C. Microsomal half-lives, reflecting depletion of test compounds, were scaled to the
corresponding intrinsic clearance values using the well-stirred model (Obach, 1999). Under
these experimental conditions, the half-lives of 1, 2 and 3 in HLM were 19.1 minutes, 28.6
minutes, and 38.8 minutes, which translated into intrinsic clearance values (ml/min/kg) of 48, 32
and 24, respectively.
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Discussion
The otherwise attractive pharmacological and pharmacokinetic attributes of ghrelin inverse
agonists 1 and 2 were offset by the presence of the imidazo[2,1-b]thiazole bicycle as part of the
core structure, especially in light of previous studies by Trimble et al. (1997) who demonstrated
the microsomal S-oxidation of the thiazole portion (within the bicycle) to an electrophilic
sulfoxide metabolite that underwent a 1,4-Michael addition reaction with GSH. In addition,
evidence linking thiazole ring bioactivation with preclinical (or clinical) toxicity has been
presented for several thiazole-based xenobiotics and drugs (Kalgutkar et al., 2005). For
example, the clinical hepatotoxicity noted with the nonsteroidal anti-inflammatory drug
sudoxicam (supplementary Figure S-6) has been attributed to a metabolic process involving
thiazole ring scission to a toxic thiourea metabolite (Obach et al., 2008). The structurally-related
anti-inflammatory agent (and marketed drug) meloxicam does not possess the hepatotoxic
liability associated with sudoxicam. Although introduction of a methyl group at the C5 position
of the thiazole ring in meloxicam is the only structural difference, the change dramatically alters
the metabolic profile such that oxidation of the C5 methyl group to the alcohol metabolite (see
Supplemental Figure 7) constitutes the principal metabolic fate of meloxicam in humans with
virtually no detectable thiazole ring opening (Chesné et al., 1998; Obach et al., 2008).
Taking into consideration the above mentioned structure-toxicity relationship and the
growing evidence linking immune-mediated drug toxicity with reactive metabolite formation
(Stepan et al., 2011; Park et al., 2011; Thompson et al., 2012; Sakatis et al., 2012), we decided to
examine the bioactivation potential of 1 and 2 in HLM. Incubation of 1 and 2 in NADPH- and
GSH-supplemented HLM led to the formation of reactive species that were trapped with GSH.
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Metabolism (including bioactivation) of both 1 and 2 was blocked when the HLM incubations
were conducted in the presence of ketoconazole (a selective CYP34 inhibitor), which implicated
a role for CYP3A4 in the metabolism of the two imidazo[2,1-b]thiazole derivatives. LC-MS/MS
characterization of the GSH conjugates suggested that the site of bioactivation in both
compounds was on the imidazo[2,1-b]thiazole bicycle. In the case of the imidazo[2,1-b]thiazole
derivative 1, the formation of the diol metabolite M1-1 was consistent with a bioactivation
pathway involving thiazole ring oxidation by CYP enzyme(s) at the C2-C3 position to yield the
electrophilic epoxide intermediate 12, which is hydrolyzed to the diol M1-1 or can potentially
react with GSH to yield the sulfydryl conjugate 13 (Figure 15, pathway A). An alternate
pathway that leads to M3-1 involves CYP-mediated oxidation on the thiazole sulfur to the S-
oxide metabolite M5-1, followed by the Michael addition of GSH to yield adduct M3-1 (see
Figure 15, pathway B) as outlined previously with the imidazo[2,1-b]thiazole analog L-766,112
(Trimble et al., 1997). NMR analysis of the crude M3-1 isolate indicated that the GSH adduct
was derived from addition of the thiol nucleophile to the electrophilic S-oxide M5-1 and not
from the ring opening of epoxide 12. The hypothesis is further strengthened by the observation
that addition of GSH to NADPH-supplemented HLM incubations of 1 significantly diminished
the levels of M5-1 formed in the incubations. Reason(s) for the lack of formation of the GSH
adduct 13 shown in Figure 15, pathway A are not apparent from this analysis. It is possible that
hydrolytic ring opening of 12 proceeds at a faster rate than the corresponding reaction with GSH.
Apart from the mono-GSH adduct M3-1, we also noted the formation of a novel bis-GSH
conjugate M2-1 with two molecules of GSH attached to the imidazo[2,1-b]thiazole framework.
A plausible mechanism outlining the formation of M2-1 is presented in Figure 15 (pathway C),
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and involves the initial addition of a GSH molecule to the putative S-oxide metabolite of 1 (i.e.,
M5-1) to generate M3-1 followed by addition of a second molecule of GSH across the electron
deficient C5 position on the imidazole ring system in M3-1, which results in imidazo[2,1-
b]thiazole-S-oxide ring scission. The outlined mechanism is certainly not beyond the realm of
possibilities considering the well known chemical (and glutathione transferase-mediated)
displacement reaction of GSH with electron deficient heteroaromatic rings (e.g., pyridine,
pyrimidine, etc) that contain alkylsulfoxide and/or alkylsulfone as leaving groups (Clapp, 1956;
Colucci and Buyske, 1965; Conroy et al., 1984; Graham et al., 1989; Yang et al., 2012). A
literature example that bears much commonality to the present situation is evident with the
proton pump inhibitor pantoprazole wherein, the benzimidazole-2-sulfoxide motif undergoes
nucleophilic attack by GSH at the electron-deficient C2 position on the benzimidazole ring
(Zhong et al., 2005). Certainly, one can view M5-1 (in Figure 15, pathway C) as an electron-
deficient heterocyle (imidazole ring) with an excellent leaving group (alkylsulfoxide) at the C5
position, which is poised for nucleophilic displacement by GSH.
In the case of 2, LC-MS/MS data on the GSH and N-acetylcysteinyl conjugates M5-2 and
M6-2, respectively, were also consistent with thiazole ring bioactivation. From a SAR
standpoint, the C-2 methyl group on the imidazo[2,1-b]thiazole ring in 2 did not prevent thiazole
ring bioactivation to reactive metabolite(s), which contrasts the structure-toxicity relationship
noted for sudoxicam and meloxicam. From a mechanistic perspective, we speculate that the rate-
limiting step in the formation of M5-2 and M6-2 proceeds via a CYP-mediated oxidation on the
thiazole ring in 2 to generate epoxide 14, which upon hydrolysis would yield the diol metabolite
M3-2 or undergo ring scission to a iminium species 15 (Figure 16). A two-electron reduction of
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the iminium bond in 15, possibly mediated by GSH, would lead to the 2-mercaptoimidazole 16,
which may be trapped by GSH or N-acetylcysteine via a developing ring-opened isothiocyanate
17 to afford sulfydryl conjugates M5-2 and M6-2, respectively. Overall the mechanistic
possibility is compatible with the results of the 18O labeling studies, wherein, lack of
incorporation of 18O in M5-2 indicates that the source of the carbonyl oxygen in M5-2 is from
molecular oxygen (i.e., from a CYP-mediated oxidation).
To examine whether iminium species 15 is indeed formed as an intermediate in the pathway
leading to M5-2, 2 was incubated in HLM in the presence of NADPH and excess potassium
cyanide, which is typically used to trap electrophilic iminium ions generated via a two-electron
oxidation of amines (Rose and Castagnoli, 1983; Argoti et al., 2005). The accurate mass of the
metabolite M7-2 detected in these incubations was consistent with the addition of cyanide across
15, which provided additional support for our overall mechanistic hypothesis. One can
rationalize the formation of M7-2 to occur via the initial addition of cyanide across the iminium
bond in 15 to yield the cyano adduct 18, which upon cyclization would lead to M7-2 (see Figure
16). With reference to the step involving the reduction of the iminium bond in 15 (to yield 2-
mercaptoimidazole 16), we invoked the involvement of GSH as a reducing agent since, two-
electron reduction of imines by GSH is a well precedented reaction. For example, N-acetyl-p-
benzoquinone imine, the oxidation product of acetaminophen, can react with GSH in a 1,4-
Michael fashion to yield a stable GSH adduct or undergo a two-electron reduction to the parent
drug in the presence of the thiol (Rosen et al., 1984; Potter and Hinson, 1986). In the present
situation, we anticipate that the thioaminal conjugation product obtained through addition of
GSH across the imine double bond in 15 will be highly unstable and spontaneously decompose
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back to 15. Clearly, a more rigorous experimental analysis of these individual mechanistic steps
will be needed to fully validate our hypothesis on the oxidative metabolism/bioactivation of
these unique bicyclo-heterocylic compounds. Towards this end, the imidazo[2,1-b]thiazole
intermediates used in the synthesis of the title compounds 1 and 2 (see supplementary section)
could provide much utility in testing certain mechanistic hypothesis (e.g., the use of deuterium
exchange to distinguish S-oxidation from mono-hydroxylation).
Overall, given this information, it appeared reasonable to attempt to eliminate bioactivation
liability in 1 and 2 via small structural modifications of the thiazole ring that would preserve the
physicochemical properties (e.g., molecular weight, lipophilicity) largely responsible for
pharmacologic potency and HLM stability. Consequently, the 2-methylimidazo[2,1-
b][1,3,4]thiadiazole analog 3 was synthesized to prevent thiazole ring epoxidation and/or
addition of GSH to an S-oxide metabolite. An identical medicinal chemistry strategy had been
successfully utilized to abrogate thiazole ring bioactivation with nonpeptidyl thrombopoietin
receptor agonists containing the 2-aminothiazole motif (Kalgutkar et al., 2007). As such, the
absence of the GSH conjugate formation in NADPH- and GSH-supplemented HLM incubations
with 3 indicates that the thiadiazole derivative is devoid of the reactive metabolite liability
associated with 1 and 2, and demonstrates the success of this medicinal chemistry tactic.
Compound 3 also retained the potent in vitro ghrelin inverse agonism and HLM stability
characteristics discerned with lead compounds 1 and 2. In summary, we have demonstrated the
human liver microsomal bioactivation of the imidazo[2,1-b]thiazole functionality present as part
of the core structure in novel ghrelin inverse agonists. Characterization of the GSH adducts
using LC-MS/MS and NMR techniques provided indirect information on the structures of the
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reactive species, thereby providing insight into the bioactivation mechanism. The information
gained from these studies was used to direct medicinal chemistry efforts in the design of a
compound with the attractive pharmacology and disposition characteristics observed with the
prototypes while avoiding potential safety concerns associated with the bioactivation of the
imidazo[2,1-b]thiazole functionality. Further lead optimization efforts on 3, which culminated in
the discovery of the clinical candidate will be reported in due course.
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Authorship Contributions
Participated in research design: Kalgutkar, Goosen, Lapham, Ryder, Walker, and Eng
Conducted in vitro experiments: Ryder, Walker, and Eng
Contributed new reagents or analytic tools: Orr and Cabral
Performed data analysis: Kalgutkar, Ryder, Walker, Orr, Eng, and Goosen
Wrote or contributed to the writing of the manuscript: Kalgutkar, Ryder, Walker, Orr,
and Eng
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Figure Legends
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.
FIG. 2. Extracted ion chromatograms of incubation mixtures of 1 (panel A) and 2 (panel B) in
NADPH- and GSH-supplemented HLM conducted at 37 °C for 60 minutes.
FIG. 3. CID spectra of 1 (panel A) and M1-1 (panel B).
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.
FIG. 5. CID spectrum of GSH conjugate M3-1. Structures of diagnostic fragment ions (and
corresponding theoretical exact masses) that allow an insight into the structure are depicted.
FIG. 6. Abbreviated 1H-1H TOCSY (panel A) and 1H-13C HSQC spectrum (panel B) of M3-1.
Red cross peaks in panel B indicate methine, whereas the blue cross peaks indicate the
methylene resonances.
FIG. 7. Extracted ion chromatograms of monohydroxylated metabolites (MH+ = 498.1473) of 1
in NADPH-supplemented HLM in the absence (panel A) and presence (panel B) of GSH.
FIG. 8. CID spectrum of M5-1 derived from an NADPH-supplemented HLM incubation of 1.
FIG. 9. CID spectra of 2 (panel A) and M3-2 (panel B).
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FIG. 10. CID spectra of the GSH conjugate M5-2 (panel A) and N-acetylcysteine conjugate M6-
2 (panel B).
FIG. 11. Full 1H spectrum of M5-2 and 2.
FIG. 12. Abbreviated 1H-13C HMBC spectrum of M5-2 (panels A and B represent specific 1H
correlations highlighted in the text).
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.
FIG. 14. CID spectrum of the cyanide conjugate M7-2 derived from NADPH- and potassium
cyanide-supplemented HLM incubations of 2.
FIG. 15. Proposed bioactivation pathways for imidazo[2,1-b]thiazole derivative 1.
FIG. 16. Proposed bioactivation pathway for 2-methylimidazo[2,1-b]thiazole derivative 2.
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Figure 1
R N
O
N
ClNN
N
1
S
N N
S
N N
H3C2
NS
N N
3H3C
Human GHS R1a IC50 = 8.0 nM Human GHS R1a IC50 = 4.7 nM Human GHS R1a IC50 = 2.6 nMHLM CLint = 24 ml/min/kgHLM CLint = 48 ml/min/kg HLM CLint = 32 ml/min/kg
R = R = R =
N
NS
SO2CH3
L-766,112
CYP N
NS
SO2CH3
O
GSH N
NS
SO2CH3
O
GSN
S
N
N
L-768,277 (devoid of bioactivation)
SO2CH3
S-Oxide
M.W. = 482, clogD = 1.48 M.W. = 497, clogD = 0.81M.W. = 496, clogD = 1.26
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Figure 2
Time (min)
6 8 10 12 14 16 18 20 220
20
40
60
80
100
Re
lati
ve A
bu
nd
anc
e
18.82
16.4717.70
1
M1-1M3-1 M2-1
5 10 15 20 25 30 35 400
20
40
60
80
10019.47
17.1921.46
15.89
2
M2-2
M5-2
M3-2M4-2 M1-2
(A)
(B)
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Figure 3
150 200 250 300 350 400 4500
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
318.1476
262.1004192.0318 464.1407301.1210165.0112
150 200 250 300 350 400 450 500m/z
0
10
20
30
40
50
60
70
80
90
100318.1476
192.0318 309.1578
N
NS
NO
N Cl
N NN
MH+ = 516.1579
318.1480
HO
HO
M1-1
(A)
(B)
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Figure 4
300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
967.2753
838.23357
1078.3061650.1351
823.22137949.2654694.1805
318.1468 458.1514 805.2108506.0823414.1077855.09903
N
N
NO
NH Cl
N NN
HS
HS
S
HN
O
NHO
HO
O
NH2
O
OH
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Figure 5
250 300 350 400 450 500 550 600 650 700 750 800
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
676.1872
787.2189318.1476
472.1308532.1342
658.1761359.0471730.1973
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Figure 6
SG15 14
N13
2021
16
17
N18
19
11
O12
1022
2328
27
26
25
24
7
8
N6
5
N4
3
2
S1
Cl29
N30
N31
32
33
N34
O9
3 2b
3
2b
A B
2a
2a
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Figure 7
M5-1
M4-1
M4-1
(A)
5 10 15 20 25 30 35 40
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Re
lati
veA
bu
nd
ance
20.26
18.8720.74
18.83
20.2316.96
(B)
M5-1
Normalized levels to base peak: 1.63 x 107
Normalized levels to base peak: 5.33 x 105
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Figure 8
150 200 250 300 350 400 450 500m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
318.1470
192.0317
278.0949
437.1295210.0326 395.1369138.0003181.0060 452.1401221.0582
372.0309
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Figure 9
150 200 250 300 350 400 450 5000
10
20
30
40
50
60
70
80
90
100 318.1473
276.1159192.0317 478.1560301.1207 342.0561
Rel
ativ
e A
bund
ance
200 250 300 350 400 450 500m/z
0
10
20
30
40
50
60
70
80
90
100 318.1474
350.1371213.0322192.0317 416.1479 510.1446
(A)
(B)
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Figure 10 R
elat
ive
Abu
ndan
ce
200 250 300 350 400 450 500 550 600 650m/z
01020
3040
5060708090
100 514.1792
318.1483 606.0683189.3719 561.5912407.892
250 300 350 400 450 500 550 600 650 700 750 8000
10
20
30
40
50
60
70
80
90
100 658.2308
341.0908 514.1776318.1474 769.2621
470.1331640.2197452.1230395.1015308.0692 695.7148542.1427
(B)
(A)
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Figure 11
1H spectrum of M5-2
1H spectrum of 2
2
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Figure 12
ppm
6.87.0 ppm
200
150
100
50
ppm
4.85.0 ppm
200
150
100
50
ppm
2.02.2 ppm
200
150
100
50
8 4 6
87 82
48
42
45
64
65
A B C
11
O12
N13
20
21
16
17N18
22
23
28
27
26
25
24
Cl29
N30N
31
32
33
N34
1915
14
2N3
N
8
4
7
5
CH36
O
SGR
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Figure 13
150 200 250 300 350 400 450 500m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
318.1480
320.1450215.0369 352.1426192.0322 418.1516 512.1449276.1160
294.5069
N NN
Cl
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Figure 14
150 200 250 300 350 400 450 500 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
318.1473
222.0326
194.0376
357.6116262.7421287.8470
236.7467
N N
N
O
N
ClNN
N
M7-2MH+ = 539.1739
318.1480
SH2N
O
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Figure 15
N
NS
RO
1 (MH+ = 482.1524)
CYPN
NS
ORO
GSH
H2O
N
NS
ROHO
HO
12
M1-1 (MH+ = 516.1579)
N
NS
ROS
HO
13 (MH+ = 805.2312)
HN
ONH
OH
O
O
H2N
OHO
CYP
N
NS
OR
M5-1O
GSH
N
NS
RO
O
S
HN NH
O
O
OHOH2N
OOH
M3-1 (MH+ = 805.2312)(B)
(A)
GSHH
GSHN
N
ROS
HN NH
O
O
OHOH2N
OOH
HS
S
HN
O
NHO
HO
O
NH2
O
OH
M2-1 (MH+ = 1096.3200)
H2O
(C)
N N Cl
N NN
R =
GSH
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Figure 16
N
NS
RO
2 (MH+ = 496.1680)
CYP N
NS
ORO
14
H3C H3CO
NHN
R
S
OCH3
N N Cl
N NN
R =
H2O
N
NS
ROHO
H3CHO
M3-2
KCN
CN
O
NHN
R
S
OCH3
CN
18
H
H
O
NN
R
SHN
H3CO
H
H
O
NN
R
SH2N
H3CO
M7-2 (MH+ = 539.1739)
2GSH GSSG
O
NHN
R
S
OCH3
15 16
O
NN
R
OCH3
S
HNR2
O
M5-2: R1 = CO(CH2)2CHNH2COOHR2 = NHCH2COOH
RSH
RSH
M6-2: R1 = COCH3; R2 = OH
R1
(MH+ = 530.1735)
(MH+ = 787.2747)(MH+ = 643.2212)
O
HN
R
OCH3
17
NCS
O
HN
R
OCH3
N
RS
SH H
- H2S
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