Demonstration of the innate electrophilicity of 4-(3-(Benzyloxy)phenyl)-2- (ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a small-molecule positive allosteric modulator of the glucagon-like peptide-1 receptor

Article abstract of DOI:10.1124/dmd.113.052183

4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagonlike peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH-and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R. Copyright

Full text of DOI:10.1124/dmd.113.052183

1521-009X/41/8/1470–1479$25.00
http://dx.doi.org/10.1124/dmd.113.052183
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 41:1470–1479, August 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Demonstration of the Innate Electrophilicity of 4-(3-
(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine
(BETP), a Small-Molecule Positive Allosteric Modulator of the
Glucagon-Like Peptide-1 Receptor
Heather Eng, Raman Sharma, Thomas S. McDonald, David J. Edmonds, Jean-Philippe Fortin,
Xianping Li, Benjamin D. Stevens, David A. Griffith, Chris Limberakis, Whitney M. Nolte,
David A. Price, Margaret Jackson, and Amit S. Kalgutkar
Pharmacokinetics, Dynamics and Metabolism–New Chemical Entities, Groton, Connecticut (H.E., R.S., T.S.M.) and
Cambridge, Massachusetts (A.S.K.); Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry, Cambridge (D.J.E.,
B.D.S., D.A.G., W.M.N., D.A.P.) and Groton (C.L.); and Cardiovascular, Metabolic and Endocrine Diseases Research Unit,
Cambridge (J.-P.F., X.L., M.J.) Pfizer, Inc.
Received March 28, 2013; accepted May 7, 2013
ABSTRACT
4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine
a facile degradation of BETP. Failure to detect metabolites in plasma
(BETP) represents a novel small-molecule activator of the glucagon- and liver microsomal incubations in the absence of NADP was
like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent suggestive of a covalent interaction between BETP and a protein
insulin secretion in rats following i.v. (but not oral) administration. To amino acid residue(s) in these matrices. Incubations of BETP with
explore the quantitative pharmacology associated with GLP-1R glutathione (GSH) in buffer revealed a rapid nucleophilic displace-
agonism in preclinical species, the in vivo pharmacokinetics of BETP ment of the ethylsulfoxide functionality by GSH to yield adduct M1,
were examined in rats after i.v. and oral dosing. Failure to detect which indicated that BETP was intrinsically electrophilic. The
BETP in circulation after oral administration of a 10-mg/kg dose in structure of M1 was unambiguously identified by comparison of its
rats was consistent with the lack of an insulinotropic effect of orally chromatographic and mass spectral properties with an authentic
administered BETP in this species. Likewise, systemic concentra- standard. The GSH conjugate of BETP was also characterized in
tions of BETP in the rat upon i.v. administration (1 mg/kg) were NADPH- and GSH-supplemented liver microsomes and in plasma
minimal (and sporadic). In vitro incubations in bovine serum albumin, samples from the pharmacokinetic studies. Unlike BETP, M1 was
plasma, and liver microsomes from rodents and humans indicated inactive as an allosteric modulator of the GLP-1R.
Introduction
been demonstrated with the s.c. administered agents exenatide in
a twice-daily formulation (marketed as Byetta; Bristol-Myers Squibb
Company, New York, NY and AstraZeneca Pharmaceuticals LP,
Wilmington, DE) or once-weekly formulation (marketed as Bydureon;
Bristol-Myers Squibb Company and AstraZeneca Pharmaceuticals LP)
and liraglutide in a once-daily formulation (marketed as Victoza; Novo
Nordisk A/S, Denmark) (Bode, 2011; Murphy, 2012; Jespersen et al.,
2013). The efficacies of these agents have been demonstrated in
multiple studies, which consistently reported clinically relevant impro-
vements in glycemic control (i.e., reductions in hemoglobinA1c, fasting
The incretin hormone glucagon-like peptide-1 (GLP-1) is synthesized
from proglucagon-derived peptides in intestinal L-cells in response to
oral nutrient ingestion (Holst, 2007). The majority of circulating GLP-1
levels comprise the 30-amino-acid peptide GLP-1(7-36)NH2, which acts
through a seven-transmembrane-spanning, heterotrimeric, class B G-
protein-coupled receptor on pancreatic b cells to exert glucoregulatory
and insulinotropic actions (Thorens, 1992). Binding of GLP-1 to the
GLP-1 receptor (GLP-1R) activates the Gas subunit, leading to stimu-
lation of membrane-associated adenylyl cyclases and increased pro-
duction of cAMP, which enhances glucose-dependent insulin secretion plasma glucose, and postprandial plasma glucose excursions) (Madsbad
(Thorens et al., 1993; Runge et al., 2008). Therapeutic benefits in the
treatment of type 2 diabetes mellitus via agonism of the GLP-1R have
et al., 2011; Scott et al., 2013). Additional injectable GLP-1R agonists
(e.g., lixisenatide, dulaglutide, and albiglutide) are currently in late
stages of clinical development (Madsbad et al., 2011; Meier, 2012).
The success of peptide-based GLP-1R agonists for the treatment of
type 2 diabetes mellitus has also led to discovery efforts aimed at the
dx.doi.org/10.1124/dmd.113.052183.
ABBREVIATIONS: BSA, bovine serum albumin; CDCl3, deuterated chloroform; CD3OD, deuterated methanol; CHO, Chinese hamster ovary; CID,
collision-induced dissociation; compound B or BETP, 4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine; d, chemical shifts
expressed in ppm; DMSO-d6, deuterated dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; GLP-1, glucagon-like peptide-1; GLP-1R,
glucagon-like peptide-1 receptor; GSH, glutathione; HPLC, high-performance liquid chromatography; IVGTT, intravenous glucose tolerance test;
J, NMR coupling constant in Hz; LC-MS/MS, liquid chromatography–tandem mass spectrometry; mCPBA, meta-chloroperoxybenzoic acid; MS,
mass spectrometer; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; t1/2, half-life; THF, tetrahydrofuran.
1470

Intrinsic Electrophilicity of BETP
1471
identification of orally active small-molecule agonists of the GLP-1R, glutathione (GSH) in buffer revealed a rapid nucleophilic displace-
which has historically proven to be a difficult task. To a large degree, ment of the ethylsulfoxide functionality by GSH. The GSH conjugate
this difficulty has been attributed to the biochemical mechanisms of of BETP was also characterized in liver microsomes supplemented
class B G-protein-coupled receptors, which require large receptor- with NADPH and GSH and in plasma samples from the pharmaco-
ligand binding sites to induce signaling. Despite this dilemma, kinetic studies. The GSH conjugate of BETP was inactive as a positive
a diverse array of low-molecular-weight nonpeptidic ligands have allosteric modulator of the GLP-1R.
been recently reported as antagonists, agonists, and positive allosteric
modulators of GLP-1R with intrinsic efficacy (Knudsen et al., 2007;
Teng et al., 2007; Sloop et al., 2010; Willard et al., 2012a). 4-(3-
Materials and Methods
(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine
(compound B or BETP) (Fig. 1) is one such analog that has been
identified as a positive allosteric modulator of the naturally occurring
inactive GLP-1 metabolite, GLP-1(9-36)NH2, while showing little
modulation of the active, circulating form, i.e., GLP-1(7-36)NH2
(Sloop et al., 2010; Wootten et al., 2012). Likewise, the insulinotropic
effect of oxyntomodulin [glucagon(1-37)], a low-affinity full agonist
of the GLP-1R, was also markedly enhanced in the rat i.v. glucose
tolerance test (IVGTT) upon i.v. coadministration with BETP, which
is consistent with the in vitro biochemical observation of the increased
Materials. Unless specified otherwise, starting materials used in the
synthesis of BETP and its GSH conjugate are generally available from
commercial sources such as Aldrich Chemicals Co. (Milwaukee, WI) and
Acros Organics (Fair Lawn, NJ). 1H NMR spectra were recorded in deuterated
chloroform (CDCl3), deuterated methanol (CD3OD), or deuterated dimethyl-
sulfoxide (DMSO-d6) on a Varian Unity 400-MHz spectrometer (DG400-5
probe; available from Varian Inc., Palo Alto, CA) at room temperature. DMSO-d6
“100%” was obtained from Cambridge Isotope Laboratories Inc. (Andover,
MA). Chemical shifts (d) are expressed in ppm relative to residual solvent as an
internal reference. The peak shapes are denoted as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. Coupling constants (J) are expressed
GLP-1R affinity of oxyntomodulin in the presence of BETP (Willard as Hz. GSH, BSA, and NADPH were purchased from Sigma-Aldrich (St Louis,
MO). Frozen plasma in K3EDTA from Wistar Han rat (pooled males), CD-1
mouse (pooled males), and human (pooled males and females) was purchased
from Bioreclamation, Inc. (Westbury, NY). Pooled liver microsomes from
humans (pool of 50 livers from male/female), male Wistar Hannover rats, and
male CD-1 mice were purchased from BD Biosciences (Woburn, MA). Jugular
vein–cannulated/carotid artery–cannulated male Wistar Hannover rats were
purchased from Charles River (Raleigh, NC). Solvents used for analysis were
of analytical or high-performance liquid chromatography (HPLC) grade (Fisher
Scientific, Pittsburgh, PA).
et al., 2012b). In vivo, BETP demonstrated glucose-dependent insulin
secretion in the IVGTT in rats after i.v. administration. Interestingly,
oral administration of BETP failed to show insulinotropic effects
similar to those achieved via i.v. administration (Sloop et al., 2010).
One possible reason for this discrepancy is that BETP suffers from
poor oral absorption due to low aqueous solubility, low absorptive
permeability, and/or extensive first-pass metabolism in the gut and liver.
As part of our general interest in examining quantitative phar-
macology for the glucose-dependent insulin secretagogue properties of
Synthesis of BETP. A solution of 4-chloro-2-(methylthio)-6-(trifluoromethyl)
GLP-1R agonists in preclinical species, the in vivo pharmacokinetics pyrimidine (1, 348 mg, 1.52 mmol), 3-(benzyloxy)phenylboronic acid (2, 200
mg, 0.88 mmol), and cesium carbonate (252 mg, 3.88 mmol) in ethylene glycol
dimethyl ether (16 ml) and water (4 ml) was degassed with N2 gas. Tetrakis
(triphenylphosphine)palladium(0) (89 mg, 0.08 mmol) was added and the
reaction mixture was heated at 85°C for 18 hours under an N2 atmosphere. The
mixture was then diluted with ethyl acetate (50 ml), dried (sodium sulfate),
filtered, and concentrated in vacuo. The crude product was purified by silica gel
chromatography (CombiFlash, Teledyne Isco, Lincoln, NE; 0%–3% ethyl
acetate in petroleum ether) to give 4-(3-(benzyloxy)phenyl)-2-(methylthio)-6-
(trifluoromethyl)pyrimidine (3, 320 mg, 0.85 mmol, 96%) as a yellow oil. 1H
NMR (400 MHz, CDCl3) d 7.81–7.77 (m, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.62
of BETP were examined in rats after i.v. and oral dosing. To our
surprise, little to no systemic exposure of BETP could be measured in
plasma samples from both the i.v. and oral arms of the pharmaco-
kinetic study. In vitro incubations in bovine serum albumin (BSA),
plasma, and liver microsomes from rodents and humans indicated
a rapid turnover of BETP. Failure to detect metabolites in BSA,
plasma, and liver microsomes (in the absence of NADPH) was
suggestive of a covalent interaction between BETP and a protein
amino acid residue(s) in the various matrices. Consistent with this
hypothesis, incubations of BETP with the endogenous antioxidant (s, 1H), 7.54–7.33 (m, 6H), 7.18 (dd, J = 2.0, 8.5 Hz, 1H), 5.18 (s, 2H), 2.67 (s, 3H).
Fig. 1. Preparation of BETP and its GSH conjugate M1.

1472
Eng et al.
To a solution of 3 (340 mg, 0.90 mmol) in dichloromethane (10 ml) was standard. Samples were vortexed and then centrifuged at 2300g for 10 minutes.
added meta-chloroperoxybenzoic acid (mCPBA) (468 mg, 2.71 mmol) at room Supernatants were analyzed for disappearance of BETP (and concomitant
temperature. The reaction mixture was stirred at room temperature for 4 hours
and then concentrated in vacuo. The crude product purified by silica gel
chromatography (CombiFlash, 0%–15% ethyl acetate in petroleum ether) to
give 4-(3-(benzyloxy)phenyl)-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine
(4, 240 mg, 0.59 mmol, 65%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
d = 8.16 (s, 1H), 7.88 (s, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.54–7.34 (m, 6H), 7.25
(br. second., 1H), 5.20 (s, 2H), 3.47 (s, 3H).
Compound 4 (1.50 g, 3.67 mmol) was dissolved in tetrahydrofuran (THF)
(6 ml) and the mixture divided between six microwave reaction tubes. To each
tube were added sodium ethanethiolate (154 mg, 1.84 mmol) and ethanethiol
(1 ml). The vials were sealed and heated at 100°C for 20 minutes under
microwave irradiation. The six portions were recombined and concentrated, and
the crude product was purified by silica gel chromatography (CombiFlash,
0%–2% ethyl acetate in petroleum ether) to give 4-(3-(benzyloxy)phenyl)-2-
(ethylthio)-6-(trifluoromethyl)pyrimidine (5, 759 mg, 1.95 mmol, 53%) as
a yellow oil. m/z = 391.0 [M+H]+; 1H NMR (400 MHz, CDCl3) d 7.75–7.81
(m, 1H), 7.69 (d, J = 7.53 Hz, 1H), 7.61 (s, 1H), 7.32–7.51 (m, 6H), 7.18 (dd,
J = 2.26, 7.78 Hz, 1H), 5.17 (s, 2H), 3.26 (q, J = 7.36 Hz, 2H), 1.47 (t, J = 7.28
Hz, 3H).
To a solution of 5 (700 mg, 1.79 mmol) in dichloromethane (10 ml) was
added mCPBA (310 mg, 1.79 mmol) portionwise at 0°C. The reaction mixture
was stirred at 0°C for 30 minutes and quenched by addition of sodium sulfite.
The layers were separated and the aqueous portion was extracted with
dichloromethane. The combined organic layers were dried over sodium sulfate
and concentrated in vacuo. The crude product was purified by silica gel
chromatography (CombiFlash, 2%–24% ethyl acetate in petroleum ether) to
give BETP (582 mg, 1.43 mmol, 80%) as a white solid. m/z = 407 [M+H]+;
1H NMR (400 MHz, CD3OD) d 8.49 (s, 1H), 8.08 (s, 1H), 7.97 (d, J = 7.53 Hz,
1H), 7.48–7.58 (m, 3H), 7.37–7.44 (m, 2H), 7.28–7.37 (m, 2H), 5.25 (s, 2H),
3.35–3.47 (m, 1H), 3.19–3.30 (m, 1H), 1.33 (t, J = 7.53 Hz, 3H).
appearance of M1) by LC-MS/MS. The metabolic fate of BETP (10 mM) in
0.1 M phosphate buffer (pH 7.4) in the presence of GSH (5 mM) was also
examined qualitatively by LC-MS/MS after incubation at 37°C for 30 minutes.
Incubations in Liver Microsomes. Stock solutions of BETP were prepared
in a solution of 1% DMSO and 99% acetonitrile. The final concentrations of
DMSO and acetonitrile in the incubation media were 0.01% and 0.99% (v/v),
respectively. Microsomal stability assessments were determined in triplicate
after incubation of BETP (1 mM) with rat, mouse, and human liver microsomes
(cytochrome P450 concentration, 0.25 mM) in 0.1 M potassium phosphate
buffer (pH 7.4), containing 3.3 mM magnesium chloride, at 37°C. Incubations
were conducted in the presence or absence of NADPH (1.3 mM) and GSH
(5 mM). The total incubation volume was 0.6 ml. Incubations were prewarmed
at 37°C for 5 minutes before the addition of BETP. Aliquots (50 ml) of the
reaction mixture at 0, 2, 5, 10, 20, 40, and 60 minutes (time period associated
with reaction linearity) were added to acetonitrile (200 ml) containing
terfenadine (mol. wt. = 472; 0.02 mg/ml) as an internal standard. The samples
were centrifuged at 2300g for 10 minutes before LC-MS/MS analysis for the
disappearance of BETP and appearance of M1. For the purposes of qualitative
metabolite identification studies, the concentration of BETP in the liver
microsomal incubations was raised to 10 mM and that of P450 in rat, mouse,
and human liver microsomes was raised to 0.5 mM. After quenching the
incubation mixtures with acetonitrile (1 ml), the solutions were centrifuged
(2300g, 15 minutes) and the supernatants were dried under a steady nitrogen
stream. The residue was reconstituted with the mobile phase and analyzed for
metabolite formation by LC-MS/MS.
Animal Pharmacokinetic Studies. Rat studies were conducted at Pfizer; all
animal care and in vivo procedures conducted were in accordance with guidelines
of the Pfizer Animal Care and Use Committee. Jugular vein–cannulated male
Synthesis of the GSH Conjugate of BETP (M1). To a solution of BETP
(100 mg, 0.25 mmol) in a mixture of THF (2.5 ml) and water (1.0 ml) at room
temperature were added GSH (154 mg, 0.50 mmol) and diisopropylethylamine
(175 ml, 1.0 mmol), and the mixture was stirred for 20 hours at room
temperature. The solution was concentrated in vacuo and the crude residue was
purified by preparative HPLC to afford M1 (165 mg, 0.25 mmol) as a white
solid. The preparative HPLC conditions were as follows: HPLC Column:
DIKMA (Lake Forest, CA) Diamonsil(2) C18 5 mm, 200 Â 20 mm. Gradient
elution: 30% acetonitrile in water (0.1% trifluoroacetic acid) to 50% acetonitrile
in water (0.1% trifluoroacetic acid). The purified product was assessed as
.95% purity by analytical HPLC and 1H NMR. m/z = 636.0 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 8.65 (t, J = 5.52 Hz, 1H), 8.56 (d, J = 8.53 Hz, 1H),
8.30 (s, 1H), 8.01–8.04 (m, 1H), 8.00 (d, J = 8.03 Hz, 1H), 7.46–7.54 (m, 3H),
7.37–7.44 (m, 2H), 7.31–7.37 (m, 1H), 7.27 (dd, J = 2.01, 8.03 Hz, 1H), 5.24
(s, 2H), 4.73 (dt, J = 4.77, 8.91 Hz, 1H), 3.96 (dd, J = 4.52, 13.55 Hz, 1H),
3.67–3.80 (m, 2H), 3.55 (t, J = 6.78 Hz, 1H), 3.30 (dd, J = 9.54, 13.55 Hz, 1H),
2.34 (t, J = 7.28 Hz, 2H), 1.82–2.04 (m, 2H).
Incubations in Plasma and BSA. Stock solutions of BETP were prepared
in DMSO. BETP (final concentration = 1 mM) was incubated in 0.1 M
potassium phosphate buffer (pH 7.4) supplemented with BSA (10 mg/ml) or in
plasma from rat (n = 3), mouse (n = 3), and human (n = 3) at 37°C (pH 7.4).
Total incubation volume was 0.6 ml and the final DMSO concentration in the
incubations was 1% (v/v). Plasmas were thawed and adjusted to pH 7.4.
Incubation matrices (594 ml) were prewarmed to 37°C and maintained at that
temperature for 5 minutes before adding substrate (6 ml). Periodically (0–60
minutes), aliquots (50 ml) of the incubation mixtures were added to acetoni-
trile (200 ml) containing terfenadine (mol. wt. = 472; 0.02 mg/ml) as an
internal standard. Samples were vortexed and then centrifuged at 2300g for 10
minutes. Supernatants were analyzed for disappearance of BETP by liquid
chromatography–tandem mass spectrometry (LC-MS/MS).
Incubations in Buffer. BETP (final concentration = 1 mM) was incubated in
0.1 M potassium phosphate buffer (pH 7.4) in the absence or presence of GSH
(5 mM) at 37°C (n = 3). Total incubation volume was 0.6 ml and the final
DMSO concentration in the incubations was 5% (v/v). Periodically (0–60
minutes), aliquots (50 ml) of the incubation mixtures were added to acetonitrile
(200 ml) containing terfenadine (mol. wt. = 472; 0.02 mg/ml) as an internal
Fig. 2. (A) Depletion of BETP and (B) appearance of GSH conjugate M1 in
incubations of BETP in buffer with and without GSH (n = 3). Mean values and
standard deviations are shown.

Intrinsic Electrophilicity of BETP
1473
Fig. 3. Extracted ion chromatogram of an incubation mixture of BETP
(10 mM) and GSH (5 mM) in 0.1 M potassium phosphate buffer at 37°C
at t = 0 minutes (top) and t = 30 minutes (bottom).
Wistar Hannover rats (0.25–0.27 kg) were used for pharmacokinetic analysis. For [10:50:40 (v/v/v)] and 0.5% (w/v) methylcellulose with 2% (v/v) DMSO for the
oral pharmacokinetic studies, animals were fasted overnight before dosing, i.v. and oral studies, respectively. After dosing, serial plasma samples were
whereas access to water was provided ad libitum. BETP was administered i.v. via collected at appropriate times via the jugular vein cannula and kept frozen at
the jugular vein of rats (n = 2). For oral studies, BETP was administered by oral 220°C until LC-MS/MS analysis for presence of BETP and M1.
gavage to rats (n = 2). BETP was administered at 1.0 mg/kg i.v. and 10 mg/kg
LC-MS/MS Methodology for Quantification of BETP and M1. Con-
orally. Orally dosed rats were fed after collection of the 4-hour blood samples. centrations of BETP and its GSH adduct (M1) in various matrices (buffer,
BETP was formulated as a solution in DMSO–polyethylene glycol 400–water plasma, and/or liver microsomes) were determined by LC-MS/MS. Briefly,
Fig. 4. CID spectrum of BETP (A) and GSH conjugate M1 (B).

1474
Eng et al.
TABLE 1
samples/sample extracts were injected by a fixed-loop CTC PAL Auto-sampler
onto a Shimadzu LC-20AD HPLC system coupled to an AB Sciex API4000
triple quadrupole mass spectrometer (MS) fitted with a TurboIonspray source
operating in positive ion mode (AB Sciex, Framingham, MA). Chromatographic
separation was performed by gradient elution on a Waters HSS T3 XP (30 Â
2.1 mm, 2.5 mm) reverse-phase column (Waters Corporation, Milford, MA),
using a binary solvent mixture consisting of 0.1% (v/v) formic acid in water
(solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B) at a flow rate
of 600 ml/min. Quantitation was performed by multiple reaction monitoring
mode with transitions of 407.2 → 379.2 (BETP), 636.2 → 507.1 (M1), and
472.2 → 436.2 (internal standard: terfenadine). Standards of BETP and M1 in
each matrix were fit by least-squares regression, and unknown concentrations
were determined from the resultant best-fit equation.
LC-MS/MS Methodology for Metabolite Identification Studies. Separa-
tion of BETP and metabolites was achieved using an ACQUITY UPLC system
(Waters Corporation) with a 2.1 Â 150 mm, 1.8-mm ACQUITY UPLC HSS
C18 column, maintained at a column temperature of 40°C. The mobile phase
consisted of water with 0.1% formic acid (A) and acetonitrile (B). The flow rate
was 0.3 ml/min and the gradient was as follows: 5% solvent B (0 minutes),
80% solvent B (5 minutes), and 5% solvent B (5.2–8.0 minutes). The injection
volume was 15 ml. Detection of BETP and metabolites was performed on
a SYNAPT G2 (Waters MS Technologies, Manchester, UK) orthogonal
acceleration quadrupole time-of-flight MS. The MS was operated in positive
ion mode using electrospray ionization. The desolvation gas was set to 700 l/h
at a temperature of 350°C. The cone gas was set to 30 l/h and the source
temperature to 150°C. The capillary voltage was set to 4 kV, the cone voltage
to 28 V, and the extraction cone to 7 V. The SYNAPT G2 was operated in V
optics mode (sensitivity mode) with resolution greater than 10,000 at full width
at half maximum. The data acquisition rate was 0.10 s/scan; data were collected
from 1 to 8 minutes in MSe acquisition mode with a collision energy ramp of
25–45 V for high-energy scans. The MS was calibrated to a mass accuracy
under 5 mDa. Data were collected in continuum mode from m/z 100 to 900.
In vitro stability of BETP in liver microsomes (n = 3) in the presence and absence of NADPH
(1.3 mM) and GSH (5 mM)
Species
NADPH
GSH
t1/2 6 S.D.
min
Human
2
2
+
2
+
2
+
2
+
2
+
2
+
2
47 6 5.0
,0.5a
8.4 6 0.3
+
,0.5a
Mouse
Rat
2
2
+
5.6 6 1.3
,0.5a
0.81 6 0.05
+
,0.5a
2
2
+
8.8 6 0.3
,0.5a
2.2 6 0.2
+
+
,0.5a
at1/2 could not be determined; samples were below the limit of analytical quantitation for
BETP at all time points.
Results
Preparation of BETP and Its GSH Conjugate M1. BETP was
prepared as shown in Fig. 1A. Suzuki coupling (Suzuki, 2005) of
chloropyrimidine (1) and boronic acid (2) derivatives yielded sulfide
3, which was oxidized with excess mCPBA to yield the corresponding
sulfone 4. Displacement with sodium ethanethiolate introduced the
ethyl sulfide (compound 5), which was oxidized to afford BETP using
one equivalent of mCPBA. An authentic sample of M1 was prepared
by reacting BETP with GSH in aqueous THF in the presence of
MassLynx version 4.1, SCN 803 software (Waters Corporation) was used for diisopropylethyl amine (Fig. 1B).
data processing.
Cell Culture and cAMP Assay. Chinese hamster ovary (CHO) cells stably
expressing the human GLP-1R (CHO-GLP1R cells) were maintained in
a Dulbecco’s modified Eagle’s medium–F-12 mixture (Invitrogen #11330032;
Invitrogen, Carlsbad, CA) supplemented with 500 mg/ml G418 (Invitrogen
#10131035) and 10% heat-inactivated fetal bovine serum. Cells were grown at
37°C in a 95% humidified atmosphere consisting of 5% CO2. A cell-based
time-resolved fluorescence resonance energy transfer (FRET) assay (Cisbio
Bioassays #62 AM4PEJ; Cisbio Bioassays, Codolet, France) was used to
measure receptor-mediated cAMP production. This method is based on
generation of a FRET signal upon the interaction between 1) an anti-cAMP
antibody coupled to a FRET donor (cryptate) and 2) a cAMP derivative
coupled to a FRET acceptor (d2). Endogenous cAMP produced by cells
competes with labeled cAMP for binding to the cAMP antibody, thus reducing
the FRET signal. Briefly, CHO-GLP1R cells were dissociated from tissue
culture plates using enzyme-free cell dissociation buffer and resuspended in an
appropriate volume of assay buffer [1Â Hanks’ balanced salt solution (Gibco
#14025-092; Invitrogen), 1 M HEPES (Gibco #15630-080)] supplemented with
500 mM 3-isobutyl-1-methylxanthine. A total of 2500 cells/well was dispensed
into white 384-well plates (BD Falcon # 353988; BD Biosciences, San Jose,
CA). The metabolite GLP-1(9-36)NH2 was serially diluted in assay buffer
containing BETP (10 mM), M1 (10 mM), or the corresponding vehicle DMSO.
Ligands (5 ml of 2Â concentration) were added to the appropriate wells, and
plates were incubated 30 minutes at room temperature. Labeled cAMP (5 ml) and
anti-cAMP antibody (5 ml) were then added to each well, and the plates were
further incubated at 37°C for 1 hour. Time-resolved FRET signal was measured
using an Envision 2103 Multilabel Plate Reader (PerkinElmer, Waltham, MA)
with a laser excitation at 337 nm and dual emissions at 665 nm and 590 nm. A
cAMP standard curve diluted in assay buffer was included and used to calculate
the amount of cAMP produced, as specified by the manufacturer.
Data Analysis. Substrate disappearance half-lives (t1/2s) were calculated
using E-WorkBook 2011 (IDBS, Guildford, Surrey, UK). Sigmoidal curve
fitting of ligand concentration-response curves was executed using GraphPad
Prism software version 5.02 (GraphPad, San Diego, CA). The same software
package was used for calculating the EC50 values, an index of ligand potency.
Fig. 5. Disappearance of BETP in rat, mouse, and human liver microsomes in the
absence (A) and presence (B) of NADPH (1.3 mM) (n = 3). Mean values are plotted
and standard deviations are indicated with error bars.

Intrinsic Electrophilicity of BETP
1475
Fig. 6. Extracted ion chromatogram of an incubation mixture of
BETP (10 mM) in rat liver microsomes in the absence (A) and
presence (B) of NADPH conducted at 37°C for 60 minutes.
Plasma and BSA Stability of BETP. To examine the interspecies phosphate buffer supplemented with 10 mg/ml BSA at 37°C also indicated
stability in plasma, BETP at a concentration of 1 mM was incubated in rat, a decline of the parent compound with a t1/2 of 54 6 5.0 minutes.
mouse, and human plasma at 37°C; periodically, aliquots of the incubation
Stability of BETP in Potassium Phosphate Buffer in the
mixture were examined for depletion of BETP. The t1/2 for depletion of Presence and Absence of GSH. BETP (1 mM) was stable in
BETP in rat, mouse, and human plasma was 35 6 3.0, 55 6 19, and 60 6 phosphate buffer (pH 7.4) at 37°C (t1/2 . 120 minutes). However,
13 minutes, respectively. Incubation of BETP (1 mM) in potassium inclusion of GSH (5 mM) in the incubation mixture resulted in a rapid
Fig. 7. CID spectra of metabolites M2 (A) and M3 (B) generated upon incubating BETP (10 mM) in rat liver microsomes in the presence of NADPH at 37°C for 60 minutes.

1476
Eng et al.
disappearance of BETP, with a t1/2 of ,0.5 minute (Fig. 2A). LC-MS/MS consistent with a monohydroxylation of M2. A proposed structure for
analysis of a reaction mixture comprising BETP (10 mM) in potassium M3 that is compatible with the fragmentation pattern is shown in Fig.
phosphate buffer and GSH (5 mM), incubated at 37°C for 30 minutes, 7A. Incubation mixtures of BETP (10 mM) in NADPH- and GSH-
revealed the formation of a single metabolite denoted as M1 (Fig. 3). supplemented rat, mouse, and human liver microsomes revealed the
Under reversed-phase HPLC conditions, M1 eluted before BETP exclusive (and quantitative) conversion to M1 following a 60-minute
(BETP: retention time (tR) = 6.00 minutes; M1: tR = 4.69 minutes). incubation at 37°C (data not shown).
The collision-induced dissociation (CID) spectra of BETP and M1 are
Pharmacokinetic Studies in Rats. The in vivo formation of M1
depicted in Fig. 4, A and B, respectively. M1 displayed a protonated was examined in rats after administration of single i.v. (1 mg/kg) and
molecular ion (MH+) at m/z 636.1734, an addition of 229.0698 Da to oral (10 mg/kg) doses of BETP. Figure 8 illustrates the mean observed
the molecular weight of BETP (MH+ = 407.1036). The CID spectrum plasma concentration– versus–time profiles of BETP and M1 after i.v.
of M1 yielded a diagnostic fragment ion at m/z 507.1302, which (panel A) or oral dosing (panel B). A small quantity (#114 ng/ml,
corresponds to the neutral loss of the pyroglutamate component in 0.28 mM) of BETP was observed after i.v. dosing, while none was
GSH (i.e., 129.0426 Da), suggesting that M1 was a GSH adduct. detected after oral dosing. The formation of M1 was observed in both
Furthermore, the occurrence of the fragment ion at m/z 363.0772 is dose groups. The rapid appearance of M1 in rat plasma (as early as 1.0
consistent with the presence of an aromatic thioether motif in M1 minute with peak total plasma concentrations of ;3.0 mM) after i.v.
(Baillie and Davis, 1993). A proposed structure of M1 that is dosing indicates the efficiency of the reaction between BETP and
compatible with the observed fragmentation pattern is depicted in Fig. GSH in rats. As such, we were unable to estimate pharmacokinetic
4B. To unambiguously prove the proposed structure, an authentic parameters for BETP due to small and sporadic amounts measurable in
standard of M1 was synthesized via an independent route. The the plasma samples.
LC-MS/MS attributes (tR and CID spectrum) of the M1 synthetic
standard were identical to the one generated in the chemical reaction 1R. Recent work demonstrated that coincubation with BETP markedly
between BETP and GSH in buffer (unpublished data). enhances the activity of truncated metabolite of GLP-1(7-36)NH2,
Activity of BETP and its GSH Conjugate at the Human GLP-
Based on the qualitative metabolite identification studies, incuba- i.e., GLP-1(9-36)NH2, at the GLP-1R (Wootten et al., 2012). Here, we
tions of BETP in GSH-supplemented phosphate buffer were si- took advantage of the latter procedure to test whether the GSH
multaneously monitored for the disappearance of BETP and the conjugate of BETP (i.e., M1) retains positive allosteric modulator
appearance of the GSH adduct, respectively (Fig. 2B). In phosphate properties of BETP. GLP-1(9-36)NH2 failed to activate the GLP-1R in
buffer containing GSH, following the 60-minute incubation period, the presence of M1 (10 mM) or corresponding DMSO vehicle (Fig. 9).
the amount of BETP remaining was 9.0 6 1.0 nM, resulting in a In contrast, consistent with previous observations, a dose-dependent
492 6 40 nM consumption of BETP when compared with 0 minutes
(500 6 41 nM). At 60 minutes, the amount of GSH adduct was 667 6
6.0 nM, resulting in similar loss of parent substrate BETP and for-
mation of the GSH conjugate.
Liver Microsomal Stability of BETP. To examine liver mi-
crosomal stability, BETP at a concentration of 1 mM was incubated in
rat, mouse, and human liver microsomes at 37°C for 60 minutes in the
presence and absence of NADPH cofactor and in the presence and
absence of GSH; periodically, aliquots of the incubation mixture were
examined for depletion of BETP and the appearance of the GSH
adduct of BETP (in liver microsomal incubations supplemented with
the thiol nucleophile) (Table 1). The t1/2 for depletion of BETP in rat,
mouse, and human liver microsomes in the absence of NADPH and
GSH was 8.8 6 0.3, 5.6 6 1.3, and 47 6 5.0 minutes, respectively
(Fig. 5A). In the presence of NADPH (but absence of GSH), the t1/2
for depletion of BETP in rat, mouse, and human liver microsomes was
2.2 6 0.2, 0.81 6 0.05, and 8.4 6 0.3 minutes, respectively (Fig. 5B).
In the presence of both NADPH and GSH, the t1/2s for depletion of
BETP in rat, mouse, and human liver microsomes were ,0.5 minute.
Metabolite Identification Studies. No metabolites were detected
upon qualitative LC-MS/MS examination of incubation mixtures of
plasma (rat, mouse, and human) and BSA with BETP (10 mM)
conducted at 37°C for 60 minutes. Likewise, no metabolite formation
was discerned upon incubation of BETP (10 mM) with rat, mouse, and
human liver microsomes in the absence of NADPH at 37°C for 60
minutes. LC-MS/MS analysis of incubation mixtures of BETP (10
mM) with rat, mouse, and human liver microsomes in the presence of
NADPH at 37°C for 60 minutes revealed the formation of two
metabolites (M2 and M3) in each species. A representative chromato-
gram of a rat liver microsomal incubation with BETP (6 NADPH) is
shown in Fig. 6. The CID spectra of M2 and M3 are depicted in Fig. 7,
A and B, respectively. M2 (tR = 4.54 minutes) displayed a MH+ at m/z
Fig. 8. Mean concentration-versus-time profiles of BETP and its GSH conjugate in
317.0566, which is consistent with O-dealkylation in BETP (Fig. 7A).
rat plasma (n = 2) after i.v. (A) or oral (B) dosing at 1 mg/kg and 10 mg/kg,
M3 (tR = 4.23 minutes) displayed a MH+ at m/z 333.0515, which is respectively. The error bars represent concentration range.

Intrinsic Electrophilicity of BETP
1477
a recurring structural theme in these examples is the presence of the
methylsulfone/sulfonamide and/or halide leaving group, which is
attached to an electron-deficient heteroaromatic ring system (e.g.,
pyridine, pyridone, benzothiazole, thiadiazole, benzofuran, indole,
etc.) (Clapp, 1956; Colucci and Buyske, 1965; Conroy et al., 1984;
Graham et al., 1989; Woltersdorf et al., 1989; Graham et al., 1990;
Kishida et al., 1990; Huwe et al., 1991; Zhao et al., 1999; Teffera
et al., 2008; Inoue et al., 2009; Litchfield et al., 2010). More recently,
Yang et al. (2012) have also demonstrated the susceptibility of 2-
(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles to
undergo nucleophilic displacement with GSH in human liver micro-
somes. The requirement of NADPH cofactor in the GSH displacement
reactions suggested that the rate-limiting step involved oxidation of the
alkylthio functionality to the corresponding electrophilic sulfoxide and
sulfone metabolites, followed by nucleophilic displacement of the
formed sulfoxide and/or sulfone by GSH. In the present work, we did
not observe further oxidation of the S-oxide motif in BETP to the
corresponding sulfone metabolite in liver microsomal incubations
supplemented with the cytochrome P450 cofactor NADPH.
Fig. 9. Allosteric modulation of the human GLP-1R by BETP and the GSH
conjugate M1. Ligand-stimulated cAMP production was measured in CHO-GLP1R
cells. Concentration-response curves are shown for the weak partial agonist GLP-
1(9-36)NH2 in the presence or BETP, the metabolite M1, or the vehicle DMSO. In
contrast to BETP, M1 failed to positively modulate the activity of GLP-1(9-36)NH2.
All data represent the mean 6 S.E.M. of three independent experiments conducted
in quadruplicate.
Our studies also revealed that BETP was unstable in BSA and
plasma from rat, mouse, and human, which is contrary to a previous
speculation that BETP is stable in plasma (Willard et al., 2012a).
Similar to the experience with BSA/plasma, incubations of BETP in
rat, mouse, and human liver microsomes in the absence of NADPH led
to a steady decline in BETP concentrations. Failure to detect products/
metabolites in these incubations suggests that the mechanism of BETP
depletion proceeds via a covalent displacement reaction between
BETP and a nucleophilic amino acid residue(s) in plasma and liver
microsomal proteins, similar to the pathway depicted with GSH in Fig.
10. Inclusion of NADPH and GSH in liver microsomal incubations led
to an even more rapid decline of BETP and the quantitative conversion
to GSH adduct M1, which is indicative of a detoxifying metabolic
pathway that competes with protein covalent binding. The propensity
of GSH to reduce microsomal covalent binding has been noted with
several drugs that are bioactivated to electrophilic species (Zhao et al.,
2007; Obach et al., 2008).
Although the protein (plasma/liver microsome) covalent adduction
theory has not been proven with a radiolabeled version of BETP, our
hypothesis is reasonably supported by literature reports. There are
numerous published accounts of covalent interactions between plasma
and/or liver microsomal proteins and electrophilic xenobiotics, in-
cluding drugs. Covalent binding of electrophilic acyl glucuronide
metabolites has been demonstrated in plasma, notably to albumin, and
has been detected in vivo in humans for a number of acyl
glucuronide–forming drugs (Smith and Wang, 1992; Ding et al.,
1993, 1995; Sallustio et al., 1997). Likewise, covalent modification of
lysine residues in human serum albumin has been noted with the
increase in cAMP production was observed when CHO-GLP1R cells
were stimulated with GLP-1(9-36)NH2 in the presence of BETP
(10 mM).
Discussion
Our present studies establish the electrophilic nature of BETP by
virtue of its facile chemical reaction with the endogenous nucleophile
GSH, which affords the corresponding sulfydryl conjugate M1. A
likely mechanism (Fig. 10) for the formation of M1 involves
nucleophilic attack of GSH on the C2 pyrimidine carbon in BETP
to yield the negatively charged s-complex or Meisenheimer complex
followed by elimination of the alkylsulfoxide group as the
corresponding sulfenic acid species. The electron-withdrawing sub-
stituents (pyrimidine nitrogen atoms in positions 1 and 3 and the
trifluoromethyl substituent at position 6) serve to increase the elec-
trophilicity of the C2 carbon via resonance and/or inductive sta-
bilization of the transition state, and favor reaction with the nucleophilic
thiol. Certainly, the role of the trifluoromethyl group in accelerating the
nucleophilic displacement of 2-halopyridines has been studied (Schlosser
and Rausis, 2005). In hindsight, our finding on the innate electrophilicity
of BETP is not surprising when one examines the plethora of
publications dealing with seemingly “chemically inert” compounds,
which are prone to nucleophilic displacement by GSH under non-
enzymatic (pH 7.4, phosphate buffer, 37°C) and/or enzymatic con-
ditions (mediated by glutathione transferases in liver microsomes
and/or liver cytosol). From a structure-activity relationship perspective,
Fig. 10. Nucleophilic displacement of the ethylsulfoxide moiety in BETP by GSH.

1478
Eng et al.
Clapp JW (1956) A new metabolic pathway for a sulfonamide group. J Biol Chem 223:207–214.
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Ovary, 1961; Yvon and Wal, 1988; Yvon et al., 1990; Bertucci et al.,
2001). Such covalent reactions have also been reported to occur in
patients treated with high dosages of b-lactam antibiotics, and are
thought to be responsible for the adverse effects associated with this
class of compounds (Batchelor et al., 1965; Ahlstedt and Kristofferson,
1982; Lafaye and Lapresle, 1988). Finally, the plasma instability
observed with the loop diuretic ethacrynic acid and HKI-272 (an
irreversible, covalent inhibitor of tyrosine kinase) have also been
attributed to a covalent interaction of their respective a,b-unsaturated
carbonyl moieties with amino acid residues in plasma proteins
(Bertucci et al., 1998; Bertucci and Domenici, 2002; Chandrasekaran
et al., 2010; Wang et al., 2010). With respect to covalent binding to
liver, both NADPH-dependent and -independent covalent interactions
have been demonstrated between liver microsomal proteins and
xenobiotics (Evans et al., 2004; Shin et al., 2007).
Colucci DF and Buyske DA (1965) The biotransformation of a sulfonamide to a mercaptan and to
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The failure to detect BETP in circulation following oral ad-
ministration to rats comes as no surprise considering the in vitro
chemical/biochemical instability of this electrophilic molecule, and the
corresponding impact this attribute can have on oral absorption. As
such, the lack of an insulinotropic effect of orally administered BETP
in the rat IVGTT (Sloop et al., 2010) parallels our inability to detect
systemic concentrations of BETP upon administration by the oral
route. With reference to the glucose-dependent insulin secretion noted
over a course of ;20 minutes after a single i.v. bolus dose of BETP at
10 mg/kg (Sloop et al., 2010), it is possible that enough BETP
systemic exposure was achieved at the i.v. dose of 10 mg/kg to cover
the in vitro EC50 of 0.75 mM of BETP against the rat GLP-1R (Sloop
et al., 2010). Based on our present work, systemic concentrations of
BETP in the rat i.v. pharmacokinetic study at the 1-mg/kg dose were
minimal (and sporadic), but did yield total plasma concentrations of
;0.28 mM. The detection of the GSH conjugate M1 at total
circulating concentrations significantly higher than BETP in the i.v.
pharmacokinetic study (;3.0 mM) also led us to examine its role in
the positive allosteric modulation of GLP-1R. However, unlike
BETP, M1 failed to enhance the activity of GLP-1(9-36)NH2 at the
GLP-1R.
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Obach RS, Kalgutkar AS, Soglia JR, and Zhao SX (2008) Can in vitro metabolism-dependent
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An analysis of 18 drugs with consideration of intrinsic clearance and daily dose. Chem Res
Toxicol 21:1814–1822.
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Authorship Contributions
Participated in research design: Kalgutkar, Eng, Sharma, McDonald,
Griffith, Stevens, Fortin, Jackson.
Conducted in vitro experiments: Eng, Sharma, McDonald, Li, Fortin, Nolte.
Contributed new reagents or analytic tools: Edmonds, Stevens, Griffith,
Limberakis, Price.
Performed data analysis: Kalgutkar, Eng, Sharma, McDonald. Fortin.
Wrote or contributed to the writing of the manuscript: Kalgutkar, Eng,
Sharma, McDonald, Fortin, Griffith.
Shin NY, Liu Q, Stamer SL, and Liebler DC (2007) Protein targets of reactive electrophiles in
human liver microsomes. Chem Res Toxicol 20:859–867.
Sloop KW, Willard FS, Brenner MB, Ficorilli J, Valasek K, Showalter AD, Farb TB, Cao JX,
Cox AL, and Michael MD, et al. (2010) Novel small molecule glucagon-like peptide-1
receptor agonist stimulates insulin secretion in rodents and from human islets. Diabetes 59:
3099–3107.
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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