Insights into the novel hydrolytic mechanism of a diethyl 2-phenyl-2-(2-arylacetoxy)methyl malonate ester-based microsomal triglyceride transfer protein (MTP) inhibitor

Article abstract of DOI:10.1021/tx300243v

Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4′- (trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)methyl) -2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of ¹⁸O incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H₂¹⁸O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1, 1′-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group. Unlike the case with JTT-130, ¹⁸O was readily incorporated into 1 during the enzymatic hydrolysis of 3, suggestive of a mechanism involving direct hydrolytic cleavage of the ester group in 3. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl] -2-ylcarboxamido)phenyl)acetate (4), which possessed an N,N-diethyl-2- phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved to be resistant to the hydrolytic cleavage/decarboxylative fragmentation pathway that yielded 1, a phenomenon that further confirmed our hypothesis. From a toxicological standpoint, it is noteworthy to point out that the liberation of the electrophilic acrylic acid species as a byproduct of JTT-130 hydrolysis is similar to the bioactivation mechanism established for felbamate, an anticonvulsant agent associated with idiosyncratic aplastic anemia and hepatotoxicity.

Full text of DOI:10.1021/tx300243v

Article
pubs.acs.org/crt
Insights into the Novel Hydrolytic Mechanism of a Diethyl
2‑Phenyl-2-(2-arylacetoxy)methyl Malonate Ester-Based Microsomal
Triglyceride Transfer Protein (MTP) Inhibitor
Tim Ryder,† Gregory S. Walker,† Theunis C. Goosen,† Roger B. Ruggeri,‡ Edward L. Conn,§
Benjamin N. Rocke,§ Kimberly Lapham,† Claire M. Steppan,† David Hepworth,‡ and Amit S. Kalgutkar*,∥
†Pharmacokinetics, Dynamics, and Metabolism−New Chemical Entities, Pfizer Worldwide Research and Development, Groton,
Connecticut 06340, United States
‡Cardiovascular Metabolic and Endocrine Diseases Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge,
Massachusetts 02139, United States
§Cardiovascular Metabolic and Endocrine Diseases Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton,
Connecticut 06340, United States
∥Pharmacokinetics, Dynamics, and Metabolism−New Chemical Entities, Pfizer Worldwide Research and Development, Cambridge,
Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Inhibition of intestinal and hepatic microsomal
triglyceride transfer protein (MTP) is a potential strategy for
the treatment of dyslipidemia and related metabolic disorders.
Inhibition of hepatic MTP, however, results in elevated liver
transaminases and increased hepatic fat deposition consistent
with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-
4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)-
phenyl)acetoxy)methyl)-2-phenylmalonate (JTT-130) is an
intestine-specific inhibitor of MTP and does not cause in-
creases in transaminases in short-term clinical trials in patients
with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific
carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts
around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of 18O
incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H218O suggested that
hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a
metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups
followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species.
Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were
supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies
on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-
phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene
group. Unlike the case with JTT-130, 18O was readily incorporated into 1 during the enzymatic hydrolysis of 3, suggestive of a
mechanism involving direct hydrolytic cleavage of the ester group in 3. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-
phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetate (4), which
possessed an N,N-diethyl-2-phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved
to be resistant to the hydrolytic cleavage/decarboxylative fragmentation pathway that yielded 1, a phenomenon that further
confirmed our hypothesis. From a toxicological standpoint, it is noteworthy to point out that the liberation of the electrophilic
acrylic acid species as a byproduct of JTT-130 hydrolysis is similar to the bioactivation mechanism established for felbamate, an
anticonvulsant agent associated with idiosyncratic aplastic anemia and hepatotoxicity.
shown to play a crucial role in the assembly of triglyceride (TG)-
rich lipoproteins, such as chylomicrometers in the intestine and
INTRODUCTION
■
Microsomal triglyceride transfer protein (MTP) is an intra-
cellular lipid-transfer protein localized in the endoplasmic reti-
culum of hepatocytes and enterocytes. With its broad dis-
tribution in both intestinal and hepatic tissue, MTP has been
Received: June 2, 2012
Published: September 18, 2012
© 2012 American Chemical Society
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Figure 1. Structures of the intestine-specific MTP inhibitor JTT-130 and related ester derivatives 3 and 4.
very low density lipoprotein (VLDL) in the liver.1,2 Patients with
the genetic disorder abetalipoproteinemia or Bassen-Kornzweig
syndrome have loss-of-function mutations in the MTP gene,3,4
resulting in extremely low plasma concentrations of cholesterol
and TGs and the absence of chylomicrometers, VLDL and LDL-
cholesterol.5 The elucidation of the mechanistic basis for this
disease led to the concept that small-molecule inhibitors of MTP
could be beneficial in the treatment of dyslipidemia and various
other metabolic disorders. Indeed, preclinical studies in animal
models showed that inhibition of MTP significantly reduced
serum cholesterol levels and slowed the formation of
atherosclerotic plaques.6−8 Furthermore, in a clinical setting,
MTP inhibition was shown to significantly reduce LDL-
cholesterol levels in patients with homozygous familial hyper-
cholesterolemia.9 Unfortunately, blockade of hepatic MTP is
accompanied by a significant decrease in the ability of the liver to
eliminate fat, and administration of systemic MTP inhibitors
universally resulted in unacceptable increases in liver trans-
aminases and increased hepatic fat deposition consistent with
hepatic steatosis or “fatty liver”.8,10−13 Consistent with these
toxicological findings were the clinical observations associating
genetic polymorphisms in the MTP gene with an increased
incidence of steotosis in type II diabetic and the abetalipopro-
teinemic patient population.14,15 Subsequently, it has been
proposed that nonsystemically absorbed MTP inhibitors could
alleviate the hepatotoxicity concerns by selectively inhibiting
lipid transport within the enterocyte, without significantly affect-
ing the export of hepatic TGs as VLDL, which is the underlying
cause of steotosis.16−19
Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-
[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)methyl)-2-
phenylmalonate (JTT-130) has been recently described as a
potent MTP inhibitor (Figure 1), which, upon oral admin-
istration to preclinical species, selectively inhibits MTP activity in
the small intestine.20−23 Furthermore, in contrast with non-
selective MTP inhibitors, JTT-130 did not cause elevations in
transaminase levels in the course of a three-month clinical trial in
patients with dyslipidemia.20 As such, selective inhibitory effects
of JTT-130 on intestinal MTP are a function of a facile (and
rapid) hydrolysis of its ester linkage by liver-specific carbox-
ylesterase(s) to yield 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoro-
methyl)-[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetic acid
(1), a pharmacologically inactive metabolite (see Figure 1).20
In conjunction with our internal tissue-specific MTP inhibitor
program,8,24,25 we conducted a detailed in vitro mechanistic
assessment of the ester bond cleavage in JTT-130 using human
liver microsomes. Lack of incorporation of 18O in 1 that was
generated upon incubating JTT-130 in human liver microsomes
suggested that hydrolysis did not proceed via a simple ester bond
cleavage to yield carboxylic acid 1 and the alcohol byproduct 2
as shown in Figure 1. The characterization of atropic acid
(2-phenylacrylic acid) as a metabolite of JTT-130 in the microsomal
incubations suggested a pathway involving the hydrolysis of one
of the pendant malonate ethyl ester groups followed by
decarboxylative fragmentation to 1. Additional support for our
hypothesis was obtained from mechanistic studies on diethyl
2-(2-(2-(3-(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1,1′-bi-
phenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalo-
nate (3) and 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phe-
nylpropyl 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-
[1,1′-biphenyl]-2-ylcarboxamido)phenyl)acetate (4) (see Figure 1).
In particular, ester 3 cannot participate in hydrolysis via the
fragmentation pathway because of the presence of an additional
methylene group. Unlike the case with JTT-130, 18O was readily
incorporated in 1 during the enzymatic hydrolysis of 3, sug-
gestive of a mechanism involving direct hydrolytic cleavage of the
ester linkage. Compound 4, which possessed an N1,N3-diethyl-2-
phenylmalonamide substituent (in lieu of the diethyl-2-phenyl-
malonate motif in JTT-130) proved to be resistant to the
hydrolytic cleavage pathway that yielded 1, a finding that pro-
vided further credence to our hypothesis. The collective findings
from these studies are summarized, herein, with a particular
emphasis on the JTT-130 hydrolysis mechanism that yields
electrophilic α,β-unsaturated carbonyl derivatives as byproduct.
MATERIALS AND METHODS
■
Materials. Unless specified otherwise, starting materials used in the
chemical synthesis are available from commercial sources such as Sigma
Aldrich (Milwaukee, WI), Lancaster Synthesis, Inc. (Windham, NH),
Acros Organics (Fairlawn, NJ), Maybridge Chemical Company, Ltd.
(Cornwall, England), Mallinckrodt Baker (Phillipsburg NJ), and EMD
(Gibbstown, NJ). Atropic acid was obtained from Apollo Scientific Ltd.
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(Cheshire, England). Solvents used for analysis were of analytical or
HPLC grade (Fisher Scientific, Pittsburgh, PA, USA). Dimethyl
sulfoxide (DMSO)-d6 “100%” was purchased from Cambridge Isotope
Laboratories (Andover, MA, USA). Chemical shifts are expressed in
parts per million (ppm) relative to the residual solvent as an internal
reference. The peak shapes are denoted as follows: s, singlet; d, doublet;
dd, doublet of doublets; t, triplet; td, triplet of doublets; q, quartet; m,
multiplet and brs, broad singlet. Coupling constants (J) are given in
hertz (Hz). Glutathione (GSH) and NADPH were obtained from Sigma
Aldrich (St. Louis, MO). Human liver microsomal fractions pooled from
53 individual donors were purchased from BD Gentest (Woburn, MA).
JTT-130, 1, 3, and 4 were synthesized using the general methods
described in the Japan Tobacco patent.26 JTT-130: 1H NMR (400 MHz,
CDCl3) δ 1.22 (t, J = 7.1 Hz, 6 H), 2.81 (brs, 3 H), 2.94 (brs, 3 H), 3.52
(s, 2 H), 4.22 (q, J = 7.1 Hz, 4 H), 4.83 (s, 2 H), 7.04 (d, J = 1.9 Hz, 1 H),
7.21 (dd, J = 8.7, 2.2 Hz, 1 H), 7.28−7.34 (m, 5 H), 7.40 (dd, J = 7.5, 1.1
Hz, 1 H), 7.47−7.57 (m, J = 14.7, 6.7, 6.7, 6.6 Hz, 2 H), 7.62 (s, 4 H),
7.70 (dd, J = 7.4, 1.2 Hz, 1 H), 8.36 (d, J = 8.5 Hz, 1 H), 9.18 (s, 1 H).
Liquid chromatography−mass spectrometry (electrospray ionization
(ESI)): MH+ = 719.3 (100%).
percentage of substrate remaining versus time was fitted to first-order
decay functions to yield in vitro t1/2 values. If the test substrate
demonstrated nonlinearity on log percentage remaining versus time
curves, only those initial time points wherein log-linearity was observed
were used to determine t1/2 values. t1/2 values reported are the mean of
two independent experiments.
Metabolite Identification in Human Liver Microsomes.
Incubations were carried out at 37 °C for 0 and 30 min in a shaking
water bath. The incubation volume was 1 mL and consisted of
microsomes (protein concentration = 1.0 mg/mL) and test compounds
(10 μM) in 0.1 M potassium phosphate buffer (pH 7.4) and MgCl2
(3.3 mM). The formation of GSH conjugates of reactive species derived
from JTT-130 metabolism was examined via the inclusion of 1 mM GSH
in the human liver microsomal incubations. Reactions were terminated
by the addition of ice-cold acetonitrile (3 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 of mobile phase and analyzed for
metabolite formation using LC−MS/MS.
18O Incorporation Studies. The source of oxygen incorporated in
the carboxylic acid metabolite 1 was characterized using 97% H218O
(Cambridge Isotope Laboratories Inc., Andover, MA). For H218O
studies, 15 mL glass centrifuge tubes containing 1.0 mL of 100 mM
potassium phosphate (pH 7.4) were concentrated to dryness using an
evaporative centrifuge. H218O (0.925 mL) was added to the dried
centrifuge tube containing phosphate buffer. Potassium phosphate (pH
7.4) buffered H218O was used to create 1.0 mL incubations containing
test compounds JTT-130 and 3 (at a concentration of 10 μM), and
3.3 mMMgCl2 (20 μL), in human liver microsomes (protein concentration =
1.0 mg/mL). The isotopic enrichment of H218O was ∼89.7%. The
sample was placed in a shaking water bath set to 37 °C. After 30 min, the
reactions were quenched with 3.0 mL of acetonitrile and centrifuged at
3000g for 5 min. The supernatant was transferred to a clean 15 mL
centrifuge tube 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 photo-
diode array detector (Thermo Electron Corporation, Waltham, MA).
Chromatography was performed on a Phenomenex Hydro RP
column (4.6 mm × 150 mm, 4 μm). 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
15% B for 5 min, was ramped linearly to 90% B over 35 min, held at 90%
B over 10 min, returned to the initial condition over 1.0 min, and allowed
to equilibrate for 4.0 min. Postcolumn flow passed through the PDA
detector to provide UV (λ = 254 nm) detection prior to being split to the
mass spectrometer such that the 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 min exclusion duration
after 3 successive product ion scans with an early exclusion if the
precursor ion falls below a signal-to-noise of 20. Additional data
dependent scanning was performed on select samples at unit mass
resolution for product ions of the two most intense ions found in the full
scan mode.
Compound 1. 1H NMR (400 MHz, CDCl3) δ 2.84 (brs, 3 H), 2.96
(brs, 3 H), 3.54 (s, 2 H), 7.09 (d, J = 2.0 Hz, 1 H), 7.24−7.28 (m, 1 H),
7.39 (d, J = 7.5 Hz, 1 H), 7.45−7.56 (m, 2 H), 7.59−7.64 (m, 4 H), 7.68
(dd, J = 7.5, 1.0 Hz, 1 H), 8.25 (d, J = 8.5 Hz, 1 H), 9.08 (s, 1 H). Liquid
chromatography−mass spectrometry (ESI) MH+ = 471.4 (100%).
1
Compound 3. H NMR (400 MHz, CDCl3) δ 1.25 (t, J = 7.1 Hz,
6 H), 2.62 (t, J = 7.2 Hz, 2 H), 2.86 (brs, 3 H), 2.95 (brs, 3 H), 3.47 (s,
2 H), 4.08 (t, J = 7.2 Hz, 2 H), 4.18−4.30 (m, 4 H), 7.09 (d, J = 2.1 Hz,
1 H), 7.25 (dd, J = 8.5, 2.0 Hz, 1 H), 7.28−7.41 (m, 6 H), 7.49 (td, J =
7.4, 1.5 Hz, 1 H), 7.54 (td, J = 7.5, 1.6 Hz, 1 H), 7.59−7.64 (m, 4 H), 7.70
(dd, J = 7.5, 1.3 Hz, 1 H), 8.36 (d, J = 8.5 Hz, 1 H), 9.15 (s, 1 H). Liquid
chromatography−mass spectrometry (ESI) MH+ = 733.4 (100%).
Compound 4. 1H NMR (500 MHz, CDCl3) δ 1.08 (t, J = 7.32 Hz, 6
H), 2.85 (brs, 3 H), 2.96 (brs., 3 H), 3.20−3.31 (m, 4 H), 3.55 (s, 2 H),
4.84 (s, 2 H), 7.04 (d, J = 1.71 Hz, 1 H), 7.12 (t, J = 4.90 Hz, 2 H), 7.16−
7.24 (m, 3 H), 7.29−7.35 (m, 3 H), 7.41 (d, J = 7.32 Hz, 1 H), 7.50 (td,
J = 7.60, 1.20 Hz, 1 H), 7.55 (td, J = 7.60, 1.22 Hz, 1 H), 7.58−7.65 (m,
4 H), 7.70 (d, J = 6.83 Hz, 1 H), 8.39 (d, J = 8.54 Hz, 1 H), 9.19 (s, 1 H).
Liquid chromatography−mass spectrometry (ESI) MH+ = 717.5
(100%).
Hydrolytic Stability in Human Liver Microsomes. Stock
solutions of test compounds were prepared in acetonitrile. Compounds
(final concentration = 1 μM) were incubated with human liver
microsomes (cytochrome P450 (P450) concentration = 0.27 μM and
protein concentration = 0.76 mg/mL) in 0.05 M potassium phosphate
buffer (pH 7.4) at 37 °C in a shaking water bath. The total incubation
volume was 0.5 mL. Aliquots (25 μL) of the reaction mixture at 0, 2, 4, 6,
8, 10, 20, and 30 min (time period associated with reaction linearity)
were added to acetonitrile (200 μL) containing a proprietary internal
standard (MW = 704, 0.1 μg/mL) and were analyzed for disappearance
of the substrate by liquid chromatography tandem mass spectrometry
(LC-MS/MS). Analyte concentrations were monitored on a Sciex API
3000 LC-MS/MS triple-quadrupole mass spectrometer. Analytes were
chromatographically separated using a Shimadzu LC-20 AD HPLC
system. An autosampler was programmed to inject 20 μL on a Synergi
Polar RP 2 μm, 30 × 2.0 mm column using a mobile phase consisting of
95:5 water/acetonitrile containing 0.1% formic acid (solvent A) and
95:5 acetonitrile/water (solvent B). The LC conditions were
programmed as follows: 10% acetonitrile for 0.5 min, increasing to
90% over the next 1.8 min and then decreasing back to 10% (i.e., the
original condition) over the final 3 min, at a flow rate of 0.5 mL/min.
Ionization was conducted in the positive ion mode at an ion-spray
interface temperature of 400 °C, using nitrogen as the nebulizing and
heating gas. The ion-spray voltage was 5.0 kV, and the orifice voltage was
optimized at 30 eV. JTT-130, 3 and 4, and the internal standard were
analyzed in the multiple-reaction monitoring mode using the transitions
[MH]+ to product ion m/z + H+ 719 → 674, 733 → 688, and 717 →
672, respectively. For half-life (t1/2) determinations for substrate
depletion in microsomes, substrate/internal standard peak height ratios
were determined and normalized to the value obtained at time t = 0. The
Biosynthesis and NMR Analysis of the “m/z 363” Metabolite
M2 Obtained from the Enzyme-Catalyzed Hydrolysis of JTT-
130. Metabolite M2 detected in the course of JTT-130 hydrolysis was
biosynthesized via a scale-up of the human microsomal incuba-
tions (protein concentration = 10 mg/mL, 1 h at 37 °C) of JTT-130
(100 μM) in a total of four 50 mL polypropylene centrifuge tubes (10 mL
incubation/tube). Cold acetonitrile (20 mL) was added to each tube,
and samples were centrifuged at 3000g for 15 min. The supernatants
were transferred to clean 50 mL polypropylene centrifuge tubes and
concentrated to dryness. The residue in each tube was reconstituted in
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250 μL of 10% acetonitrile in water. Reconstituted residues were
combined in a 2 mL HPLC vial and were injected onto an HPLC
semipreparative system using three injection volumes of approximately
330 μL. The semipreparative system consisted of a Shimadzu SiL-HTC
autosampler, two LC-20AD solvent pumps, an SPD-M20A diode array
detector, and a FRC-10A fraction collector. Separation was performed
on a Zorbax RX C8, 9.4 × 250 mm, 5 μM semipreparative HPLC
column. The mobile phase was composed of 5 mM ammonium formate
buffer (pH 3.0) (solvent A) and acetonitrile (solvent B). The flow rate
was 4.0 mL/min. The LC gradient started at 15% B for 5 min, was
ramped linearly to 90% B over 35 min, held at 90% B over 10 min,
returned to the initial condition over 1.0 min, and allowed to equilibrate
for 4.0 min. HPLC fractions were collected throughout the run at 1 min
intervals into glass culture tubes. Aliquots (100 μL) of fractions at the
retention time (tR) of the UV peak believed to be the unknown
metabolite M2 (tentatively assigned as MH+ = 363) were analyzed by
LC/MS for verification. Fractions containing the peak (MH+ = 363)
were combined into a single 15 mL glass centrifuge tube and
concentrated to dryness. The residue was then evacuated in a drybox
for about two hours prior to being reconstituted in DMSO-d6 for NMR
studies. NMR spectra 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 of DMSO-d6.
All spectra were referenced using residual DMSO-d6 (δ = 2.5 ppm
relative to TMS, δ = 0.00). 1D spectra were recorded using a sweep
width of 8000 Hz and a total recycle time of 3.6 s. The resulting time-
averaged free induction decays were transformed using an exponential
line broadening of 1.0 Hz to enhance signal-to-noise. Multiplicity edited
heteronuclear single quantum coherence (HSQC) and heteronuclear
multiple bond correlation (HMBC) data were recorded using the
standard pulse sequences provided by Bruker. 2D NMR data were
Figure 2. LC-UV (λ = 254 nm) chromatograms of incubations of JTT-
130 (10 μM) in human liver microsomes (in the absence of NADPH) at
t = 0 min (panel A) and at t = 30 min (panel B). Incubations were
conducted at 37 °C in a shaking water bath.
Figure 3. LC-UV (λ = 254 nm) chromatogram (panel A) and mass spectrum (panel B) of isolated M2 (tR = 18.31 min). Structures depict the fragment
ions discerned in the mass spectrum of M2.
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Figure 4. Expanded aromatic region of the 1H−13C multiplicity edited
HSQC (panel A) and HMBC (panel B) spectra of M2.
typically acquired using a 1K × 128 data matrix with 16 dummy scans.
The data was zero-filled to a size of 1K × 1K. Unless otherwise noted, for
2D NMR experiments, a relaxation delay of 1.5 s was used between
transients.
Figure 5. Comparison of the HMBC NMR spectra of an authentic
standard of atropic acid (panel A) and isolated M2 (panel B).
RESULTS
■
Stability of JTT-130, 3, and 4 in Human Liver
Microsomes. To examine microsomal stability, test compounds
(JTT-130, 3, and 4) at a concentration of 1 μM were incubated in
human liver microsomes at 37 °C in the presence or absence of
NADPH cofactor; periodically, aliquots of the incubation
mixture were examined for substrate depletion. The t1/2 for
depletion of JTT-130, 3, and 4 in human liver microsomes in the
absence of NADPH were 1.2 min, 1.4 min, and >60 min,
respectively. The t1/2 for the depletion of JTT-130, 3, and 4 in
human liver microsomes in the presence of NADPH were 1.3,
1.4, and 2.4 min, respectively. The rapid degradation of JTT-130
in human liver microsomes ( NADPH) appears to be in general
agreement with the previous results of Mera et al.,20 wherein,
∼ 85% of JTT-130 was consumed in a 5 min incubation with the
human liver S9 fraction.
Metabolite Identification in Human Liver Microsomes.
Metabolism of JTT-130. LC-UV/MS traces of human liver
microsomal incubations of JTT-130 (10 μM), conducted at
37 °C for 30 min are shown in Figure 2. Examination of the
metabolic profile of human liver microsomal incubations of
JTT-130 in the absence of NADPH revealed near complete
conversion of the parent compound and the appearance of two
metabolites (designated as M1 and M2). A similar metabolic
profile was discerned in JTT-130 incubations in liver microsomes
in the presence of NADPH (data not shown). The tR (24.44 min)
and collision-induced dissociation (CID) spectrum for metab-
olite M1 (MH+ at m/z 471.1526 → 426.0947 (100%), loss
of dimethylamine) were identical to those discerned with
an authentic standard of the carboxylic acid 1 (Figure 1S,
Supporting Information). Metabolite M2 (tR = 18.31 min)
displayed a MH+ at m/z 363.05886, which does not correspond
with the protonated molecular mass of the primary alcohol
metabolite 2 that can be obtained via the JTT-130 hydroly-
sis route depicted in Figure 1 or with possible downstream
ester hydrolysis products of 2, namely, 3-ethoxy-2-(hydrox-
ymethyl)-3-oxo-2-phenylpropanioic acid (MH+ = 239.0914) and
Figure 6. LC-UV (λ = 254 nm) chromatograms of incubations of 3
(10 μM) in human liver microsomes at t = 30 min in the absence of
NADPH (panel A) and t = 30 min in the presence of NADPH (panel B).
Incubations were conducted at 37 °C in a shaking water bath. The peak
at tR = 22.30 min is background noise.
2-(hydroxymethyl)-2-phenylmalonic acid (MH+ = 211.0601).
Furthermore, attempts to generate a CID spectrum of M2 were
unsuccessful, raising the question of whether m/z 363 was the
actual protonated molecular mass of the metabolite.
Isolation and Structural Characterization of M2.
Unknown metabolite M2 was isolated by semipreparative
HPLC fraction collection of human liver microsomal incubations
of JTT-130. MS data of the M2 isolate was complex with no clear
single MH+ ion (Figure 3). The spectra contained repeating ions
differing by 170.0348 Da starting at m/z 193.0236 with the last
observable ion at m/z 1213.2350. Exact mass data suggested
that M2 (m/z = 363.05886) could be the product of 2-phenyl-
acrylic acid (i.e., atropic acid) with m/z 193 being the diso-
diated singly charged species and the 170 amu intervals being
the neutral addition of subsequent sodium 2-phenylacrylate
molecules (Figure 3). 1H NMR analysis of this material indicated
that the isolate contained five aromatic resonances, two olefinic
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resonances, and a complete absence of aliphatic resonances.
Multiplicity edited HSQC and HMBC NMR spectra positively
identified two olefinic protons proximal to the carbonyl carbon
and an aromatic ring, which is consistent with the structure of
atropic acid (Figure 4). Unambiguous confirmation of the struc-
ture was obtained via comparison of the NMR characteristics of
Table 1. LC-MS/MS Data for 3 and Its Metabolites in Human Liver Microsomes
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Table 1. continued
a
b
Human liver microsomal incubations with 2 in the presence or absence of NADPH cofactor. Under the HPLC conditions noted in Materials and
Methods.
isolated M2 with the commercially available atropic acid standard
(Figure 5, panels A and B). The finding that atropic acid formed
ion clusters under the electrospray ionization conditions was
not altogether surprising. Ion cluster formation is common in
soft ionization techniques such as electrospray ionization and
has been noted with carboxylic acid derivatives under similar
conditions.28
Metabolism of 3. LC-UV/MS traces of human liver
microsomal incubations of 3 (10 μM), conducted at 37 °C for
30 min, are shown in Figure 6. Table 1 reveals the molecular
weights, tR, CID spectral data, and proposed assignments for 3
and its metabolites. In the absence of the NADPH cofactor, the
metabolic profile revealed complete consumption of the parent
ester 3 (MH+ = 733.2731, tR = 37.0 min), and the concomitant
Figure 7. LC-UV (λ = 254 nm) chromatograms of incubations of 4
formation of four metabolites, carboxylic acid 1 and M3−M5
(10 μM) in human liver microsomes at t = 30 min in the absence of
(Figure 6, Panel A). M3 (tR = 35.99 min) displayed a MH+ at m/z
NADPH (panel A) and t = 30 min in the presence of NADPH (panel B).
661.2521, which corresponds to a loss of one of the pendant ethyl
ester moieties (−72.021 Da) in 3. The CID spectrum of
M3 displayed a diagnostic fragment ion at m/z 616.1945
Incubations were conducted at 37 °C in a shaking water bath.
corresponding to the loss of the N,N-dimethylamine motif,
which supported the proposed structural assignment for M3,
shown in Table 1. Metabolite M4 (tR = 30.5 min) displayed a
MH+ at m/z 633.2207, which corresponds to a loss of the ethyl
group (−28.0314 Da) from metabolite M3. The CID spectrum
of M4 displayed a characteristic fragment ion at m/z 588.1632
corresponding to the loss of the N,N-dimethylamine motif,
which supported the proposed structural assignment for M4,
shown in Table 1. The mass spectral data (MH+ at m/z 471.1526 →
426.0947 loss of dimethylamine) for the metabolite, which
eluted at tR = 24.7 min was identical to the one discerned for M1
(i.e., the carboxylic acid derivative 1). Finally, the structure of
metabolite M5 (tR = 17.7 min), which displayed a MH+ at m/z
209.1172, was consistent with ethyl 4-hydroxy-2-phenylbuta-
noate, the alcohol obtained upon direct hydrolysis of the ester
linkage in M3.
In the presence of the NADPH cofactor, LC-UV/MS analy-
sis also revealed near complete consumption of ester 3 and
formation of carboxylic acid 1, M3, and M4. Trace levels
of downstream oxidative metabolites of 1, M3, and M4, i.e.,
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M6−M10, were also detected in the presence of the NADPH
cofactor (Figure 6, panel B). Metabolites M6 (tR = 37.5 min)
and M10 (tR = 26.9 min) displayed protonated molecular ions
at m/z 647.2363 and 457.1369, which corresponded to the
loss of 14.015 Da from the molecular masses of M3 and 1,
respectively. The presence of diagnostic fragment ions at m/z
616.1946 and 426.0945 in the CID spectra of M6 and M10
established that they were N-demethylated metabolites of
M3 and 1, respectively. Metabolites M7 (tR = 34.5 min) and M8
(tR = 33.8 min) indicated protonated molecular masses at m/z
677.2469 implying that they were isomers derived from the
monohydroxylations of M3. Both metabolites possessed a
common fragment ion at m/z ∼ 632.189, which ruled out the
N,N-dimethylamine motif as the site(s) of hydroxylation. The
presence of a fragment ion at m/z 614.1792 in the CID spectrum
of M8 was consistent with a loss of a water molecule, suggesting
that the site of oxidation was on an aliphatic hydrogen (i.e., on
one of the two methylene or methine hydrogens) in the alcohol
Table 2. LC-MS/MS Data for 4 and Its Metabolites in Human Liver Microsomes
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Table 2. continued
a
Under the HPLC conditions noted in Materials and Methods.
Figure 8. Examination of 18O incorporation in the carboxylic acid metabolite 1 obtained from the hydrolysis of JTT-130 (panel A) and 3 (panel B) in
human liver microsomes in the presence of H218O.
substituent of M3. We speculate that the site of oxidation in M8 is
on the methine hydrogen based on the proposed mechanism
leading to the fragment ion m/z 426.0949 (see Table 1). The
absence of m/z 614 and m/z 426 fragment ions in the CID
spectrum of M7 suggested that M7 was derived from an aromatic
hydroxylation on the alcohol unit in M3. Metabolite M9 (tR =
28.5 min) was a monohydroxylated metabolite of M4 with
a MH+ at m/z 649.2156 (15.9949 Da higher than the MH+
observed for M4) and fragment ions at m/z 604.1582, 586.1474,
and 426.0950. The fragment ions at m/z 604.1582 and 426.0950
ruled out hydroxylation on the substituent attached to the
carboxylic acid portion in M4, whereas the ion at m/z 586.1474
derived from the loss of a water molecule suggested that the site
of oxidation was on a aliphatic hydrogen (i.e., on one of the two
methylene or methine hydrogens) in the alcohol substituent in
M4. Similar to the situation with M8, we propose the methine
hydrogen as the likely site for P450 oxidation in M9 based on
the proposed mechanism leading to the fragment ion at m/z
426.0950 (Table 1).
Metabolism of 4. LC-UV/MS traces of human liver
microsomal incubations of 4 (10 μM), conducted at 37 °C for
30 min, are shown in Figure 7. Table 2 reveals the molecular
weights, tR, CID spectral data, and proposed structural assign-
ments for 4 and its metabolites. In the absence of the NADPH
cofactor, 4 was resistant to metabolic turnover in human liver
microsomes (Figure 7, panel A). In the presence of the NADPH
cofactor, the metabolic profile revealed the formation of six
metabolites M11−M16 (alongside the parent compound 4)
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Figure 9. Extracted ion chromatogram of the glutathione conjugates of atropic acid (panel A) and its ethyl ester (panel B) obtained upon incubating
JTT-130 in human liver microsomes in the presence of GSH.
(Figure 7, panel B). M11 (tR = 34.1 min) revealed a MH+ at m/z
701.2581, which is 16.0313 Da lower than the MH+ of 717.2894
for the parent compound 4. The fragment ion at m/z 670.2159 in
the CID spectrum of M11 is 2.0156 Da lower than the corres-
ponding fragment ion (m/z = 672.2315) in the mass spectrum of
4, implying that M11 is derived from an (a) N-demethylation of
the N,N-dimethylcarboxamide motif and (b) desaturation of one
of the pendant N-ethylamide substituents. M12 (tR = 33.0 min)
revealed a MH+ at m/z 703.2738, which was 14.0156 Da lower
than the MH+ of 717.2894 for the parent compound 4. Further-
more, M12 displayed a fragment ion at m/z 672.2315 that is
identical to the one observed for 4. This finding suggests
that M12 is derived from an N-demethylation of the N,N-
dimethylcarboxamide motif in 4.
In the case of M13 (tR = 29.9 min), the protonated molecular
and fragment ions at m/z 675.2425 and m/z 644.2004 were
28.031 Da lower than the corresponding values noted with M12,
indicating that M13 is obtained from the N-deethylation of one
of the pendant N-ethylamide groups in M12. The protonated
molecular and fragment ions at m/z 715.2738 and 670.2157 for
metabolite M14 (tR = 27.7 min) were consistent with oxidative
desaturation on one of the pendant ethyl amide substituents in 4
to yield the corresponding enamide metabolite. Metabolites M15
(tR = 27.6 min) and M16 (tR = 25.1 min) displayed protonated
molecular ions at m/z 689.2581 and 661.2268, which corres-
ponded to the loss of 28.0313 and 56.0626 Da, respectively, from
the MH+ of 717.2894 for the parent compound 4. The presence
of fragment ions at m/z 644.2004 and 616.1690 in the CID
spectra of M15 and M16 suggested that these metabolites were
derived from sequential N-deethylation of the two pendant
amide groups in 4. Importantly, the carboxylic acid metabolite 1
was not detected as a metabolite of 4 in the human liver micro-
somal incubations.
18O Incorporation Studies. Feasibility of direct ester
hydrolysis in JTT-130 via the pathway depicted in Figure 1
was probed by using H218O in microsomal incubations. LC-UV/
MS analysis of a human liver microsomal incubation of JTT-130
(10 μM) conducted at 37 °C for 30 min in the presence of H218O
(89.7%) did not reveal 18O incorporation into the carboxylic
acid metabolite 1 (Figure 8, panel A). 18O incorporation, how-
ever, was noted in the atropic acid metabolite M2 (Figure 2S,
Supporting Information) that was generated in the JTT-130/
human liver microsomal incubations. Lack of 18O incorporation
in 1 suggested that the carboxylic acid metabolite is not formed
by direct ester hydrolysis of JTT-130. Incorporation of 18O in M2
served as an internal control for direct hydrolysis of the pendant
ester group(s) in JTT-130. In contrast with the observations with
JTT-130, human liver microsomal incubations with 3 in the
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Figure 10. CID spectra of the glutathione conjugates of atropic acid (panel A) and its ethyl ester 5 (panel B).
presence of H218O showed incorporation of 18O in 1 demon-
strating direct ester hydrolysis of 3 (Figure 8, panel B).
Reactive Metabolite Trapping Studies. Human liver
microsomal incubation of JTT-130 in the presence of
exogenously added GSH indicated the formation of the GSH
conjugates of atropic acid (tR = 8.82 min, MH+ at m/z 456.1435)
and its ethyl ester derivative 5 (tR = 14.23 min, MH+ at m/z
484.1748) in trace quantities (Figure 9, panels A and B). The
fragment ions at m/z 327.1001 (Figure 10, panel A) and m/z
355.1322 (Figure 10, panel B) in the CID spectra of the GSH
conjugates of atropic acid and 5, respectively, are derived from
the characteristic loss of the pyroglutamic acid component of
GSH.27
differences in ester substrate specificities for the human serine
esterases, carboxylesterase 1 (hCE-1), and carboxylesterase 2
(hCE-2), respectively. Although both hCE-1 and hCE-2 are
present in several organs, the hydrolase activity of the liver and
small intestine is predominantly attributed to hCE-1 and hCE-2,
respectively.29,30 Furthermore, the substrate specificity of hCE-1
and hCE-2 is significantly different; hCE-2 recognizes a substrate
with a large alcohol group and small acyl group. In contrast, hCE-1
mainly hydrolyzes a substrate with a small alcohol group and
large acyl group.29,30 On the basis of the JTT-130 structure (the
presence of a large acyl group and a small alcohol group), it is
plausible that hydrolysis of JTT-130 to 1 in human liver
microsomes is mediated by hCE-1. The finding that JTT-130
is resistant to hydrolytic cleavage (t1/2 > 90 min) in human
intestinal microsomes (Goosen, T., and Lapham K., unpublished
observations) appears to corroborate this hypothesis.
Although the formation of 1 in JTT-130 incubations in human
S-9 fractions has been previously noted, there is no information
pertaining to the formation of the alcohol metabolite 2 in these
incubations.20 Our present work establishes that JTT-130 is not
hydrolyzed in a straightforward fashion to yield carboxylic acid 1
and alcohol 2 as judged from the lack of incorporation of 18O in 1
in human liver microsomal incubations of JTT-130 in the pre-
sence of H218O. We rationalize JTT-130 hydrolysis to proceed
DISCUSSION
■
The metabolism-guided approach leading to the discovery of
JTT-130 constitutes an elegant tactic toward delivering a
selective inhibitor of intestinal MTP.20 JTT-130 was designed
to be rapidly hydrolyzed and inactivated (to the carboxylic
acid metabolite 1) via hydrolytic cleavage of the ester group
immediately after intestinal absorption. The success of this
strategy is evident from the lack of elevated transaminase levels in
phase I clinical trials with JTT-130. From a drug design per-
spective, it is likely that Mera et al.20 capitalized on the known
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Figure 11. Mechanism of hydrolytic cleavage of JTT-130.
via the initial hydrolytic cleavage of one of the pendant malonic
acid ethyl ester substituents to yield the carboxylic acid
intermediate 6 that would spontaneously undergo decarbox-
ylative fragmentation to yield 1 and the ethyl ester of atropic acid
5 (Figure 11, pathway A). Although 5 was not detected as a
metabolite in the microsomal incubations with JTT-133, its
formation was inferred from the characterization of the
corresponding GSH adduct. Furthermore, the MS and NMR
characterization of atropic acid, which can be obtained via the
hydrolysis of 5 in the JTT-130/human liver microsomal incuba-
tions, provides additional credence to our theory.
To further test our hypothesis, compounds 3 and 4 were
synthesized. Ester 3 possesses an additional methylene group on
the alcohol substituent (diethyl 2-(2-hydroxyethyl)-2-phenyl-
malonate in 3 instead of diethyl 2-(hydroxymethyl)-2-phenyl-
malonate in JTT-130) and cannot undergo the decarboxylative
fragmentation following hydrolysis of one of the pendant malo-
nate ester groups. The formation of 1 from 3 can only arise
through a direct hydrolysis of the ester linkage (Figure 11,
pathway B), and this was reflected in the 18O incorporation in 1
following an incubation of 3 in microsomes in the presence of
H218O. Ester 4, which incorporated an N1,N3-diethyl-2-phenyl-
malonamide substituent (instead of the diethyl-2-phenylmalonate
motif in JTT-130) failed to generate 1 in human liver micro-
somes. Although the malonamide motif was deliberately incor-
porated to block decarboxylative elimination, we did not anti-
cipate the complete resistance of 4 toward hydrolysis. This
finding implies that the central ester linkage in 4 (and for
that matter JTT-130) is sterically hindered and cannot undergo
hydrolytic cleavage by hCE-1. From a structure−activity
relationship perspective, it is interesting to note that the mere
addition of the extra methylene linker in 3 drastically lessens
the steric hindrance around the central ester linkage thereby
increasing its susceptibility toward hydrolytic cleavage. As such,
these observations also highlight the importance of the pendant
malonate ester groups in the efficient detoxication of JTT-130 to
the inactive acid metabolite 1 in the liver.
From a toxicological perspective, it is tempting to draw
parallels between the JTT-130 hydrolysis process resulting in the
liberation of electrophilic α,β-unsaturated carbonyl derivative
(atropic acid and its corresponding ethyl ester 5) byproduct and
the bioactivation pathway proposed for the anticonvulsant
felbamate, which is associated with significant incidences of
aplastic anemia and hepatotoxicity.31,32 The rate-limiting step in
felbamate bioactivation (Figure 12) is believed to involve an
amidase-mediated hydrolysis of one of the carbamate groups
to 2-phenyl-1,3-propanediol monocarbamate (7), which is sub-
sequently oxidized by alcohol dehydrogenase to 3-carbamoyl-
2-phenylpropionaldehyde (8). Aldehyde 8, which exists in
equilibrium with the cyclized 4-hydroxy-5-phenyl-tetrahydro-
1,3-oxazin-2-one (9) tautomer, is highly unstable (t1/2 < 30 s)
under physiological conditions and spontaneously degrades to
the electrophilic 2-phenylpropenal (atropaldehyde, 10) metab-
olite that adducts with GSH under nonenzymatic conditions
(pH 7.4 buffer) and in the presence of glutathione transferases
(Figure 12, pathway a).33−37 In addition, GSH conjugates of
atropic acid (also characterized as a metabolite of JTT-130)
have been characterized in human liver S9 incubations with the
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Figure 12. Bioactivation of the anticonvulsant felbamate to reactive species.
synthetic oxazin-2-one (9) metabolite of felbamate in the
presence of NAD+ and GSH (Figure 12, pathway b).38 In vivo
evidence for the occurrence of these bioactivation pathways has
been obtained via the characterization of urinary mercapturic
acid conjugates of atropic acid (and its reduced alcohol form)
following felbamate administration to rats and humans.33−35
Whether the sulfydryl conjugates of atropic acid observed in vivo
are derived from direct conjugation of GSH to the α,β-
unsaturated carboxylic acid or through initial adduction with
10 followed by further oxidation to atropic acid remains unclear.
As such, chemical reactivity studies with GSH reveal atropic acid
to be a significantly weaker electrophile compared to atro-
paldehyde, although the role of glutathione transferases as
catalysts of this reaction has yet to be explored.38 Regardless of
the differences in the relative Michael acceptor properties of the
α,β-unsaturated-aldehyde and -carboxylic acid, the formation of
GSH conjugates of atropic acid and the corresponding ethyl ester
5 in the course of JTT-130 hydrolysis can be viewed as an
undesirable feature from a toxicological perspective. The
formation of GSH conjugate(s), an indicator of reactive species
formation, is of particular concern due to the cumulative evi-
dence supporting the hypothesis that reactive metabolites can be
involved in direct organ toxicity as well as in idiosyncratic adverse
drug reactions, leading to suspension from clinical trials, black
box warnings, or, in some cases, withdrawal from the market.39
The potential for idiosyncratic immune-mediated toxicity arising
through adduction of atropic acid or the ethyl ester 5 to proteins
is too high of a risk, especially in a disease area with diverse
therapeutic options (e.g., statins and fibrates). In conclusion, the
mechanistic insights gathered from the present work on JTT-130
hydrolysis can be potentially utilized in the design of ester-based
gut MTP inhibitors that are incapable of yielding the electrophilic
acrylate species upon hydrolysis in the liver.
ASSOCIATED CONTENT
* Supporting Information
■
S
CID spectrum for the carboxylic acid metabolite (M1) and
extracted ion chromatograms depicting 18O incorporation in the
atropic acid metabolite obtained upon incubation of JTT-130 in
human liver microsomes. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: (617)-551-3336. E-mail: amit.kalgutkar@pfizer.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
MTP, microsomal triglyceride transfer protein; TG, triglyceride;
VLDL, very low density lipoprotein; JTT-130, diethyl 2-((2-(3-
(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-
ylcarboxamido)phenyl)acetoxy)methyl)-2-phenylmalonate; 1,
2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-bi-
phenyl]-2-ylcarboxamido)phenyl)acetic acid; 2, diethyl
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collisions of vibrationally excited cationic dicarboxylic acid clusters with
2-(hydroxymethyl)-2-phenyl malonate; 3, diethyl 2-(2-(2-(3-
(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1,1′-biphenyl]-2-
ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalonate; 4,
3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-
(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-
ylcarboxamido)phenyl)acetate; J, coupling constant; ESI,
electrospray ionization; GSH, glutathione; P450, cytochrome
P450; LC-MS/MS, liquid chromatography tandem mass
spectrometry; t1/2, half-life; tR, retention time; CID, collision-
induced dissociation; HSQC, multiplicity edited heteronuclear
single quantum coherence; HMBC, heteronuclear multiple bond
correlation; hCE-1, human carboxylesterase-1; hCE-2, human
carboxylesterase-2
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