Metabolic Study of Stable Isotope Labeled Indolinone Derivative in Hepatocyte Cell by UPLC/Q TOF MS

Article abstract of DOI:10.1021/jasms.1c00146

The aggregation process of α-synuclein (α-syn) is substantial in the pathogenesis of Parkinson's disease. Indolinone derivatives are inhibitors of α-syn aggregates and can be used as PET-based radiotracers for imaging α-syn fibrils. However, no investigations on the metabolism of indolinone derivatives have been reported until now. In the present research, a 13C and 15N isotope labeling strategy was developed to synthesize compound [13C2,15N]-(Z)-1-(4-aminobenzyl)-3-((E)-(3-phenyl)allylidene)indolin-2-one (M0′), which was then used in a study of metabolism in hepatocytes. The metabolites were characterized using accurate mass and characteristic ion measurements. In the metabolic system, compound M0′ was the main component (accounting for 97.5% of compound-related components) after incubation in hepatocytes for 3 h, which indicated that compound M0′ possessed great metabolic stability. Seven metabolites have been successfully verified by UPLC/Q TOF MS in metabolic studies, including hydroxyl M0′ (M1′), hydroxyl and methylated M0′ (M2′), N-acetylated M0′ (M3′), sulfate of hydroxyl M0′ (M4′), the glucose conjugate of M0′ (M5′), glucuronide conjugate of M0′ (M6′), and glucuronide conjugate of hydroxyl M0′ (M7′). The study on metabolism provides the important information to develop effective α-syn aggregate inhibitors and new PET-tracer-related indolinone derivatives.

Full text of DOI:10.1021/jasms.1c00146

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Research Article
Metabolic Study of Stable Isotope Labeled Indolinone Derivative in
Hepatocyte Cell by UPLC/Q TOF MS
Jixia Yang, Gongzheng Zhang, Zhaoyang Wang, Jian Meng,* and Hongliang Wen*
Cite This: J. Am. Soc. Mass Spectrom. 2021, 32, 1538−1544
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ABSTRACT: The aggregation process of α-synuclein (α-syn) is substantial in the pathogenesis of Parkinson’s disease. Indolinone
derivatives are inhibitors of α-syn aggregates and can be used as PET-based radiotracers for imaging α-syn fibrils. However, no
investigations on the metabolism of indolinone derivatives have been reported until now. In the present research, a 13C and 15N
isotope labeling strategy was developed to synthesize compound [13C2,15N]-(Z)-1-(4-aminobenzyl)-3-((E)-(3-phenyl)allylidene)-
indolin-2-one (M0′), which was then used in a study of metabolism in hepatocytes. The metabolites were characterized using
accurate mass and characteristic ion measurements. In the metabolic system, compound M0′ was the main component (accounting
for 97.5% of compound-related components) after incubation in hepatocytes for 3 h, which indicated that compound M0′ possessed
great metabolic stability. Seven metabolites have been successfully verified by UPLC/Q TOF MS in metabolic studies, including
hydroxyl M0′ (M1′), hydroxyl and methylated M0′ (M2′), N-acetylated M0′ (M3′), sulfate of hydroxyl M0′ (M4′), the glucose
conjugate of M0′ (M5′), glucuronide conjugate of M0′ (M6′), and glucuronide conjugate of hydroxyl M0′ (M7′). The study on
metabolism provides the important information to develop effective α-syn aggregate inhibitors and new PET-tracer-related
indolinone derivatives.
KEYWORDS: indolinone derivatives, α-synuclein, [13C2, 15N]-isotope labeling, metabolite, UPLC/Q TOF MS
different molecule weights, which is conducive to detection by
mass spectrometry.11−13 Stable isotope labeling is an effective
method of identifying drug metabolites.14−16 Based on the
INTRODUCTION
■
The metabolites generated in vivo have similar structures to the
parent drug and may possess potential pharmacological activity
or toxicity, so it is essential to identify metabolites in the early
stage of drug development.1−4 The liver is the most important
organ for metabolism in humans, and the hepatocytes contain
more metabolic enzymes compared with liver microsomes,
liver cytosolic fractions, and pooled human liver S9 fractions
and also can allow small-molecular-weight drugs (MW ∼ 500
Da) to enter in a variety of ways, such as passive diffusion,
active transport of transporters, and so on.5−7 Metabolic
studies are therefore usually carried out in vitro with human
hepatocytes before clinical studies.
It is crucial to identify metabolites for drug development, but
it is also difficult to detect and predict the presence and nature
of unknown metabolites due to endogenous interference from
proteins and lipids.8−10 Stable-isotope-labeled drugs could
retain the requisite physical and chemical properties but have
2
elements found in many drugs, stable isotopes H, 13C, 15N,
and 18O are usually candidates.17,18 However, significant
2
kinetic isotope effects with H may affect, or even change,
the metabolic pathway of drugs, and the 18O of any carbonyl
group might be exchanged with the 16O in H2O in the form of
harmonic alcohol.9,19 Thus, 13C and 15N are more suitable for
Received: April 23, 2021
Revised: May 14, 2021
Accepted: May 14, 2021
Published: May 24, 2021
© 2021 American Society for Mass
Spectrometry. Published by American
Chemical Society. All rights reserved.
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a
Scheme 1. Synthesis of M0′
a
Key: (a) DCM, DMF, 0 °C to rt, 4 h; (b) [15N]-aniline, DCM, triethylamine, 0 °C to rt, 1 h; (c) anhydrous alchlor, 230 °C, 15 min; (d)
cinnamaldehyde, piperidine, ethanol, reflux, 1 h; (e) 4-nitrobenzyl bromide, 60% NaH, DMF, rt, 0.5 h; (f) Fe/HCl, methanol, reflux, 2 h.
use in stable isotope labeling than 2H and 18O for the
identification of metabolites.
The preparations of compounds 1−3 were described in our
previous report.26 The detailed synthetic steps for compounds
4−6 and M0′ are shown in S1. Compounds 4−6 and M0′
were purified by column chromatography, and the structures of
compounds 4−6 and M0′ were further confirmed by 1H NMR
and HRMS (Figures S1−S4). The isotopic enrichment in
compound M0′ was approximate 99%.
Parkinson’s disease (PD) is the second most common
progressive neurodegenerative disorder, which threatens
approximately 2% of the population over the age of 60.20−22
The aggregation of α-syn is key to PD pathogenesis.23,24
Indolinone derivatives showed activity and selectivity toward
α-syn fibrils as demonstrated by Mach et al. Furthermore, the
indolinone analogues were also developed as PET-based
radiotracers for imaging α-syn fibrils.25 Although indolinone
derivatives showed potential in the treatment of PD, the
metabolite study of indolinone derivatives has never been
reported before. (Z)-1-(4-Aminobenzyl)-3-((E)-(3-phenyl)-
allylidene)indolin-2-one (M0) is our candidate compound
developed for treatment of PD, which can inhibit α-syn fibrils
to a significant extent in terms of measured activity (data not
published). The present study is to synthesize and characterize
the stable labeled indolinone derivative [13C2,15N]-(Z)-1-(4-
aminobenzyl)-3-((E)-(3-phenyl)allylidene)indolin-2-one
(M0′) and then profile the metabolites in human hepatocytes
using the stable labeled compound by UPLC/Q TOF MS.
In Vitro Samples Incubation and Pretreatment.
Hepatocytes metabolism tests in vitro were carried out by
undergoing the method from ref 7. Briefly, the hepatic cell line
was cultured in William’s Medium E (pH 7.4) at a density of
1.0 × 106 cells/mL under a volume of 100 μL. Hepatocytes
were treated with 10 μM labeled or unlabeled compound in a
37 °C incubator. After 3 h, cold acetonitrile (200 μL) was
added to quench the reaction. The test samples were stored at
−70 °C.
The incubated hepatocytes were vortexed and centrifuged at
14000 rpm for 5 min. The supernatant was completely
collected and blown dry with nitrogen stream at 40 °C. The
dried residues were redissolved in acetonitrile−water (200 μL,
50:50, v/v). The labeled and unlabeled samples were taken to
analyze by UPLC/Q TOF MS.
UPLC/Q TOF MS Analysis. Chromatographic separation
for the metabolite profiling and identification were conducted
on an Acquity UPLC HSS T3 column (100 × 2.1 mm, i.d., 1.8
μm) at 45 °C using an Acquity UPLC system (Waters,
Milford, MA). The mobile phase consisted of 0.02% formic
acid in ammonium acetate (5 mM, elution phase A) and
acetonitrile (elution phase B). The elution duration was set as
30 min, and elution gradient was as follows: 90% A−10% B
from 0 to 20 min, 30% A−70% B from 20 to 25 min, 10% A-
90% B from 25 to 27 min, 10% A-90% B from 27 to 30 min.
MS detection was achieved using a quadrupole TOF 5600+
MS/MS system (AB Sciex, Concord, Ontario, Canada) in the
positive electrospray ionization (ESI) mode. The mass range
was set at m/z 50−1000. The parameters were set as follows:
ion spray voltage, 5500 V; declustering potential, 60 V; ion
source heater, 550 °C; curtain gas, 40 psi; ion source gas 1, 55
psi; and ion source gas 2, 55 psi. For the TOF MS scans, the
EXPERIMENTAL SECTION
■
Chemicals and Reagents. [13C2]-Chloroacetic acid with
isotopic enrichment for 99% and [15N]-aniline with isotopic
enrichment for 100% were purchased from Cambridge Isotope
Laboratories (Andover, MA). Cryopreserved human hepato-
cytes for the metabolite incubation were obtained from BD
Gentest (Woburn, CA). All solvents for the ultraperformance
liquid chromatography (UPLC)/quadrupole time-of-flight
mass spectrometry (Q TOF MS) analyses were of high-
performance liquid chromatography grade (Merck, Darmstadt,
Germany). Ultrapure water was generated using a Milli-Q
Gradient system (Millipore Corp., Molsheim, France). Other
reagents were analytical grade and used without further
purification.
Synthesis of Compound M0′. As shown in Scheme 1,
compound M0′ was prepared through a six-step reaction with
[13C2]-chloroacetic acid and [15N]-aniline as starting materials.
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Figure 1. MS and MS/MS spectra of M0 and M0′. (A) MS spectrum of M0, (B) MS/MS spectrum of M0, (C) MS spectrum of M0′, (D) MS/MS
spectrum of M0′.
Figure 2. Metabolites profiling of M0′ after incubation with human hepatocytes for 3 h at 37 °C by UPLC/Q TOF MS. The inset is the expanded
chromatogram in the region of 7−18 min.
collision energy was 10 eV. For product ion scans, the collision
energy was 15 eV, with a spread of 15 eV in the MS/MS
experiment. Information-dependent acquisition (IDA) was
used to trigger the acquisition of MS/MS spectra for ions
matching the IDA criteria. A real-time multiple mass defect
filter was used for the IDA criteria. The automatic calibration
delivery system (CDS) was used to correct of MS and MS/MS
by external standard method. The resolutions for MS or MS/
MS analysis were maintained above 30000 or 15000−20000.
Data Analysis and Metabolites Identification. Metab-
olites were identified and characterized by Metabolite Pilot 2.0
software with multiple postprocessing functions. Comparing of
the raw data, we could acquire the information on probable
metabolites products, such as biotransformation, molecular
formula, retention time, m/z values, and relative error (ER) by
Metabolite Pilot 2.0 software (AB Sciex, USA). According to
the full-scan MS spectra and MS/MS spectra of the probable
metabolites and special focus on the mass shift resulted from
stable labeling, the structure characterization of compound
M0′ was analyzed and the probable chemical structures of the
metabolites were proposed.
material. Therefore, the [13C2,15N] isotope labeling strategy
was developed to aid in the detection and identification of the
metabolites of the indolinone derivative. Both labeled and
unlabeled compounds were treated with hepatocytes, respec-
tively. The molecular formulas of potential metabolites could
be determined according to accurate high-resolution full scan
mass data. Then the observed product ions were initially
compared with those of parent compounds to assess structural
similarities and changes within the metabolites. These tests
were performed with the aid of the mass difference resulting
from the stable-isotope labeling.
First, the standards of compounds M0′ and M0 were
analyzed by UPLC/Q TOF MS in positive-ion mode. Figure 1
shows the MS and MS/MS spectra of compounds M0 and
M0′, in which M0 and M0′ gave a protonated molecular ion at
m/z 353.1666 and 356.1702, indicating that their elemental
compositions were C24H21N2O and 13C2C22H2115NNO,
respectively. The unlabeled compound M0 produced three
major characteristic product ions at m/z 260.1078, 248.1086,
and 106.0650; the labeled compound M0′ produced
corresponding product ions at m/z 263.1117, 251.1122, and
106.0654. The product ions at m/z 263.1117 and 251.1122
were formed by the loss of the aniline moiety and the p-
toluidine group, respectively. The product ion at m/z 106.0654
was obtained from the p-toluidine group.
RESULTS AND DISCUSSION
■
Strategy for Metabolite Discovery and Identification.
The identification of metabolites in complex biological
matrices is challenging due to the presence of an excess of
proteins, lipids, and other endogenous materials which
potentially interfere with the detection of drug-derived
After incubation with human hepatocytes, the metabolites of
compounds M0′ and M0 were detected by UPLC/Q TOF MS
and seven metabolites were identified (Figures 2 and S5,
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Figure 3. MS (A), MS/MS spectra (B), and fragmental pathways (C) of M1′.
Figure 4. MS (A), MS/MS spectra (B), and fragmental pathways (C) of M2′.
Figure 5. MS (A), MS/MS spectra (B), and fragmental pathways (C) of M4′.
respectively). M1′ was eluted at 13.17 min, with a protonated
molecular mass of m/z 372.1622 (Figure 3). The elemental
composition of M1′ was 13C2C22H2115NNO2, indicating the
addition of an oxygen to M0′. The fragment ion of M1′ at m/z
106.0651 was the same as that of M0′. The fragment ions at
m/z 279.1069 and 267.1096 were 16 Da bigger than those of
M0′. According to the proposed fragmental pathways, M1′
could be formed through the aromatic hydroxylation or the
oxidation of the alkene to an epoxide. However, the sulfate of
hydroxyl M0′ (M4′) and the glucuronide of hydroxyl M0′
(M7′) were detected as downstream metabolites of M1′;
therefore M1′ was proposed to be the phenol metabolite of
M0′, but the precise oxidative location could not be
determined. As expected, M1 formed from M0 was also
detected with the protonated molecular mass at m/z 369.1580
(a mass-shift of 3 Da from m/z 372.1622) and the product
ions at m/z 276.1032 and 264.1016 (a mass-shift of 3 Da from
m/z 279.1069 and 267.1096, respectively) and 106.0535 (no
mass-shift) (Figure S6). Furthermore, M1′ and M1 shared the
same retention time; therefore, M1′ was further confirmed as a
phenol metabolite.
M2′ was eluted at 16.95 min, with a protonated molecular
mass of m/z 386.1785 (Figure 4). The elemental composition
of M2′ was 13C2C23H2315NNO2, indicating that the addition of
OCH2 to M0′. The fragment ion of M2′ at m/z 106.0655 was
the same as that of M0′. The fragment ions at m/z 293.1229
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Figure 6. Metabolic pathways of M0′.
Table 1. Summarized Information of Metabolites from M0′
metabolite code
metabolic pathways
parent compound
percent (%)
tR (min)
molecular formula
13C2C22H2115NNO+
exact mass (m/z)
observed mass (m/z)
M0′
M1′
M2′
M3′
M4′
M5′
M6′
M7′
97.5
0.48
0.50
0.09
0.24
0.45
0.60
0.14
17.21
13.17
16.95
16.39
9.71
356.1722
372.1671
386.1827
398.1827
452.1239
518.2250
532.2043
548.1993
356.1702
372.1622
386.1785
398.1783
452.1166
518.2186
532.1977
548.1919
13C2C22H2115NNO2
13C2C23H2315NNO2
13C2C24H2315NNO2
+
oxidation
+
+
oxidation and methylation
acetylation
oxidation and sulfation
glucosylation
13C2C22H2115NNO5S+
13C2C28H3115NNO6
13C2C28H2915NNO7
13C2C28H2915NNO8
+
12.89
11.53
7.68
+
glucuronidation
+
oxidation and glucuronidation
and 281.1217 were 30 Da bigger than those of M0′. The
formation of the fragment ion at m/z 145.0658 was attributed
to cleavage of the 13C−C double bond; therefore, the
formation of M2′ was attributed to O-methylation following
the aromatic hydroxylation of M0′, and the result was also
verified by the detection of M2 from M0. Similarly, M3′, M5′,
and M6′ were identified as the metabolites formed via N-
acetylation, N-glucosylation, and N-glucuronidation of M0′,
respectively (Figures S7 and S6).
Therefore, seven metabolites of M0′ were identified after the
verification of the detected metabolites of M0. The proposed
metabolic pathways of M0′ are illustrated in Figure 6 and a
summary of information pertaining to those metabolites
identified is presented in Table 1. After incubation with
human hepatocytes for 3 h at 37 °C, there was approximately
97.5% of the M0′ detected, demonstrating the metabolic
stability of M0′. In vitro, M0′ was primarily metabolized by
hydroxylation, and secondarily by methylation, acetylation,
sulfation, glucosylation, and glucuronidation, etc.
M4′ was eluted at 9.71 min, with a protonated molecular
mass of m/z 452.1166 (Figure 5). The elemental composition
of M4′ was 13C2C22H2115NNO5S, indicating that the addition
of SO4 to M0′. M4′ was proposed as the sulfate of the
hydroxyl M0′. The fragment ion of M4′ at m/z 106.0637 was
the same as that of M0′. The fragment ion at m/z 279.1069
was 16 Da bigger than that of M0′. The fragment ions at m/z
359.0615 and 347.0627 were 96 Da bigger than those of M0′,
suggesting that M4′ was not the N-sulfate, but the O-sulfate of
M0′. After verification of the detected M4 having been formed
from M0 (Figure S6), M4′ was identified as the O-sulfate of
the hydroxyl M0′. Similarly, M7′ was identified as the O-
glucuronide of the hydroxyl M0′ (Figure S7).
CONCLUSIONS
■
Compound M0′ was synthesized and used in the investigation
of metabolism in hepatocytes for the first time. The
metabolites were studied by UPLC/Q TOF MS, which were
verified by unlabeled compound M0. Seven metabolites were
preliminary discovered and confirmed, including hydroxyl M0′
(M1′), hydroxyl and methylated M0′ (M2′), N-acetylated
M0′ (M3′), sulfate of hydroxyl M0′ (M4′), the glucose
conjugate of M0′ (M5′), glucuronide conjugate of M0′ (M6′),
and glucuronide conjugate of hydroxyl M0′ (M7′). After
incubation with human hepatocytes for 3 h at 37 °C,
compound M0′ was the main component, accounting for
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good metabolic stability. This study contributed to establishing
an efficient and rapid method for analysis and identification of
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jasms.1c00146.
■
sı
Detailed synthetic steps for compounds 4−6 and M0′;
1H NMR and HRMS of compounds 4−6 and M0′;
metabolites profiling of M0 after incubation with human
hepatocytes for 3 h at 37 °C by UPLC/Q TOF MS; MS
and MS/MS spectra of the metabolites formed from
M0; MS, MS/MS spectra, and fragmental pathways of
the metabolites formed from M0′(PDF)
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AUTHOR INFORMATION
Corresponding Authors
Jian Meng − Shanghai Institute of Materia Medica Chinese
Academy of Sciences, Shanghai 201203, P.R. China;
Email: goodwill_123@163.com
■
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Hongliang Wen − Key Laboratory of Medical Molecule
Science and Pharmaceutics Engineering, Ministry of Industry
and Information Technology, School of Chemistry and
Chemical Engineering, Beijing Institute of Technology, Beijing
102488, P.R. China; Email: wen.hongliang@bit.edu.cn
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Authors
Jixia Yang − Key Laboratory of Medical Molecule Science and
Pharmaceutics Engineering, Ministry of Industry and
Information Technology, School of Chemistry and Chemical
Engineering, Beijing Institute of Technology, Beijing 102488,
P.R. China; orcid.org/0000-0003-0446-231X
Gongzheng Zhang − Key Laboratory of Medical Molecule
Science and Pharmaceutics Engineering, Ministry of Industry
and Information Technology, School of Chemistry and
Chemical Engineering, Beijing Institute of Technology, Beijing
102488, P.R. China
Zhaoyang Wang − Key Laboratory of Medical Molecule
Science and Pharmaceutics Engineering, Ministry of Industry
and Information Technology, School of Chemistry and
Chemical Engineering, Beijing Institute of Technology, Beijing
102488, P.R. China
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Tracer Techniques to Increase Efficiency of Clinical Pharmacokinetic
Trials in Oncology. Drugs R&D 2020, 20 (2), 147−154.
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jasms.1c00146
(17) Berry, D.; Stecher, B.; Schintlmeister, A.; Reichert, J.;
Brugiroux, S.; Wild, B.; Wanek, W.; Richter, A.; Rauch, I.; Decker,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Beijing Institute of Technology Analysis &
Testing Center for spectral data of H NMR and HRMS.
■
1
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