High-resolution mass spectrometry-based approach for the
identification and profiling of the metabolites of taletrectinib
formed in liver microsomes
Yongbin Ye | Xiaojuan Guo | Xin He | Mingwan Zhang | Huiqing He |
Dafa Qiu | Ziwen Guo
Department of Hematology, Zhongshan
Hospital of Sun Yat-Sen University &
Zhongshan City People’s Hospital, Zhongshan,
China
Correspondence
Ziwen Guo, Department of Hematology,
Zhongshan Hospital of Sun Yat-Sen
University & Zhongshan City People’s
Hospital, No. 2 East Sunwen Road. Zhongshan
528403, China.
Email: [email protected]
Abstract
Taletrectinib is a potent, orally active, and selective ROS1/NTRK kinase inhibitor.
The aim of this study was to study the metabolism of taletrectinib in rat, dog, and
human liver microsomes. The biotransformation of taletrectinib was carried out using
rat, dog, and human liver microsomes supplemented with nicotinamide adenine
dinucleotide phosphate tetrasodium salt (NADPH) and uridine diphosphate
glucuronic acid (UDPGA). The microsomal incubations were conducted at 37
C for
60 min. The formed metabolites were identified by ultrahigh performance
liquid chromatography coupled to high-resolution tandem mass spectrometry
(UHPLC-HRMS) using electrospray ionization in the positive ion mode. They were
identified by accurate masses and MS/MS spectra and based on their fragmentation
pathways. With UHPLC-HRMS, a total of 10 metabolites including one glucuronide
conjugate (M7) were structurally identified. M9 and M10 were unambiguously identi￾fied as taletrectinib alcohol and taletrectinib ketone, respectively, using reference
standards. The phase I metabolic pathways of taletrectinib involved N-dealkylation,
O-dealkylation, oxidative deamination, and oxygenation; the phase II metabolic
pathways referred to glucuronidation. The current study investigated the in vitro
metabolic fate of taletrectinib in animals and human species, which would bring us
considerable benefits for the subsequent studies focusing on the pharmacological
effect and toxicity of this drug.
KEYWORDS
liver microsomes, metabolic pathways, metabolite identification, Taletrectinib
1 | INTRODUCTION
Taletrectinib is a novel and new generation ROS1/NTRK kinase inhibi￾tor, which is a potent and selective ROS1 and TRK family inhibitor
capable of inhibiting ROS1 G2032R and other crizotinib-resistant
ROS1 mutants.1 It has been demonstrated that taletrectinib displayed
an enzymatic inhibition (IC50) against ROS1, NTRK1, NTRK2, and
NTRK3 at 0.207, 0.622, 2.28, and 0.98 nM, respectively.1 Currently,
taletrectinib is under clinical development in United States and
Japan.2 A clinical experiment showed that taletrectinib was well toler￾ated in Japanese patients with nonsmall cell lung cancer (NSCLC) and
effective in crizotinib treatment-native patient,2 and the maximum tol￾erated dose was 800 mg once daily.3 Taletrectinib may be an effective
agent for the pretreatment NSCLC patients with ROS1 fusions.2
There are limited reports regarding the identification and profiling
Yongbin Ye and Xiaojuan Guo are authors who contributed equally to this work. of the metabolites of taletrectinib. Drug metabolism plays crucial role
Received: 10 November 2020 Revised: 25 January 2021 Accepted: 27 January 2021
Drug Test Anal. 2021;1–9. wileyonlinelibrary.com/journal/dta © 2021 John Wiley & Sons, Ltd. 1
pharmacokinetics and drug safety assessment as some metabolites may
be pharmacologically active, reactive, or toxic.4–6 Therefore, fast and
accurate detection and identification of the metabolites of taletrectinib
is of great importance for further development. Mass spectrometry￾based approach is widely used in detecting and identifying drug metabo￾lites in vitro and in vivo.7 High-resolution mass spectrometers, such as
Orbitrap mass spectrometers, can provide accurate molecular weight
and reliable structural information of drugs and their metabolites, which
facilitates the metabolite identification and profiling.8–11
The main aims of this study was (1) to perform detailed
metabolite profiling of taletrectinib and to detect and characterize its
metabolites produced in rat, dog, and human liver microsomes using
high-resolution mass spectrometry (HRMS) combined with ultrahigh
performance liquid chromatography (UHPLC); (2) to propose the
possible metabolic pathways in vitro; and (3) to disclose the species
difference of the metabolism of taletrectinib between animals and
humans. Under the current conditions, a total of 10 metabolites were
detected and identified. As far as we know, this is the first report on
the metabolite identification and profiling of taletrectinib.
2 | MATERIALS AND METHODS
2.1 | Chemicals and reagents
Taletrectinib (purity >98%) was purchased from BiochemPartner
(Shanghai, China). The reference standards of taletrectinib alcohol
(M9) and taletrectinib (M10) ketone were synthesized in our labora￾tory, of which the structures were confirmed by nuclear magnetic res￾onance spectroscopic analysis. The 13C-NMR data of M9 and M10
were compared with those of taletrectinib. M9 and M10 showed simi￾lar 13C-NMR data to taletrectinib, except for the side chain. Compared
with taletrectinib (ω-1 carbon, δc 48.5), the signal of ω-1 carbon of
M10 and M9 downshifted to δc 205.1 and to 67.3, respectively,
suggesting the presence of carbonyl and alcohol groups in the mole￾cules of M10 and M9, respectively. Sprague–Dawley rat (RLM), beagle
dog (DLM), and human (HLM) liver microsomes were purchased from
BD Gentest (Woburn, MA, USA). Nicotinamide adenine dinucleotide
phosphate tetrasodium salt (NADPH), uridine diphosphate glucuronic
acid (UDPGA), alamethicin, and MgCl26H2O were purchased from
Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile was pur￾chased from Merck (Darmstadt, Germany). LC–MS (Liquide chroma￾tography tandem mass) grade formic acid was supplied by Thermo
Fisher Scientific. Deionized water was prepared by a Milli-Q purifica￾tion system (Millipore Corp., Bedford, MA, USA).
2.2 | Microsomal incubation
The liver microsomes (0.5 mg protein/ml) were suspended in 100 mM
phosphate buffer (pH 7.4) containing NADPH (2 mM), UDPGA (2 mM),
MgCl2 (3 mM), and alamethicin (25 μg/ml). The total incubation volume
was 0.2 ml. The mixtures were pre-incubated at 37
C for 5 min, and
then taletrectinib (3 μM) was added to the incubations to start the reac￾tions. Incubations without taletrectinib served as blank controls. The
total volume of organic solvent was less than 0.5% (v/v). After incuba￾tion at 37
C for 60 min, the reactions were terminated by adding 1.0 ml
of ice-cold acetonitrile. Then the samples were centrifuged at 20,000 g
for 10 min, and the resulting supernatants were transferred into another
tubes and dried under nitrogen gas. The residues were reconstituted
using 0.2 ml of acetonitrile–water solution (1:9, v/v). An aliquot of 2 μl
was submitted to UHPLC-HRMS system for analysis.
2.3 | Instrument and analytical conditions
LC–MS analyses were conducted on a Dionex Ultimate UHPLC system
(Thermo Fisher Scientific) coupled to a Q-Exactive Orbitrap tandem mass
spectrometer (Thermo Fisher Scientific) equipped with an electrospray
ionization source (ESI) operated in positive ion mode. The UHPLC
system was supplied with a set of binary solvent manager, column man￾ager, on-line degasser, and sample manager. The effective chromato￾graphic separation was executed on an ACQUITY UPLC BEH C18
column (100 × 2.1 mm; Waters Corporation, MA, USA) kept at a con￾stant temperature of 45
C, using a gradient elution of 0.1% formic acid
in water (solvent A) and acetonitrile (solvent B) at a flow rate of
0.25 ml/min. The gradient conditions were optimized as follows: 5% B at
0–1 min, 5%–20% B at 1–5 min, 20%–30% B at 5–10 min, 30%–90% B
at 10–13 min, 90% B at 13–15 min, and finally 5% B at 15–17 min. The
autosampler was kept at 10
C, and the injection volume was 2 μl.
The ESI source parameters were optimized as follows: spray volt￾age, 3.0 kV; capillary temperature, 200
C; sheath gas flow rate,
40 arb; auxiliary gas flow rate, 10 arb; sweep gas flow rate, 5 arb;
sheath gas heater temperature, 150
C; and S-Lens voltage, 50 V. The
data were obtained in the m/z range of 50–1000 Da in centroid mode
with a resolution of 70,000 FWHM (Full width at half maxima). The
collision energy was 30 V. All the data were acquired with Xcalibur
software (Version 2.3.1, Thermo Fisher Scientific).
2.4 | Data processing and analysis
The acquired data were processed by MetWorks software (version
1.3 SP3, Thermo Fisher Scientific) whereby mass defect filter (MDF)
and background subtraction analyses were performed by comparing
the total ion chromatograms of taletrectinib-containing samples with
those of controls. The tolerance of MDF was within 5 ppm.
3 | RESULTS AND DISCUSSIONS
3.1 | UHPLC-HRMS analysis of taletrectinib
First, the parent compound taletrectinib was subjected to
UHPLC-HRMS for analysis. Taletrectinib was detected at the
retention time of 6.63 min with a protonated molecular ion [M + H]+
2 YE ET AL.
at m/z 406.2045 (1.7 ppm, elemental composition C23H25FN5O). Main
fragment ions (Figure 1a) were obtained at m/z 349.1465, 284.1511,
227.0930, 212.0812, and 123.0608 in product ion scan mode. The
possible fragmentation pathways were illustrated in Figure 1b. The
fragment ion at m/z 349.1465 was formed by the cleavage of
isopropylamine moiety (-C3H7N). The fragment ion at m/z 284.1511
was formed by the loss of 1-ethyl-3-fluorobenzene moiety (-C8H8F).
This ion further produced the ions at m/z 227.0930 through the
loss of isopropylamine moiety (-C3H7N). The fragment ion at m/z
123.0608 was attributed to 1-ethyl-3-fluorobenzene moiety (C8H8F).
These fragment ions were the indicative fragment ions of taletrectinib,
which can be used to rapidly identify the metabolites of this drug.
3.2 | UHPLC-HRMS analysis of the microsomal
incubation samples
Metabolites were generated by incubating taletrectinib with rat, dog, and
human liver microsomes. Metabolic reactions catalyzed by CYP and uri￾dine diphosphoglucuronosyltransferases (UGT) enzymes were followed.
The reaction mixtures were analyzed by UHPLC-HRMS in positive ion
mode for the metabolites identification and profiling. The total ion chro￾matograms of incubation mixtures were compared with those of incuba￾tions without taletrectinib. Figure 2 displayed the MDF chromatograms
of the metabolites from different species. With high-resolution mass, the
accurate masses of the metabolites and their fragment ions were deter￾mined and elemental compositions measured. Table 1 summarized the
detailed information on the metabolites detected in different species.
The identities of the metabolites were proposed on the basis of
chromatographic retention times, MS/MS fragment ions, and elemental
composition inferred through HRMS. The identities of metabolites M9
and M10 were verified with reference standards.
3.3 | Structural elucidation
3.3.1 | Metabolite M1
M1 was detected at the retention time of 2.45 min. The protonated
molecular ion [M + H]+ was observed at m/z 284.1512, suggesting
FIGURE 1 MS/MS spectrum (a) and proposed fragmentation patterns (b) of taletrectinib
YE ET AL. 3
FIGURE 2 Ultrahigh performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS)
chromatograms of taletrectinib and the metabolites in liver microsomes from different species
TABLE 1 Summary of the metabolites of taletrectinib in microsomal incubations detected by UHPLC-HRMS
M1 2.45 N-dealkylation C15H17N5O 284.1512 2.1 105,669,782 5,629,706 6,979,924
M2 5.24 N-dealkylation and oxidative
deamination
C15H16N4O2 285.1351 1.8 91,440,675 6,233,722 6,536,320
M3 5.33 N-dealkylation, oxidative
deamination and
oxygenation
C15H14N4O2 283.1194 1.4 6,842,767 N. D. N. D.
M4 5.67 Oxygenation C23H24FN5O2 422.1994 1.7 20,254,234 N. D. N. D.
M5 7.12 Oxygenation C23H24FN5O2 422.1995 1.9 256,562,987 9,579,848 91,460,055
M6 8.31 N-dealkylation, oxidative
deamination and
oxygenation
C15H17N5O2 300.1460 1.7 5,599,874 6,936,475 3,698,474
M7 9.04 Oxidative deamination and
glucuronidation
C29H31FN4O8 583.2207 1.4 N. D. 7,456,397 N. D.
M8 9.91 O-dealkylation C20H17FN4O 349.1465 1.7 25,367,858 12,695,481 9,025,485
M9 10.84 Oxidative deamination C23H23FN4O2 407.1885 1.7 36,987,851 66,984,789 45,863,364
M10 11.15 Oxidative deamination and
oxygenation
C23H21FN4O2 405.1728 1.7 365,488,776 56,875,299 10,982,254
Parent 6.63 Parent C23H24FN5O 406.2045 4.2 345,517,737 987,598,991 141,490,537
Abbreviations: UHPLC-HRMS, Ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry; N.D., not detected.
4 YE ET AL.
that this metabolite was N-dealkylation product of parent. The
MS/MS spectrum (Figure 3a) showed an indicative fragment ion at
m/z 227.0931, which was formed by the cleavage of isopropylamine
moiety (-C3H7N).
3.3.2 | Metabolite M2
M2 was detected at the retention time of 5.24 min. The protonated
molecular ion [M + H]+ was observed at m/z 285.1351, 2.0157 Da
higher than that of M3, suggesting that M2 was derived from M3
through reduction (+2H). The MS/MS spectrum (Figure 3b) showed
an indicative fragment ion at m/z 226.0853, which was formed by the
cleavage of isopropyl alcohol moiety (-C3H7O).
3.3.3 | Metabolite M3
M3 was detected at the retention time of 5.33 min. The protonated
molecular ion [M + H]+ was observed at m/z 283.1194, 1.0318 Da
lower than that of M1, suggesting that M3 was derived from M1
through oxidative deamination (-NH2 + OH-2H). The MS/MS
spectrum (Figure 3c) showed an indicative fragment ion at m/z
226.0853, which was formed by the cleavage of acetone moiety
(-C3H5O).
3.3.4 | Metabolite M4
M4 was detected at the retention time of 5.67 min. The protonated
molecular ion [M + H]+ was observed at m/z 422.1994, 15.9949 Da
higher than that of parent, suggesting that M4 was hydroxylation
product of parent. The MS/MS (Figure 3d) spectrum showed three
informative fragment ions at m/z 365.1414, 284.1510, and 227.0931,
which were identical to those of parent, suggesting that hydroxylation
occurred at 1-ethyl-3-fluorobenzene moiety.
3.3.5 | Metabolite M5
M5 was detected at the retention time of 7.12 min. The protonated
molecular ion [M + H]+ was observed at m/z 422.1995, 15.9950 Da
higher than that of parent, suggesting that M5 was oxygenation
product of parent. The MS/MS spectrum (Figure 4a) showed three
FIGURE 3 MS/MS spectra of M1 (a), M2 (b), M3 (c), and M4 (d)
YE ET AL. 5
informative fragment ions at m/z 348.1386, 300.1460, and 243.0880,
which were 16 Da higher than those of parent, suggesting that the
moieties of 1-ethyl-3-fluorobenzene and isopropylamine remained
unmodification. It should be noted that M5 was eluted latter than par￾ent, suggesting the decreased alkalinity. Therefore, M5 was proposed
as the lactam derivative of the parent.
3.3.6 | Metabolite M6
M6 was detected at the retention time of 8.31 min. The protonated
molecular ion [M + H]+ was observed at m/z 300.1460, 15.9948 Da
higher than that of M1, suggesting that M6 was oxygenation product
of parent. The MS/MS spectrum (Figure 4b) showed an informative
fragment ion at m/z 243.0880, which was 16 Da higher than that of
M1, suggesting that M6 was oxygenation product of M1.
3.3.7 | Metabolite M7
M7 was detected at the retention time of 9.04 min. The protonated
molecular ion [M + H]+ was observed at m/z 583.2207, 176.0322 Da
higher than that of M9, suggesting that M7 was glucuronidation prod￾uct of M9. The MS/MS spectrum (Figure 4c) showed characteristic
neutral loss of glucuronyl (−176 Da), resulting in the fragment ion at
m/z 407.1882. Therefore, M7 was identified as glucuronidation prod￾uct of M9.
3.3.8 | Metabolite M8
M8 was detected at the retention time of 9.91 min. The proton￾ated molecular ion [M + H]+ was observed at m/z 349.1465,
57.0580 Da lower than that of the parent, suggesting that M8 was
O-dealkylation product of the parent. The MS/MS spectrum
(Figure 4d) showed two indicative fragment ions at m/z 227.0931
and 123.0606, which further confirmed the loss of isopropylamine
moiety.
3.3.9 | Metabolite M9
M9 was detected at the retention time of 10.84 min. The protonated
molecular ion [M + H]+ was observed at m/z 407.1885 (elemental
FIGURE 4 MS/MS spectra of M5 (a), M6 (b), M7 (c), and M8 (d)
6 YE ET AL.
composition C23H24FN4O2), 2.0157 Da higher than that of M10,
suggesting that M9 was reduction (+2H) product of M10. The MS/MS
spectrum (Figure 5a) showed a characteristic ion at m/z 349.1460,
which was formed by the loss of isopropyl alcohol moiety (-C3H7O).
The fragment ion at m/z 285.1352 was formed by the loss of 1-ethyl-
3-fluorobenzene moiety (-C8H8F). To further confirm the structure,
the reference standard of M9 was synthesized. M9 showed identical
retention time, accurate mass, and fragment ions to those of
taletrectinib alcohol under the same analytical conditions. Therefore,
M9 was unambiguously identified as taletrectinib alcohol.
FIGURE 5 MS/MS spectra of M9 (a) and M10 (b)
FIGURE 6 Proposed metabolic pathways of taletrectinib in vitro
YE ET AL. 7
3.3.10 | Metabolite M10
M10 was detected at the retention time of 11.15 min, which
displayed a protonated molecular ion [M + H]+ at m/z 405.1728
(elemental composition C23H22FN4O2), 1.0317 Da lower than that of
the parent, suggesting that M10 was oxidative deamination
(-NH2 + OH-2H) product of the parent. The MS/MS spectrum
(Figure 5b) showed a characteristic fragment ion at m/z 283.1193,
which was formed by the loss of 1-ethyl-3-fluorobenzene moiety
(-C8H8F). The ion at m/z 348.1385 was formed by the loss of
acetone moiety (-C3H5O). To further confirm the structure, the
reference standard of M10 was synthesized. M10 showed
identical retention time, accurate mass, and fragment ions to those of
taletrectinib ketone under the same analytical conditions. Therefore,
M10 was unambiguously identified as taletrectinib ketone.
3.4 | Metabolite profile and metabolic pathways
Based on the UHPLC-HRMS, a total of 10 metabolites from rat, dog,
and human liver microsomes exposure to taletrectinib were
structurally proposed. The metabolites M1, M4, M5, M8, and M10
were primary metabolites, while M2, M3, M6, M7, and M9 were
secondary metabolites. The metabolic pathways were proposed in
Figure 6. The first pathway is oxidative deamination, resulting in the
formation of ketone derivative (M10), which is further subject to
reduction to form alcohol derivative (M9) and the latter further
undergoes glucuronidation to yield glucuronide conjugate (M7). The
second pathway is N-dealkylation, yielding the formation of
N-dealkylated taletrectinib (M1). This metabolite further undergoes
oxidative deamination (M2 and M3) or oxygenation (M6). The third
pathway is O-dealkylation to form O-dealkylated taletrectinib. The
fourth pathway is oxygenation to produce oxygenated metabolites
(M4 and M5). In the current study, sulfation pathway was not
evaluated. Further study will be conducted using hepatocyte to
evaluate whether taletrectinib undergoes sulfation.
After 1 h incubation, the remaining of taletrectinib was 28%,
66%, and 41% for rat, dog, and human, respectively. The present
study provides insight into the metabolic profiles of taletrectinib in
liver microsomes from different species. The species difference
between animals and humans was found, as summarized in Table 1.
In rat, a total of nine metabolites (M1–M6 and M8–M10) were
detected, including two rat-specific metabolites (M3 and M4).
Among these metabolites, M1, M2, M5, and M10 were the most
abundant metabolites (Table 1). In dog, eight metabolites (M1–M2
and M5–M10) were identified. All the metabolites were minor, and
M7 was dog specific. In human, seven metabolites (M1–M2,
M5–M6, and M8–M10) were identified. Among these metabolites
(M5) was the most abundant metabolite, and no human-specific
metabolites were found. The observed metabolic difference would
be attributed to the different isoform compositions, expression, and
catalytic activities of drug-metabolizing enzymes between animals
and humans. The findings in the present study would be helpful for
the experimental design and assessment of metabolism-mediated
toxic risk of taletrectinib.
4 | CONCLUSIONS
In summary, in vitro metabolism of taletrectinib in liver microsomes
was investigated via UHPLC-HRMS approach. Ten metabolites includ￾ing nine phase I and one phase II metabolites were identified and
structurally characterized according to accurate mass measurements,
MS/MS fragment ions, and retention times. The identities of M9
and M10 were verified by comparing with reference standards.
Taletrectinib is metabolized mainly through oxidative deamination,
O-dealkylation, N-dealkylation, oxygenation, and glucuronidation. This
study is the first to elucidate the metabolic fate of taletrectinib in liver
microsomes, which may guide subsequent studies on taletrectinib
in vivo metabolism and toxicity assessment.
ACKNOWLEDGEMENTS
This work was financially supported by the Natural Science and Tech￾nology Research Program of Zhongshan City (Grant No.: 2016B1023),
Major Medical and Health Projects of Zhongshan City (Grant
No. 2017B1002), and Guangdong Medical Science andTechnology
Research Foundation (Grant No.: A2016043).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Ziwen Guo https://orcid.org/0000-0003-2414-8803
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How to cite this article: Ye Y, Guo X, He X, et al. High￾resolution mass spectrometry-based approach for the
identification and profiling of the metabolites of taletrectinib
formed in liver microsomes. Drug Test Anal. 2021;1–9.
YE ET AL. 9