Zeyun Li Lizhen Zhang Yongliang Yuan Zhiheng Yang. Abstract 1 INTRODUCTION RESEARCH ARTICLE

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1 Received: 16 May 2018 Revised: 20 July 2018 Accepted: 2 August 2018 DOI: /dta.2477 RESEARCH ARTICLE Identification of metabolites of evobrutinib in rat and human hepatocytes by using ultra high performance liquid chromatography coupled with diode array detector and Q Exactive Orbitrap tandem mass spectrometry Zeyun Li Lizhen Zhang Yongliang Yuan Zhiheng Yang Department of Pharmacy, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Correspondence Zhiheng Yang, Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou , China. dmpk_2018@126.com Abstract Evobrutinib is a highly selective inhibitor of Bruton's tyrosine kinase (BTK) which may be clinically effective in treating certain autoimmune diseases. The purpose of the present study was to investigate the metabolism of evobrutinib in rat and human hepatocytes. Evobrutinib was incubated with rat and human hepatocytes at 37 C for 2 hours after which the samples were analyzed by ultra high performance liquid chromatography with diode array detection and Q Exactive Orbitrap tandem mass spectrometry (UPLC DAD Q Exactive Orbitrap MS). The acquired data were processed by MetWorks software using mass effect filter and background subtraction functions. Under these conditions, 23 metabolites were detected and their identities proposed. Among these metabolites, M13 and M15 were identified by comparison of their retention times, accurate masses, and fragment ions with those of authentic reference standards. The metabolic pathways of evobrutinib were proposed accordingly. Our results demonstrated that evobrutinib was metabolized via hydroxylation, hydrolysis, O dealkylation, glucuronidation, and GSH conjugation. Species related metabolic differences between rat and human hepatocytes were observed. M1 M4 were rat specific metabolites. M13 (hydroxyl evobrutinib) was the major metabolite whereas M15 (evobrutinib diol) was a minor metabolite in rat hepatocytes. On the other hand, M6, M11, M16, M17, and M19 were human specific metabolites. M15 was the most abundant metabolite whereas M13 was the minor metabolite in human hepatocytes. This study provides preliminary information regarding the metabolism of evobrutinib that may be helpful in understanding the pharmacology of evobrutinib. KEYWORDS evobrutinib, hepatocytes, human, metabolite, rat 1 INTRODUCTION Bruton's tyrosine kinase (BTK) is an important regulator of the B cell receptor (BCR) pathway. 1 A deficiency of BTK prevents B cell maturation whereas inhibition of BTK can block BCR signaling and induce apoptosis. 2 BTK has emerged as a promising therapeutic target in autoimmune diseases and multiple cancers related to B lymphocytes. 3,4 Recently, various BTK inhibitors have been discovered. Among these inhibitors, evobrutinib, 1 (4 (((6 amino 5 (4 phenoxyphenyl) pyrimidin 4 yl)amino)methyl)piperidin 1 yl)prop 2 en 1 one, is a highly selective BTK inhibitor with potential anti neoplastic activity. 5,6 Evobrutinib can inhibit the Drug Test Anal. 2019;11: wileyonlinelibrary.com/journal/dta 2018 John Wiley & Sons, Ltd. 129

2 130 LI ET AL. activity of BTK and prevent the activation of the BCR signaling pathway. 7 Drug metabolism studies play important roles in identifying lead compounds and optimizing drug discovery and development. 8,9 In the early stages of drug development, results of drug metabolism studies provide rationales for the selection of lead compounds with desirable absorption, distribution, metabolism, excretion, and toxicity profiles, and later, drug metabolism data aid in the design clinical experiments and interpretation of clinical outcomes. 9 On one hand, drug metabolism is expected to produce more polar metabolites relative to the parent substance. On the other hand, drug metabolism affects a drug's duration in biofluids, bioavailability, safety, and toxicity Regulatory agencies recommend that metabolites be given full consideration in the safety assessment of drugs Undesirable metabolic and pharmacokinetic profiles are among the primary reasons for discontinuation of new drugs. Toxic drug metabolites pose a safety concern, especially in the case of reactive metabolites (RMs). In some cases, RMs are associated with hepatotoxicity and genotoxicity, which often contribute to drug development failure and withdrawal of post market drugs One of the purposes of pre clinical drug metabolism studies is to predict human metabolic profiles. In some cases, this prediction is very challenging because species related metabolic difference may exist between animals and humans. Therefore, comparative metabolism studies of drugs in animal and human models may be of use in understanding interspecies differences in toxicity and pharmacologic effects. However, the detection and identification of drug metabolites in biological matrices is a challenge because (a) the concentrations of metabolites may be at or below the lower limit of detection of available methodology; (b) different compounds have different metabolic profiles and identities of metabolites may be hard to predict, and (c) biosamples are complex mixtures of substances. 17 Liquid chromatography combined with high resolution mass spectrometer (LC HRMS) is one of the reliable analytical techniques that are frequently used for metabolite detection and identification. 17 HRMS can provide exact molecular weight of metabolite as well as the fragment ions, which benefits the structural characterization. To the best of our knowledge, the metabolic profiles of evobrutinib have not been reported. Hence, the aim of the current study was (a) to identify metabolites of evobrutinib in rat and human hepatocytes by liquid chromatography combined with diode array detection and Q Exactive Orbitrap tandem mass spectrometry (UPLC DAD Q Exactive Orbitrap MS); (b) to propose the metabolic pathways of evobrutinib; and (c) to compare metabolites between rat and human. 2 MATERIALS AND METHODS 2.1 Chemicals and reagents Evobrutinib (purity >98%) was purchased from Med Chem Express (Shanghai, China). Authentic standards of evobrutinib diol and hydroxyl evobrutinib were synthesized in our laboratory; purities were determined by high performance liquid chromatography (HPLC) and structures were verified by nuclear magnetic resonance (NMR). Cryopreserved Sprague Dawley rat hepatocytes (pooled from 12 donors) and human hepatocytes (pooled from 10 donors) were obtained from the Research Institute for Liver Diseases (Shanghai) Co., Ltd (Shanghai, China). InVitroGRO HT Medium and InVitroGRO KHB were purchased from Bioreclamation IVT (Brussels, Belgium). HPLC grade formic acid was purchased from Sigma Aldrich (St Louis, MO, USA). Acetonitrile was of HPLC grade and purchased from Thermo Fisher Scientific Co. (Santa Clara, CA, USA). Deionized water was prepared using a Milli Q Water Purification System (Millipore Corp., MA, USA). All other chemicals and reagents were of analytical grade and commercially available. 2.2 Metabolism of evobrutinib in hepatocytes Evobrutinib was dissolved in acetonitrile at a concentration of 10mM and then diluted in Krebs Henseleit buffer to a concentration of 20 μm. Cryopreserved hepatocytes were carefully thawed using InVitroGRO HT Medium according to the manufacturer's instructions. Thawed rat and human hepatocytes were suspended in Krebs Henseleit buffer to yield a final cell density of 2 million cells/ ml. Then, 100 μl of hepatocyte suspension and 100 μl of evobrutinib in Krebs Henseleit buffer were added into 48 well plates and incubated in a humidified CO 2 incubator at 37 C; the concentration of acetonitrile in the incubation mixture was 0.5% (v/v). Incubation mixtures without evobrutinib served as blank controls. The total volume of each incubation mixture was 200 μl and each sample was prepared in duplicate. After incubation for 2 hours, the incubations were quenched by the addition of 400 μl of ice cold acetonitrile to each well. Then, the samples were centrifuged at rpm for 10 minutes. The supernatant solutions from two replicates were pooled and evaporated to dryness under nitrogen gas. Each residue was dissolved in 100 μl of 20% acetonitrile and a 2 μl aliquot of each sample was injected into the UPLC DAD Q Exactive Orbitrap MS system. 2.3 LC HRMS conditions A Thermo Dionex Ultimate 3000 LC system (Thermo Fisher Scientific, Santa Clara, CA, USA) consisting of a vacuum degasser, a dual pump, a column compartment, an auto sampler, and a diode array detector was employed in this study. Chromatographic separation was carried out on an Acquity UPLC BEH C 18 column ( mm, i. d., 1.7 μm) by gradient elution of 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was 0.3 ml/min. The gradient procedure was performed as follows: 10% B at 0 1 minutes, 10% 30% B at 1 5 minutes, 30% 50% B at 5 9 minutes, 50% 75% B at 9 13 minutes, 75% 90% Bat13 16 minutes, and 10% B at minutes. The auto sampler and column were held at 4 C and 40 C, respectively. The wavelength range of the diode array detector was set to nm. The relative concentrations of the major metabolites were estimated by the UV peak area (λ = 270 nm) normalization method. High resolution mass detection was performed on a Q Exactive Orbitrap tandem mass spectrometer (Thermo Fisher Scientific, Santa Clara, CA, USA) equipped with a heated electrospray ionization source

3 LI ET AL. 131 (ESI) operated in positive ion mode. The ESI source parameters were optimized as follows: Capillary voltage, 3.0 kv; capillary temperature, 300 C; sheath gas heater temperature, 250 C; sheath gas flow rate, 35 arbitrary units; auxiliary gas flow rate, 10 arbitrary units. Full mass spectra were obtained from m/z 50 to 1000 with a resolution of , while data dependent MS 2 (dd MS 2 ) spectra were acquired at a resolution of with ramp collision energy at 25, 35, and 45 ev. Xcalibur software (Version 2.3.1, Thermo Fisher Scientific, Santa Clara, CA, USA) was used to control the LC HRMS system and for data acquisition. Post data processing was performed with MetWorks software (Version 1.3 SP3, Thermo Fisher Scientific, Santa Clara, CA, USA). 3 RESULTS AND DISCUSSIONS 3.1 Analytical strategy LC HRMS is one of the most frequently used tools for metabolite profiling and identification. In this study, an efficient and reliable analytical strategy (Figure 1) based on UPLC DAD Q. Exactive Orbitrap MS combined with post acquisition data processing has been developed for profiling and identifying the metabolites of evobrutinib. Firstly, high resolution full mass scans were obtained in positive ion mode on the Q Exactive Orbitrap mass spectrometer and MS 2 data were simultaneously acquired using the dd MS 2 data scan mode. Secondly, the mass defect filter (MDF) function and background subtraction program were applied for postacquisition data processing in order to obtain the accurate mass of the protonated molecular ion of potential metabolites. Thirdly, by comparing m/z values of fragment ions of each metabolite with those of evobrutinib, the metabolic site(s) were localized to a certain region of the molecule and probable structures of metabolites were proposed. Lastly, major metabolites were unambiguously identified by comparing their retention times, accurate masses, and fragment ions with those of authentic standards. Then, metabolic pathways of evobrutinib were proposed based on tentative and confirmed structures of metabolites. 3.2 Mass fragmentation behavior of evobrutinib To facilitate elucidation of metabolites structures, the mass spectrometric characteristics of evobrutinib were investigated using UPLC DAD Q Exactive Orbitrap MS analysis in positive ionization mode. Under the current conditions, evobrutinib formed a protonated molecular ion [M + H] + at m/z ( 0.5 ppm, elemental composition C 25 H 28 N 5 O 2 ) (Figure 2A). The protonated molecular ion produced a series of structurally indicative fragment ions at m/z , , , , , , and , as shown in Figure 2B. The fragment ions at m/z and were formed by the breakage of the amide bond. The fragment ion at m/z produced the most abundant fragment ion at m/z Fragment ions at m/z and derived from the breakage of a C N bond. The fragment ion m/z may further produce fragment ions at m/z and through the breakage of amide bonds. Based on the results above, the fragmentation pathways shown in Figure 2C were proposed. 3.3 Identification of metabolites of evobrutinib in hepatocytes By comparing the total ion chromatograms of drug containing samples with those of blank samples, a total of 23 metabolites were found and identified using the developed strategy based on UPLC DAD Q Exactive Orbitrap MS analysis. Among these metabolites, 20 metabolites were found in human hepatocytes and 19 metabolites were detected in rat hepatocytes. The measured and theoretical masses, mass errors, and characteristic fragment ions of the proposed metabolites are summarized in Table 1. The LC UV chromatograms FIGURE 1 Workflow of the developed strategy and methodology [Colour figure can be viewed at wileyonlinelibrary.com]

4 132 LI ET AL. FIGURE 2 MS and MS 2 spectra of and evobrutinib and its fragmentation patterns [Colour figure can be viewed at wileyonlinelibrary.com] (λ = 270 nm) of evobrutinib and its metabolites in rat and human hepatocytes are displayed in Figure 3. As shown in Figure 3, M13 appeared to be the most abundant metabolite in rat hepatocytes whereas M15 was the major metabolite in human hepatocytes, indicating the presence of species differences Metabolite M1 M1 was characterized by a retention time of 2.27 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 0.3 ppm, elemental composition C 29 H 41 N 8 O 8 S), Da higher than that of M2, which suggested that M1 was the GSH conjugate of M2. In its MS 2 spectrum, a diagnostic product ion at m/z was observed, which was associated with the loss of a glutamyl moiety ( Da) from the precursor ion. This neutral loss is a characteristic of GSH conjugates in positive ion mode. 18,19 The product ions at m/z and ( Da, GSH) further demonstrated the presence of the GSH moiety. 20 Therefore, M1 was proposed as a GSH conjugate of and that GSH conjugation was through the α, β unsaturated ketone Metabolite M2 M2 was characterized by a retention time of 3.99 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 0.2 ppm, elemental composition C 19 H 24 N 5 O 2 ), Da lower than that of evobrutinib, suggesting that M2 was the O dealkylated metabolite of evobrutinib. Its MS 2 spectrum (Figure 4A) showed three product ions at m/z , and , which were identical to those of evobrutinib. The product ions at m/z and demonstrated the absence of phenyl moiety. Therefore, M2 was identified as the O dealkylated metabolite of evobrutinib.

5 LI ET AL. 133 TABLE 1 Identified metabolites of evobrutinib in rat and human hepatocytes by UPLC DAD Q Exactive Orbitrap MS Peak ID RT (min) Mass Change Theo. m/z Meas. m/z Error [M + H] + [M + H] + (ppm) MS 2 Fragment Ions Metabolite Description Source M , , Dealkylation and GSH conjugation M , , , Dealkylation , M , , , M , , , M , , , , M , , , , , , Oxygenation and dehydrogenation Oxygenation and GSH conjugation Oxygenation and glucuronide conjugation M , , ,, M , , ,, M , , Oxygenation and GSH conjugation M , , , M , , , , , , M , , , , M , , , , , M , , , , M , , , , M , , , , , , M , , , , , , M , , , , , , Oxygenation and glucuronide conjugation GSH conjugation Oxygenation GSH and dehydrogenation Oxygenation and hydrolysis M , , Oxygenation H M , , , , , , M , , Oxygenation Evobrutinib , , , , , , Parent M , , Oxygenation M , , , Dehydrogenation , Note: RT: retention time; R, rat; H, human. R R R R H H H H Metabolite M3 M3 was characterized by a retention time of 4.88 minutes and an accurate mass of the protonated molecular ion [M + H] + at m/z (0.4 ppm, elemental composition C 25 H 26 N 5 O 3 ), suggesting that M3 was an hydroxylation and dehydrogenation metabolite of evobrutinib. Its MS 2 spectrum was characterized by product ions at m/z and, which were 2 Da lower than those of evobrutinib, suggesting that dehydrogenation occurred on the methyl

6 134 LI ET AL. FIGURE 3 The LC UV chromatograms (λ = 270 nm) of evobrutinib and its metabolites in A, rat and B, human hepatocytes [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 4 MS 2 spectra of A, M1 and B, M5 and their fragment ions [Colour figure can be viewed at wileyonlinelibrary.com] piperidine moiety. The product ions at m/z and were identical to those of M13. Hence, M3 is identified as a dehydrogenation product of M Metabolite M4 M4 was characterized by a retention time of 5.05 minutes and an accurate mass of the protonated molecular ion [M + H] + at m/z (0 ppm, elemental composition C 35 H 45 N 8 O 9 S), Da higher than that of evobrutinib, suggesting that M4 resulted from hydroxylation with GSH conjugation of evobrutinib. Its MS 2 spectrum was characterized by a product ion at m/z , which was attributed to the neutral loss of a glutamyl moiety ( Da). 18,19 Furthermore, the product ion at m/z was attributed to the loss of a GSH residue ( Da) from

7 LI ET AL. 135 the precursor ion, further suggesting the presence of a GSH moiety. 20 Considering that hydroxylation of the para position of the aromatic ring is the primary metabolic fate, M4 was tentatively proposed as GSH conjugate of M13 and that GSH conjugation was through the α, β unsaturated ketone Metabolite M5 M5 was characterized by a retention time of 5.14 minutes and an accurate mass of the protonated molecular ion [M + H] + at m/z ( 0.3 ppm, elemental composition C 31 H 36 N 5 O 9 ), Da higher than that of M13, suggesting that M5 was the glucuronide conjugate of M13. Its MS 2 spectrum (Figure 4B) was characterized by a fragment ion at m/z , which was associated with the loss of glucuronyl moiety ( Da) from the precursor ion, a typical neutral loss for glucuronide conjugates. 21 The other fragment ions at m/z , , , and were identical to those of M13. Therefore, M5 was tentatively identified as a glucuronide conjugate of M Metabolites M6, M11, M16, M17, M18, and M20 M6, M11, M16, M17, M18, and M20 were characterized by retention times of 5.16, 5.95, 6.52, 6.64, 6.72, and 6.91 minutes, respectively, and accurate masses of the protonated molecular ions [M + H] + at m/z ( 2.1 ppm, elemental composition C 25 H 28 N 5 O 4 ), Da higher than that of evobrutinib, suggesting all of these metabolites were di hydroxylation metabolites. Their MS 2 spectra were characterized by product ions at m/z and , which suggested that hydroxylation occurred on the 1 (4 (aminomethyl)piperidine 1 yl)prop 2 en 1 one moiety. In addition, two minor product ions at m/z and were observed. These ions were attributed to the loss of H 2 O and bis H 2 O, respectively, from the product ion at m/z further indicating that di hydroxylation occurred on the piperidine moiety Metabolites M7 and M8 M7 and M8 were characterized by retention times of 5.41 and 5.80 minutes, respectively, and accurate masses of the protonated molecular ions [M + H] + at m/z (0 ppm, elemental composition C 25 H 28 N 5 O 4 ), suggesting that both of the metabolites were di hydroxylation metabolites of evobrutinib. In the MS 2 spectra, product ions were found at m/z and, indicating that one hydroxylation reaction occurred on the piperidine moiety. Another product ion at m/z was identical to those of M13. Considering that hydroxylation of the para position of the aromatic ring is the primary metabolic fate, M7 and M8 were tentatively identified as resulting from hydroxylation of M Metabolite M9 M9 was characterized by a retention time of 5.84 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z (0 ppm, elemental composition C 35 H 45 N 8 O 9 S), Da higher than that of evobrutinib, suggesting that M9 resulted from hydroxylation with GSH conjugation of evobrutinib. Its MS 2 spectrum was characterized by a product ion at m/z , which was associated with the cleavage of the glutamyl moiety ( Da) from the precursor ion, a typical neutral loss of GSH in the positive ion mode. 18,19 The hydroxylation may have occurred on the piperidine moiety because the product ion at m/z was derived from m/z by the loss of H 2 O. This deduction was further confirmed by the presence of a product ion at m/z. Hence, M9 was tentatively identified as a GSH conjugate of M19 or M21 or M22 and that GSH conjugation was through the α, β unsaturated ketone Metabolite M10 M10 was characterized by a retention time of 5.84 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 0.7 ppm, elemental composition C 31 H 36 N 5 O 9 ), Da higher than that of evobrutinib, suggesting that M10 resulted from hydroxylation of evobrutinib followed by glucuronidation. Its MS 2 spectrum was characterized by a fragment ion at m/z , resulting from neutral loss of a glucuronyl moiety ( Da), a typical MS 2 fragmentation of a glucuronide conjugate. 21 The other fragment ions at m/z , ,, and revealed that hydroxylation occurred on the piperidine moiety. Therefore, M10 was tentatively identified as the glucuronide conjugate of M19 or M21 or M Metabolite M12 M12 was characterized by a retention time of 6,10 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 0.2 ppm, elemental composition C 35 H 45 N 8 O 8 S), Da higher than that of evobrutinib, suggesting that M12 was a GSH conjugate of evobrutinib. Its MS 2 spectrum (Figure 5) was characterized by a product ion at m/z , which originated from the precursor ion through the loss of the glutamyl moiety ( Da). The product ion at m/z was formed by the cleavage of the GSH moiety ( Da), further indicating the presence of GSH moiety. Therefore, M12 was identified as the GSH conjugate of evobrutinib and that GSH conjugation was through the α, β unsaturated ketone Metabolite M13 M13 was characterized by a retention time of 6.28 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 1.0 ppm, elemental composition C 25 H 28 N 5 O 3 ), Da higher than that of evobrutinib, suggesting an hydroxylated metabolite. Its MS 2 spectrum (Figure 6A) contained fragment ions at m/z , and that are identical to those of evobrutinib, indicating that the 1 (4 (aminomethyl)piperidine 1 yl)prop 2 en 1 one moiety is unmodified in M13. The product ions at m/z , , and demonstrated hydroxylation. In order to confirm the site of hydroxylation, a standard was synthesized for comparison. The retention time, accurate mass, and fragment ions of M13 were the same as those of standard, thereby unambiguously identifying M13 as hydroxyl evobrutinib. This metabolite was the most abundant metabolite in rat hepatocytes. Our results suggested that hydroxylation at the para position of the aromatic ring is the primary

8 136 LI ET AL. FIGURE 5 MS 2 spectrum of A, M12 and B, its fragmentation pathways metabolic pathway of evobrutinib in the rat, while the ortho and meta positions of evobrutinib were less susceptible to hydroxylation Metabolite M14 M14 was characterized by a retention time of 6.34 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 1.8 ppm, elemental composition C 35 H 43 N 8 O 8 S), Da higher than that of evobrutinib, suggesting that M14 was dehydrogenated followed by GSH conjugation of evobrutinib. Its MS 2 spectrum was characterized by a product ion at m/z , which was derived from the precursor ion by the loss of the glutamyl moiety ( Da), a typical neutral loss for GSH conjugates. 18,19 The product ion at m/z was formed by loss of the GSH moiety ( Da), which further demonstrated its presence in M The product ion at m/z was identical to that of evobrutinib; the product ions at m/z and suggested that dehydrogenation took place on the piperidine moiety. Therefore, M14 was tentatively identified as the dehydrogenated and GSH conjugated metabolite of evobrutinib and that GSH conjugation was through the α, β unsaturated ketone Metabolite M15 M15 was characterized by a retention time of 6.38 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 1.5 ppm, elemental composition C 25 H 30 N 5 O 4 ), Da higher than that of parent, suggesting that M15 originated from evobrutinib through the addition of an oxygen atom followed by hydrolysis. Its product ions (Figure 6B) at m/z , , , , and were identical to those of evobrutinib, suggesting that addition of an atom of oxygen followed by hydrolysis occurred at the α, β unsaturated ketone moiety. This metabolite may be formed via an epoxide intermediate followed by hydrolysis. In order to confirm the structure, the standard was synthesized. The retention time, accurate mass, and fragment ions of the standard were the same as M15 thereby unambiguously identifying it as evobrutinib diol Metabolites M19, M21, and M22 M19, M21, and M22 were characterized by retention times of 6.85, 7.33 and 8.97 minutes, respectively, and accurate masses of the protonated molecular ions [M + H] + at m/z ( 2.0 ppm, elemental composition C 25 H 28 N 5 O 3 ), Da higher than that of evobrutinib, suggesting that these metabolites resulted from mono hydroxylation of evobrutinib. Their product ions at m/z , , and were identical to those of evobrutinib, indicating that the hydroxylation presented at piperidine moiety.

9 LI ET AL. 137 FIGURE 6 MS 2 spectra of A, M13 and B, M15 and their fragment ions [Colour figure can be viewed at wileyonlinelibrary.com] Metabolite M23 M23 was characterized by a retention time of 9.25 minutes and accurate mass of the protonated molecular ion [M + H] + at m/z ( 1.2 ppm, elemental composition C 25 H 26 N 5 O 2 ), Da lower than that of evobrutinib, indicating it was a dehydrogenated metabolite. The product ion at m/z was identical to that of evobrutinib, indicating that dehydrogenation occurred on the piperidine moiety. Other product ions at m/z , , and supported this assignment of structure for M Metabolic pathways of evobrutinib and interspecies comparison Based on the identified metabolites, the metabolic pathways of evobrutinib in rat and human hepatocytes are proposed in Figure 7. In general, the metabolic pathways of evobrutinib can be summarized as follows: the first metabolic pathway is O dealkylation to yield M2 which can be conjugated with GSH to form M1. The second metabolic pathway is hydroxylation, yielding mono hydroxylated metabolites M13, M19, M21, and M22, which are subject to hydroxylation to form di hydroxylation metabolites M6 M8, M11, M16 M18, and M20; or conjugated with glucuronide to form M5 and M10; or conjugated with GSH to form M4 and M9. The third metabolic pathway involves formation of an epoxide, which is unstable and undergo hydrolysis to the diol metabolite M15 (evobrutinib diol). The fourth pathway involved formation of the dehydrogenation metabolite M23, which is conjugated with GSH, yielding M14. The last metabolic pathway is conjugation of evobrutinib with GSH to form M12. A comparison of metabolic profiles between rat and human hepatocytes indicated that M1 M4 were rat specific whereas M6, M11, M16, M17, and M19 were human specific. In the rat, M13 (hydroxyl evobrutinib) was the major metabolite (approximately 65%) and M15 (evobrutinib diol) was the minor metabolite. On the other hand, M15 was the most abundant metabolite (approximately 43%) whereas M13 was the minor metabolite. These metabolic difference between species may result in differences in toxicity as well as pharmacokinetics/pharmacodynamics of evobrutinib. 4 CONCLUSIONS A total of 23 metabolites were detected and structurally proposed in rat and human hepatocytes by using the developed strategy based on the UPLC DAD Q Exactive Orbitrap MS method. The proposed strategy appears to be practically applicable for metabolite characterization. The structures of the metabolites were characterized based

10 138 LI ET AL. FIGURE 7 Proposed metabolic pathways of evobrutinib in rat and human hepatocytes on their accurate masses, mass fragmentations as well as chromatographic retention times. M13 (hydroxyl evobrutinib) was the most abundant metabolite in rat, whereas M15 (evobrutinib diol) was the major metabolite in human. The metabolic pathways of evobrutinib mainly involved hydroxylation, hydrolysis, O dealkylation, glucuronidation, and GSH conjugation. Species related metabolic difference between rat and human was observed. This study provides preliminary information regarding the metabolism of evobrutinib that may be helpful in understanding the pharmacological effects of evobrutinib. CONFLICT OF INTEREST All authors declare that they have no competing interests.

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