Identification and synthesis of metabolites of the new 4.5-dihydroisoxazol-5-carboxamide derivate

Introduction: 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide is new antirheumatic drug. It is necessary to identify and synthesize the biotransformation products for its complete pharmacokinetic study. Material and Methods: A biotransformation study was carried out by intraperitoneal administration of the drug to Wistar rats and Soviet Chinchilla breed rabbits. Animal blood sampling was performed before the injection and 0.5 h, 1 h, 2 h, 4 h, 24 h after the injection of the investigated compound. The samples were immediately centrifuged for plasma separation. Urine was simultaneously collected from rats before the administration and at intervals of 0-2 h, 2-4 h, 4-6 h, 6-24 h after administration, faeces – before administration and at intervals of 0-12 h and 12-24 h after administration. The samples were analyzed by HPLC-MS/MS after immediate preparation by adding acetonitrile. Results and Discussion : 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid and 4-methoxy-3-(trifluoromethyl)aniline – hydrolysis products of the active substance were found during the analysis of plasma, urine and fecal samples. The 4,5-dihydro-1,2-oxazole-5-carboxylic acid derivative has been synthesized. The second metabolite is a raw material for production of active pharmaceutical substance. During comparative tests, no significant difference between the retention times, ratio areas of chromatographic peaks at the main MRM-transitions and mass spectra of these metabolites on chromatograms of standard and animal samples was found, which indicates the correct identification of biotransformation products. Conclusion: The studied drug undergoes biotransformation by hydrolysis to form two main metabolites: 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid and 4-methoxy-3-(trifluoromethyl)aniline. The structure of the metabolites was confirmed by comparison with the synthesized standard samples using HPLC-MS/MS.

Identification and synthesis of metabolites of the new 4.5-dihydroisoxazol-5-carboxamide derivate Introduction Rheumatoid arthritis (RA) is characterized by chronic autoimmune process with joint destruction and systemic damage of internal organs.This disease is widespread: up to 1.0% of the population suffers from it.The morbidity of RA increases with age (Scherer et al. 2020).The inhibition of PAR-2 receptors of cell membranes of the immune system is one of the promising targets for the treatment of RA.Both experimental peptide molecules and low molecular weight compounds are used for realization of this action mechanism (Romanycheva et al. 2023).3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N- [4methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2oxazole-5-carboxamide (R004) (Fig. 1) is a new selective inhibitor of PAR-2 receptors.This drug has proven its pharmacological activity on a formaldehyde edema model in rats (Korsakov et al. 2023).Thus, R004 surpassed diclofenac sodium in its anti-inflammatory effect.Bioanalytical studies of this molecule have not been conducted before.Therefore, the main metabolites must be identified and synthesized before starting the full pharmacokinetic investigation.The biochemical reactions using microsomes, S9 fractions or hepatocytes, and the administration of drugs to laboratory animals are used for this purpose.The detection of biotransformation products is carried out in samples obtained within these experiments (Khokhlov and Pyatigorskaya 2019).The main analytical method for achieving these aims is highperformance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS).In this case, it is possible to study the structure of metabolites by measuring the exact values of the mass-to-charge ratio of molecular and daughter ions, using multi-stage fragmentation of analytes (MS n ) using ion traps, according to predicted MRM-transitions using fragmentation data of the drug molecular ion (Reddy et al. 2021).The metabolism study of this compound is complicated by the presence of an amide bond in the structure, which may be undergoes to hydrolysis (On Approval of the Rules for Conducting Bioequivalence Studies on Medicines in the Eurasian Economic Union.Decision of the Council of the Eurasian Economic Commission No 85, 2016; Mironov (Ed.) 2014).Therefore, it is necessary to demonstrate the stability of R004 in biological samples in order to distinguish decomposition and biotransformation products.

Graphical abstract
Thus, the aim of the study is identification and synthesis of biotransformation products of 3-

Analytical equipment and reagents
The investigation was performed using HPLC-MS/MS system (Analyst 1.6.2software (AB Sciex, USA)) including an Agilent 1260 Infinity chromatograph (Agilent Technologies, Germany) and an AB Sciex QTRAP5500 tandem mass spectrometric detector (AB Sciex, Singapore).MultiQuant 3.0.5 software was applied for chromatograms integration.Predicted MRMtransitions of putative metabolites was created by LightSight 2.3 software (AB Sciex, USA).

Sample preparation and analysis
An aliquot of 100 µL of acetonitrile was added to 20 µL of stabilized plasma and urine samples.The mixture was centrifugated at 10000 rpm and +4°C for 5 min after vortexing.Supernatant in volume 1 µL was injected to the chromatography system.Homogenates with acetonitrile in a ratio of 1:1 (weight/volume) were prepared from feces.They were centrifugated at 10000 rpm and +4°C for 5 min, and 200 µL of acetonitrile was added to 50 µL of the supernatant.The mixture was also centrifugated and analyzed by HPLC-MS/MS.The reversed phase gradient chromatography using Poroshell 120EC-C18 (50*3.0mm, 2.7 microns) chromatographic column with Zorbax Eclipse Plus C18 pre-column (12.5*2.1 mm, 5.0 microns) was applied for sample analysis (Table 1).HPLC-MS-grade formic and acetonitril was used for mobile phase preparation.Mass spectrometric detection was performed in MRM-mode with positive polarity.Eluate was ionized by electrospray under voltage 5500V and source temperature 650° C.
Sensitivity check of the HPLC-MS/MS-system was carried out by analysis spiked plasma, urine and feces samples at concentration of R004 of 1 ng/mL before beginning the batch.The signal-to-noise ratio of the R004 chromatographic peaks at MRM-transitions 445→198 m/ z, 445→226 m/z, 445→281 m/z, 445→387 m/z was at least 10:1.
The concentration of the R004 in the samples for stability tests was 500 ng/mL (500 ng/g for feces).It was achieved by adding dimethylsulfoxide standard analyte solutions to blank matrix in a volume ratio of 1:19.Solutions of CH3COONH4 with pH = 4.0; 5.0; 6.0; 8.0 were used in stability study.The pH value of 250 mM ammonium acetate solutions was adjusted by adding glacial acetic or aqueous ammonia.

Animals
The 6 Wistar rats weighing 243.67±22.73g (M±SD) and 6 Soviet Chinchilla rabbits weighing 2.59± 0.23 kg (M±SD) from SMK Stezar LLC Nursery (Russian Federation) was used for biotransformation study of R004.Three males and three females of each species were included in the experimental group.Catheterization of rat into the right jugular vein was performed before drug administration.The rabbit blood samples were collected from the ear vein.
The study was approved by the Ethics Committee of YSPU named after K.D. Ushinsky (Minutes №1 of 29 January 2024).

Dosing of the drug and sample collection
The substance of R004 was injected intraperitoneally.Drug suspension was obtained using solution consisting of 1% tween-80 and 0.9% sodium chloride.The dosage was equal for rats and rabbits -50 mg/kg.Blood was collected in volume of 0.2 mL in capillary K3EDTA-tube.There were following time points for both species: before administration and 0.5 h, 1 h, 2 h, 4 h, 24 h.The sample was centrifuged for 10 min at 2500 rpm and a temperature of +4°C.Obtained plasma was stabilized by addition of ammonium acetate solution 250 mM (pH=4.0) in volume ratio 1:5 (stabilizer: plasma).It was necessary to prevent in-vitro hydrolysis of R004.
Rat excreta sampling was performed using metabolic cells.Urine was collected before administration and at intervals of 0-2 h, 2-4 h, 4-6 h, 6-24 h after administration of the drug; feces were collected before administration and at intervals of 0-12 h and 12-24 h after administration of the drug.Feces were immediately homogenized by acetonitrile without any stabilizers.Ammonium acetate solution 250 mM (pH=4.0) was added to urine samples in volume ratio 1:5 (stabilizer: urine) There were two groups, which included 3 individuals of each species.The first group was used for the preliminary detection of biotransformation products.The experiment on the second group was conducted for confirmation of the coincidence of the structure of the found metabolites with those of the synthesized substances.Analysis of fresh samples of second stage was also necessary due to hydrolytic instability of R004.
In total, the following samples were analyzed: 36 rat plasma samples, 30 rat urine samples, 18 rat feces samples, and 36 rat rabbit samples.Every group of samples included 6 control samples.The total number of observations was 120 (including 30 control observations).

Statistical analysis
All statistical calculations were carried out using Microsoft Excel 2016 (Microsoft Corporation, USA).The matching of quantitative parameters of metabolite and synthesized substances was carried out by comparison of their mean values (formula 1): Note: parameter -retention times of chromatographic peaks; ratio of chromatographic peak areas at the main MRM-transitions.
Dispersion measure of the data shown in the table 6 is standard deviation (SD).
Calculations during stability study of R004 in plasma, urine and feces was performed by external standard method (formula 2): the mean value of chromatographic peak area of R004 (MRM: 445→226 m/z) at test sample (Mean Stest sample) was compared with the mean value of chromatographic peak area of R004 in fresh samples (Mean Sfresh sample):

Сriteria of comparison of analytical signals of metabolites and synthesized substances
Comparison of metabolites in animal and standard spiked samples was performed by the following parameters (GPA.1.2.1.2.0001.15The Chromatography; GPA.1.2.1.1.0008.15The Mass-spectrometry.The State Pharmacopoeia of Russian Federation.XV edition 2023): • Coincidence of presence of chromatographic peaks on main MRM-transitions.In this case, chromatographic peaks in the blank sample should be absent (qualitative parameter); • Deviation of retention times of chromatographic peak (tR) of standard should be no more than ±1% from tR of the metabolite (quantitative parameter -% of match -99-101%); • Deviation of ratio of chromatographic peak areas at the main MRM-transitions of standard should be no more than 20% from the area ratio of the metabolite (quantitative parameter -% of match -80-120%); • Matching of MS2-mass spectra of metabolite and standard should be no less than 85%.
The matching of quantitative parameters of metabolite and synthesized substances was carried out by comparison of their mean values (formula 3): Note: parameter -retention times of chromatographic peaks; ratio of chromatographic peak areas at the main MRM-transitions.
Calculations during stability study of R004 in plasma, urine and feces was performed by external standard method (formula 4): the mean value of chromatographic peak area of R004 (MRM: 445→226 m/z) at test sample (Mean Stest sample) was compared with the mean value chromatographic peak area of R004 in fresh samples (Mean Sfresh sample):

Results and Discussion
The selection of sampling and storage conditions was carried out due to the instability of the R004 molecule to hydrolysis in order to distinguish biotransformation products from the in-vitro decomposition products.Tests were performed on the short-term stability (STS) of R004 at room temperature, stability during 3 freeze/thaw cycles (FTS), and stability of prepared samples in an autosampler (ASS) in each studied biological object (Khokhlov and Pyatigorskaya 2019).The usage of K3EDTA, lithium heparin and a mixture of sodium fluoride and potassium oxalate in plasma studies did not completely prevent the hydrolysis of the analyte.Application of combination of sodium fluoride and potassium oxalate with high antiesterase activity (Khokhlov and Pyatigorskaya 2019) provided the best results of STS (74.98±8.02% of the initial value) and FTS (77.05±5.45% of the initial value) (Fig. 2).However, these concentration values were not within the required range of 85-115% of the initial value.
The addition of ammonium acetate solution with pH=4.0 in a volume ratio of 1:5 was allowed to stop hydrolysis using all anticoagulants.K3EDTA was selected for further research, because 0.2 mL capillary tubes with this compound are the most commercially available.The short-term stability of R004 in whole blood was also studied.The analyte content remained within the required range for at least 30 minutes (Fig. 2).This time is sufficient for plasma separation.R004 was more stable in urine samples: after 24 hours of storage at room temperature, 77.06±3.95% of the initial concentration remained in the samples, and after 3 cycles of freezing/defrosting -81.52±1.01% of the initial concentration (Fig. 3).The addition of acidified solutions of ammonium acetate with pH=4.0 and pH=5.0 completely stopped hydrolysis (Fig. 3).The solution with pH=4.0 was also chosen to stabilize the samples, as well as for plasma.Decomposition of R004 in acetonitrile homogenates of feces was not observed: the results of the STS and FTS tests were within the permissible range of 85-115% of the initial value.Therefore, auxiliary measures for its storage were not required.All prepared samples with acetonitrile were also stable in the autosampler: the addition of the organic solvent completely stopped the hydrolysis of R004.
The identification of metabolites was carried out after confirmation of the absence of decomposition of R004 in the collected samples.Product ions 198 m/z, 281 m/z, 226 m/z, 387 m/z, which reflect the main fragments of the R004 structure, were selected to create the screening method (Fig. 4).Changing m/z of these ions makes it possible to accurately determine the localization of the modification.The following biotransformation ways were predicted: hydroxylation and dihydroxylation, Noxidation, demethylation, demethylation with hydroxylation, and dehydration of 4,5-dihydro-1,2oxazole cycle and hydrolysis (Table 2).3).The stabilization measures taken allow asserting that these compounds were formed during biotransformation and were not products of in-vitro decomposition.Both the active substance and its metabolites were identified in plasma samples of rats and rabbits (Fig. 5).M1 and trace amounts of M2 were identified in urine (Fig. 6).Therefore, fecal samples were analyzed to determine the pathway of excretion or possible products of M2 biotransformation.M2, unchanged R004 as well as M1, were found in rat feces (Fig. 6).At the same time, the analytical signal M2 in fecal samples was much higher than in urine samples, and the analytical signal M1 was much lower than in urine samples (Fig. 6).An additional MRM method was created to identify possible biotransformation products of M1 and M2 (Table 4).Glucuronidation, sulfonation, methylation, addition of hydroxyl group were predicted for metabolites.Acetylation and demethylation of M2 w e r e a d d i t i o n a l l y s t u d i e d .F o r m a t i o n o f benzoquinonymine derivate after demethylation as in paracetamol biotransformation (Athersuch et al. 2018) was also checked.New metabolites have not been detected.
Preparation of 3-(2-butyl-5-chloro-1H-imidazol-4yl)-4,5-dihydro-5-isoxazolecarboxylic acid (M1) was performed on next stage.Ester (Fig. 7-4) (13.94 g (0.04 mol)) was dissolved in 150 mL of 80% ethyl alcohol, and 7.14 g (0.127 mol) of sodium hydroxide was added to the resulting mass.The reaction mixture was stirred at 50°C for 12 h, then cooled to 0°C.The reaction mass was acidified with concentrated sulfuric acid to pH=3.0 and left overnight to cool.The precipitate that formed was filtered off, washed with 20 mL of chilled water, and dried at 50°C for 24 hours.We obtained 9.35 g of 3-(2-butyl-5c h l o r o -1 H -i m i d a z o l -4 -y l ) -4 , 5 -d i h y d r o -5isoxazolecarboxylic acid (Fig. 7-5) in the form of a beige powder.The product yield was 74%.The results of structure confirmation of all synthezed compounds are shown in Table 5.
The second stage of the study was carried out after the successful synthesis of M1 and confirmation of the structure of M2.Samples of each biological object with the addition of metabolites were prepared for comparison with animal samples.The results are presented in Table 6.The retention time of M1 in the test and spiked samples coincided at 99.79-100.18%,and tR M2 coincided by 99.91-100.14%.Thus, the differences of this parameter did not exceed the maximum acceptable interval of 1%.
The discrepancy in the area ratios of M1 chromatographic peaks at MRM-transitions with the most intense signal 272→200 m/z and 272→157 m/z was 99.64-101.41%.It was within the valid limit of ±20%.The percentage of coincidence of this parameter in M2 was within the range of 94.67-100.25%,which also meets the established requirements.The MS2-mass spectra of molecular ions 272 m/z (M1) and 192 m/z (M2) were compared to additional confirmation of the structure of the metabolites.The determination was carried out at points with the most intense analytical signal of the studied compounds: 4 h and 6 h -for rat and rabbit plasma, in the intervals of 2-4 h and 4-6 h -for rat urine, and in the interval of 12-24 h -for rat feces.Thus, the mass spectra of M1 matched by at least 90%, and the mass spectra of M2 -by 87% (Fig. 8).The low percentage of coincidence of mass spectra of M1 in fecal samples and mass spectra of M2 in urine samples was due to their low contents in these objects.Thus, 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2oxazole-5-carboxamide undergoes biotransformation by hydrolysis.The structure of its metabolites has been proven by comparison with synthetic substances.It was also found that the molecule of R004 is not stable in plasma and urine after collection.Therefore, acidification of samples is necessary for conduction of bioanalytical studies of this drug, as well as for study of statins (Khokhlov and Pyatigorskaya 2019).

%
of m a tch = Mea n of p ar a m eter × test a ni m a l sa m ple Mea n of p ar a m eter × spi k ed st a n d ar d sa m ple × 100 % (1) % o f i n i t i a l c o n ce n t r a t i o n = Mea n S t es t s a m pl e Mea n S f r esh s a m pl e × 100 % (2) % of m a tch = Mea n of p ar a m eter × test a ni m a l sa m ple Mea n of p ar a m eter × spi k ed st a n d ar d sa m ple × 100 % (3) % o f i n i t i a l c o n ce n t r a t i o n = Mea n S t es t s a m pl e Mea n S f r esh s a m pl e × 100 % (4)

Figure 2 .Figure 3 .
Figure 2. The results of stability study of R004 in plasma and blood samples.Note: ACA -ammonium acetate solution; STS -short-term stability; FTS -stability during 3 freeze/thaw cycles; ASS -stability of prepared samples in an autosampler; Results -mean values of percent