Pyridazinones: YWS01125 (purity of 98.83%) was supplied by the Institute of Materia Medica, Shandong Academy of Medical Sciences (Jinan, China); MGL-3196 (internal standard, IS) was purchased from Selleck Corporation (Beijing, China). The structural equations of the two compounds are shown in Figure 1. Nicotinamide adenine dinucleotide sodium phosphate (NADP), glucose-6-phosphate disodium salt (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-PDH) were purchased from Sigma (Sigma, Germany). The reagent was purchased from Sigma Company (St. Louis, MO) and was chromatographically pure. The rat and human liver microsomes were purchased from Red Company (Shanghai, China). HPLC-grade methanol was supplied by Thermo Fisher Scientific Co. Ltd. (Shanghai, China); HPLC-grade acetonitrile was offered by Thermo Fisher Scientific Co. Ltd. (Shanghai, China); HPLC-grade formic acid was provided by Tianjin Kermel Chemical Reagent Co. Ltd. (Tianjin, China); purified water used for the all analysis was employed by Wahaha Co. Ltd. (Hangzhou, China).

The synthetic route of pyrazinone compound YWS01125 is as depicted in Scheme 1. Synthesis of 1-(3,5-dichloro-4-((5-cyclobutyl-6-oxo-1,6-dihydropyridazin-3-yl) oxy) phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (YWS01125).

Synthesis of ethyl (2-cyano-3-methoxyacryloyl) carbamate (1).

Ethyl (2-cyanoacetyl) carbamate (3.12 g, 20 mmol), trimethyl orthoformate (3.5 ml, 40 mmol), acetic anhydride (20 ml), and acetonitrile (40 ml) were added to a 100-ml flask. The solution was stirred at 80°C for 4 h, and then the reaction stopped. The solvent was evaporated. Diethyl ether (50 ml) was added to the mixture, and the resulting suspension was kept at 2–8°C overnight. The suspension was filtered and rinsed several times with ether (3 × 50 ml) to give target compound 1 as a white solid (2.20 g, 56%). ESI m/z: C₈H₁₁N₂O₄ (M + H)+m/z = 199.1; molecular weight, 198.1780.

Synthesis of 3,5-dichloro-4-((6-chloro-5-cyclobutylpyridazin-3-yl) oxy) aniline (2).

The solution of 6-dichloro-4-cyclobutylpyridine (1.62 g, 8.00 mmol), 2, 6-dichloro-4-aminophenol (1.42 g, 8.00 mmol), and potassium carbonate (2.21 g, 16.00 mmol) in N, N-dimethylformamide (20 ml) was stirred at 120°C for 3 h. The reaction was stopped. An appropriate amount of diatomite was added into the sand core funnel, and the reaction solution was pumped and filtered to collect the filtrate. The filtrate was washed with 10% sodium chloride aqueous solution and saturated sodium chloride and was dried with anhydrous sodium sulfate. The solvent was evaporated to give a crude product which was purified by column chromatography (petroleum ether/ethyl acetate = 4/1) to obtain compound 2 as a light-brown solid (1.36 g, 50%). ESI m/z: C₁₄H₁₃Cl₃N₃O (M + H)+344.0; molecular weight, 344.6200.

Synthesis of 6-(4-amino-2,6-dichlorophenoxy)-4-cyclobutylpyridazin-3(2H)-one (3).

In a 100-ml round-bottom flask, the solution of compound 2 (1.36 g, 3.97 mmol), sodium acetate (1.30 g, 15.86 mmol), and glacial acetic acid (20 ml) was stirred at 120°C for 24 h. The reaction was stopped and the mixture cooled to room temperature. Then, 10 ml of water was added. The pH was adjusted to 9–10 with 1N HCl. The aqueous phase was extracted with ethyl acetate (25 ml × 3). The organic phase was combined, washed with brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to give a crude product which was used directly in the next step without further purification.

The above obtained solid was dissolved in methanol (24 ml), and NaOH solution (1N, 24 ml) was added. The resulting mixture was stirred at 100°C for 24 h. The reaction was stopped. The solvent was evaporated. The suspension was extracted with ethyl acetate (25 ml × 3), and then the organic phase was combined, washed with brine, and dried over anhydrous sodium sulfate. The solution was concentrated under pressure and purified by column chromatography (petroleum ether/ethyl acetate = 2/1) to give compound 3 as a gray solid (0.57 g, 44%). ESI m/z: C₁₄H₁₄Cl₂N₃O₂ (M + H)+326.0; molecular weight, 326.1770.

Synthesis of ethyl (2-cyano-3-((3,5-dichloro-4-((5-cyclobutyl-6-oxo-1,6-dihydropy-ridazin-3-yl) oxy) phenyl) amino) acryloyl) carbamate (4).

To a 10-ml reaction tube were added 1 (71 mg, 0.36 mmol), 3 (98 mg, 0.30 mmol), and chloroform (5 ml). The reaction mixture was stirred at reflux for 1 h and the reaction stopped. The mixture was then cooled to room temperature. The suspension was filtered and rinsed with ether. The filtrate cake was dried at 50°C. Compound 4 was obtained as a white solid (80 mg, 54%). ESI m/z: C₂₁H₂₀Cl₂N₂O₅ (M + H)+492.1; molecular weight, 492.2130.

1-(3,5-dichloro-4-((5-cyclobutyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (YWS01125).

Compound 4 (80 mg, 0.17 mmol) was added into a 10-ml microwave reaction tube, followed by the addition of potassium acetate (33 mg, 0.34 mmol) and N, N-dimethylacetamide (2 ml). The mixture reacted at 140°C for 30 min, then stopped. The reaction solution was diluted with ethyl acetate (20 ml), transferred into a 150-ml separating funnel, and diluted with water (10 ml). The organic phase was separated, washed with brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to give a crude mixture which was purified by column chromatography (methylene chloride/methanol = 30/1 to 10/1) to give the target compound YWS01125 as a light-yellow solid (37 mg, 52%). 1H NMR (600 MHz, DMSO-d6) δ 12.31 (s, 1H), 12.22 (s, 1H), 8.90 (s, 1H), 7.88 (s, 2H), 7.50 (s, 1H), 3.59–3.53 (m, 1H), 2.30–2.24 (m, 2H), 2.19–2.10 (m, 2H), 2.07–1.97 (m, 1H), 1.85–1.80 (m, 1H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 160.9, 160.0, 154.7, 151.5, 151.4, 149.5, 145.9, 136.4, 128.9 (2C), 128.7 (2C), 120.0, 114.4, 89.2, 35.5, 27.3 (2C), 18.3 ppm; ESI-HRMS m/z calculated for C₁₉H₁₄Cl₂N₅O₄ (M + H)+446.0423; molecular weight, 446.0420 (Figure 2).

A total of 60 male C57BL/6J mice, weighting 23 ± 5 g, were obtained from Jinan Peng Yue Experimental Animal Breeding Co. Ltd. (Jinan, China). The permit number was SCXK (Lu) 20190003. The mice were allowed free laboratory sterile feed and sterile water and were housed in controlled environment conditions since at least 1 week before the experiment. The adjustable temperature was set at 20 ± 5°C and the moisture was 55 ± 15%, with a 12-h dark–light cycle. The tested mice were carried out according to the National Institute of Health Guideline for the Care and Use of Laboratory Animals, and performed by the Animal Ethics Committee of Institute of Materia Medica, Shandong Academy of Medical Sciences (Jinan, China). Before the pharmacokinetic study, all experimental mice had fasted for 12 h with free drinking water.

The stock solution of YWS01125 was prepared by accurately weighing a lot of the medicine, then dissolving it in methanol, yielding a final concentration of 1 mg/ml, and storing at 4°C. It was gradually diluted before use into a series of working solutions at concentrations of 4–400 ng/ml with acetonitrile. MGL-3196 powder was prepared as a 1 mg/mL internal standard stock solution and stored in refrigerator at 4°C. Immediately before use, use chromatographic acetonitrile to dilute step by step to prepare an internal standard solution with a concentration of 2 ng/ml MGL-3196, and placed in 4°C standby. These standards of the quality control (QC) samples were prepared by diluting the stock solution of YWS01125 to the blank plasma at low, medium, and high concentrations of 10, 50, and 300 ng/ml, respectively, and by depositing them at 4°C until analysis. All solutions were kept at 4°C before use.

Hypersil GOLD C18 column (4.6 × 150 mm, 5 μm, Thermo). A 0.025% ammonia aqueous solution in water (solvent A) and acetonitrile (solvent B) were used as mobile phases with gradient elution. The gradient was as follows: 0.01 min, 15% B; 5 min, 20% B; 12 min, 80% B; 16 min, 80% B; 16.01 min, 15% B; 19 min, 15% B; and 19.01 min, stopped. The timing interval was 19 min, and the flow rate was set at 1 ml/min.

Sorvall Biofuge Stratos high-speed refrigerated centrifuge was bought from the Thermo Fisher Scientific Co. Ltd. (Shanghai, China); EL204 electronic balance was selected from Shanghai Mettler-Toledo Instruments Co. Ltd. (Shanghai, China); IKA Vortex Genius 3 Vortex Mixer was purchased from Aika Instrument Equipment Co. Ltd. (Guangzhou, China); and the test was carried out using an AB SCIEX™ 5500 Q-Trap^® mass spectrometer (Applied Biosystems, United States) coupled to a Shimadzu Prominence UPLC (ultra-performance liquid chromatography, Shimadzu, United States) system. It was equipped with a binary pump, a degasser, an auto sampler, and an automatic thermostatic column compartment. The column was utilized from Agela Technologies UHP Innoval C18 (2.1 × 50 mm, 1.9 μm; Agela Technologies; Made in China). The whole process with a gradient elution program is as follows: 0.00–0.50 min, 5% B; 0.50–1.50 min, 90% B; 1.50–3.00 min, 90% B; 3.00–3.10 min, 15% B; and 3.10–5.00 min, 15% B. The mobile phase was made up of 0.10% formic acid in water (A) and acetonitrile (B), and the velocity of flow was 0.50 ml/min. The auto sampler and column temperature were set at 15 and 40°C, respectively. The injection volume was 1 μl. The Q-Trap mass spectrometer was operated with positive electrospray ionization source (ESI) and scanned using the multiple-reaction monitoring (MRM) mode. The Analyst^® software version 1.6.3 (Applied Bio-systems, AB SCIEX™, United States) was used to control the UPLC-MS/MS system. The best of the YWS01125 parameters was curtain gas, 35 psi; collision gas, medium; ion spray voltage, 5500 V; temperature, 500°C; ion source gas 1, 50 psi; and ion source gas 2, 50 psi. The quantitative analysis ion pairs of YWS01125 and MGL-3196 (IS) were m/z 446.2 → 375 and m/z 435.2 → 182, individually. The suitable collision energy (CE) of YWS01125 and IS was 42 and 50 eV, respectively, and the decluttering voltage (DP) was 160 and 180 V, respectively.

The frozen mice plasma samples and tissue homogenates were thawed at room temperature, vortexed for 1 min, then taken out in 10-μl aliquots in a 1.50-ml centrifuge tube, and this was then mixed with 1 μl of standard solution and 50 μl of internal standard (IS) solution, which was then dissolved and diluted with acetonitrile. Subsequently, it was vortexed for 3 min and at 14,000 rpm centrifugation for 5 min. The adopted 1 μl of the supernatant was used for sample injection analysis.

The validation was carried out by the US Food and Drug Administration bioanalytical method guidance (Bioanalytical Method Validation, 2018; US Food and Drug Administration, 2018; Chen et al., 2019), namely evaluating selectivity, linearity, accuracy and precision, recovery, matrix effect, and stability. Selectivity was assessed by contrasting the normal blank plasma samples' and analyst plasma samples' chromatograms from six different specimens. Linearity was appraised by different concentrations of the standard medicated plasma samples for 3 days continuously. The peak area was recorded (Booth et al., 2015), and weighted (1/x²) least-squares analysis was used to acquire the standard curve equations. The accuracy and precision were assessed by three different (low, medium, and high) QC samples by diluting with acetonitrile; each concentration had six samples and was continuously accessed for 3 days, and the accuracy, intra-day precision, and inter-day precision were obtained. Recovery was evaluated by comparing the peak area of protein precipitated with acetonitrile first and finally using it to calculate the recovery of the analyst. The matrix effect was measured by comparing the analyst peak area with the saline solution samples and QC samples at the three concentrations. The stability was assessed by defining the three QC levels in several different storage conditions: at room temperature for 6 h, injector stability at 15 °C for a whole night, and after three freeze–thaw cycles.

The incubation system was carried out in 0.05 M Tris/KCl buffer (PH = 7.4) and included the following: pyrazinone compounds dissolved with dimethyl sulfoxide (DMSO), mouse liver microsomes and human liver microsomes at 0.50 mg/ml, 1.30 mM nicotinamide adenine dinucleotide sodium phosphate (NADP), 3.30 mM glucose-6-phosphate disodium salt (G-6-P), 0.40 units/mL glucose-6-phosphate dehydrogenase (G-6-PDH), and 3.30 mM magnesium chloride. The volume of the organic solvent did not exceed 1% of the reaction system. After preincubation for 3 min in an 850-rpm blender (37°C), liver microsomes were added to initiate the incubation reaction (37°C). The incubation time was 0, 30, 60, and 90 min. After incubation, an equal volume of cold acetonitrile was added to stop the reaction, and an internal standard (0.05 mg/ml acetaminophen solution) was added. The samples were centrifuged at 10,000 rpm for 10 min to precipitate proteins (Day et al., 2017; Manne et al., 2018), and the 10 µL of the supernatant was taken for HPLC analysis.

A total of 18 C57BL/6J mice were given YWS01125 by oral administration with a dose of 10, 20, and 40 mg/kg, respectively (Lally et al., 2019; Kelly et al., 2014; Motamed et al., 2017) (with the concentration of drug administration selected according to the study by Kelly et al.). The blood samples of 20 μl were collected and placed in heparinized tubes from the ophthalmic venous plexus before and at 0.03, 0.08, 0.16, 0.25, 0.50, 1, 2, 4, 6, 8, 12, and 24 h. The samples were immediately centrifuged at 14,000 rpm for 10 min at 4°C. The plasma was separated and stored below −80°C until UHPLC-MS/MS analysis.

A total of 24 male C57/BL6J mice were divided into 4 groups at random, with 6 mice in a group for tissue distribution at 0.25, 1, 6, and 24 h. They were given YWS01125 after oral administration of 20 mg/kg, and then sacrificed at 0.25, 1, 6, and 24 h. Tissues from the heart, liver, spleen, lungs, kidneys, stomach, small intestine, colon, fat, muscle, testes, and brain were taken and rinsed with saline solution to remove the blood and connective tissues (Geng et al., 2015; He et al., 2017; Jiang et al., 2017), then put on the filter paper and weighed, quickly. Each tissue sample was homogenized by using five times of the tissue weight saline (w/v). All homogenates were stored at−80 °C until analysis.

In this study, the comparison of the pharmacokinetic data between the normal and experimental groups was done using IBM SPSS Statistics 22.0 (Statistical Product and Service Solutions, United States) with an independent-samples t-test. The value of p < 0.05 was considered statistically significant.

The liver microsomal metabolic system was established. Phenacetin was used as a positive control, and its metabolite paracetamol were used for liquid phase analysis. The concentration of phenacetin peaked at 12.50 min, and its metabolite paracetamol peaked in about 5 min. In addition, phenacetin was added into the metabolic system and cultured for 0, 30, and 60 min. The results showed that its metabolite acetaminophen appeared in a time-dependent manner. However, YWS01125 had no metabolites during the culture time in mice and human liver microsomes, suggesting that YWS01125 was not metabolized in the liver microsomal system (Figure 3).

Under the above chromatographic conditions, the retention time for YWS01125 and MGL-3196 (IS) was 1.60 and 1.80 min, respectively. The production scan MS spectra of MGL-3196 and YWS01125 is shown in Figure 4. Figure 5 shows YWS01125 of 4 and 400 ng/ml, and IS of 2 ng/ml UPLC chromatographic images. During optimization of the mass spectrometric parameters, a higher response was observed in the positive mode than in the negative mode for all of the analysts. The deprotonated precursor molecular ions (M − H)− were chosen to be monitored. The quantitative analysis was carried out in the MRM mode at m/z 446.2 → 336 for YWS01125 and m/z 435.2 → 182 for MGL-3196 (IS). Meanwhile, the transitions were regarded as qualitative analysis. Finally, for YWS01125, the decluttering potential (DP), entrance potential (EP), cell exit potential (CXP), and collision energy (CE) were 160, 42, 15, and 28 V, respectively, while for MGL-3196, the DP, EP, CXP, and CE were 180, 50, 43, and 20 V, respectively.

The validated method was applied to the pharmacokinetic study of YWS01125 in normal C57BL/6J mice after oral administration, at doses of 10, 20, and 40 mg/kg suspensions with sodium carboxymethyl cellulose (CMCNa). YWS01125 was measured in the plasma until the last blood collection time (24 h). The plasma concentration–time profiles of YWS01125 following the single oral administration in C57/BL6J are presented in Figure 8. The main pharmacokinetic parameters of YWS01125 were processed by a no compartmental model using the DAS 2.0 software package, and the pharmacokinetic parameters are summarized in Table 4. As can be seen, the peak concentration (C_max) of each concentration of YWS01125 in the mice plasma is 188.50 ± 30.03 ng/ml at 55.00 ± 12.25 min for 10 mg/kg, 293.42 ± 60.43 ng/ml at 60.00 ± 0.01 min for 20 mg/kg, and 381.58 ± 30.55 ng/ml at 55 ± 12.25 min for 40 mg/kg. From a comparison of the maximum plasma concentration (C_max), the time to reach C_max (T_max) and areas under the plasma drug concentration–time curve (AUC0–t and AUC0–∞) of YWS01125 in the C57BL/6J mice were markedly increased, and there were statistically significant differences in these parameters (p < 0.05).

The tissue distributions of YWS01125 were investigated in C57BL/6J mice at 0.25, 1, 6, and 24 h after oral administration. The examined organs or tissues included the heart, liver, spleen, lungs, kidneys, stomach, small intestine, colon, fat, muscle, testes, and brain. The results are presented in Figure 9. The linear ranges, correlation coefficients, weight factors, and regression equations for all analytes in the mouse plasma and tissue homogenates are shown in Table 5. The concentration of each tissue at 0.25 h is indicated as stomach > kidneys > liver > colon > small intestine > testes > heart > spleen > lungs > fat; concentration of each tissue at 1 h is small intestine > colon > liver > stomach > kidneys > lungs > heart > fat > testes; concentration of each tissue at 6 h is small intestine > colon > stomach > liver > kidneys > heart; and concentration of each tissue at 24 h is that the whole tissues were not detected (Table 6). After oral administration, the concentrations of YWS01125 were not detected in the brain and muscle, suggesting that the blood–brain barrier may block the entry of the drug into the brain. The content of the compound in the stomach was much higher than that in any other organ. The highest levels were reached 0.25 h after oral administration, then decreased thereafter. For the heart, liver, kidneys, lungs, small intestine, colon, and fat, the highest level was reached 1 h after oral administration, and it reached the highest levels at 0.25 h in the spleen, stomach, and testes. These data show that YWS01125 is quickly distributed throughout the mice body after oral administration; however, long-term accumulation might not be observed.