Compound 9f exhibited IC50 values of 2.3 nM over EGFR and 234 nM over HER2, which is 38-fold of staurosporine and 10-fold of TAK-285 over EGFR. Compound 9f also showed high selectivity profile when tested over a small kinase panel. Compounds 9a–h showed IC50 values in the range of 1.0–7.3 nM and 0.8–2.8 nM against PC3 and 22RV1 prostate carcinoma cell lines, respectively. Cell cycle analysis, apoptotic induction, molecular docking, dynamics, and MM-GBSA studies confirmed the plausible mechanism(s) of compound 9f as a potent EGFR/HER2 dual inhibitor with an effective antiproliferative action against prostate carcinoma.Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) protein tyrosine kinases co-expressed in various cancers such as ovarian, breast, colon, and prostate subtypes. Herein, new TAK-285 derivatives ( The IC50 values of the highly active candidate were evaluated over both kinases. In addition, to verify the selectivity of the most active candidate, a small kinase panel was then employed. Cell-based antiproliferative evaluation was carried out over two prostate carcinoma cell lines. Moreover, comprehensive simulation analyses were accomplished to recognise the binding affinities and direction of the final small molecules.Up to date, many potential antitumor small molecule inhibitors bearing 2-nitroimidazole moiety have been discovered2a–d via reacting 2-nitroimidazole (1) with different 1,n-dibromoalkanes in DMF solvent and K2CO3 at 60 °C. In 3) reacted with 3,4-dichlorophenol in the presence of K2CO3 and acetonitrile solvent for 3 h at 85 °C to afford 1,2-dichloro-4-(2-chloro-4-nitrophenoxy)benzene (4). The nitro group of intermediate 4 was then reduced via 10% platinum on carbon and H2 gas at room temperature to afford 3-chloro-4-aniline (5). The second aniline reagent (3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline) was purchased. In 6) reacted separately with the two aniline reagents to form intermediates 7a and 7b. Intermediates 8a and 8b were then produced by hydrolysis of the acetate group of compounds 7a and 7b utilising 28% aqueous ammonia in methanol solvent. The free phenolic group in compounds 8a and 8b was allowed to react with the imidazoles 2a–d in DMF solvent and K2CO3 at 80 °C to generate the desired TAK-285 derivatives (9a–h) (s (9a–h) .9a–h, various spectroscopic approaches were used including 1H NMR, 13C NMR, and HRMS. Also, their purity was acquired via the HPLC system where they showed purity higher than 95%. Since all final compounds have a methoxy group, a singlet peak at the 3.95–3.92 ppm range was observed in the 1H NMR spectra. In addition, the specific peak of C2 position proton of quinazoline scaffold was found at 8.54–8.51 ppm (see supporting information). Furthermore, the extended alkyl linkers of all final compounds (n = 2–5) were confirmed where their protons and carbons were found in relatively low chemical shifts. In the 1H NMR spectrum, compound 9a showed the C2 of quinazoline at 8.52 ppm, and three protons of the methoxy group at 3.92 ppm as a singlet peak. Additionally, the O-alkylation reaction between the OH group and the imidazole linker (step vi) was proved by the disappearance of the OH peak. Like compound 9a, the target compounds 9b, 9c, and 9d have also shown a similar pattern, and their extending protons and carbons were found in the range of 2.04–1.34 ppm in 1H NMR spectra and 80.00–20.00 ppm in 13C NMR spectra, respectively. The 3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline derivative 9e was identified by one proton of benzene ring at 8.22 ppm as a doublet peak with a J coupling constant of 4.0 Hz. In addition, there is a singlet peak at 3.95 ppm attributable to the three protons of the methoxy group in the 1H NMR spectrum, while its carbon was acquired at 56.35 ppm in the 13C NMR spectrum. These findings provided evidence proving the successful synthesis of the desired TAK-285 derivatives 9a–h.To elucidate the chemical structure of compounds 9a–h) were assessed over EGFR and HER2 kinases at Reaction Biology Co.  via “HotSpotSM” assay at 10 µM concentration in the presence of 10 µM of ATP. The results were obtained as the % remaining kinase activity of test samples in comparison to the DMSO vehicle. The rates of % kinase inhibition of compounds 9a–h over both kinases were computed and described in 9a–h over EGFR kinase. Incorporation of 3-chloro-4-anilino group in compounds 9a–d demonstrated an inhibitory range of 88.90–94.25%, which is lower than the inhibitory range expressed by derivatives with the hydrophobic moiety of TAK-285 (3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline, 9e–h) that demonstrated an outstanding range of % inhibition over EGFR (96.75–99.33%). In the case of HER2, it was observed that most tested compounds showed lower inhibition rates. Nevertheless, a comparable SAR model was revealed over HER2 kinase. While derivatives 9a–d exhibited a modest inhibitory range of 69.83–80.26%, TAK-285 derivatives 9e–h revealed a higher inhibitory activity range (81.74–97.95%). Among all, compound 9f exhibited the best dual inhibition values over EGFR and HER2 with 99.33 and 97.95% inhibition over both kinases, respectively. Accordingly, compound 9f was selected for further investigations.All the newly synthesised TAK-285 derivatives  encouraged us to do further assessment of compound  kinases . The IC59f), an in vitro screening assay was performed over a small panel of cancer-related kinases including fibroblast growth factor receptor 1 (FGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), cyclin-dependent kinase 2 (CDK2), c-mesenchymal-epithelial transition factor (c-MET), and p38α mitogen-activated protein kinase (MAPK14) in a single-dose concentration of 10 µM. As shown in 9f with its nanomolar potency against both kinases (EGFR and HER2) compared to the other tested kinase targets.To assess the selectivity and the kinase inhibition profile of the most active dual inhibitor of this new series  was measured using MTT assay against two human prostate carcinoma cell lines (PC3 and 22RV1). 9a–h showed IC50 values in the range of 1.0–7.3 nM and 0.8–2.8 nM against PC3 and 22RV1, respectively. Compound 9f was the most potent derivative over PC3 cell line with an IC50 value of 1.0 nM, while compound 9e showed the best IC50 value over 22 RV1 cell line (0.8 nM). The potent nanomolar cell-based activity of this series of compounds over the tested prostate carcinoma cell line could be attributed to other possible targets in addition to EGFR and HER2 receptors. The presence of the 2-nitroimidazole moiety in the chemical structure of compounds 9a–h could develop possible covalent bonds between the nitroimidazole moiety and some cellular proteinsSome inhibitors of the ErbB (EGFR/HER2/ErbB3/ErbB4) family, notably the dual EGFR/HER2 inhibitor lapatinib, failed in phase II clinical trials despite overexpression of this family in castration-resistant prostate cancer (CRPC)50 values.Three independent experiments were performed in triplicate. Standard deviation (±SD) of the mean was also obtained for all experiments along with the IC9f, was chosen for testing its outcomes on the cell cycle profile and apoptosis. PC3 and 22RV1 cells were treated with compound 9f at its IC50. The comparison data in 9f (test 2) arrested the cell cycle of 22RV1 and PC3 cells at the G2/M phase by 62.74% and 49.43%, respectively (9f treated sample has arrested it at G2/M phases as indicated by higher number of counts (%) in these phases of both cell cycle studies.Cell cycle progression is responsible for normal cell growth and proliferation. DNA damage can result in apoptosis, which causes cell death, or DNA repair. At specific checkpoints that serve as control mechanisms to guarantee correct cell division, the state of the cells is evaluated. Checkpoints in the cell cycle include the G1 (restriction), S (metaphase), and G2/Mectively . Also, t9f-treated 22RV1 cells underwent early apoptosis (13.20%), and simultaneously, some cells had prominently progressed to the late apoptosis phase (15.38%) (9f-treated PC3 cells went through early apoptosis (6.27%), and late apoptosis phase (16.94%)  . We foun(15.38%) . Moreove16.94%)  and 6. L.94%  and(16.94%)  and PC3 (16.94%)  cells coAnnexin V conjugated to fluorochromes (PE) retains its high affinity for phosphatidylserine (PS), making it a sensitive probe for flow cytometric analysis of apoptotic cells. PE Annexin V staining occurs prior to the loss of membrane integrity that occurs in the final stages of cell death caused by either apoptotic or necrotic processes. As a result, PE Annexin V staining is typically used in conjunction with a vital dye such as propidium iodide (PI) or DAPI to allow the investigator to identify early apoptotic cells . Viable cells with intact membranes exclude DAPI, whereas dead and damaged cells’ membranes are permeable to DAPI. When apoptosis is measured over time, cells can often be tracked from PE Annexin V and DAPI negative , to PE Annexin V positive and DAPI negative (early apoptosis), and finally to PE Annexin V and DAPI positive (end stage apoptosis and death). The progression of cells through these three stages suggests apoptosis.9a–h) towards EGFR ) of EGFR and HER2 were inserted as reference standards. Since all docked compounds (9a–h) showed promising results, derivatives 9f and 9g, which were biologically superior, were selected for deep investigations compared to the co-crystallised inhibitor in each case.To validate the dual inhibitory activities of the newly designed TAK-285 derivatives (9f showed the formation of one H-bond with Lys692 (2.95 Å) and three pi–H bonds with Cys773, Val702, and Arg817 . Its binding score was found to be −8.79 kcal/mol (RMSD = 1.13), besides compound 9g binding score was −8.46 kcal/mol (RMSD = 1.45), indicating superior binding affinities for both derivatives compared to that of the docked AQ4 inhibitor. Also, compound 9g formed one H-bond with Met769 (3.16 Å) and two pi–H interactions with Cys773 and Leu694 , as depicted in The binding pocket of EGFR receptor  achieved two H-bonds with Met801 and Cys805 . Besides, compound 9g  got stabilised through the formation of four H-bonds with Met801 (1), Lys753 (2), and Phe731 (1) at 3.52, 3.01, 3.20, and 3.39 Å, respectively, as represented in 9f and 9g members) towards the binding pockets of both EGFR and HER2 receptors.On the other hand, the binding site of the HER2 receptor  and HER2 (3RCD) tyrosine kinase domains. The protein conformational change was monitored via the difference in the position of the Cα atoms of the protein backbone and was reported in Å. The positional change of the Cα for both protein complexes was plotted as a function of simulation time in Molecular dynamic simulations were applied to simulate the behaviour of the hit compounds in a cell-like environment. Compounds 9f-1M17 complex showed an RMSD of about 3.00 Å, which is considered acceptable for such protein. The 9g-1M17 and the Co-1M17 complexes showed a fluctuation at around 4.00 and 5.00 Å, respectively; such instability is due to the presence of the hinge as it is playing a critical rule whenever the ligand tries to orient itself inside the active site, the salt bridge between the residue Glu734 and Lys851 will break, the protein subunits will move apart, and the hinge will open which results in higher fluctuation. The distance between the Glu734 and Lys851 for the 9g-1M17 complex is depicted in 9f, the co-crystal is illustrated in Figure SI 1 (SI).The RMSD of the 1M17 complexes is plotted in 9f-3RCD and the Co-3RCD were stable and fluctuated at around 2.00 Å. On the other hand, the 9g-3RCD complex fluctuates at around 4.00 Å, which comes from the unfolded loop of the Arg756, Glu757, Asn758, Thr759, and Ser760, coloured red in In the case of the 3RCD complexes, the 9g was unstable inside the active site. This instability is a result of the moving of the imidazole ring out of the active site, and this leads to relocating the ligand within the active site. The butyl bridge rotatability plays a critical role in this instability. Compound 9f, on the other hand, showed much more stability; the compound was stable during the simulation time, at around 155–170 ns, a fluctuation of 8.00 Å was observed; this fluctuation is again due to the presence of the alkyl bridge, the imidazole group moved out of the protein active site during this period, before it goes back inside the active site as described in With regards to 1M17, compound 9f showed a fluctuation between 2.00 and 4.00 Å; the compound imidazole group was shifting its position inside the active site, which led to this RMSD. Compound 9g also showed similar behaviour, and the pentyl bridge gives the imidazole flexibility to move out/in the active site, which results in an RMSD of 3.00–4.00 Å. The co-crystal ligand holds tight inside the active site with an RMSD of ∼2.00 Å during the simulation.In the case of 3RCD, compound 9f-1M17 and 9g-3RCD with the active site residues was carried out. The interactions of these ligands with protein residue were plotted using the simulation interaction diagram panel of Maestro software.Further, a deep analysis of the interactions of the compounds Figure SI 3. Compound 9f interactions histogram , Phe731 (95%), Ala751 (75%), Phe1004 (45%), and Leu852 (40%). In addition, Thr862 formed a water bridge hydrogen bond almost 65% of the simulation time, along with Lys753 (35%). The residues that were able to form interactions more than 30% of the simulation time are reported in Figure SI 5.In the case of 3RCD, compound istogram , the Metistogram . Compoun9f and 9g showed lower binding energy compared to the co-crystal ligand in the case of 1M17, while in the case of 3RCD, compound 9g showed superiority over 9f, which in turn, showed higher binding energy compared to the co-crystal ligand.The average MM-GBSA binding energy over the last 50 ns was generated using the thermal_mmgbsa.py python script provided by Schrödinger, which also generates Coulomb energy, covalent binding energy, van der Waals energy, lipophilic energy, generalised Born electrostatic solvation energy, and hydrogen-bonding energy. All the obtained data are shown in 2a–d were synthesised in our recently published report4 and 5 were synthesised following the earlier report1H NMR spectra were added in the Supplementary File.The general protocols utilised for the chemical synthesis, structure elucidation, and purity of the synthesised compounds were provided in the Supplementary File. Compounds 6, 1.0 equiv.) and the appropriate aniline reagent (1.2 equiv.) in isopropyl alcohol (i-PrOH). The mixture was refluxed for 4 h and cooled to rt after completion of the reaction. The reaction mixture was filtered using i-PrOH to give the target intermediate, which was used in the next step without further purification.A round-bottom flask was charged with 4-chloro-7-methoxyquinazolin-6-yl acetate phenyl)amino)-7-methoxyquinazolin-6-yl acetate (7a). Ivory solid. Yield: 100% . 1H NMR  δ 11.10 , 8.92 , 8.61 , 8.13 , 7.76 , 7.64 , 7.43 , 7.35 , 7.31 , 6.97 , 3.99 , and 2.38 .4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-7-methoxyquinazolin-6-yl acetate (7b). Ivory solid. Yield: 100% . 1H NMR  δ 10.99 , 8.91 , 8.57 , 8.15 , 7.78 , 7.63 , 7.51 , 7.41 , 7.36 , 7.29–7.24 , 3.99 , and 2.38 .7, 3–4 mmol) and an excess amount of aqueous ammonia solution (28% in H2O) in methanol (CH3OH) were put into the round-bottom flask and stirred for 4 h at rt. The excess solvent of the resulting mixture was partially concentrated, filtered with chilled methanol, and dried to give the required intermediates without further purification.The appropriate acetate intermediate phenyl)amino)-7-methoxyquinazolin-6-ol (8a). Ivory solid. Yield: 94.3% . 1H NMR  δ 9.70 , 9.52 , 8.48 , 8.29 , 7.91 , 7.79 , 7.60 , 7.29 , 7.22–7.21 , 6.91 , and 3.96 . 13C NMR  δ 157.25 (quinazoline-C4), 156.21 (quinazoline-C7), 154.41 (Ar-C), 152.35 (quinazoline-C2), 147.25 (quinazoline-C6), 146.73 (quinazoline-C8a), 145.12 (Ar-C), 138.83 (Ar-C), 132.46 (Ar-C), 132.00 (Ar-C), 125.27 (Ar-C), 125.23 (Ar-C), 123.10 (Ar-C), 122.95 (Ar-C), 121.96 (Ar-C), 118.70 (Ar-C), 117.05 (Ar-C), 110.10 (quinazoline-C4a), 107.65 (quinazoline-C8), 105.74 (quinazoline-C5), and 56.40 (OCH3). HRMS (ESI) m/z calculated for C21H15Cl3N3O3 [M + H]+: 462.0179, found: 462.0170.4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-7-methoxyquinazolin-6-ol (8b). Ivory solid. Yield: 64% . 1H NMR  δ 9.71 , 9.53 , 8.49 , 8.30 , 7.92 , 7.79 , 7.60 , 7.45 , 7.30 , 7.21–7.18 , 3.96 . 13C NMR  δ 158.23 (quinazoline-C4), 156.23 (quinazoline-C7), 154.41 (Ar-C), 152.36 (quinazoline-C2), 147.25 (quinazoline-C6), 146.73 (quinazoline-C8a), 145.11 (Ar-C), 138.79 (Ar-C), 131.85 (Ar-C), 131.28 (Ar-C), 125.43 (Ar-C), 123.14 (Ar-C), 123.08 (Ar-C), 122.81 (Ar-C), 122.04 (Ar-C), 120.49 (Ar-C), 119.76 (Ar-C), 113.11 (Ar-C), 110.10 (quinazoline-C4a), 107.65 (quinazoline-C8), 105.75 (quinazoline-C5), and 56.39 (OCH3). HRMS (ESI) m/z calculated for C22H16ClF3N3O3 [M + H]+: 462.0832, found: 462.0821.8a, 1.0 equiv.) in DMF, potassium carbonate (2.0 equiv.) and the appropriate bromoalkyl imidazole  were added at rt. The reaction mixture was stirred for 4 h at 80 °C and partitioned using EtOAc and water. The collected organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The crude was purified by flash column chromatography (CH2Cl2:CH3OH = 20:1) to afford the desired final TAK-285 derivatives.To the intermediate phenyl)-7-methoxy-6-(3-(2-nitro-1H-imidazol-1-yl)propoxy)quinazolin-4-amine (9a). Yellow solid . m.p.: 237–238 °C. HPLC purity: 96.95% . 1H NMR  δ 9.55 , 8.52 , 8.18 , 7.85 , 7.81 , 7.67 , 7.62 , 7.32 , 7.23 , 7.21 , 7.17 , 6.93 , 4.62 , 4.19 , 3.92 , 2.42–2.35 . 13C NMR  δ 157.14 (quinazoline-C4), 156.38 (quinazoline-C7), 155.02 (Ar-C), 153.18 (imidazole-C2), 148.40 (quinazoline-C2), 147.66 (quinazoline-C6), 145.60 (quinazoline-C8a), 138.35 (Ar-C), 132.48 (Ar-C), 132.04 (Ar-C), 128.34 (Ar-C), 128.24 (imidazole-C5), 125.36 (imidazole-C4), 125.27 (Ar-C), 123.69 (Ar-C), 122.87 (Ar-C), 122.47 (Ar-C), 118.87 (Ar-C), 117.21 (Ar-C), 109.23 (quinazoline-C4a), 107.81 (quinazolie-C8), 103.35 (quinazoline-C5), 66.54 (propyl-CH2), 56.36 (OCH3), 47.43 (propyl-CH2), and 29.61 (propyl-CH2). HRMS (ESI) m/z calculated for C27H22Cl3N6O5 [M + H]+: 615.0717, found: 615.0723.N-phenyl)-7-methoxy-6-(4-(2-nitro-1H-imidazol-1-yl)butoxy)quinazolin-4-amine (9b). Yellow solid . m.p.: 101–102 °C. HPLC purity: 96.17% (RT = 15.333 min). 1H NMR  δ 9.56 , 8.52 , 8.19 , 7.86 , 7.81 , 7.76 , 7.62 , 7.32 , 7.24 , 7.21 , 7.19 , 6.93 , 4.50 , 4.18 , 3.92 , 2.04–1.97 , and 1.86–1.79 . 13C NMR  δ 157.14 (quinazoline-C4), 156.35 (quinazoline-C7), 155.00 (Ar-C), 153.07 (imidazole-C2), 148.66 (quinazoline-C2), 147.49 (quinazoline-C6), 145.59 (quinazoline-C8a), 138.36 (Ar-C), 132.48 (Ar-C), 132.04 (Ar-C), 128.35 (Ar-C), 128.31 (imidazole-C5), 125.36 (imidazole-C4), 125.26 (Ar-C), 123.71 (Ar-C), 122.85 (Ar-C), 122.50 (Ar-C), 118.86 (Ar-C), 117.21 (Ar-C), 109.30 (quinazoline-C4a), 107.77 (quinazoline-C8), 103.15 (quinazoline-C5), 68.96 (butyl-CH2), 56.36 (OCH3), 49.70 (butyl-CH2), 27.15 (butyl-CH2), and 25.88 (butyl-CH2). HRMS (ESI) m/z calculated for C28H24Cl3N6O5 [M + H]+: 629.0874, found: 629.0881.N-phenyl)-7-methoxy-6-((5-(2-nitro-1H-imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (9c). Yellow solid . m.p.: 94–95 °C. HPLC purity: 97.95% (RT = 15.902 min). 1H NMR  δ 9.55 , 8.51 , 8.19 , 7.89 , 7.80 , 7.71 , 7.62 , 7.32 , 7.24 , 7.20 , 7.18 , 6.93 , 4.43 , 4.13 , 3.92 , 1.92–1.83 , and 1.51–1.44 . 13C NMR  δ 157.15 (quinazoline-C4), 156.33 (quinazoline-C7), 154.98 (Ar-C), 153.02 (imidazole-C2), 148.79 (quinazoline-C2), 147.45 (quinazoline-C6), 145.57 (quinazoline-C8a), 138.38 (Ar-C), 132.48 (Ar-C), 132.04 (Ar-C), 128.35 (Ar-C), 128.30 (imidazole-C5), 125.35 (imidazole-C4), 125.26 (Ar-C), 123.72 (Ar-C), 122.85 (Ar-C), 122.51 (Ar-C), 118.85 (Ar-C), 117.20 (Ar-C), 109.31 (quinazoline-C4a), 107.74 (quinazoline-C8), 102.89 (quinazoline-C5), 69.04 (pentyl-CH2), 56.33 (OCH3), 49.75 (pentyl-CH2), 30.00 (pentyl-CH2), 28.56 (pentyl-CH2), and 23.00 (pentyl-CH2). HRMS (ESI) m/z calculated for C29H26Cl3N6O5 [M + H]+: 643.1030, found: 643.1031.N-phenyl)-7-methoxy-6-((6-(2-nitro-1H-imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (9d). Yellow solid . m.p.: 90–91 °C. HPLC purity: 95.31% (RT = 16.266 min). 1H NMR  δ 9.55 , 8.51 , 8.19 , 7.87 , 7.79 , 7.69 , 7.61 , 7.32 , 7.23 , 7.19 , 7.16 , 6.92 , 4.38 , 4.12 , 3.92 , 1.86–1.77 , 1.54–1.46 , and 1.41–1.34 . 13C NMR  δ 157.15 (quinazoline-C4), 156.33 (quinazoline-C7), 154.99 (Ar-C), 153.00 (imidazole-C2), 148.85 (quinazoline-C2), 147.44 (quinazoline-C6), 145.57 (quinazoline-C8a), 138.39 (Ar-C), 132.48 (Ar-C), 132.04 (Ar-C), 128.28 (Ar-C), 128.25 (imidazole-C5), 125.35 (imidazole-C4), 125.25 (Ar-C), 123.72 (Ar-C), 122.85 (Ar-C), 122.52 (Ar-C), 118.85 (Ar-C), 117.21 (Ar-C), 109.32 (quinazoline-C4a), 107.74 (quinazoline-C8), 102.86 (quinazoline-C5), 69.12 (hexyl-CH2), 56.33 (OCH3), 49.80 (hexyl-CH2), 30.17 (hexyl-CH2), 28.92 (hexyl-CH2), 26.05 (hexyl-CH2), and 25.56 (hexyl-CH2). HRMS (ESI) m/z calculated for C30H28Cl3N6O5 [M + H]+: 657.1187, found: 657.1184.N-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-7-methoxy-6-(3-(2-nitro-1H-imidazol-1-yl)propoxy)quinazolin-4-amine (9e). Yellow solid . m.p.: 232–233 °C. HPLC purity: 98.08% (RT = 14.336 min). 1H NMR  δ 9.58 , 8.54 , 8.22 , 7.88 , 7.84 , 7.69 , 7.63 , 7.49 , 7.36 , 7.25–7.23 , 7.19 , 4.64 , 4.22 , 3.95 , and 2.43–2.38 . 13C NMR  δ 158.14 (quinazoline-C4), 156.39 (quinazoline-C7), 155.02 (Ar-C), 153.18 (imidazole-C2), 148.39 (quinazoline-C2), 147.65 (quinazoline-C6), 145.58 (quinazoline-C8a), 145.20 (Ar-C), 138.31 (Ar-C), 131.89 (Ar-C), 131.30 (Ar-C), 130.98 (imidazole-C5), 128.34 (imidazole-C4), 128.23 (Ar-C), 125.45 (CF3), 123.75 (Ar-C), 123.02 (Ar-C), 122.55 (Ar-C), 120.65 (Ar-C), 119.87 (Ar-C), 113.22 (quinazoline-C4a), 109.23 (Ar-C), 107.80 (quinazoline-C8), 103.34 (quinazoline-C5), 66.54 (propyl-CH2), 56.35 (OCH3), 47.43 (propyl-CH2), and 29.61 (propyl-CH2). HRMS (ESI) m/z calculated for C28H23ClF3N6O5 [M + H]+: 615.1371, found: 615.1371.N-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-7-methoxy-6-(4-(2-nitro-1H-imidazol-1-yl)butoxy)quinazolin-4-amine (9f). Yellow solid . m.p.: 103–104 °C. HPLC purity: 95.07% (RT = 14.794 min). 1H NMR  δ 9.57 , 8.52 , 8.20 , 7.87 , 7.82 , 7.75 , 7.61 , 7.47 , 7.34 , 7.23–7.19 , 4.50 , 4.18 , 3.92 , 2.03–1.97 , and 1.85–1.79 . 13C NMR  δ 157.60 (quinazoline-C4), 155.83 (quinazoline-C7), 154.47 (Ar-C), 152.55 (imidazole-C2), 148.12 (quinazoline-C2), 146.98 (quinazoline-C6), 145.04 (quinazoline-C8a), 144.52 (Ar-C), 137.78 (Ar-C), 131.35 (Ar-C), 130.75 (Ar-C), 130.43 (imidazole-C5), 127.80 (imidazole-C4), 127.76 (Ar-C), 124.89 (CF3), 123.24 (Ar-C), 122.47 (Ar-C), 122.06 (Ar-C), 120.11 (Ar-C), 119.33 (Ar-C), 112.62 (quinazoline-C4a), 108.75 (Ar-C), 107.26 (quinazoline-C8), 102.62 (quinazoline-C5), 68.41 (butyl-CH2), 55.82 (OCH3), 49.15 (butyl-CH2), 26.60 (butyl-CH2), and 25.33 (butyl-CH2). HRMS (ESI) m/z calculated for C29H25ClF3N6O5 [M + H]+: 629.1527, found: 629.1528.N-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-7-methoxy-6-((5-(2-nitro-1H-imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (9g). Yellow solid . m.p.: 90–91 °C. HPLC purity: 95.59% (RT = 15.254 min). 1H NMR  δ 9.56 , 8.51 , 8.20 , 7.87 , 7.80 , 7.71 , 7.61 , 7.47 , 7.34 , 7.22–7.18 , 4.43 , 4.13 , 3.92 , 1.91–1.83 , and 1.52–1.44 . 13C NMR  δ 158.15 (quinazoline-C4), 156.34 (quinazoline-C7), 154.98 (Ar-C), 153.03 (imidazole-C2), 148.79 (quinazoline-C2), 147.45 (quinazoline-C6), 145.56 (quinazoline-C8a), 145.00 (Ar-C), 138.35 (Ar-C), 131.89 (Ar-C), 131.30 (Ar-C), 130.98 (imidazole-C5), 128.34 (imidazole-C4), 128.29 (Ar-C), 125.43 (CF3), 123.78 (Ar-C), 123.00 (Ar-C), 122.60 (Ar-C), 120.65 (Ar-C), 119.86 (Ar-C), 113.20 (quinazoline-C4a), 109.31 (Ar-C), 107.75 (quinazoline-C8), 102.90 (quinazoline-C5), 69.04 (pentyl-CH2), 56.32 (OCH3), 49.75 (pentyl-CH2), 30.00 (pentyl-CH2), 28.56 (pentyl-CH2), and 23.00 (pentyl-CH2). HRMS (ESI) m/z calculated for C30H27ClF3N6O5 [M + H]+: 643.1684, found: 643.1693.N-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-7-methoxy-6-((6-(2-nitro-1H-imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (9h). Yellow solid . m.p.: 84–85 °C. HPLC purity: 96.10% (RT = 15.657 min). 1H NMR  δ 9.56 , 8.51 , 8.20 , 7.88 , 7.81 , 7.69 , 7.61 , 7.47 , 7.33 , 7.22–7.20 , 7.16 , 4.38 , 4.13 , 3.93 , 1.87–1.78 , 1.54–1.47 , and 1.42–1.34 . 13C NMR  δ 158.15 (quinazoline-C4), 156.33 (quinazoline-C7), 154.97 (Ar-C), 153.00 (imidazole-C2), 148.84 (quinazoline-C2), 147.44 (quinazoline-C6), 145.54 (quinazoline-C8a), 144.98 (Ar-C), 138.36 (Ar-C), 131.87 (Ar-C), 131.30 (Ar-C), 130.98 (imidazole-C5), 128.28 (imidazole-C4), 128.23 (Ar-C), 125.43 (CF3), 123.77 (Ar-C), 122.97 (Ar-C), 122.59 (Ar-C), 120.63 (Ar-C), 119.88 (Ar-C), 113.16 (quinazoline-C4a), 109.32 (Ar-C), 107.73 (quinazoline-C8), 102.84 (quinazoline-C5), 69.11 (hexyl-CH2), 56.30 (OCH3), 49.80 (hexyl-CH2), 30.17 (hexyl-CH2), 28.92 (hexyl-CH2), 26.06 (hexyl-CH2), and 25.56 (hexyl-CH2). HRMS (ESI) m/z calculated for C31H29ClF3N6O5 [M + H]+: 657.1840, found: 657.1841.SM service from Reaction Biology Co.  was used to screen the tested compounds. The HotSpot assay platform includes specific kinase/substrate pairs and required cofactors. The base reaction buffer consisted of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. The tested compounds were dissolved in 100% DMSO to a specific concentration, and serial dilution was conducted. The reaction mixture containing the tested compound and 33P-ATP was incubated at room temperature for 2 h, and radioactivity was detected using the filter-binding method.Enzyme inhibitory assays were carried out as described in our previous reports in the literature2 with a 95% humid atmosphere.Prostate cancer cell lines (PC3 and 22RV1) of American Type Culture Collection (ATCC) were obtained from Korean Cell Center (KCL). PC3 and 22RV1 cell lines were cultured in Roswell Park Memorial Institute Medium (RPMI) 1640 and Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 1% penicillin–streptomycin and 10% foetal bovine serum (FBS). The cells were maintained at 37 °C in a 5% CO4 cells per well) and then incubated for 24 h at 37 °C in a 5% CO2-containing atmosphere. Subsequently, the medium from the culture plate was removed, and 100 µL of RPMI media containing different concentrations of compounds  were then added into the cell lines in each well and incubated for 24 h in a 5% CO2 humidified incubator at 37 °C. Afterwards, MTT solution  was added to each well, replacing the media-containing compounds. After 4 h of incubation, the MTT reagent was removed, and 200 μL of DMSO was then added to dissolve the formazan crystals, and colour intensity was measured at a wavelength of 540 nm using a BioTek Synergy H1 instrument . IC50 was calculated according to the equation of Boltzmann sigmoidal concentration–response curve using Graph Pad Prism 5 .To further analyse the intracellular cytotoxicity effect of the as-synthesised compounds, MTT assay was performed. Moreover, the experiments were divided into subcategories. In each group, cancer cells were first seeded in 96-well plates (4 × 106 cells per well) and then incubated for 24 h at 37 °C in a 5% CO2-containing atmosphere. Subsequently, the medium from the culture plate was removed, and 3 mL of RPMI and DMEM media containing concentrations of compound 9f at its IC50 value were then added into the cells in each well and incubated for 24 h in a 5% CO2 humidified incubator at 37 °C. Afterward, the media was removed following the washing once with PBS and the 25 µL cell sample mixtures were stained with 25 µL PI stain. 25 µL of sample mixture was loaded into ADAMII LS assay slide, then the slide was incubated at room temperature for 1 min in the dark and run on the ADAMII LS fluorescence cell analyser. Cell cycle distribution was calculated using ADAMII LS software .To determine the effect of the tested compound on the cell cycle distribution of PC3 and 22RV1 cell lines. Cell cycles can be analysed with checking the cell cycle change compared to the control group. Control cells without any treatment were used as a reference point for determining the cell cycle arrest phase for the test sample. Cell cycle analysis was performed using the Cell Cycle Analysis Kit . Briefly, cancer cells were first seeded in six-well plates  after washing with phosphate-buffered saline (PBS). The cell was resuspended in 100 µL 1X Annexin V binding buffer, and 5 µL Annexin V-PE reagent was added afterward and incubated for 15 min at room temperature. The sample was subsequently centrifuged, and the pallet was resuspended again with 500 µL 1X Annexin V binding buffer and added 1.25 µL DAPI reagent. The slide loaded with the prepared sample was incubated at room temperature for 1 min in the dark and run on the ADAMII LS fluorescence cell analyser. The comparative data including dot plot graphs and images were calculated and analysed using ADAMII LS software .The apoptotic effect of a potent compound on PC3 and 22RV1 cell lines was assessed using apoptosis analysis with Annexin V-PE, DAPI solution . Early and late apoptotic effects were analysed compared to the control. Cells were seeded in a six-well plate with cells density 0.8 × 10The statistical analysis of the data was done by standard deviations and all values represent the mean ± SD of three independent experiments.9a–h) towards the two target receptors (EGFR and HER2) using the MOE 2019.0102,9a–h) were sketched using the ChemDraw program, transferred into the MOE window, and prepared to be inserted into the same database besides the co-crystallised inhibitor in each case, as previously discussed,,,Two molecular docking processes were performed for the newly designed and synthesised derivatives (The MD simulations and MM-GBSA study were performed following same procedures found in our earlier report9a–h) were synthesised and assessed as EGFR/HER2 dual inhibitors. Applying “HotSpotSM” assay at 10 µM concentration in the presence of 10 µM of ATP, all compounds 9a–h exhibited encouraging percentage inhibition ranges against both kinases. The most active candidate (9f) showed nanomolar IC50 values of 2.3 nM over EGFR and 234 nM over HER2. A kinase selectivity panel of derivative 9f indicated a potential selective profile. Potent nanomolar IC50 values of compounds were obtained against PC3 and 22RV1 cell lines. The cell cycle of 22RV1 and PC3 cells was inhibited by compound 9f by 62.74 and 49.43% in the G2/M phase, respectively. Also, cells treated with compound 9f underwent early apoptosis. Molecular simulation studies revealed insights about the binding mode of the designed compounds. Overall, we report compound 9f as a new potent dual EGFR/HER2 inhibitor with significant cytotoxic activity against 22RV1 and PC3 prostate carcinoma cell lines and could be a potent anticancer drug for future medication.EGFR and HER2 have been co-expressed and recognised in numerous solid tumours. Successfully, new TAK-285 derivatives (Click here for additional data file.


The method yielded the expected α-azidomethyl styrene 4, in good yield, via a dearomative rearrangement.There is a growing interest in developing more efficient synthetic alternatives for the synthesis of nitrogen-containing allylic compounds. This article presents a straightforward two-step protocol to produce 5-(3-azidoprop-1-en-2-yl)benzo[ In ad2.E)-allylic compounds such as (E)-allylic azide 3 (4 (α-azidomethyl styrene) dioxole (2a) was obtained in a 22% yield, while 5-benzo[d]dioxole (2b) was isolated in a 63% yield. 1,3-dibromide 2b was then reacted under our standard azidation conditions [d]dioxole 4] was obtained in a 91% yield -alstyrene) . At the styrene) . Therefoperature , albeit ectively . Hence, nditions  using so1% yield . The speE)-5-(3-azidoprop-1-en-1-yl)benzo[d]dioxole 3 [d]dioxole 4, without the requirement of a transition metal catalyst. If needed, accessing allylic azide 3  provided both dibromo products (2a and 2b) as separable mixture of regioisomers with 1:1.75 ratio (2a:2b)Compounds terature ,29: To a1H NMR  6.79–6.69 , 5.98 , 4.45 , 3.85 , 3.72 , 3.60 , 3.00 . 13C NMR  δ 147.7, 147.22, 129.78, 114.83, 112.85, 111.47, 101.84, 51.13, 43.01, 36.93. IR : 2896, 1619, 1473, 1407, 1230, 1033, 964, 852. LRMS (EI) Calcd for C10H10Br2O2 [M], 321.90. Found: 320[M − 2], 322[M], 324[M + 2]. The analytic data are in accordance with those reported in the literature , 321.90. Found: 320[M − 2], 322[M], 324[M + 2]. The spectroscopic data are in accordance with those reported in the literature [3.3.d]dioxole 4 was synthesized via a modification of a procedure described in the literature  provided the pure product 4 as colorless oil .The new compound 5-(3-azidoprop-1-en-2-yl)benzo, 203.07. Found: 203 [M]. Elemental analysis calculated (%) for C10H9N3O2: C 59.11, H 4.46, N 20.68. Found: C 59.09, H 4.44, N 20.63.4.d]dioxole 4 from safrole. The method is straightforward. The obtained yield in our study compares favorably with that of existing strategies based on allylic C-H activation. Notably, our protocol exclusively yields the expected styrene product. This methodology offers a milder, cost-effective, selective, and simpler alternative for efficiently constructing styrene azide scaffolds from electron-rich systems.In summary, we have introduced a milder two-step approach for the synthesis of 5-(3-azidoprop-1-en-2-yl)benzo[SI


Introduction: Usnic acid (UA) and barbatic acid (BA), two typical dibenzofurans and depsides in lichen, have a wide range of pharmacological activities and hepatotoxicity concerns. This study aimed to clarify the metabolic pathway of UA and BA and illuminate the relationship between metabolism and toxicity.Methods: An UPLC-Q-TOF-MS method was developed for metabolite identification of UA and BA in human liver microsomes (HLMs), rat liver microsomes (RLMs), and S9 fraction (RS9). The key metabolic enzymes responsible for UA and BA were identified by enzyme inhibitors combined with recombinant human cytochrome P450 (CYP450) enzymes. The cytotoxicity and metabolic toxicity mechanism of UA and BA were determined by the combination model of human primary hepatocytes and mouse 3T3 fibroblasts.Results: The hydroxylation, methylation, and glucuronidation reactions were involved in the metabolic profiles of UA and BA in RLMs, HLMs, and RS9. CYP2C9, CYP3A4, CYP2C8, and UGT1A1 are key metabolic enzymes responsible for metabolites of UA and CYP2C8, CYP2C9, CYP2C19, CYP1A1, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 for metabolites of BA. UA and BA did not display evident cytotoxicity in human primary hepatocytes at concentrations of 0.01–25 and 0.01–100 µM, respectively, but showed potential cytotoxicity to mouse 3T3 fibroblasts with 50% inhibitory concentration values of 7.40 and 60.2 µM.Discussion: In conclusion, the attenuated cytotoxicity of BA is associated with metabolism, and UGTs may be the key metabolic detoxification enzymes. The cytotoxicity of UA may be associated with chronic toxicity. The present results provide important insights into the understanding of the biotransformation behavior and metabolic detoxification of UA and BA.  Usnea have been widely used in fodder, dyes, food, perfumery, cosmetics, pharmaceuticals, preservatives, deodorants, ecological applications, and miscellaneous purposes throughout the world, particularly in Europe and East Asian countries, such as China, Japan, and India  , longiusnine (−)-placodiolic acid, depsides barbatic acid (BA), evernic acid, diffractaic acid, and ramalic acid  , antipyracteria) , anti-inacteria) , antitumacteria)  and antiacteria) , for theacteria) , photopracteria) , antioxiacteria) . All thelic acid . Figure However, recent reports associated with liver-related adverse events of UA-containing products aroused widespread concern , and theHepatic enzymes, especially cytochrome P450 (CYP450), commonly have an important role in the metabolism of drugs or chemicals. UA can significantly induce P450 activity in HepG2 cells at concentrations of 10 µM or higher, and slight UA metabolism by CYP1A1 may be present . One stuin vivo and in vitro. However, no liver drug enzymes responsible for UA metabolism have been identified and confirmed, which resulted in an incomplete understanding of UA metabolites and metabolic pathways. In addition, there is an absence of direct evidence of the relationship between metabolism and attenuated toxicity of UA. For BA, another important depside component in Usnea, our previous pharmacokinetic results showed that the exposure of BA in rats was comparable to that of UA after oral administration of Usnea extract , rat liver microsomes (RLMs), and S9 fraction (RS9) combined with UPLC with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology to analyze and identify the metabolites and metabolic pathways of UA and BA. The method of enzyme inhibitor combined with recombinant human CYP450 enzymes was used to identify and confirm the metabolic enzymes responsible for UA and BA. Finally, the co-incubation model of HLMs and mouse 3T3 fibroblasts was used to evaluate the correlation between UA and BA metabolism and hepatocyte toxicity by the Cell Counting Kit-8 test and clarify the mechanism of toxicity induced or attenuated by metabolism. The results of this study will provide a basis for the systematic understanding of metabolic profiles and potential toxic mechanisms of UA and BA.This study aimed to use UA  with a purity of >98% was obtained from Chengdu Desite Biotechnology Co., Ltd.  and BA  with a purity of >98% was obtained from Wuxi Apptec Co., Ltd. . D-Glucose 6-phosphate (G-6-P) disodium salt hydrate, glucose-6-phosphate dehydrogenase (G-6-P-DH) from leuconostoc mesenteroides, β-nicotinamide adenine dinucleotide phosphate disodium salt (NADP+), uridine 5′-diphospho-glucuronidation trisodium salt (UDPGA), 3′-phosphoadenosine-5′-phosphosulfate (PAPS), α-naphthoflavone, 8-methoxypsoralen, orphenadrine, sulfaphenazole, quinidine, 4-methylpyridine, neutral red, tamoxifen, and LC-MS-grade formic acid were purchased from Sigma-Aldrich . Furafylline, nootkatone, and ketoconazole were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. . Quercetin and alamethicin were provided by J&K Scientific . Montelukast was obtained from TCI Development Co., Ltd. . Tris-base was obtained from Shanghai Majorbio Bio-pharm Technology Co., Ltd. . Bradford reagent was purchased from Sangon Biotech Co., Ltd. . cDNA-expressed recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 Bactosomes were acquired from Cypex Limited . Dimethyl sulfoxide, MgCl2, and HCl were purchased from Sinopharm Chemical Reagent Co., Ltd. . Mouse 3T3 fibroblasts were provided by CobioerBiosciences Co., Ltd. . RLMs and their liver S9 fractions (RLS9) were prepared from pooled liver tissues by standard differential ultracentrifugation . HLMs, pThe metabolic profiling study was carried out using a Shimadzu 30A UPLC system  coupled with AB SCIEX Triple TOF™ 5600+ system  equipped with an electrospray ionization (ESI) source. Mass spectrometric detection was performed in negative mode. Nitrogen was used as the nebulizer and auxiliary gas. The flow rates of nebulizer gas (GS 1), heater gas (gas 2), and curtain gas were 55, 55, and 35 psi, respectively. The turbo spray temperature was 550°C. The ion spray voltage floating was −4500 V, and the declustering potential was −100 V. The collision energy (CE) was −35 eV, and the CE spread was set to 15 eV. The TOF scan range was set from m/z 100 to m/z 1200.Chromatographic separations were conducted on an ACQUITY UPLC HSS T3 column . The mobile phase was a gradient system consisting of 0.1% formic acid in water (A) and acetonitrile (B) with a gradient elution system: 10% B (0–1.5 min), 10%–40% B (1.5–3 min), 40%–95% B (3–10 min), 95% B (10–11.5 min), 95%–5% B (11–11.6 min), and 5% B (11.6–12 min). The column temperature was set to 40°C, the flow rate to 0.3 mL/min, and the injection volume to 5 µL.For the phase I metabolism study, the incubation mixture, with a total volume of 200 μL, included liver microsomes (rat: 10.0 mg protein/mL or human: 21.9 mg protein/mL), 50 mM Buffer , NADPH-generating system , and substrate (1 µM). For the phase II metabolism study, the liver microsomes were first mixed with alamethicin (25 μg/mL), which was used as a perforating agent for 5 min preincubation at 37°C in a shaking water bath. The incubation system contained an additional 5 mM UDPGA, and the other reagents were the same as those in phase I. For the RLS9 metabolism study, as for the phase II metabolism study, the RLS9 was first mixed with alamethicin (25 μg/mL) for 5 min preincubation at 37°C in a shaking water bath. The incubation system contained additional 100 µM PAPS, and the other reagents were the same as those in phase I. The substrates were previously dissolved in methanol, and the final methanol concentration was below 1% (v/v) in the system. The reaction was initiated by the addition of a NADPH-generating system. After 60 min incubation at 37°C in a shaking water bath, the reaction was terminated by the addition of 1.0 mL ice-cold acetonitrile. The mixture was kept on ice until it was centrifuged at 12,000 × g for 10 min at 4°C. The supernatants were transferred and dried under a gentle stream of nitrogen at 37°C and then dissolved with 100 µL methanol for analysis. Blank control samples without substrates and negative control samples without NADP+ (for phase I metabolism study) or NADP+ and UDPGA (for phase II metabolism study) or NADP+ and PAPS (for RLS9 metabolism study) were included. Each group was prepared in triplicates.Chemical inhibitor methods together with recombinant CYP and UGT enzymes were used to identify the CYP and UGT isozymes involved in the metabolic pathways of UA and BA.Chemical inhibition studies were performed by the addition of different human CYP inhibitors to the incubation mixture before the addition of the NADPH-generating system. The liver microsomes were first mixed with different concentrations of inhibitors and preincubated at 37°C for 5 min. The incubation system was similar to the phase I liver microsomal reaction system. The selective inhibitors of 10 major CYPs were as follows: α-naphthoflavone for CYP1A1, furafylline for CYP1A2, 8-methoxypsoralen for CYP2A6, orphenadrine for CYP2B6, quercetin for CYP2C8, sulfaphenazole for CYP2C9, quinidine for CYP2D6, nootkatone for CYP2C19, 4-methylpyridine for CYP2E1, and ketoconazole for CYP3A4. The concentrations of the 10 selective inhibitors were 5, 10, 20, 50, and 100 µM.For the recombinant CYP and UGT enzyme method, the incubation system was similar to the phase I and II liver microsomal reaction systems. The liver microsomes or RLS9 were substituted with recombinant enzymes. The 10 recombinant CYP enzymes were as follows: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2C19, CYP2E1, and CYP3A4. The 12 recombinant UGT enzymes were as follows: UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17.The subsequent steps were performed as described in 2 at an initial density of 7 × 105 cells/mL per well at 37°C until the formation of a single-cell layer. The plating medium was changed to an incubation medium, and incubation was continued overnight. Mouse 3T3 fibroblasts were incubated under 5% CO2 at 37°C for 24 h. Different concentrations of UA or BA (100 µL) were incubated with human primary hepatocytes or mouse 3T3 fibroblasts for 48 h, respectively. At the end of incubation, the medium was removed quickly, and cells were washed once with 250 µL Hank’s balanced salt solution (HBSS). A total of 250 µL neutral red (250 μg/mL) was added for staining for 3 h. After neutral red was removed, 100 µL chromogenic solution was added, and shaking was carried out for 30 min at room temperature. The optical density (OD) of each well was read at 570 and 690 nm using a microplate reader, and the relative cell viability  was calculated. Control samples without UA or BA and the positive control group were included. The amount of organic solvent was below 1% (v/v) in the system, and each group was prepared in triplicates.The cytotoxicity of UA and BA was assessed in primary human hepatocytes and mouse 3T3 fibroblasts seeded in 96-well plates, respectively. The concentrations of UA were 0.01, 0.1, 1, 10.0, and 25 μM, those of BA were 0.01, 0.1, 1, 10, and 100 μM, and tamoxifen (50 µM) was set as a positive control. The primary hepatocytes were incubated under 5% CO2 at 37°C overnight. At different concentrations of UA or BA, blank incubation medium (90 µL) as the control group and tamoxifen as the positive control group 1 were incubated with mouse 3T3 fibroblasts for 4 h. The HLMs were preincubated with different concentrations of UA or BA, NADPH, NADPH and UDPGA, or NADPH and UDPGA and inhibitors of CYP2C8, CYP2C9, and CYP3A4 at 37°C for 1 h. Positive control group 2 was also preincubated with HLMs together with NADPH and UDPGA. After preincubation, 90 µL supernatant was incubated with mouse 3T3 fibroblasts under 5% CO2 at 37°C for 4 h. The amount of organic solvent was below 1% (v/v) in the system, and each group was prepared in triplicates.To determine the effect of metabolism on the cytotoxicity of UA and BA, fibroblast NIH mouse 3T3 fibroblasts were assessed and seeded in 96-well plates. The concentrations of UA were 1, 2, 5, 10, 25, and 50 μM, those of BA were 1, 10, 20, 50, 100, and 200 μM, and that of tamoxifen as positive control was 50 µM. Montelukast, sulfaphenazole, and ketoconazole were selected as exclusive inhibitors of CYP2C8, CYP2C9, and CYP3A4, respectively, and the concentrations were 100, 10, and 5 μM, respectively. Mouse 3T3 fibroblasts were incubated under 5% COThe subsequent steps were performed as described in All values were expressed as mean ± standard deviation (SD). The correlated fitting analyses were performed with Analyst TF 1.7.1, PeakView 2.2 , and Graph pad Prism 6.0 .The cell viability was calculated as follows:50) was calculated by Graph pad Prism as follows:The 50% inhibitory concentration . UA provided abundant fragment ions at [M-H-CH3]- m/z 328.0610  [M-H-2CH3]- m/z 313.0366  [M-H-2CH3-CH2]- m/z 299.0944  [M-H-CH3-C3HO2]- m/z 259.0635 , and [M-H-CH3-C3HO2-CO]- m/z 231.0678 . To identify its metabolites, we first investigated the MS/MS fragmentation behaviors of UA by UPLC/ESI-QTOF-MS. UA was eluted at 10.141 min and showed [M-H]in vitro. Metabolites M1-1 (tR = 5.181 min), M1-2 (tR = 5.789 min), M1-3 (tR = 7.276 min), M1-4 (tR = 8.262 min), and M1-5 (tR = 10.127 min) showed similar quasi-molecular of C18H15O8− (m/z 359.0772 [M-H]-), which was 15.9949 Da higher than that of UA. In the MS/MS spectra, the [M-H]- of M1-1 showed fragment ions at [M-H-C4H4O3-CO]- m/z 231.0711 (C13H11O4−) and [M-H-C11H8O4]- m/z 155.0363 (C7H7O4−). The [M-H]- of M1-2 exhibited fragment ions at [M-H-CH3]- m/z 344.0534 (C17H12O8−) [M-H-CH3-C2H2O]- m/z 302.0453 (C15H10O7−), and [M-H-CH3-C4H6O2]- m/z 258.0190 (C13H6O6−). The [M-H]- of M1-3 revealed fragment ions at [M-H-CH3]- m/z 344.0562 (C17H12O8−) [M-H-OH-H]- m/z 341.0686 (C18H13O7−) [M-H-CH3-OH-H]- m/z 326.0448 (C17H10O7−) [M-H-CH3-OH-H-CO]- m/z 298.0495 (C16H10O6−) [M-H-C4H6O3]- m/z 257.0481 (C14H9O5−), and [M-H-C4H6O3-CO]- 229.0490 (C13H9O4−). The [M-H]- of M1-4 presented fragment ions at [M-H-CH3]- m/z 344.0568 (C17H12O8−) [M-H-2CH3]- m/z 329.0334 (C16H9O8−) [M-H-CH3-OH-H-CO]- m/z 298.0522 (C16H10O6−) [M-H-C2H5O2]- m/z 285.0437 (C16H10O6−) [M-H-C4H4O2]- m/z 275.0589 (C14H11O6−) [M-H-C4H6O3]- m/z 257.0476 (C14H9O5−), and [M-H-C4H6O3-CO]- m/z 229.0542 (C13H9O4−). The [M-H]- of M1-5 showed fragment ions at [M-H-CH3]- m/z 344.0504 (C17H12O8−) [M-H-2CH3-CO]- m/z 301.0324 (C15H9O7−) [M-H-CH2OH-CH3-2CO]- m/z 257.0518 (C14H9O5−), and [M-H-C4H6O3-CO]- m/z 229.0509 (C13H9O4−). The proposed fragmentation pathway of these metabolites, including the loss of methyl radical and retro Diels–Alder (RDA) transform, is similar to that of UA. In addition, the fragmentation behaviors between these metabolites were very similar. As no more information was observed, the specific structure of each compound could not be established from the mass spectrum data alone. Five hydroxylation metabolites of UA were identified in the microsomal incubation system M1-6 (tR = 4.483 min), M1-7 (tR = 4.791 min), M1-8 (tR = 5.561 min), M1-9 (tR = 5.841 min), M1-10 (tR = 7.170 min), M1-11 (tR = 8.159 min), and M1-12 (tR = 10.050 min) showed similar quasi-molecular ion of C18H15O9− (m/z 375.0721 [M-H]-), which was 31.9898 Da higher than that of UA. In the MS/MS spectra, the [M-H]- of M1-6 showed fragment ions at [M-H-CH3]- m/z 360.0503 (C17H12O9−) and [M-H-CH3-C2H4O2]- m/z 300.0265 (C15H8O7−). The [M-H]- of M1-7 exhibited fragment ions at [M-H-CH3]- m/z 360.0493 (C17H12O9−) [M-H-CH3-CH2-CO]- m/z 318.0382 (C15H10O8−), and [M-H-CH3-CH2-CO-C2H2O]- m/z 259.0258 (C13H7O6−). The [M-H]- of M1-8 presented fragment ions at [M-H-OH-H]- m/z 357.0603 (C18H13O8−) [M-H-OH-H-CH3]- m/z 342.0385 (C17H10O8−), and [M-H-2CH2-2OH]- m/z 313.0380 (C16H9O7−). The [M-H]- of M1-9 displayed fragment ions at [M-H-CH3]- m/z 360.0564 (C17H12O9−) [M-H-CH2-CO]- m/z 333.0579 (C16H13O8−), and [M-H-CH3-CH2-CO]- m/z 302.0489 (C15H10O7−). The [M-H]- of M1-10 manifested fragment ions at [M-H-OH-H]- m/z 357.0656 (C18H13O8−) [M-H-CO-2OH]- m/z 313.0715 (C17H13O6−), and [M-H-2CH3-CO-OH-H]- m/z 299.0150 (C15H7O7−). The [M-H]- of M1-11 showed fragment ions at [M-H-O]- m/z 359.0776 (C18H15O8−) [M-H-CO-O]- m/z 331.0914 (C17H15O7−) [M-H-C2H6O-O]- m/z 313.0345 (C16H9O7−), and [M-H-CO-O-C4H4O2]- m/z 247.0649 (C13H11O5−). The [M-H]- of M1-12 indicated fragment ions at [M-H-C6H5O43-]- m/z 235.0623 (C12H11O5−) [M-H-C6H7O43-]- m/z 233.0468 (C12H9O5−) [M-H-C6H5O43--CO]- m/z 207.0597 (C11H11O4−), and [M-H-C12H11O53-]- m/z 141.0211 (C6H5O4−). M1-6 to M1-12 were tentatively identified as a metabolite of 2 × hydroxylation. Metabolites M1-13 was detected at 7.329 min and showed quasi-molecular at m/z 347.1150 . In the MS/MS spectra, the [M-H]- of M1-13 showed fragment ions at [M-H-C4H4O2-CH3-OH]- m/z 231.0616  and [M-H-C14H14O5]- m/z 85.0290 . The proposed fragmentation pathway of M1-13 revealed similar characteristics to that of UA, including the RDA transform. Metabolite M1-14 was detected at 5.409 min and showed quasi-molecular at m/z 373.0594 . In the MS/MS spectra, the [M-H]- of M1-14 exhibited fragment ions at [M-H-OH-H]- m/z 355.0445  [M-H-CH3-CO-H]- m/z 329.0334  [M-H-CO-OH-H]- m/z 327.0495  [M-H-C3H4O-CO-OH-H]- m/z 271.0197 , and [M-H-C3H4O-2CO-OH-H]- m/z 243.0248 . M1-14 was tentatively identified as a metabolite of hydroxylation and oxidation. M1-14.Metabolite M1-15 presented a quasi-molecular ion at m/z 519.1149  and could be detected at 5.357 min, which was 176.0321 Da higher than that of UA m/z 343.0851  and indicates the characteristic loss of glucuronic acid group (C6H10O7-H2O). The MS/MS spectra exhibited fragment ions at [M-H-C6H8O6]- m/z 343.0838  [M-H-C6H8O6-CH3]- m/z 328.0622 , and [M-H-C6H8O6-CH3-C3HO2]- m/z 259.0636 . The proposed fragmentation pathway of metabolites M1-15 was similar to that of UA, including the loss of methyl radical and RDA transform. As no more information was observed, the specific structure could not be established from the mass spectrum data alone. M1-15.Metabolite M1-16 showed a quasi-molecular ion at m/z 462.0858  and could be detected at 5.107 min. The MS/MS spectra displayed fragment ions at [M-H-CH3]- m/z 447.0627  [M-H-C3H5NO2S-2H]- m/z 341.0663  [M-H-CH3-C3H5NO2S-2H]- m/z 326.0430 , and [M-H-C3H5NO2S-2H-CH3-C3HO2]- m/z 257.0468 . The proposed fragmentation pathway of metabolites M1-16 was similar to that of UA, including the loss of methyl radical and RDA transform. As no more information was observed, the specific structure could not be established from the mass spectra data alone. M1-16.Metabolite M1-17 manifested the quasi-molecular ion at m/z 535.1092  and could be detected at 4.663 min.The MS/MS spectra revealed fragment ions at [M-H-OH-H]- m/z 517.1005  [M-H-OH-H-C6H8O6]- m/z 341.0666  [M-H-OH-H-C6H8O6-CH3]- m/z 326.0424  [M-H-2OH-C6H8O6-CO]- m/z 297.0799 , and [M-H-OH-H-C6H8O6-C3HO2-CH3]−m/z 257.0451 . The proposed fragmentation pathway of metabolite M1-17 was similar to that of UA, including the loss of methyl radical and RDA transform. As no more information was observed, the specific structure could not be established from the mass spectra data alone. M1-17.Metabolite M1-18 unveiled a quasi-molecular ion at m/z 478.0836  and could be detected at 4.726 min. The MS/MS spectra showed fragment ions at [M-H-C3H5NO2S-OH-H]– m/z 341.0665 , and [M-H-C8H8O3]- m/z 326.0339 . As no more information was observed, the specific structure could not be established from the mass spectra data alone. M1-18.Metabolite in vitro were speculated . BA provided abundant fragment ions at [M-H-C10H10O3]- m/z 181.0506  [M-H-C10H12O4]- m/z 163.0390  [M-H-C10H10O3-COO]- m/z 137.0601 , and [M-H-C10H12O4-COO]- m/z 119.0494 . To identify the metabolites of BA, the MS/MS fragmentation behaviors of BA were first investigated by UPLC/ESI-QTOF-MS. BA was eluted at 9.354 min and showed [M-H]M2-1 showed a quasi-molecular ion at m/z 375.1109 and could be detected at 6.832 min.The MS/MS spectra presented fragment ions at [M-H-C10H10O4]- m/z 181.0514  [M-H-C10H12O5]- m/z 163.0411  [M-H-C10H10O4-COO]- m/z 137.0614 , and [M-H-C10H12O5-COO]- m/z 119.0503 . The proposed fragmentation pathway of metabolites M2-1 was similar to that of BA. As no more information was observed, the specific structure could not be established from the mass spectrum data alone. M2-1.Metabolite M2-2 presented a quasi-molecular ion at m/z 389.1260  and could be detected at 6.436 min.The MS/MS spectra exhibited fragment ions at [M-H-C11H12O4]- m/z 181.0515  [M-H-C11H14O5]- m/z 163.0394  [M-H-C11H12O4-COO]- m/z 137.0602 , and [M-H-C11H14O5-COO]- m/z 119.0512 . The proposed fragmentation pathway of metabolite M2-2 was similar to that of BA. As no more information was observed, the specific structure could not be established from the mass spectrum data alone. M2-2.Metabolite M2-3 showed the quasi-molecular ion at m/z 389.1260  and could be detected at 6.436 min.The MS/MS spectra revealed fragment ions at [M-H-COO]- m/z 345.1334  [M-H-COOH-CH2-OH]- m/z 313.1101  [M-H-COOH-CH2-OH-CH3]- m/z 298.0836  [M-H-COO-CH3-CH2-2OH]- m/z 283.0972  [M-H-COO-2CH3-CH2-2OH]- m/z 268.0761  [M-H-C10H8O5]- m/z 181.0854  [M-H-C10H8O5-CH2O]- m/z 151.0773 , and [M-H-C10H8O5-2CH2O]- m/z 121.0658 . M2-3.Metabolite M2-4 presented a quasi-molecular ion at m/z 359.0786  and could be detected at 6.688 min.The MS/MS spectra displayed fragment ions at [M-H-OH-H]- m/z 341.0671  [M-H-CHO-OH]- m/z 313.0701  [M-H-COOH-OH]- m/z 297.0765 , and [M-H-COOH-CHO-CH3]- m/z 270.0519 . As no more information was observed, the specific structure could not be established from the mass spectrum data alone. M2-4.Metabolite M2-5 manifested the quasi-molecular ion at m/z 361.0904  and could be detected at 6.248 min.The MS/MS spectra presented fragment ions at [M-H-C9H8O3]- m/z 197.0484  [M-H-C9H8O4]- m/z 181.0495  [M-H-C9H10O5]- m/z 163.0411  [M-H-C9H8O4-COO]- m/z 137.0604 , and [M-H-C9H10O5-COO]- m/z 119.0492 . The proposed fragmentation pathway of metabolite M2-5 was similar to that of BA. As no more information was observed, the specific structure could not be established from the mass spectrum data alone. M2-5.Metabolite in vitro. Metabolites M2-6 (tR = 5.388 min), M2-7 (tR = 6.064 min), and M2-8 (tR = 7.173 min) showed the same quasi-molecular of C25H27O13− (m/z 535.1452 [M-H]-), which was 176.0321 Da higher than that of BA m/z 359.1147 . This finding indicated the characteristic loss of the glucuronic acid group (C6H10O7-H2O). In the MS/MS spectra, the [M-H]- of M2-6 showed fragment ions at [M-H-C6H8O6]- m/z 359.1139  [M-H-C6H8O6-COO]- m/z 315.1228  [M-H-C6H8O6-C10H10O3]- m/z 181.0511  [M-H-C6H8O6-C10H12O4]- m/z 163.0404  [M-H-C6H8O6-C10H10O3-COO]- m/z 137.0610 , and [M-H-C20H22O10]- m/z 113.0241 . The [M-H]- of M2-7 presented fragment ions at [M-H-C6H8O6]- m/z 359.1136  [M-H-C6H8O6-C10H10O3]- m/z 181.0512  [M-H-C6H8O6-C10H12O4]- m/z 163.0405  [M-H-C6H8O6-C10H10O3-COO]- m/z 137.0608 , and [M-H-C20H22O10]- m/z 113.0241 . The [M-H]- of M2-8 exhibited fragment ions at [M-H-C6H8O6]- m/z 359.1099  [M-H-C6H8O6-C10H10O3]- m/z 181.0495 , and [M-H-C6H8O6-C10H12O4]- m/z 163.0419 . The proposed fragmentation pathway of metabolites M2-6 to M2-8 was similar to that of BA. Moreover, the fragmentation behaviors between these metabolites were very similar. As no more information was observed, the specific structure could not be established from the mass spectrum data alone. Five glucuronidation metabolites of BA were identified in the microsomal incubation system M1-1, and CYP2A6 exhibited a weak inhibition effect only at 5 µM with RLMs. CYP2B6 presented a strong inhibitory effect on M1-3, followed by CYP1A1, 2A6, 2C9, 2C19, and 3A4 with RLMs. As for M1-4, CYP2B6, 2A6, 1A1, 3A4, 2C9, 2C19, 2D6, 2E1,1A2, and 2C8 showed high to low levels of inhibition effect with RLMs. CYP2B6 and 1A1 with RLMs showed a stronger inhibition effect on M1-6 than CYP3A4, 2D6, 2C9, 2C19, 2A6, 1A2, and 2C8. CYP2B6 and 1A1 with RLMs also displayed a stronger inhibition effect on M1-7. In addition, CYP3A4, 2A6, 2C9, 2D6, 2C19, 2C8, 1A2, and 2E1 exhibited high to low levels of inhibition effect with RLMs. As for M1-8, only CYP2C8, 2C19, and 1A1 with RLMs showed low inhibition effects. CYP2C19, 2C8, and 2B6 with RLMs revealed a weak inhibition effect on M1-9. For M1-10, CYP2B6, 1A1, 2A6, and 2C9 presented high to low levels of inhibition effect with RLMs, and CYP3A4 and 2C19 with RLMs showed weak inhibition effects only at 100 µM. For M1-11, CYP2B6, 2A6, 1A1, 2C9, 2C19, 3A4, 2D6, 2E1, and 1A2 with RLMs showed high to low levels of inhibition effects.Chemical inhibition studies reported the different effects of CYP inhibitors on the biotransformation of UA. CYP3A4, 1A1, and 2C9 showed low inhibition effects on M1-1. CYP1A1 and 2A6 showed weak inhibition effects only at 5 µM. For M1-3, CYP2C9, 2E1, 2D6, 2B6, 2C19, 2C8, 3A4, 1A2, 1A1, and 2A6 with HLMs displayed high to low levels of inhibition effects. For M1-6, CYP2C9, 2E1, 2D6, 2B6, 1A1, 2C19, 1A2, and 2A6 with HLMs exhibited high to low levels of inhibition effects, and CYP2C19 with HLMs showed weak inhibition effect only at 5 µM. For M1-8, CYP3A4, 2E1, 2C9, 2B6, 2C19, 1A2, 2C8, and 2D6 with HLMs manifested high to low levels of inhibition effects, and CYP2A6 and 1A1 with HLMs showed a mild inhibition effect only at 5 µM. For M1-10, CYP2C9 presented a strong inhibition effect at all concentrations with HLMs. In addition, CYP2E1, 2D6, 2B6, 1A2, 1A1, 3A4, 2C19, 2C8, and 2A6 showed moderate inhibition effects with HLMs. For M1-13, CYP2E1, 2D6, 2B6, 3A4, 2C8, 2C19, 2A6, 1A2, 1A1, and 2C9 presented high to low levels of inhibition effects with HLMs. For M1-14, CYP2E1, 2D6, 3A4, 2C19, 2B6, 2C9, 2C8, 2A6, 1A1, and 1A2 showed moderately high to low inhibition effects with HLMs. Compared with the control group, the inhibitors of CYP2B6, 1A1, and 2A6 with RLMs inhibited UA metabolism the most  of 0.01 µM–100.00 µM BA in human primary hepatocytes  ranged fTo confirm the contribution of UA and BA or their metabolites to the cytotoxicity of mouse 3T3 fibroblasts, we studied the effects of UA or BA incubation with HLMs, NADPH and UDPGA, and montelukast, sulfaphenazole, and ketoconazole, the exclusive inhibitors of CYP2C8, CYP2C9, and CYP3A4, by measuring cell viability. After incubation with mouse 3T3 fibroblasts for 4 h, the cell viability of UA groups from 1 μM to 50 µM ranged from 80.04% to 106.16%. None of the five groups exhibited cytotoxicity. The results showed that the cytotoxicity mechanism of UA may be related to long-term toxicity as it did not show cytotoxicity within 4 h of incubation in each group .Based on previous experiments, the relative cell viability showed that 100 µM BA exhibited cytotoxicity after incubation with mouse 3T3 fibroblasts for 24 h (26.87%) but not after 4 h (93.35%). Meanwhile, 200 µM BA displayed cytotoxicity after incubation with mouse 3T3 fibroblasts for 4 h (63.07%), which indicated that the cytotoxicity of BA increased in a time- and concentration-dependent manner. When incubated with HLMs, 20 and 200 µM BA showed cytotoxicity toward mouse 3T3 fibroblasts . The relative cell viability of these groups had an overall downward trend from 85.27% to 69.73%. After incubation with HLMs and NADPH or HLMs, NADPH, UDPGA, and inhibitors of CYP2C8, CYP2C9, and CYP3A4 for 4 h, the relative cell viability (% of NC) of 100 and 200 μM BA, which exhibited cytotoxicity toward mouse 3T3 fibroblasts, ranged from 68.26% to 76.84%. BA showed no cytotoxicity to mouse 3T3 fibroblasts with HLMs, NADPH, and UDPGA only . These rDrug-induced liver injury is one of the most common liver diseases. As the earliest lichen compound has been commercialized and developed, recent reports associated with liver-related adverse events of UA and other lichen compounds contained in products aroused widespread concern. Furthermore, the toxicity and mechanism of UA and other lichen compounds that induce hepatotoxicity remain unclear and have become a highly valued topic.®, which is composed of norephedrine hydrochloride (25 mg), sodium usniate (100 mg), 3,5-diiodothyronine (100 μg), yohimbine hydrochloride (3 mg), and caffeine (100 mg)  . In addi(100 mg) . The toxMost cellular studies showed that UA can cause cell necrosis and affects mitochondrial function . Early sCurrently, several scholars suggest that when CYP1A is inhibited, the metabolism of UA will decrease and result in its accumulation, which leads to the excessive inhibition of mitochondrial respiration, insufficient ATP, and cell necrosis. Therefore, several CYP enzyme inhibitors may increase the cytotoxicity of UA on rat hepatocytes, implying that UA might have a metabolic detoxification mechanism . AnotherIn this study, the metabolic profiles of UA and BA were investigated in RLMs, HLMs, and RS9. A total of 14 phase I metabolites and 4 phase II metabolites of UA and 4 phase I metabolites and 6 phase II metabolites of BA were identified. The results revealed that the metabolism of UA and BA with RLMs and HLMs was induced by CYP450 or UGTs and mediated by hydroxylation, methylation, and glucuronidation reactions. These metabolites showed similar fragmentation patterns in MS/MS spectra and only differed in fragment abundance.M1-2 and M1-3 were partially metabolized by CYP1A2. The metabolites of M1-1, M1-3, M1-4, and M1-9 were mainly influenced by CYP3A4, and M1-15 was regulated with UGT1A1. CYP2C9, CYP3A4, CYP2C8, and UGT1A1 are the main metabolic enzyme subtypes responsible for several metabolites of UA.UA is metabolized primarily by CYP1A2, UGT1A1, UGT1A3, and UGT1A8 . In addiin vivo and in vitro. Therefore, this study presents novel results that indicate that CYP2C8, CYP2C9, CYP2C19, CYP1A1, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 are the main metabolic enzymes responsible for several metabolites of BA.It is worth mentioning that there are no studies on enzymes responsible for BA metabolism The identification of metabolic enzymes is a challenging study and is influenced by numerous factors and research strategies, such as the selection of target metabolites, activity of metabolic enzymes, specificity and inhibitory intensity of inhibitors, co-participation of multiple metabolic enzymes, and composition of the incubation system. In general, the results confirmed by recombinant isozymes are more reliable. To determine the major metabolic enzymes responsible for a compound, scholars must first identify the major metabolites from multi-metabolites or identify the metabolites related to metabolic activation or metabolic detoxification and then determine metabolic enzymes based on specific metabolites. However, only metabolites and the metabolic pathways of UA and BA were identified and analyzed in this research paper. Given the lack of separation, preparation, and structure confirmation of metabolites, quantitative determination of metabolites is impossible, which leads to the failure of identifying major and key metabolites based on existing results. Therefore, the identification of the key metabolic enzymes responsible for UA and BA is extremely difficult. This work will be further carried out in follow-up studies.50 values of 7.40 and 60.2 µm. The cytotoxicity of UA may be associated with chronic toxicity. The cytotoxicity of BA is associated with metabolism, and UGTs may be the main metabolic detoxification enzymes.The cytotoxic mechanism of drugs is usually based on their effect on hepatic drug-metabolizing enzymes , such asUsnea are due to the coexisting compounds contained in the lichen. Considering a mixture extract of plant herb or polyherbal compound, drug-drug interaction between UA and BA or other coexisting components must be taken into account. In particular, attention should be paid to the inhibitory and inductive effects of different compounds on drug-metabolizing enzymes and on transporters that play key roles in absorption and excretion processes  of rocesses .in vitro experiments, the present results provide important insights into the understanding of the biotransformation behavior and metabolic detoxification of UA and BA. They will provide important references for the research of metabolism behavior and clinical pharmacology of UA, BA, and other similar lichen compounds in vivo.Despite the difficulty of determining the exact structure of metabolites through MS only and confirming the specific mechanism of metabolic detoxification completely through In conclusion, hydroxylation, methylation, and glucuronidation reactions were involved in the metabolic profiles of UA and BA in RLMs, HLMs, and RS9 mediated by CYP450 and UGT. CYP2C9, CYP3A4, CYP2C8, and UGT1A1 were the key metabolic enzymes responsible for the metabolism of UA. CYP2C8, CYP2C9, CYP2C19, CYP1A1, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 were the main metabolic enzymes responsible for the metabolism of BA. UA and BA did not display evident cytotoxicity toward human primary hepatocytes but did so toward mouse 3T3 fibroblasts. The attenuated cytotoxicity of BA was associated with metabolism, and UGTs may be the key metabolic detoxification enzymes. The cytotoxicity of UA may be associated with chronic toxicity. Overdose and long-term consumption of UA and BA, as well as the medicinal materials containing these compounds, still have potential risks of liver toxicity, and their application and safety deserve to be studied and receive more attention.


N-Boc azaketones and oxoetherswith the aim of obtaining N-protected azalactonesand their analogues with oxygen in place of nitrogen. N-Boc-4-piperidinone and all the cyclic oxoethers were successfullyoxidized to lactones, while the azacyclic ketones with nitrogen α-positionedto carbonyl were univocally transformed into N-Boc-ω-aminoacids and N-Boc-N-formyl-ω-aminoacids operating in alkaline water and DMF, respectively.Therecently reported efficient conversion of cyclic ketones tolactones by Oxone in neutral buffered water is extended to heterocyclicketones, namely, cyclic  Within the wide arsenal of oxidants,great attention has been directed to trichloroisocyanuric acidN-Boc protectedcyclic 3- and 4-oxo-amines, namely N-Boc-3-azetidinone(1), N-Boc-3-pyrrolidinone (2), N-Boc-3-piperidinone (3), and N-Boc-4-piperidinone (4), and the correspondingcyclic 3- and 4-oxo-ethers, namely 3-oxetanone (5), 3-oxo-tetrahydrofuran(6), tetrahydropyran-3-one (7), and tetrahydropyran-4-one(8)  1.N-Boc derivatives, was inspired by the interestin azalactones that could result, in principle, from their BV oxidation(4 to N-Boc-4-azacaprolactone (9) by BV oxidationwith m-chloroperoxybenzoic acid (MCPBA) in DCM andthe subsequent ring-opening polymerization of the azalactone to poly(β-aminoester)have been reported.9 Instead, the ringexpansions of 1–3 to azalactonesby BV oxidation are not described. Indeed, preparations of N-Boc-3-azabutyrolactone and N-Boc-4-azavalerolactoneare reported, but not by BV oxidation of 1 and 2 respectively, while N-Boc-3-azavalerolactoneand N-Boc-3- and N-Boc-5-azacaprolactoneare not described 10 and tetrahydropyran-4-one (8).11 Analogously to azalactones, some lactones derivablefrom cyclic oxo-ethers are useful monomers to develop degradable synthetichomo- and copolymers, such as polys and poly(ether-ester)s.12The choice of cyclic oxo-ethers 4 into 9, which has been recently accomplishedunder classical BV conditions(MCPBA in DCM) in 73% yield,9 to have immediatefeedback on the performance of our procedure based on the use of Oxonein neutral water environment. After 24 h of reaction at room temperatureand standard workup, we isolated 9 by flash chromatographywith 75% yield, in line with the literature datum and in confirmationof the stability of the ester function we had previously observedfor the corresponding carba-lactone . This product became predominant in overnightor 48 h reactions or by increasing the reaction temperature to 40°C. After 1 h of reaction at room temperature, we detected alsoa third product, N-Boc-N-formyl-γ-aminobutyricacid (11), whose quantity became close to that of 10 in a 24 h reaction, when N-Boc-3-azacaprolactonewas reduced to about 10%. Anyway, regardless of when the reactionwas stopped, N-Boc-3-azacaprolactone was chromatographicallyisolated with poor yield and always in mixture with a nonnegligibleamount (>10%) of 10, which increased during storage.The intrinsic instability of the azalactone, reasonably due to thehemiaminal ether substructure, and, on the other hand, our interestin simple Oxone BV oxidation protocols leading to a unitary productprompted us to develop reaction conditions selecting the other twoproducts, namely, 10 and 11. The reactionwith Oxone in phosphate buffer did not allow product selectivity,while that in 1 M NaOH quantitatively provided 10 in30 min at room temperature. High-yield conversion of 3 into 11 was instead accomplished with Oxone in DMFin 1 h at room temperature  and N-Boc-N-formylglycine(15). Otherwise, to efficiently convert 2 into N-Boc-β-alanine (12), itwas necessary to oxidize the substrate with Oxone in phosphate bufferand to treat the resultant mixture of oxidation products with 1 MNaOH at room temperature for 30 min, while the oxidation to N-formyl-N-Boc-β-alanine (13) was performed similarly as with 1 and 3  resultingfrom 5–7, were stable enough to bechromatographically purified. The two unsymmetrically substitutedketones 6 and 7 displayed the same BV regioselectivitywith preferential migration of the O-linked methylene resulting inthe formation of the lactone acetals 17 and 18, according to what is reported in the literature for 7 analyses were carried out on alumina sheets precoatedwith silica gel 60 F254. High Resolution Mass Spectra (HRMS) wereacquired by direct infusion on a ThermoScientific Orbitrap Elite  operated in positiveElectroSpray Ionization (ESI+).Heterocyclic ketones N-tert-Butyloxycarbonyl-4-oxopiperidinewas added to a solution of Oxone  in 2 M NaH2PO4/Na2HPO4 buffer . The reaction was stirred at room temperature for 24 h. Themixture was extracted with EtOAc (3 × 10 mL). The combined organiclayers were dried (Na2SO4) and evaporated underreduced pressure. Purification by flash column chromatography (Petroleumether/EtOAc 1:1) gave 9 as a white amorphous solid; 75%yield; Rf (Petroleum ether/EtOAc 1:1 stainedwith KMnO4) 0.38; 1H NMR  δ 4.30–4.21 , 3.84–3.72 ,3.72–3.58 , 2.85–2.75 , 1.47 ; 13C{1H} NMR  δ 174.0,154.4, 81.1, 69.5, 47.7, 41.2, 37.6, 28.4.2SO4 and concentrated.Purification by flash column chromatography on silica gel (DCM/MeOH95:5) gave the corresponding products.The appropriate ketone (1 equiv) wasadded to a solution of Oxone (2 equiv) in 1 M NaOH. The reaction wasstirred at room temperature for 30 min. The mixture was acidifiedwith 1 M HCl and extracted with EtOAc (3 × 10 mL). The combinedorganic layers were dried in NaN-tert-butoxycarbonyl-3-piperidone and Oxone  in1 M NaOH (40 mL)at room temperature for 30 min, following the general procedure reportedabove. Isolated as a yellow solid after column chromatography; 88%yield; Rf (DCM/MeOH 95:5 stained withKMnO4) 0.3; mp 49.2–52.5 °C; 1HNMR  δ 10.01 , 6.10 , 4.79 , 3.26–3.01 , 2.36 , 1.88–1.70 , 1.42 ; 13C{1H} NMR  δ 178.4,157.9 (minor rotamer), 156.3 (major rotamer), 81.0 (minor rotamer),79.6 (major rotamer), 41.0 (minor rotamer), 39.9 (major rotamer),31.4, 28.4, 25.2.Obtainedfrom N-tert-butoxycarbonyl-3-azetidinone  andOxone  in 1 M NaOH (10 mL) at room temperaturefor 30 min, following the general procedure reported above. Isolatedas white solid after column chromatography; 92% yield; Rf (DCM/MeOH 95:5 stained with KMnO4) 0.16;mp 87–89.5 °C; 1H NMR  δ 8.87 , 6.70 , 5.05 ,4.10–3.74 , 1.45 ; 13C{1H} NMR  δ 174.5 (major rotamer),174.0 (minor rotamer), 157.4 (minor rotamer), 156.2 (major rotamer),81.9 (minor rotamer), 80.5 (major rotamer), 43.4 (minor rotamer),42.3 (major rotamer), 28.3.Obtained from N-tert-Butoxycarbonyl-3-pyrrolidone was added to a solution of Oxone in 2 M NaH2PO4/Na2HPO4 buffer . The reaction mixture was stirredat room temperature for 2 h and extracted with EtOAc (3 × 5 mL).The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give a residue that wastreated with 1 M NaOH (10 mL) under stirring at room temperature for30 min. The mixture was acidified with 1 M HCl and extracted withEtOAc (3 × 5 mL). The combined organic layers were dried (Na2SO4) and concentrated. Purification of the residueby flash column chromatography (DCM/MeOH 97:3) afforded the titleproduct as a white solid: 76% yield; Rf (DCM/MeOH 95:5 stained with KMnO4) 0.21; mp 73–75°C; 1H NMR  δ 11.29, 6.28 , 5.17 , 3.52–3.14, 2.74–2.33 , 1.41 ; 13C{1H} NMR  δ 177.5 (major rotamer),176.5 (minor rotamer), 157.7 (minor rotamer), 156.1 (major rotamer),81.2 (minor rotamer), 79.8 (major rotamer), 37.3 (minor rotamer),36.0 (major rotamer), 34.7, 28.4.2O (10–20 mL) andthen extracted with EtOAc (3 × 10–20 mL). The combinedorganic layers were dried in Na2SO4 and concentratedunder reduced pressure. Purification by flash column chromatographyon silica gel (DCM/MeOH 95:5) afforded the corresponding products.The appropriate ketone(1 equiv) was added to a suspension of Oxone (2–6 equiv) inDMF. The resulting suspension was stirred at the specified temperature.The mixture was quenched with HN-tert-butoxycarbonyl-3-piperidone  and Oxone  in DMF (25mL) at room temperature for 1 h, following the general procedure reportedabove. Isolated as a yellow oil after column chromatography; 78% yield; Rf (DCM/MeOH 95:5 stained withKMnO4) 0.32; 1H NMR  δ 9.17 , 3.66 ,2.37 , 1.96–1.78 ,1.54 ; 13C{1H} NMR  δ 178.4, 163.4, 152.5, 84.4, 39.8, 31.3, 28.1, 23.3.Obtained from N-tert-butoxycarbonyl-3-pyrrolidone  andOxone  in DMF (10 mL) at room temperature for 16h, following the general procedure reported above. Isolated as a brownsolid after column chromatography; 77% yield; Rf (DCM/MeOH 95:5 stained with KMnO4) 0.25; mp 68,2–70,3 °C; 1H NMR  δ 9.15 , 3.91 , 2.61 , 1.55; 13C{1H} NMR  δ 177.0, 163.1, 152.1, 84.7, 36.2, 32.7, 28.1.Obtained from N-tert-butoxycarbonyl-3-azetidinone and Oxone  inDMF (10 mL) at 60 °C for 5 h, following the general procedurereported above. Isolated as a white solid after column chromatography;84% yield; Rf (DCM/MeOH95:5 stained with KMnO4) 0.19; mp 101,2–103,6 °C; 1H NMR  δ 9.20 , 4.39, 1.54 ; 13C{1H} NMR  δ 173.5, 162.6, 151.6, 85.3, 41.3, 28.0. HRMS(ESI+) m/z calcd. forC8H13NO5Na [M + Na]+ 226.06914,found 226.06833.Obtained from 2PO4/Na2HPO4 buffer  at room temperature or 0 °C. The solutionwas stirred 3–40 min. EtOAc (15–30 mL) was added, andthe mixture was stirred vigorously. The phases were separated, andthe water phase was extracted again with EtOAc (2 × 15–30mL). The combined organic phases were washed with brine, dried overNa2SO4, and concentrated. Purification of theresultant residue by flash chromatography on silica gel (gradientof petroleum ether/EtOAc from 0% to 50% EtOAc) afforded the correspondingproducts.The appropriate ketone(1 equiv)was added to a solution of Oxone (2–4 equiv) in NaH8, 0.5mL, 5.4 mmol) and Oxone  in 1 M NaH2PO4/Na2HPO4 buffer (21.7 mL) atroom temperature for 40 min, following the general procedure reportedabove. Concentration of the organic extracts gave 16  as a unitary product with only traces of impurities (see 1H NMR spectrum of the crude), which was isolated as an amorphoussolid by column chromatography with 82% final yield; Rf (Petroleum ether/EtOAc 6:4 stained with KMnO4) 0.19; 1H NMR  δ 4.33–4.24, 3.94–3.84 , 3.84–3.77 , 2.93–2.83; 13C{1H} NMR  δ 174.1, 70.7, 70.3, 64.7, 39.2.Obtained from tetrahydropyran-4-one and Oxone  in 2 M NaH2PO4/Na2HPO4 buffer (13 mL) at 0 °Cfor 10 min, following the general procedure reported above. Concentrationof the organic extracts gave 17  as a unitaryproduct with only traces of impurities (see 1H NMR spectrumof the crude), which was isolated as a colorless oil by column chromatographywith 75% final yield; Rf (Petroleum ether/EtOAc6:4 stained with KMnO4) 0.22; 1H NMR  δ 5.24 , 4.04–3.92 ,2.91–2.76 , 2.00–1.82 ; 13C{1H} NMR  δ 174.2, 93.3, 73.5,34.1, 24.8.Obtained from tetrahydropyran-4-one and Oxone  in2 M NaH2PO4/Na2HPO4 buffer(26 mL) at 0 °C for 10 min, following the general procedure reportedabove. Concentration of the organic extracts gave 18  as a unitary product with only traces of impurities (see 1H NMR spectrum of the crude), which was isolated as a colorlessoil by column chromatography with a 61% final yield; Rf (Petroleum ether/EtOAc 6:4 stained with KMnO4) 0.19; 1H NMR  δ 5.38, 4.10 , 2.78 ; 13C{1H} NMR  δ 166.5, 94.1, 64.1, 31.0.Obtained from 3-oxo-tetrahydrofuran and Oxone  in 2 M NaH2PO4/Na2HPO4 buffer (31.2 mL) at 0 °C for3 min, following the general procedure reported above. Due to thehigh volatility of 19, the organic extracts were concentratedto a 40% w/w residual content of ethyl acetate. The 1HNMR spectrum of the concentrated extracts (915 mg) showed the presenceof 19 as a unique product, allowing calculation of an80% yield. After column chromatography, 19 was isolatedas a colorless oil with a 61% final yield; Rf (Petroleum ether/EtOAc 6:4 stained with KMnO4)0.53; 1H NMR  δ 5.54 , 4.22 ; 13C{1H} NMR  δ 171.3, 96.2, 62.6.Obtainedfrom oxetan-3-one (