L-amino Acid Carbamate Prodrugs Of Scutellarin And Its Aglycone: Synthesis,Caco-2 Cell Permeability And In Vitro Antioxidative Activity Evaluation

Objective To design and synthesize a series of novel L-amino acid carbamate conjugates with improved physiochemical properties, absorption and Pharmacological activity by using scutellarin, scutellarein and scutellarein-7-methyl ether as the lead compounds. Methods (1) Scutellarin methyl ester was prepared the methyl ester of scutellarin by the reaction of scutellarin with excess methanol in DMF under the catalysis of hydrogen chloride. L-Amino acid tert-butyl ester hydrochlorides were treated with triphosgene [bis (tri-chloromethyl) carbonate (BTC) in anhydrous CH2Cl2 at low temperature to afford L-amino acid ester isocyanate, Subsequently, treated with scutellarin methyl ester in anhydrous THF/DMF in the presence of Et3N to afford tert-butyl ester carbamate of scutellarin methyl ester. Removal of tert-butyl protecting group with trifluoroacetic acid (TFA) at low temperature gave the first series compounds as yellow solids(1a-1h); Scutellarin was treated with 8% dilute sulfuric acid aqueous solution at 90℃, followed by filtration, drying and extraction with acetone gave scutellarein in good yield, which was then treated with diphenyl dichloromethane in diethylene glycol dimethyl ether (DME) to provide 6,7-dihydroxy ketal protection intermediate of scutellarein. Amino acids were dissolved in anhydrous THF, Bis(4-nitrophenyl) carbonate(NPC) and N,N-diisopropylethylamine(DIPEA) added to this solution, and the reaction mixture was stirred at room temperature. After 12 h,6,7-dihydroxy ketal protection scutellarein was added, and the mixture was stirred for 12 h at room temperature. Lastly removal of protecting group with TFA at low temperature gave the second series compounds (2a,2b);Scutellarein was fully acetylated with NaOAc and an excess of acetic anhydride which were heated to boiling for 10 min, and then mixed with methyl iodide, potassium carbonate and dry acetone reflux 20h to provide O-methyl and O-acetylated derivatives. The deacetylation of O-methyl derivative was performed with ammonia saturated methanolic (NH3/MeOH) at low temperature to give intermediates compound 7-O-methyl Scutellarein, which then coupled with L-amino acid tert-butyl ester isocyanate to afford scutellarein-7-methyl ether-4’-L-amino acid carbamate tert-butyl ester. Lastly deprotected with trifluoroacetic acid (TFA) at low temperature to yield the third series(3a-3c). (2) The physiochemical properties include PBS stability, plasmastability and aqueous solubility were performed by UPLC-MS/MS. (3) Caco-2 cell model was used to evaluate the absorption of compounds. We tested compounds Caco-2 cell monolayer uptake in various conditions, including the effect of concentrations, time, temperature and pH. In the Caco-2 cell transport experiment, we measured TEEP, AKP and the transmittance of fluorescein sodium and studied the bilateral Permeability of first series compounds. Papp values and efflux rate(ER) values were calculated. (4) PC 12 cells oxidative damage were established by exposure to 800μM H2O2 6h and VE was used as positive drug. The cell viability were determined by MTT assay and the first series compounds’ anti-oxidative activity were tested by this way. General morphology was observed by inverted phase contrast microscope. Kits were used to assay SOD active and MDA content. The changes of ROS and MMP were measured by flow cytometry. By these indicators anti-oxidative mechanism were presumed. Result (1)Scutellarin methyl ester 4’-L-amino acid carbamate conjugates (1a-1h) were synthesized, yield 22.4-34.11%. Scutellarein-4’-L-amino acid carbamate conjugates(2a,2b) and scutellarein-7-methyl ether-4’-L-amino acid carbamate conjugates (3a-3c) were obtained, yield 8.7-9.94% and 3.22-3.6% respectively. The structure of synthesized thirteen target compounds were confirmed by 1H-NMR, ESI-MS and HRMS. (2) Scutellarein and scutellarein-7-methyl Compounds are instability. Scutellarin methyl ester 4’-L-amino acid carbamate conjugates (1a-1h) are relatively more stable (t1/2>72 h) in acidic condition (pH 2) than in neutral condition (pH 7.4) (t1/21.0-9.0 h). Compounds 1e、 1f、1g were comparatively stable with t1/2> 30min in plasma. Except for compound 1d aqueous solubility of all target compounds (22.54-426.51 mg/mL) were higher than that of scutellarin(15.24 mg/mL), especially the solubilities of compound 1e (426.51 mg/mL) 1a(412.78 mg/mL) and 1b (365.80 mg/mL), which were 28,27 and 24 times higher than that of scutellarin respectively. (3) The uptake of compounds lc、1f、1g were more than Scutellarin and 1g showed remarkable increase in uptake.The relative permeability (Papp A to B) of lc, 1f and 1g were 8,7 and 13 times higher than that of scutellarin, among them compound 1g had highest Pap Papp A to B value(1.85±0.16×10-6 cm/s). The ratios of Papp BL to AP to Paap AP to BL(ER) of target compounds lb-lg were within the range of 0.56-1.68, which were less than that value of scutellarin (1.93), suggesting an efflux effect for scutellarin is higher than that of target compounds 1b-1g. Among them, efflux rate (ER) values for lc, Id and 1f were higher than 1.0, while ER values for lb and 1g were less than 1.0, suggesting greater permeability in the BL to AP direction for compounds 1c, 1d and 1f than that for compounds 1b and 1g. (4) Anti-oxidative activity assay showed that tested compounds exhibit protective activity against cytotoxicity produced by H2O2 in concentration of 1-10μM; When treated with H2O26 h, PC 12 cells morphological alterations were observed. The deformed and shrinkage cells in VE and 1g group were less than H2O2 group and cell density were higher. The SOD activity was improved and MDA content were decreased when pretreated with 5μM 1g or VE. Furthermore 1g can decreased the content of ROS and rised the MMP like as VE. Conclusion Scutellarin methyl ester 4’-L-amino acid carbamate conjugates have improved solubility and appropriate stability both in PBS and plasma. It releases scutellarin according to the expected process, which consistent with prodrug design. The introduction of L-amino acid carbamate fragment can improve permeability. Scutellarin methyl ester 4’-L-amino acid carbamate have anti-oxidative effect and involve a succession of factors: H2O2-induced ROS production is inhibited and enhances SOD activity, eventually cell injury is decreased. The reduced damage and maintained function are can be reflected by the decreased MDA content and elevated MMP. 1g has good chemical stability, high permeability and clear anti-oxidation mechanism, which warrants further development as a scutellarin prodrug.