Design, Synthesis And In Vitro Bioactivity Studies On 4’-N-substituted(Aminomethyl)Benzoate-7-nicotinic Acid Ester Derivatives Of Scutellarein

Objective: To design and synthesis 4’-N-substituted(aminomethyl) benzoate-7-nicotinic acid ester conjugates of scutellarein with improved physiochemical properties, in vitro anti-oxidative activity and BBB permeability by using scutellarein as lead compound. Methods:(1) Scutellarin was used as starting material by hydrolysis with 8% sulfuric acid solution to obtain scutellarein, which then was treated with diphenyldichloromethane to provide 6,7-dihydroxy ketal protection of scutellarein intermediate, which then was treated with 4’-N-substituted(aminomethyl) benzoic acids, following acetyl chloride/methanol treatment in low temperature to afford 4’-N-substituted(aminomethyl) benzoate derivatives of scutellarein. After that the obtained compounds coupled with substituted nicotinic acid to obtain target compounds.(2) The physiochemical properties of target compounds including PBS stability and aqueous solubility were identified by HPLC protocols.(3) In vitro anti-oxidative activity of target compounds was investigated in H2O2-induced PC12 cell injury model by using MTT value, LDH leakage rate, SOD / MDA content as index of investigation.(4) Caco-2 cell monolayer permeability for target compounds was assayed in established Caco-2 cells monolayer model, bilateral permeability(Papp AP to BL/Papp BL to AP) and ER(Papp BL to AP/Papp AP to BL) were calculated.(5) In vitro BBB permeability evaluations for target compounds were investigated in the established triple co-culturing rat brain microvascular endothelial cells, pericytes and astrocytes model(EPA) model. Bilateral permeability(Papp AP to BL/Papp BL to AP) and ER(Papp BL to AP/Papp AP to BL) were calculated. Results:(1) fourteen 4’-N-substituted(aminomethyl) benzoate-7-nicotinic acid ester conjugates of scutellarein(6a-6f,7a-7c,6m,6n,6w,6y,6x) were synthesized. The structures of synthesized compounds were confirmed by 1H-NMR, ESI-MS and ESI-HRMS techniques as the target ones.(2) The target compounds are relatively more stable(t1/2 >36 h) in acidic condition(p H 2) than in neutral condition(p H 7.4)(t1/2 2.9–8.3 h), except for two compounds solubility of target compounds exhibited was significantly enhanced from 65.9μg·m L-1 of scutellarin to 127.87-779.13μg·m L-1.(3) In vitro anti-oxidative assay suggested that compared with scutellarin, compounds 6b、6c、6d、6e、6f、6m、6n and 6w can alleviate H2O2-induced PC12 cell morphology damage, significantly increase cell viability and SOD content, and reduce LDH leakage rate and MDA content as compared with scutellarin(P < 0.05).(4) Oral absorption potential evaluation results showed that Papp AP to BL transporting amount of compounds 6b、6c、6d、6n and 6w(1.47-3.11×10-6 cm·s-1)is significantly higher than that of scutellarin(0.53 × 10-6cm·s-1)and scutellarein(0.83 × 10-6cm·s-1), among them compound 6c exhibit the highest transporting property(3.11 × 10-6cm·s-1).(5) In vitro BBB permeability results indicated that Papp AP to BL transporting amount of compounds 6b、6c 、 6d and 6n(0.59-1.65 × 10-6cm·s-1) is significantly higher than that of scutellarin(0.17 × 10-6cm·s-1) and scutellarein(0.18 × 10-6cm·s-1). These result suggested that introduction of nicotinic acid fragment at 7-position of 4’-N-substituted(aminomethyl) benzoate derivatives of scutellarein can enhance in vitro BBB permeability. Conclusion: The dissertation comprehensively evaluated in vitro anti-oxidative activity, oral absorption potential property and in vitro BBB permeability for 4’-N-substituted(aminomethyl) benzoate-7-nicotinic acid ester conjugates of scutellarein by established models, finally selected four target compounds(6b、6c、6d、6n)exhibiting higher anti-oxidative activity, Caco-2 cell permeability and in vitro BBB permeability than those of positive controls. The results of the present study indicated that 4’-N-substituted(aminomethyl) benzoate and 7-substituted nicotinic acid prodrug strategy can be used in modification of scutellarein to obtain target compounds with improved physiochemical properties and bioactivity.