Aromatic Structure Modification And Bone-targeting Prodrug Studies Of Matrine Derivative M19

Developing various reliable and stable new medicines by discoverying active Chinese medicine active monomers with definite pharmacological activity from Chinese traditional medicines and further chemical structure optimization is a hotspot in traditional Chinese medicine chemistry research.The thiomatrine derivative M19(13-aminomethyl-18-thiomatrine)is derived from traditional Chinese medicine and previous studies have already found that M19 showed effective anti-osteoporosis activity in vitro and in vivo.Unfortunately,it has also exhibited some problems such as unstable molecular structure,low lipid-water partition coefficient and high cytotoxicity,rendering it difficult to directly apply M19 as an anti-osteoporosis drug.In order to address the aforementioned problems,we used two different modification strategies to optimize M19.First of all,classical chemical structure modification strategy via aromatic hydrophobic groups introduction was performed.Using Discovery studio 2.5 software,a series of M19 aromatic derivatives designed based on the crystal structure were subjected to Lib Dock molecular simulation docking with target protein ribosomal protein S5.The chemically achievable target compound M54(13-dithiocarbamate benzyl-18-thiomatrine)with the highest Lib Dock score was virtually screened out and synthesized through thiolation,Michael addition and thioamidation reactions using sophoroline as starting material in a total yield of 8.8%.Structural stability experiments showed that no obvious degradation products were found in the M54 aqueous solution within 14 days,suggesting greatly improved stability compared with the prototype drug M19.Following bioactivity characterization experiments showed that compared to the prototype compound M19,the cytotoxicity of M54 was reduced while the osteoclasts inhibition activity was improved.Subsequently,we verified its target and mechanism in osteoclasts by knocking out ribosomal protein S5 using lentivirus.In vivo experiments suggested that M54 significantly improved BMD,BV/TV,Tb.N,BS/TV and other related parameters,exhibiting anti-osteoporosis activity.Considering of the low chemical yield and tedious synthesis steps during lab synthesis of M54,further research focused on synthetic technology of M54 was carried out.Dichloromethane was used to replace original reaction solvent,which led to a clear reaction system and improved yield and purity.The subsequent realization of one-pot preparation of M54-3 further simplified the operating procedures and improved chemical yield to 40.4% in two steps.Multiple solvents for refining were investigated and ethanol was finally selected to avoid the residual solvent problem,resulting in a satisfactory purity of the final products.To examine the applicability of the process,we finally performed a large-scale synthesis with hundred-grams level of starting materials and successfully obtained hundred-grams level of products with a total yield of 28%.To sum up,the aromatization modification strategy based on virtually screening has successfully improved the osteoclast inhibitory activity in vitro,reduced cytotoxicity and retained the anti-osteoporosis activity in vivo of the prototype molecule,which was proved to be an effective structural modification strategy.The synthetic technology has simplified the operation process and improved the yield and purity,which greatly improved the preparation ability of M54.It has not only addressed the raw materials shortage problem of M54,but also provided a reference for other synthetic technology studies of different alkaloids derived from various traditional Chinese medicine.Besides,this part of our work provided a useful reference for future chemical structure modification studies of other traditional Chinese medicine monomers.Secondly,a peptide-drug conjugates(PDCs)based prodrug modification strategy was studied.Coupling M19 with bone-targeting polyaspartic acid through cathepsin K dipeptide substrate(leucine-arginine),we have designed a series of M19 based PDCs which were successfully synthesized via SPPS and liquid-phase chemistry with total yields of about 30%.Structural stability experiments showed that no obvious degradation products were found in the PDCs aqueous solution within 14 days,suggesting greatly improved stability compared with the prototype drug M19.Subsequent bioactivity experiments showed that the PDCs were able to release more than 95% of prototype drug within 4 hours' treatment of cathepsin K,exhibit more than 95% binding affinity to hydroxyapatite in 80 minutes and show hydrolase protease stability.In addition,compared with the prototype drug M19,the cytotoxic effects of the PDCs were reduced and the osteoclast inhibitory activities were comparable to that of the prototype drug,except for BTM19-1.All in all,PDCs based prodrug modification has not only stabilized the molecular structure and conferred bone-targeting properties,but also reduced the cytotoxicity and maintained the osteoclasts inhibitory activity of the prototype drug,led to improved chemical and biological properties of M19.Meanwhile,this work has also provided a reference for other targeting prodrug modification studies of similar traditional Chinese medicine monomers.