Study On The Construction And Bioactivities Of Novel D-ring Modified N-heterosteroid Derivatives

Steroid compounds belong to a special class of polycyclic terpenoids,which include three parts of core ring,C₁₇ side chain and different functional group substituents.The market demand of steroid compounds in the pharmaceutical field is second only to antibiotics,with a global total value of 40 billion yuan.At present,there are more than 400 authorized steroid drugs in circulation,and the trend will be increasing in the future.Steroid derivatives always exhibit extensive biological activities,including antibacterial,anti-inflammatory,anti-Alzheimer’s disease,anti-proliferation,anti-neuritis and anti-cancer.Steroid drugs are widely used as traditional hormonal drugs,and their application in anti-tumor therapy has been one of the research fields of extensive attention in recent years.Nitrogenous heterocyclic compounds are often widely used as important structural units of pharmaceutical compounds due to their unique biological activities,high systemic nature and low toxicity.In view of the significant physiological and biological activities of steroids containing nitrogen-heterocyclic functional groups,they have unique properties superior to their parent compounds,especially in antibacterial,antiviral,anti-inflammatory and anti-tumor activities.Dehydroepiandrosterone,epiandrosterone,androsterone,4-androstenedione,9α-hydroxy-androst-4-ene-3,17-dione,1,4-androstenedione,and dienedione were used as the basic backbone of steroids,the bioactive N-heterocyclic rings such as 2-aminopyrimidine ring,thiazole ring and pyrazopyridine ring were introduced through the principle of molecular heterozygosity and structural diversity design strategy.A total of 119 compounds were obtained by using high-efficiency chemical synthesis and biocatalysis to synthesize multiple types of D-ring modified new N-heterosteroid derivatives,mainly including steroidal[17,16-d]pyrimidine derivatives,steroidal thiazoline derivatives,steroidal pyrazole[1,5-a]pyrimidine derivatives,steroidal hydrazone derivatives,etc.The obtained 119 molecules were characterized in detail and their structures were determined by ¹H NMR,¹³C NMR,ESI-MS and HPLC.The inhibitory effects of 119 compounds on the proliferation of different cancer cell lines including human gastric cancer cell line,lung cancer cell line,liver cancer cell line,melanoma cell line and breast cancer cell line were determined by MTT（3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide）colorimetric assay.The results showed that compounds 15c,16c,39a,44a,54b and 62a could induce apoptosis of A875 cells.The steroidal thiazoline derivatives 15c and 16c could induce apoptosis of A875 cells in the early stage,while the new steroidal derivatives 39a,44a,54b and 62a could induce apoptosis in the late stage.The compounds 3a,15c,16c,39a and 44a were found to inhibit the activity of Cyclin-Dependent protein Kinase 1（CDK1）.Among them,compound 39a showed a stronger inhibitory effect on ALK protein kinase.Molecular simulation docking was used to further analyze the relationship between the structure and activity of the new nitrogen heterocyclic compounds.The results showed that the core ring structure of the steroid compounds was mainly bound to the key amino acids of the target through hydrophobic interaction,and the nitrogen heterocyclic structure was connected by hydrogen bonds or functioned as an important connecting structure.Based on the structure analysis of the substituent containing the benzene ring structure,the strengthening of hydrophobic force and the strengthening of binding byπ-πstacking are important.These provide a reference for the development of new N-heterosteroid compound with targeting,high biological activity and low toxic anti-tumor activity.A total of 119 compounds were tested for their insecticidal activity against insect pests such as aphids and Diamondback moths,and the results showed that compounds20a,20b,20c,45c and 54h had some insecticidal activity against diamondback moths.These compounds were found to have 1,4-androstenedione steroid core ring backbone structure,2-chlorophenyl and thiazoline ring structural units.This new finding will provide theoretical basis for the design and development of steroid derivative pesticides.In summary,seven backbone molecules 3a,15c,16c,39a,44a,54b and 62a were found to have strong anti-tumor cell proliferation activity,and three molecules 20a,20b and 20c were found to have strong insecticide-activity against diamondback moth.These backbone molecules will provide an important reference for the design and development of new anti-tumor N-hetersteroids and insecticides.