Synthesis And Antitumor Activity Of Spinosad Derivatives

Background:At present,the traditional treatment methods for cancer are surgery,radiotherapy and chemotherapy,and they all need the adjuvant treatment of drugs.Most of the traditional chemotherapy drugs have low targeting,drug resistance or serious side effects.New anti-tumor drugs and new therapeutic targets are still the focus and hotspot of tumor therapy.ASS1 is a rate-limiting enzyme in the urea cycle.It mainly catalyzes the conversion of citrulline and aspartic acid to arginine succinic acid,which is then cleaved by lyase to arginine and fumaric acid.ASS1 is involved in multiple metabolic processes and plays an important role in the occurrence and development of tumors as a tumor suppressor and an oncogenic factor.Currently,there is no clinical drug targeting ASS1 activation,and it is of great significance to develop new drugs targeting ASS1 activation.In our previous studies,spinosad and its derivative LM-2I were found to have targeted activation of ASS1 and anti-tumor effect.Spinosad is a metabolite produced by Gram-positive filamentous bacteria in the aerobic fermentation process,with a novel and unique structure.Our preliminary structural modification of spinosad showed that its anti-tumor activity was greatly enhanced,and it is a potential new structure of anti-tumor drugs.However,the relationship between the activity and structure of this compound is not clear,so we used spinosyn A as the lead structure to design,synthesize and explore the structure-activity relationship of spinosyn A,which is of great significance for studying its mechanism of action and discovering new anti-tumor drugs targeting ASS1.Methods:18 compounds were designed and synthesized by structural modification of C5-C6 double bond,C17 and D-forosamine nitrogen atoms with spinosyn A as the lead compound.Meanwhile,11previously synthesized spinosyn A derivatives were summarized and confirmed by ¹H-NMR,¹³C-NMR and LC/MS-MS.Twenty-nine compounds were screened for their antitumor activity in A549,HT-29,Hep G2,MDA-MB-231 and LO2 cells by MTT assay.At the same time,ASS1 activation effect of all compounds was detected,and spearman analysis was used to analyze the correlation between IC₅₀ value of compounds and ASS1 activation activity,and molecular docking was used to study the interaction between compound C₅ and ASS1.Results:MTT assay was used to evaluate the antitumor activity of the compound.It was found that the loss of C17 D-forosamine and the epoxidation of C5-C6 double bond resulted in a decrease in antitumor activity.The overall activity of compounds in Series C was better than that of Series A and B.The activity of introducing amide and ester bonds on C17 position D-forosamine was weaker than that of amine side chain,while the activity of aliphatic amines with three carbon chains in amine side chain was stronger than that of aromatic amines,and the target compound C₅ had the best activity.In addition,HT-29 cell was sensitive to the derivatives.In the in vitro enzyme activity test,the ester compounds introduced by Series A and B had better enzyme activation effect,and Series C had stronger activity on ASS1 as a whole.It is indicated that the enzyme activation effect of the nitrogen atom of C17 position D-forosamine in spinosyn A derivatives substituted with fatty amines or heterocyclic amines substituents was obvious.Compared with SPA control group,most compounds in Series C significantly enhanced the activation effect of ASS1.In addition,the C17-position D-forosamine and the C5-C6double bond on spinosyn A are important groups affecting the activation effect of the enzyme.The correlation between the antitumor activity of the compounds and the activation ability of ASS1 was analyzed,and a linear negative correlation was found.It was found that compound C₅ not only had good antitumor activity,but had a strong activation effect on ASS1.Molecular docking of compound C₅ with ASS1 revealed hydrogen bond interaction between compound C₅ and ASS1.Conclusion:When spinosyn A introduced different aliphatic amine derivatives with three carbon length side chains on the the nitrogen atom of C17 position D-forosamine,the anti-tumor effect was significantly enhanced,and HT-29 cell was sensitive to these derivatives.The compounds of Series C showed the most obvious activation effect on ASS1 in vitro.Compound C₅ not only has good antitumor effect,but has the best activity to activate ASS1.Further molecular docking revealed hydrogen bond interaction between compound C₅ and ASS1.