Synthesis Of 2-Chloro-3-Alkyl-1,4-Naphthoquinone Derivatives And The Mechanism Of Anti-leukemia Activity Via Targeting Mitochondria

The occurrence and progression of human acute myeloid leukemia?AML?are associated with mitochondrial dysfunction and mutations of mitochondrial DNA?mtDNA?.The 1,4-naphthoquinone derivatives are an important class of anti-cancer molecules and mainly target in nuclear DNA.However,their effects on mtDNA have rarely been studied.Our previous studies found that 2-chloro-1,4-naphthoquinone had significant anti-leukemia activity,so we took 2-chloro-1,4-naphthoquinone as the lead structure in the present study.The research contents are as following:1.Synthesis of the title compounds:for the lead scaffold,the alkyl side chain and the nitrogen alkyl side chain were introduced at the C-3 position,and the trimethoxy groups was introduced into the benzene ring.The six derivatives were obtained.2.Evaluation of anti-proliferative activity of the title compounds?MTT method?:We measured the anti-proliferative activity of the title compound against HL-60 cells?acute myeloid leukemia cell?and normal cells WI-38?human embryonic lung fibroblasts?.The results showed that Compound 18 displayed the best activity(IC₅₀=2.85?M)and high selectivity index?SI=8.07?.3.Compound 18 induced mt DNA damage and mitochondrial dysfunction:18induced a significant arrest of S phase in HL-60 cells and a slight arrest in G2/M phase,suggesting that mtDNA damage occurred in cells after treating with compound18.The results of WB showed that 18 decreased the protein expression of mt DNA transcription factor A and mtDNA specific polymerase gamma,confirming the existence of mtDNA damage.After treating with compound 18,the anti-proliferative activity of HL-60?^o?mtDNA deletion?cells was significantly reduced with IC₅₀>10?M.In addition,18 led to a decrease in mitochondrial membrane potential and ATP production4.Compound 18 induced intracellular GSH depletion:18 caused depletion of GSH and increase of GSSG.By measuring intracellular reactive oxygen species and using UV-Vis spectroscopy and HRMS analysis,we concluded that the depletion of GSH is attributed to GSH being oxidized by reactive oxygen species to GSSG and GSH with 18 to form adducts.Collectively,weproposedthattheanti-AMLmechanismof2-chloro-1,4-naphthoquinone compounds is related to mtDNA damage and reduction of intracellular GSH.