Preliminary Discussion On The Druggability Of A Novel Antitumor Compound CNF2

Cytisine N-methylene-?5,7-dihydroxy-4'-methoxy?isoflavone?CNF2?was extracted and isolated from the Chinese herbal medicine Sophora alopecuroides L.by our research group,which is a natural active compound with the novel structure.Previous pharmacodynamic studies have shown that CNF2 has obvious anti-tumor activity and can inhibit the migration of MDA-MB-231 and 4T1 breast cancer cells.To further investigate the druggability of the compounds,this paper mainly determined the basic physicochemical properties of CNF2,established the quantitative analysis method for HPLC biological samples.The absorption and tissue distribution of CNF2 in Sprague-Dawley?SD?rats was also studied.Finally,the druggability of the compound was preliminarily evaluated.The work of this paper mainly includes the following parts:1.Studied on the Physicochemical Properties of CNF2.In this paper,the method for determining the content of CNF2 was established by ultraviolet spectrophotometry to determine the equilibrium solubility of CNF2 in different organic solvents and different p H buffers.The experimental results show that CNF2 is easily soluble in organic solvents such as acetone and methanol,and slightly soluble in water.The order of its solubility in different solvents:acetone>methanol>ethanol>isopropanol>acetonitrile>octanol>ethyl acetate>water;The solubility of CNF2 in acidic and weakly alkaline buffer solutions is large,which indicates that the compound is easily absorbed and digested by the human's gastrointestinal and is suitable for being developed into an oral preparation.Besides,the oil-water partition coefficient?log P?and dissociation constant?p Ka?of CNF2 were determined,and the results showed that the log P of CNF2 was 1.24±0.08and p Ka was 7.18.2.Established and verified of the HPLC analysis method of CNF2 in biological samples.The pretreatment method for biological samples in this paper uses acetonitrile as a protein precipitant and extractant,and the protein removal content reaches more than 97%.HPLC chromatographic conditions were as follows:Hypersil ODS C18 column?4.6×250mm,5?m?;mobile phase was methanol and triethylamine phosphate buffer?p H 7.30±0.05??48/52,V/V?,isocratic elution,with a flow rate was 1 m L/min;column temperature was35°C;detector wavelength was 290 nm;injection volume was 20?L.The HPLC in vivo quantitative analysis method established in this paper has good sensitivity,with a lower limit of quantitation of 0.4?g/m L,and a good linear relationship between 0.4 and 40?g/m L?with the correlation coefficient is 0.999?was observed.It has excellent extraction recovery,ranging from 90%to 96%.The precision and accuracy of the analytical method are within±15%,which meets the requirements of the US FDA guidelines.This method has been successfully used for the first time to study the plasma pharmacokinetics and tissue distribution of CNF2 in rats.3.Studied on Pharmacokinetics and Tissue Distribution of CNF2 in Rats.SD rats were administered by intragastric administration?60 mg/kg?and tail vein injection?6 mg/kg?,the whole blood of the orbital venous plexus was collected at different time points for pharmacokinetic studies.The established and validated HPLC analysis method was used to determine the plasma concentration at different time points,and then the plasma concentration-time curve was drawn.Plasma pharmacokinetic parameters were obtained according to the non-compartment model?Win Nonlin 5.2?,and absolute oral bioavailability was obtained by the calculation formula.Statistics of pharmacokinetic parameters are as follows:?1?intragastric administration:AUC_?0-t?was 2302.825±67.269?g/m L·min,AUC_?0-??was 2507.903±67.988?g/m L·min,MRT_?0-t?was 109.110±0.955min,MRT_inf was 611.236±0.94 min,T_max was 15 min,and C_max was 7.72±0.86?g/m L;?2?tail vein administration:AUC_?0-t?was 3480.91±173.66?g/m L·min,AUC_?0-??was 3885.56±201.4?g/m L·min,MRT_?0-t?was 184.470±3.4 min,MRT_inf was 650.440±4.8 min,and C_maxwas 12.43±2.47?g/m L.The absolute bioavailability of CNF2 in rats was 6.6%.CNF2 was administered orally to SD rats in a single-dose?60 mg/kg?.The rats were killed by eyeball removal at 5 minutes,15 minutes and 40 minutes after administration,and twelve organs including hearts,livers,spleens,lungs,kidneys,stomachs,brains,intestines,pancreas,muscles,fats and uteruses were rapidly removed.Organ tissues are flushed with normal saline,and then wiped with filter paper and weighed.The organ tissue was then mixed with physiological saline for homogenization,and the concentration of CNF2 in the above tissue was determined by the established and validated HPLC analysis method.The experimental results show that CNF2 is mainly distributed in the stomach,intestine and lung tissues,followed by the liver,fat,uterus and kidney,which are least distributed in the heart and spleen,and has no distribution in the brain tissue.4.Molecular docking simulation and druggability evaluation of CNF2.This paper uses Auto Dock Tools-1.5.6 to analyze the docking of compounds CNF2 and human epidermal growth factor receptor-2?HER2?,uses ACD/Percepta software to predict the physicochemical properties and ADME properties of CNF2 based on quantitative structure-activity relationship?QSAR?and evaluates the druggability of CNF2 based on the experimental results.Theoretical research has shown that the binding affinity of CNF2 to HER2 is stronger than that of the positive control inhibitor SYR127063,which indicates that CNF2 has a certain inhibitory effect on HER2.At the same time,CNF2 was screened according to Lipinski's Rule of Five.Therefore,CNF2 can be developed as a lead compound with a specific structure and the antitumor activity.However,experimental data indicate that CNF2 has a lower absolute bioavailability,which may be related to the first-pass effect of CNF2.