Studies On Synthesis And Antitumor Activity Of Pyxinol Urea Derivatives

Malignant tumors have high incidence rates and mortality rates,which seriously threaten the human health.The present drugs have some disadvantages such as drug resistance,side effects and so on.It is an important research direction to develop new anti-cancer drugs with high activity and low toxicity.The active components of natural products have the characteristics of novel structures,characteristic pharmacological activities,low toxicities,little drug resistance,multi-target effect and so on.Natural products become the important sources for finding the novel anti-cancer drug.Both pyxinol and its epimer,（24S）-pyxinol,are the ocotillol-type compounds.They not only exist in many plants,but also are the active metabolites of ginsenosides in the body.They have many pharmacological effects,such as anti-tumor,antibacterial,anti-inflammatory and myocardial protection.Recently,great progress had been made in the structural modification of these two lead compounds.C3-OH was also proved to be the most suitable site for structural modification.Urea groups have always played a key role in drug research and development.They are not only the key structures of some marketed drugs,but also the pharmacodynamic groups in some candidate drugs.Furthermore,the urea groups also play an important role in the structural modification of natural products.The urea derivatives could significantly improve the anticancer,antibacterial,anti convulsive,anti HIV and anti diabetes effects of the lead compounds.In order to further improve the antitumor activity of pyxinol and（24S）-pyxinol,the hydroxyl group at the carbon 3 position was replaced with urea groups according to the"bioelectronic isosteric"drug design strategy.In this paper,the urea derivatives of pyxinol or（24S）-pyxinol had been were synthesized for the first time;the inhibitory effects of each derivatives on human hepatoma cells（HepG2 cells）,human lung cancer cells（A549 cells）and human cervical cancer cells（Hela cells）were evaluated;the derivative with the best activity was screened;and the metabolomics study of the active derivative in hepatoma cells was carried out.The main achievement are as follows:1.Chemical synthesis of pyxinol and（24S）-pyxinol urea derivativesTaking pyxinol and（24S）-pyxinol as the lead compounds,and the hydroxyl group connected at the carbon 3 position as the reaction site,a series of urea derivatives were synthesized.The alkyl isocyanates,halogen aryl isocyanates and aryl isocyanates were used to introduce the urea groups.（1）Synthesis and structural identification of intermediatesCombined literature reports and pre-experiments,the steps of synthesizing urea derivatives,the target products,were designed including oxidation,oximation,amination and urea reaction.There were three kinds of intermediates:（1）With the action of pyridine chlorochromate（PCC）,the C₃-OH of pyxinol or（24S）-pyxinol was oxidized to keto carbonyl to obtain the oxidation intermediates.（2）The oximation intermediates were obtained by oximation reaction between oxidation intermediate and hydroxylamine hydrochloride.（3）The oxime groups were then reduced to amino groups by reduction reaction,and the aminated intermediates were obtained.The crude products were separated and purified by silica gel column chromatography and recrystallization technologies.And the structures were identified by high resolution mass spectrometry（HR-MS）and nuclear magnetic resonance（NMR）.The synthesized six intermediates were 3-keto-pyxinol,（24S）-3-keto-pyxinol,3-oxime-pyxinol,（24S）-3-oxime-pyxinol,3β-amino pyxinol and（24S）-3β-amino pyxinol,respectively.（2）Synthesis and structural identification of urea derivativesTaking the aminated intermediates as the starting materials,each isocyanates was added to carry out nucleophilic addition reaction,respectively.The crude products were then separated and purified by silica gel column chromatography and recrystallization technologies.And the structures were identified by HR-MS and NMR.The synthesized 36 urea derivatives（including 18 alkyl ureas,8 halogen aromatic ureas,10 other aromatic ureas）were as follows:3β-ethylurea-pyxinol（1）,3β-propylurea-pyxinol（2）,3β-propenylurea-pyxinol（3）,3β-pbutylurea-pyxinol（4）,3β-amylurea-pyxinol（5）,3β-hexylurea-pyxinol（6）,3β-isopropylurea-pyxinol（7）,3β-cyclopentylurea-pyxinol（8）,3β-cyclohexylurea-pyxinol（9）,（24S）-3β-ethylurea-pyxinol（10）,（24S）-3β-propylurea-pyxinol（11）,（24S）-3β-propenylurea-pyxinol（12）,（24S）-3β-pbutylurea-pyxinol（13）,（24S）-3β-amylurea-pyxinol（14）,（24S）-3β-hexyl-urea-pyxinol（15）,（24S）-3β-isopropylurea-pyxinol（16）,（24S）-3β-cyclopentylurea-pyxinol（17）,（24S）-3β-cyclohexylurea-pyxinol（18）,3β-（2-bromophenylurea）-pyxinol（19）,3β-（4-bromophenylurea）-pyxinol（20）,3β-chlorophenylurea-pyxinol（21）,3β-[3,5-bis（trifluoromethyl）phenylurea]-pyxinol（22）,（24S）-3β-（2-bromophe-nylurea）-pyxinol（23）,（24S）-3β-（4-bromophenylurea）-pyxinol（24）,（24S）-3β-chloro-phenylurea-pyxinol（25）,（24S）-3β-[3,5-bis（trifluoromethyl）phenylurea]-pyxinol（26）,3β-benzylurea-pyxinol（27）,3β-phenethylurea-pyxinol（28）,3β-（1-naphthylurea）-pyxinol（29）,3β-（4-methyl-parabenurea）-pyxinol（30）,3β-benzenesulfonylurea-pyxinol（31）,（24S）-3β-benzylurea-pyxinol（32）,（24S）-3β-phenethylurea-pyxinol（33）,（24S）-3β-（1-naphthylurea）-pyxinol（34）,（24S）-3β-（4-methylparabenurea）-pyxinol（35）and（24S）-3β-benzene-sulfonylurea-pyxinol（36）.All the synthesized derivatives are the new compounds.2.The effects of each urea derivative on A549,HepG2 and Hela cellsA549 cells,HepG2 cells and He La cells were used to evaluate the inhibitory effects of two lead compounds（Pyxinol and（24S）-Pyxinol）,six intermediates（intermediates 1～6）and 36 urea derivatives（compounds 1～36）by CCK-8 method.In HepG2 cells,the IC₅₀values of compounds 1,20,22,26,36 were all less than10μM.Among them,the IC₅₀values of compound 1 and of compound 20 were 5.47±0.85μM and 5.41±0.98μM,respectively.In additional,the IC₅₀values of intermediate 3 and compounds 21,23,24,29,33,35 were all less than 20μM.In A549 cells,the IC₅₀values of compounds 3,7,20,26,28 were all less than 30μM.In He La cells,the IC₅₀values of compound 20 was 5.79±0.67μM.Addtionally,IC₅₀values of compounds 14,18,20,24,25,26 were all less than 20μM.In all synthetic urea derivatives,the IC₅₀ of compound 20 on HepG2 cells and He La cells were all less than 10μM,and the IC₅₀ on A549 cells was 20.45±3.63μM.It was showed that compound 20 was the derivative with the extensive and strongest anticancer activity.The structure-activity relationship study found that most urea derivatives showed better antitumor activity than the lead compounds,indicating that the introduction of urea groups into the structures of Pyxinol and（24S）-Pyxinol was helpful to improve the antitumor activity,and the activities of aromatic urea derivatives were better than that of alkyl urea derivatives and when halogen was introduced into aromatic urea derivatives,the antitumor activity was stronger.3.Metabolomics study of Compound 20 on hepatoma cellsIn order to explore the anticancer mechanism of compound 20 from the metabolite level,the strategy of UPLC-Q/TOF-MS cell metabolomics was used.The cell samples intervened with compound 20 were analyzed to identify the endogenous metabolites and the involved metabolic pathways closely related to the anticancer activity.By analyzing the metabolic profiling differences between the control group and the compound 20 administration group,a total of 13 differential metabolites such as taurine,L-glutamic acid,uridine diphosphate-n-acetylglucosamine,uridine diphosphate glucose,citric acid,glutathione,cysteine glycine,acetylacetic acid,PE（20:0/15:0）,PC（14:1（9Z）/16:0）,PC（P-16:0/16:0）,PC（16:1（9Z）/16:1（9Z））and PC（18:1（9Z）/14:0）were identified from the HepG2 cells.Pathway enrichment analysis showed that amino acid metabolic pathways such as glutathione,alanine,aspartic acid,glutamate and D-glutamine;organic acid metabolic pathways such as butyric acid,citric acid,taurine and sub taurine;carbohydrate metabolic pathways such as amino sugar and nucleotide sugar;and the synthesis and degradation pathways of ketones were all significantly disturbed.In conclusion,a total of 36 new pyxinol and（24S）-pyxinol urea derivatives were synthesized for the first time,and the inhibitory effects of each derivative on HepG2,A549 and He La cells were evaluated.Compound 20 was screened as the best anti HepG2 cell proliferation derivative.Its potential mechanism was the intervention of metabolites such as glutamate and metabolic pathways such as amino acids.Through the study of this paper,the structural modification of pyxinol and（24S）-pyxinol was enriched,and a candidate drug with potential anti-hepatoma activity was screen out.