Research On Cytotoxiity,Structure-activity Relationships And Mechanism Of Apoptosis Induction Of 2-aryl-3,4-dihydroisoquinoline Compunds

Natural isoquinoline compounds containing C=N⁺double bond are a rare kind of low-content alkaloid with special structure.This kind of compounds show prominent anticancer,antimicrobial,insecticidal,anti-inflammatory and antioxidant effects and other important bioactivities and has therefore attracted great interests of pharmaceutical researchers especially in the anticancer field both at home and abroad.However,the potential development and utilization of these compounds is restricted to some extent because of the active chemical property,difficult synthesis and isolation and insufficient modification sites in their molecules.An important method for new drug development is to practice the structural simulations by taking natural active substances as lead compounds or template compounds.In early research of our team,a series of 2-aryl-3,4-dihydroisoquinoline compounds were designed and synthesized according to molecular simulation principle,in which two quaternary benzophenanthridine compounds containing C=N⁺double bond,sanguinarine and chelerythrine were used as the template compounds.And it was found that the2-aryl-3,4-dihydroisoquinoline compounds have significant antimicrobial and acaricidal activitiessimilartothoseofthetemplatecompounds,thusprovingthat2-aryl-3,4-dihydroisoquinoline compounds can be used the ideal secondary lead compounds for developing the quaternary benzophenanthridine compounds.This study aimed to further investigatethecytotoxicactivityandthestructure-activityrelationshipof2-aryl-3,4-dihydroisoquinolinecompounds,andprimarilyexploretheircell apoptosis-inducing mechanism in order to clarify whether this kind of compounds can be used as secondary lead compounds of the template compounds in the anticancer drug research on quaternary benzophenanthridine compounds and to lay some theoretical foundation for research on this kind of alkaloids.The main results of this are as follows.1.First of all,MTT assay was used to have determined the cytotoxic activities in two lines of tumor cells（NB4 cells and MKN-45 cells）by tow series of 1033,4-dihydroisoquinolin compounds（2-aryl-3,4-dihydro-isoquinoline bromide,class I;2-aryl-8-methoxy-3,4-dihydro-isoquinoline bromide,class II;2-aryl-8-hydroxyl-3,4-dihydro–isoquinoline bromide,class III）,one series of 25 2-aryl-1-cyano-1,2,3,4-tetreahydro-isoquinoline compounds（class I’）and one compound of class II′,2-（2-iodo-phenyl）-1-cyano-8-methoxy-1,2,3,4-tetrahydro-isoquinoline（II′-15）.The results indicated that all compounds at tested concentration≤20.0μM showed some inhibitory effects on the cell proliferation of two kinds of tumor cells and the the cytotoxic activities of all compounds to NB4 cell line were higher than those to MKN-45 cell line.And the compounds are in order of class I≈class I′>class II>class III from high to low cytotoxic activities.The cytotoxic activities of most class I and class I’compounds were higher than those of chelerythrine but lower than those of sanguinarine.The cytotoxic activities of almost all class II and class III compounds were lower than those of sanguinarine and chelerythrine.Secondly,the cytotoxic activities of 17 compounds of class I’and 28 compounds of class II were further tested since their cytotoxic activities are comparatively higher.The results showed that the IC₅₀0 values of class I’compounds to NB4 cell line were 0.16⁰.67μg/mL（0.67².24μM）,their cytotoxic activities were higher than that of 6-cyano-dihydro-chelerythrine(IC₅₀0 0.69μg/mL,1.85μM)and that of and reference drug DDP(IC₅₀0 0.72μg/mL,2.39μM),but lower than that of6-cyano-dihydro-sanguinarine(IC₅₀0 0.19μg/mL,0.53μM).The IC₅₀0 values of class I’compounds to MKN-45 cells were 0.90³.50μg/mL（2.99¹3.01μM）,their cytotoxic activities were higher than those of 6-cyano-dihydro-chelerythrine(IC₅₀0 4.74μg/mL,12.72μM)and reference drug DDP(IC₅₀0 3.41μg/mL,11.36μM),but lower than that of6-cyano-dihydro-sanguinarine(IC₅₀0 0.55μg/mL,1.53μM).The IC₅₀0 values of most class I’compounds to NB4 cell line were 0.68³.81μg/mL（1.54⁹.61μM）,their cytotoxic activities were lower than those of sanguinarine(IC₅₀0 0.51μg/mL,0.53μM),chelerythrine(IC₅₀0 0.71μg/mL,1.49μM)and DDP(IC₅₀0 0.72μg/mL,2.39μM).In the opposite,the cytotoxic activities of most class II compounds(IC₅₀<3.4μg/mL)to KMN-45 cell line were higher than those of chelerythrine(IC₅₀0 6.04μg/mL,12.71μM)and DPP but lower than that of sanguinarine(IC₅₀0 0.70μg/mL,1.51μM).The cytotoxic activity of II′-15(IC₅₀0 0.74μg/mL,2.39μM)to NB4 cell line was close to those of 6-cyano-dihydro-chelerythrine and DDP but lower than that of 6-cyano-dihydro-sanguinarine.The cytotoxic activity of II′-15(IC₅₀0 0.77μg/mL,2.50μM)was significantly higher than those of 6-cyano-dihydro-chelerythrine and DDP but lower than that of 6-cyano-dihydro-sanguinarine.Thirdly,the cytotoxic activity of two compounds of I′₂ and I′₁₇7 to two kinds of normal primary cells,fetal goat fibroblasts and fetal porcine renal cells,were tested.And the results indicated that the cytotoxic activity of the compound I′₂ to primary fetal porcine renal cells was slightly higher than that to KMN-45cell line.The cytotoxic activities of two compounds I′₂ and I′₁₇7 to abovementioned two kinds of primary normal cells were lower that of DDP,and lower than the cytotoxic activities to cancer cells,thus indicating that these two compounds can selectively inhibit the proliferation of cancer cells.2.The structure-activity relationship between the tested compounds and their cytotoxic activities were researched and the results indicated that:（1）The class I compounds and corresponding class I′compounds showed nearly the same intensity of cytotoxic activities,indicating that the class I′compounds may be the drug precursors of class I compounds.And the precursor relation also existed between sanguinarine and 6-cyano-dihydro-sanguinarine and between chelerythrine and 6-cyano-dihydro-chelerythrine.（2）The classification,number and location of the substituent in N-benzene ring significantly affected the cytotoxic activities of the compounds of class I,class I′,class II and class III to two cancer cell lines.（3）The compounds of class I and class I′showed exactly the same structure-activity relationship.Introduction of electron-withdrawing groups,such as halogens,trifluoro-methyl and nitro,could enhance their cytotoxic activities.In the opposite,the cytotoxic activities were decreased in the compounds if an electron-donating group,such as a methyl and methoxy group,was introduced into the N-benzene ring of the compounds.With respect to the class I or class I’compounds or isomers which were prepared by substitution with weakly electron-withdrawing halogen atoms,the highest cytotoxic activity appeared at the location of2’-halogeno-substitutes.Similar changes of structure-activity between the cytotoxic activities and the substitution locations also appeared in the class I or class I’isomers prepared with electron-donating groups.In addition,halides containing two halogen atoms showed higher cytotoxic activities than their corresponding halides containing one halogen atom,showing that the increase of the number of substituent halogen atoms can correspondingly increase the cytotoxic activities.In respect to the intensively electron-withdrawing trifluoro-methyl and nitro,3′-halogeno-substitute showed the highest cytotoxic activities whereas the cytotoxic activity of 2′-halogeno-substitute showed the lowest cytotoxic activity.（4）The effects of classification of the substituent on the cytotoxic activities of class II compounds were similar to but not the same as those of class I compounds.In general trends,the electron-withdrawing group could increase while the electron-donating group could decrease the cytotoxic activity of the substitute.To NB4 cell line,the cytotoxic activity of4’-halogeno-isomer was the highest among the halogeno-isomer in other locations.As for fluorine or chlorine halogeno-isomer,the cytotoxic activity of 2’-halogeno-isomer was the highest among the halogeno-isomers in all locations.However,for bromine or iodine atom as the substituent,the cytotoxic activity of 3’-halogeno-isomers was the highest among the halogeno-isomers in other locations.All classes of II₂′-halogeno-isomers showed the highest cytotoxic activities to MKN-45 cell line but the cytotoxic activities of 3’-halogeno-isomers and of 4’-halogeno-isomers depend on the classification of the halogens.The cytotoxic activities of the isomers of class II compounds containing cyano or trifluoromethyl as the substituent depend on the cell classification,thus indicating that the classification and location of intensive electron-withdrawing groups in substitutes both affected the cytotoxic activities of class II compounds to different kinds of target cells.Similar to those of class I compounds,the cytotoxic activities of halogeno-substitutes containing two halogen atoms were higher than those of halogeno-substitutes containing one halogen atom,indicating that the increase of the halogen atom could increase the cytotoxic activities of halogeno-substitutes.（5）Introduction of 8′-OH could significantly decrease the cytotoxic activities of all class I compounds to two kinds of cancer lines while the introduction of 8’-OCH₃ could increase the cytotoxic activities of some compounds,especially the cytotoxic activity to KMN-45 cell line.3.The antioxidant capacities of I₂ and I₁₇,also as two representative compounds,and of the template compounds—sanguinarine and chelerythrine were studied.The results showed that these four compounds at higher concentrations were capable of cleaning free radicals of DPPH and ABTS and protecting BSA protein from the harmful oxidation induced by Cu²⁺/H₂O₂ system.The anti-oxidation order of the four compounds is sanguinarine>chelerythrine>I₂>I₁₇7 from high to low.But the anti-oxidation capacity of four compounds was far lower than that of the reference substance Trolox.The IC₅₀0 values of anti-oxidation of the four compounds were much higher than their IC₅₀0 values to cancer cell lines,indicating that their cytotoxic activities were possibly unrelated to the anti-oxidation capacity.4.DAPI staining,AO/EB staining,ultrastructural analysis and protease activity assays indicated that I′samples could significantly induce NB4 cell line and MKN-45 cell line into apoptosis in a dose-dependent manner.The possible mechanism may lie in the excessive formation of ROS,which leads to the cell apoptosis.And flow cytometry showed that the cells in apoptosis induced by the compounds of the research were at early stage of apoptosis,and the cell cycle arrest was not affected significantly.5.QRT-PCR assay for the expression of 12 apoptosis-related genes found that the representative samples of I′₂ could significantly upregulate the mRNA expression level of CFLAR,THBS1,CCNB1 genes（all at p<0.01）.The mRNA expression levels of TNF,CYCS,CASPASE 3,CCNE2,GTSE1 were all significantly upregulated（all at p<0.01）.The mRNA expression levels of FAS,BAX and HDAC1 were elevated but on an insignificant level.The mRNA expression of BCL2 was also insignificantly downregulated.In the experiment of Western blot it was found that I′₂ sample could induce apoptosis by way of endogenous mitochondria-mediated pathway including the downregulation of the protein expression of Bcl-2/Bax,the upregulation of the protein expression of cyto-c and cleaved caspase/caspase 3,the activation of the p38MAPK signal pathway and the inhibition of the phosphorylation of Akt.In summary,2-aryl-isoquinoline compounds used in this research were highly cytotoxic in general,among which several compounds with high activity were in development and application.And analysis of the structure-activity relationship indicated that the substituent groups in the two aromatic rings of so kind of compounds affect the anticancer activities of the molecules possibly by way of comprehensive impact on molecular electron density.The results of this research were of much theoretical significance and reference value to further research on the structural optimization,the safety evaluation and the apoptosis induction mechanism of 2-aryl-isoquinoline compounds.