Synthesis And Anti-lung Cancer Activity Of Novel 4-aryl-1,4-dihydroprydines

Background Lung cancer is the most common malignancy worldwide and its mortality rate is the highest among all the malignant tumors.Patients with lung cancer generally can't be treated with surgery for they have progressed to advanced stage when diagnosis.So chemotherapy is the main therapeutic means for many lung cancer patients.Chemotherapy is a direct damage of DNA,blocking DNA synthesis or interfering with cell division by cytotoxic drugs.Although chemotherapy can successfully treat part of the cancer patients,the side effects raised from toxicity of chemotherapeutic drugs on normal tissue and multidrug resistance often lead to chemotherapy failure.Therefore,the development of cytotoxic drugs with tumor-selectivity and multidrug resistance reversal activity has great research value and broad application prospects in the treatment of cancer.1,4-dihydropyridines(1,4-DHPs)are a kind of important nitrogen-containing heterocyclic compounds.Its structural framework is widely found in natural products,drugs and molecules with biological drug activity.The 1,4-DHPs have been reported to have various bioactivity such as antihypertensive,anti-angina,anti-atherosclerotic,anti-diabetes,anti-mycobacterium tuberculosis,anticoagulation,anti-oxidation,anti-virus,anti-bacterial and anti-inflammatory effects.As a calcium channel antagonist,1,4-DHPs are clinically used for first-line treatment of hypertension,angina,and arrhythmia.For multidrug resistance(MDR)reversal agent verapamil also belongs to the calcium channel antagonist,the MDR reversal activity of1,4-DHPs has been widely studied since the 1980 s by group replacement on the relevant sites of 1,4-DHP pyridine ring.Subsequently,the cytotoxic activity of1,4-DHPs to tumor cells was researched extensively as well.Purpose In this paper,based on the reported 1,4-DHPs synthesis method and application especially antitumor activity,four novel 4-aryl-1,4-dihydropyridine derivatives were synthesized by the multi-component "one-pot" method.Then the antitumor activity of the synthesized1,4-DHPs was studied in vitro and in vivo,and the corresponding structure-activity relationship was analyzed.The main work of this paper was shown as follows.Methods and results The best known procedure for preparation of 1,4-DHPs is the classical Hantzsch reaction.We improved this method by using ammonium acetate instead of concentrated ammonia,and 4-(dimethylamino)pyridine as a highly efficient homogenous catalyst.Four novel 4-aryl-1,4-dihydroprydines(DHP-1~DHP-4)were synthesized via a one-pot three-component reaction by condensing aromatic aldehydes,ammonium acetate and ethyl acetoacetate in ethanol which was refluxed for 2-3 h with a yield of 80-98%.Compared with the conventional methods,the main advantages of the present procedure are easier to operate,milder conditions,shorter reaction time,higher yields and more friendly to the human health and the environment.Then the structures of the synthesized compounds have been deduced from infra-red(IR),1H nuclear magnetic resonance(1H NMR)and high resolution mass spectrometer(HRMS).And DHP-1 was selected for single crystal culture and crystal structure detection.The results of X-ray diffraction analysis showed that the six-member ring containing nitrogen atom was nearly planar and the N1–C10 and N1–C16 bonds were significantly shorter than the typical C–N bond which could be rationally explained that the lone-pair electrons in N atoms participate in forming a large conjugated system between the carbonyl group and ethene bond.In addition,theorientation of two carbonyl groups was completely opposite and the six-member ring was almost perpendicular to the benzene ring.DHP-1 exhibited a two-dimensional supra-molecular layer which was stabilized by the N—H…O and C—H…O hydrogen bonds in its packing structure.In the second part of this paper,we analyzed the anti-lung cancer activity of the four synthesized novel 4-aryl-1,4-dihydropyridine derivatives.In the in vitro cell experiments,we firstly investigated the calcium channel antagonistic activity of the newly synthesized 1,4-DHPs compounds.Because 1,4-DHPs are widely used as calcium channel antagonists for the treatment of cardiovascular diseases,the calcium channel antagonistic activity will be a side effect in the antitumor application.So the1,4-DHP with weak calcium channel antagonistic activity will be selected as the research object.The isolated guinea pig ileum longitudinal smooth muscle was treated with compound DHP-1~DHP-4 separately,and the RM6240 B system and tension transducer were exploited to test the contraction changes of ileum longitudinal muscle before and after 1,4-DHP treatment,which was used for examining the effects of various compounds on calcium channel.The results showed that DHP-2 and DHP-4induced the minimal changes in the longitudinal muscle contraction of isolated guinea pig ileum.Thus they were 1,4-DHP compounds with relatively weak calcium channel antagonistic activity.Then we selected five cells containing three kinds of lung cancer cells(H1975,HCC827 and A549)and two lung normal cells(BEAS-2B,MRC-5)to study the cytotoxicity of the four kinds of 1,4-DHP compounds on human lung cancer cell lines by MTT method.By comparison of the IC50 values,DHP-3 and DHP-4 were found to have similar cytotoxicity on human lung cancer cell lines with doxorubicin.But what was different was that the toxicity of these two compounds on normal lung cells was far less than that on lung cancer cells(IC50 value: 5-7 times higher).Thus,DHP-3 and DHP-4 were selectively toxic to lung tumor cells.From the antitumor screening results against three human lung cancer cell lines,some structure activity relationships can be suggested.The strong electron-withdrawing group(Cl,SO2CH3)in the phenyl ring in DHP-3 and DHP-4 favored the antitumor activity;the strong electron-donating group(OH,OCH3)in the phenyl ring in DHP-2 inactive the activity.Subsequently,human lung adenocarcinoma cell line A549 and its resistant strain A549/DDP was selected to study the multidrug resistance reversal activity of1,4-DHPs compounds.The drug resistance property of A549/DDP was verified by MTT firstly.As the drug resistance of A549/DDP cell was mainly mediated by a membrane transporter P-glycoprotein(P-gp),we examined the efflux of P-gp substrate fluorescence dye rhodamine 123(Rh-123)by A549/DDP cells using flow cytometry.The changes of fluorescence intensity when treated with the four 1,4-DHP derivatives were employed to study the inhibitory activity of each compound on P-gp.Similar to the positive control verapamil,DHP-2 and DHP-4 all showed the inhibitory activity to P-gp namely they had MDR reversal activity.However,DHP-3 showed no MDR reversal activity at any concentration which could be related to the existence of a strong electron-withdrawing group Cl on the meta position of aromatic ring at the C4 position of pyridine ring.By summarizing the above results of calcium channel antagonistic activity,MDR reversal activity and tumor cell cytotoxicity detections,we chose DHP-4 with strong anticancer activity and weak calcium channel antagonistic activity for the in vivo study.By using doxorubicin as a positive control,we analyzed the antitumor activity of DHP-4 in a mouse Lewis lung carcinoma model.During the treatment period,the tumor diameters of the treated groups were significantly lower than that of the control group,and the tumor diameter of DHP-4 was significantly smaller than that of doxorubicin.At the end of administration,the tumor weights and tumor tissue H&E staining results also showed that DHP-4 had stronger antitumor effect than doxorubicin.To study the antitumor mechanism of DHP-4,the expression of the representative tumor apoptosis genes Bcl-2 and Bax were selectively detected by immunohistochemistry in tumor tissue.Results showed that after administration,the expression of Bcl-2 in the tumor tissue was significantly decreased,the expression of Bax was significantly increased,and the ratio of Bcl-2/Bax was significantlydecreased.And the effect of DHP-4 was more significant than that of doxorubicin.Thus we concluded that the cytotoxicity of DHP-4 was related to its apoptosis promotion activity via inhibition of Bcl-2 expression and upregulating Bax expression.However,the more precise molecular mechanism of apoptosis induced by DHP-4remained to be further studied.Conclusion In summary,four novel 1,4-DHPs were synthesized by a simple and efficient multi-component one-pot method in this study.DHP-4 was found to have strong tumor specific cytotoxicity and multidrug resistance reversal activity in vitro,and its calcium channel antagonistic activity is weak.The in vivo studies showed DHP-4 had stronger anti-lung cancer activity and weaker toxicity than doxorubicin,and further study found that its antitumor effect was related to the regulation of tumor apoptosis genes.