Design,Synthesis And Anticancer Activity Evaluation Of Novel 2-Acylaminothiophene-3-Carboxamide And Coumestan Derivatives

Focusing on the discovery of anti-cancer drugs,this dissertation carried out three parts of research work.Part one.Structural modification and preliminary study on anti-cancer activity of 2-acylaminothiophene-3-carboxamide compound LGH00278As one of the indicators of mitochondrial function,mitochondrial membrane potential(MMP),involved in the cytochrome c release of apoptosis,Caspase 3 cleavage,Bcl-2 family of proteins regulation and so on,is a potential target for the discovery of anti-cancer drugs.In the Part A of this dissertation,structural modification and preliminary study on anti-cancer activity of 2-acylaminothiophene-3-carboxamide compound LGH00278,which was obtained by high-throughput screening and had the potential of reducing the MMP of L6 myocytes,were carried out to obtain a new group of compounds with a strong effect on reducing the MMP of L6 myocytes and to explore the preliminary structure-activity relationship(SAR).The new compounds with strong reducing effect on MMP of L6 myocytes were selected and their anti-cancer activities were tested.And then a further modification of the optimized lead compound was carried out and the structure-activity relationship was studied.Furthermore,the anti-cancer mechanism of this kind of compounds was preliminarily investigated.The main results were listed as follows:1.Forty novel 2-acylaminothiophene-3-carboxamide compounds were designed and synthesized on the basis of the analysis of LGH00278,and all their structures were characterized by NMR and HRMS spectra.2.The effects of all the new compounds on the MMP of L6 myocytes were tested,and the preliminary SAR was studied.The presence of the amide bond and hydrogen atom on the amide bond in area A of the structure of LGH00278 is beneficial to decrease the MMP of L6 myocytes;if the benzene ring is replaced by an aliphatic chain,the effect of MMP decrease is lost;the introduction of electron withdrawing group to the benzene ring is favorable to the MMP decrease;the position of the substituents on the benzene ring has little effect on reducing the MMP of L6 myocytes.When a six-member ring or five-member ring fused at 4,5-position of thienyl part in area B of the structure of LGH00278,their effect of reducing the MMP of L6 myocytes are equivalent;however,the compound with 4,5-dimethyl substitution or 4,5-unsubstitution on thienyl part lost their activity.In area C of the structure of LGH00278,the presence of hydrogen atom in N1 block of the amide bond,which attached to thiophene ring,is beneficial to the reducing of MMP of L6 myocytes;The modification of N2 block of the amide bond has a significant effect on the MMP regulation of L6 myocytes.The de-oxo or de-amides of the structure made the compound lose their potential on MMP decrease of L6 myocytes;When the compound in area C is substituted with a chloroacetamido group,the MMP of L6 myocytes was decreased and the effective concentration was lower than that of LGH00278,the effect was higher than that of LGH00278 at high concentration.Also,it showed significant concentration gradient relations.3.Thirty compounds,which can significantly reduce MMP of L6 myocytes,were selected to test the inhibitory activity of gastric cancer cell line MGC-803.The results showed that all the compounds with different substitutions in the area A and B of LGH00278 showed some inhibitory activities against MGC-803 with IC50=10-20?M.When modified the area C of LGH00278 with chloroacetamido group,the inhibitory activity against MGC-803 was significantly increased,and the structural modification in area A of LGH00278 obviously influenced the activity.Among all the compounds with chloroacetamido group in area C of LGH00278,2-66 with benzylaminoformyl substitution in area A inhibited MGC-803 with IC50?2.53±0.1?M.4.With compound 2-66 as a lead compound,further modification in its area A,B and C was carried out,and the inhibitory activity against MGC-803 and HCT-116 was investigated.A preliminary SAR was summarized as follows:The structural modification in area A of 2-66 and its inhibitory activity against MGC-803 and HCT-116 cells showed that the compound with a five-member ring fused at 4,5-position of thiophene part was a dominant structure.When the length of the chloroacetyl carbon chain was extended or substitution of the terminal chain with dichloro or vinyl group in area B,the inhibitory activities against MGC-803 and HCT-116 cells were decreased;The inhibitory activities were almost lost when the a-position of acyl group or the terminal chain were substituted with trichloroacetyl,carboxyl or hydroxymethyl group,respectively.When the carbon chain between the amide nitrogen and benzene ring was extended or the benzene ring was substituted in area C of 2-66,the inhibitory activities against MGC-803 and HCT-116 cells were substantially maintained.The results indicated that the area C of 2-66 may be a bioactivity insensitive region;Altering the electronegativity and position of the substituents on aromatic ring have little effect upon the activity of the compounds.5.Selected 2-74b and 2-74d,which showed good inhibitory activity against MGC-803 and HCT-116 cells,as representative compounds,the anti-cancer mechanism was preliminarily investigated.The results showed that 2-74b and 2-74d could decrease the MMP of HCT-116 cells,and the inhibitory activity was achieved by apoptosis involved Caspase 3 activation.In addition,2-74b and 2-74d could promote glucose consumption,but did not promote oxygen consumption which indicated that 2-74b and 2-74d were not mitochondrial uncouplers.Subsequent mechanisms research is ongoing.Part two.Structural modification of 2-benzamido-thiophene[2,3-d]pyrimidin-4-ones and preliminary study of their anti-cancer activityDuring the preparation of mitochondrial uncoupler 2-benzoylguanidothiophene-3-carboxylic acid,a ring-closing byproduct 2-benzamido-thiophene[2,3-d]pyrimidin-4-one was conveniently obtained,which has a significant effect on MMP reduction.Through the structural modification and anti-cancer activity test,the following results were obtained:1.Eighteen novel 2-benzamido-thiophene[2,3-d]pyrimidin-4-one compounds were designed and synthesized,and their structures were characterized by NMR and HRMS spectra.2.The inhibitory activity against leukemia cell line NB4 and its resistant cell line NB4-R1 and colon cancer cell line HCT-116 of all the compounds were tested.The results were showed as below:(1)The type and position of the substituents on the 2-benzamidobenzene ring had a significant effect on the inhibitory activity of the compounds:When substituted with 3,4-dichloro on the benzene ring(3-3c),the compounds exhibited significant inhibitory activity against NB4 and NB4-R1 with IC50=0.18±0.07 ?M and 0.18 ± 0.01 ?M;When substituted with 3-fluoro on the benzene ring(3-3a),the compound exhibited significant inhibitory activity against NB4 and NB4-R1 with IC50=0.99± 0.04 ?M and 0.78±0.05 ?M;When the substitution position of chlorine or fluorine is changed or substituted with other groups,such as 3,4,5-trifluoro group,2,3-dimethoxy and 2,4,5-trimethoxy group,no significant inhibitory activity was presented.(2)The alteration at 4,5-position of thiophene ring also had a significant effect:5-isopropyl substituted compound(3-9c)showed good inhibitory activity against NB4,NB4-R1 and HCT-116 with IC50=0.64 ± 0.01 ?M,0.48 ± 0.03 ?M and 0.29 ± 0.08?M,respectively;Increasing the steric hindrance of the substituents is unfavorable to the activity;The compound with seven-member ring fused at 4,5-position of thiophene showed better inhibitory activity against NB4 and NB4-R1,with IC50=0.33 ± 0.01 ?M and 0.69±0.02 ?M,respectively,while compared to the compounds with fused six-member or five-member ring;The inhibitory activities of the 4,5-unsubstituted or heterocyclic substituted compounds were almost lost;(3)The inhibitory activity was lost when the pyrimidine ring of the compound was replaced by oxazine unit.(4)In view of the good inhibitory activity of compound 3-3a against NB4 and NB4-R1,the inhibitory activity of 3-3a against NB4-R2 was also investigated.The results showed that the compound also had a good inhibitory activity against NB4-R2.3.Compound 3-3a was safe and less toxic to HAF and NCM460,but was toxic to L02 at a certain degree.4.The effects of compound 3-3a on the cell differentiation of NB4,NB4-R1 and NB4-R2 were investigated.The results showed that 3-3a could significantly inhibit the proliferation of NB4 cells Tumor growth.The anti-tumor mechanism of compound 3-3a was investigated:compound 3-3a significantly promoted the cell differentiation of NB4,NB4-R1 and NB4-R2.Mice in vivo experiments showed that 3-3a could significantly inhibit the growth of APL.Further molecular mechanism is ongoing.Part three.A new synthetic method for constructing Coumestans,synthesis of new Coumestan derivatives and preliminary study on their inhibitory activity1.Using 2,3-bis(2-methoxyphenyl)-3-oxo-propionaldehyde as the starting material,the "one pot" method of constructing Coumestans via the demethylation,intramolecular ring-closing,oxidation reactions was developed.The method was easy to obtain starting materials,mild reaction conditions,good substrate stability and good yield(the highest yield up to 90%).The reaction process was explored by means of variable-temperature NMR and related experimental methods,and the possible reaction mechanism was suggested.2.On the basis of the new synthethic method,mono-or bis-methoxyphenyl substituted Coumestans were obtained by Suzuki coupling of 3-or/and 9-position bromo substituted Coumestans with mono-or/and bis-methoxy-substituted phenylboronic acid.Finally,new Coumestan derivatives containing mono-/dihydroxyphenyl were successfully synthesized by demethylation reaction.3.The preliminary inhibitory activity test of the novel compounds showed that some of the Coumestan derivatives,which containing mono-or di-hydroxyphenyl groups,has good inhibitory activity against leukemia cell line NB4 and its resistant strain NB4-R1 and colon cancer cell line HCT-116.The IC50 values of 4-5b against NB4 and NB4-R1 were 0.57±0.01 ?M and 1.24±0.01 ?M,respectively.The investigation in this part enriched the method of Coumestans construction,developed new Coumestan derivatives,and also provided novel hit compounds for anti-cancer drug discovery.