Design,Synthesis And Biological Evaluation Of P-Aminosalicylic Acid Derivatives

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that can cause death in humans,and the mortality rate is second only to AIDS（HIV）.Although there are more than 10 anti-TB drugs,there are still more than 10 million new TB patients in the world in 2018.TB is still a major infectious disease that threatens human health.For drug-sensitive tuberculosis,WHO recommends the use of four first-line drugs（rifampicin,isoniazid,pyrazinamide,and ethambutol）for a 6-month combination treatment plan,and the cure rate can reach more than 85%,but the Therapy is accompanied by shortcomings such as long treatment cycle,poor patient compliance,and toxic and side effects.Therefore,research on new drugs to improve the quality of TB treatment and shorten the treatment cycle is still a challenge for drug workers.Although combination medicine is currently the most effective and economical treatment for tuberculosis,patients with combination treatment schemes take many types of drugs and have long treatment cycles,resulting in poor patient compliance.Inspired by the idea of combination medicine,we envision conjugating two or three anti-tuberculosis drugs in one molecule,hoping to obtain a multi-target high-activity single molecule,so as to replace two or three drugs with a single drug,which is convenient for patients Taking medicine to improve patient compliance.Based on the multi-target drug design ideas,exploratory research can design multi-component linked molecules.The authors found that the anti-tuberculosis drug p-aminosalicylic acid（PAS）contains three functional groups and can be connected to multiple other molecular fragments;if the PAS is used as the parent structure to design the connected multi-fragment molecule,it is both a linker（Linker）and a An anti-tuberculosis drug,the molecule designed by it can reduce the molecular weight,and under the advantage of reducing the difficulty of synthesis,it may also improve the drugability of the entire molecule.Therefore,it is determined to use PAS as the parent skeleton to design a multi-component connected molecule.Borrowing many years of drug research experience in this laboratory,this study selected fluoroquinolone（FQs）,the core therapeutic drug for drug-resistant tuberculosis,as an effective fragment of the target molecule;at the same time,it selected azoles and bis Hydroartemisinin（DHA）is another effective fragment.Using the principle of drug design,we spliced the drug effect fragments A（paraaminosalicylic acid PAS）,B（azoles,fluoroquinolone FQs）and C（dihydroartemisinin DHA,cycloserine L-Cys）through Linker,Form target molecules in two modes,A-linker-B and C-linker-A-linker-B;design target molecules after Linker selection;calculate physical parameters of each designed target molecule;select the target molecule by the"five rules"of oral drug preparation;explore experimental conditions and synthesize target molecules;conduct biological activity screening to obtain a variety of activity data.Through this research,we hope to obtain new biological activities or multi-target compounds with better biological activities than existing ones,provide new ideas for the research of new anti-tuberculosis drugs,and lay the foundation for the follow-up research of lead molecules.According to the aforementioned design idea,the author uses PAS as the skeleton structure,selects chloroacetyl chloride,succinic anhydride or 3-bromopropanol as the Linker reagent,and connects FQs,DHA,L-Cys and azoles according to the aforementioned target molecular model.14 series of 181 multi-segment target molecules were designed;the Plexus software of Eli Lilly and Company was used to calculate the physicochemical parameters of the compound,and the target molecules with good drug-like properties were selected from them.Finally,8 series（TM1-TM8）of 86 target molecules were obtained.Target molecules;optimized reaction conditions were used to achieve the complete synthesis of the designed molecules;the structure of the synthesized compounds was confirmed by ¹H NMR,¹³C NMR and HR MS.Based on the multi-target drug design ideas and the biological activity of the molecular fragments,the authors carried out the target compound against wild-type Mycobacterium smegmatis,common pathogenic bacteria（Staphylococcus aureus,E.coli,Micrococcus tenella,Pseudomonas aeruginosa Bacteria,Salmonella,Acinetobacter baumannii）,fungi（Pichia pastoris）,tumor cells（colon cancer cell HCT116,colon cancer cell SW620 and prostate cancer cell PC3）,plant pathogens（Cletotrichum gloeosporioides strain Co.3,the activity test of citrus brown spot pathogen Alternaria alternata strain Al.6 and citrus canker pathogen Xanthomonas axonopodis.pv.citri strain Xac）.（1）Antituberculosis activityThe MIC value was determined by the liquid method,and the target compound and intermediate were subjected to the MIC test to inhibit the activity of wild-type M.smegmatis.The results show that there are 4 compounds in the tested target molecule（TM2-1、TM2-4、TM5-1 and TM5-4）whose MIC value is between 0.78-6.25μg/m L,and the inhibitory activity is stronger than the positive control isoniazid and rifampicin;at the same time,3 intermediates were found MIC value is between 0.19-0.38μg/m L,the activity is better than the target compound,second only to moxifloxacin（MIC=0.156μg/m L）.（2）Antibacterial biological activityThe microbial dilution method was used to test the antibacterial activity of 86target molecules and some intermediates.The results show that 86 target compounds have a certain inhibitory activity on the 6 strains of bacteria as a whole;the inhibitory effect on Staphylococcus aureus is the best,the MIC value of 11 molecules is less than 1μg/m L,and the MIC value of 7 molecules 0.2μg/m L or 0.4μg/m L,lower than or equivalent to the positive control clinfloxacin（MIC=0.4μg/m L）,much lower than the positive control norfloxacin（MIC=12.8μg/m L）;those containing FQs fragments Target molecule,its activity is stronger than the target molecule containing azole fragments;linker with acetyl group is stronger than succinoyl group（TM2>TM3,TM5>TM6）;when fragment B is FQs,the activity of PAS methyl ester derivatives Stronger than its ethyl ester derivatives（TM2>TM5,TM3>TM6）,if it is azole,then PAS ethyl ester derivatives are stronger than methyl ester derivatives（TM4>TM1）;intermediates（IM2 and IM2’）Has a certain inhibitory effect on some strains,and IM2’has a MIC value of Salmonella as low as 0.8μg/m L,second only to the positive control clinfloxacin（MIC=0.4μg/m L）.The preliminary structure-activity analysis showed that the antibacterial activity of the compound was not only related to the pharmacophore linked to the B and C fragments,but also to the type of Linker:1)The B fragment was a structure of clinfloxacin,baloxacin and gatifloxacin When the unit is used,the compound’s antibacterial activity is generally better;2)the compound whose Linker is acetyl is better than the compound whose linker is succinyl;3)The activity of TM4 series is generally poor,and the C segment is introduced on this basis,namely TM7,the antibacterial activity has been improved.（3）Antifungal biological activityThe agar dilution method was used to determine the antifungal activity of target compounds and some intermediates.The test results show that the bacteriostatic activity of 30 molecules（MIC≤4μg/m L）is better or equivalent than the positive control fluconazole（MIC=4μg/m L）after 24 hours of cultivation;PAS ethyl ester derivatives are more active than their methyl ester derivatives.（4）Antitumor activityThe TM1 and TM4 series of target molecules and some intermediates were tested to inhibit the activity of tumor cells.The results showed that:1)against the colon cancer cell HCT116,the compounds TM1-2,TM4-6,IM2 and IM2’had an inhibition rate exceeding 50%,and the intermediate IM2’had the highest inhibition rate（about 60%）;2)targeting Colon cancer cell SW620,the inhibition activity is not good enough,the inhibition rate is around 25%;3)For prostate cancer cell PC3,the inhibition rate of compounds TM4-3,TM4-4 and IM2’is higher than 50%,and the intermediate IM2’reaches 75%.（5）Activity against plant pathogensThe inhibition rate of plant fungi（Colletotrichum gloeosporioides strain Co.3 and citrus brown spot pathogen Alternaria alternata strain Al.6）were determined for TM1,TM4 and TM7 series of target molecules to determine the strength of the antibacterial activity.The results of preliminary screening showed that:1)Compounds TM1-5,TM4-10,TM7-4,TM7-5,TM7-12,and TM7-14 inhibited Col.trictotrichum gloeosporioides strain Co.3 with an inhibition rate greater than 40%;When the test concentration is 4μg/m L,the inhibition rate of TM7-12 is as high as 53.33%,close to the positive control prochloraz（60.00%）;2)Intermediate IM2’and the target compounds TM1-2 and TM7-2 on citrus The inhibition rate of brown spot pathogen was better than that of other compounds,especially the inhibition rate of intermediate IM2’at 4μg/m L（64.29%）was higher than that of positive control prochloraz（50.00%）.The rescreening results show that TM7-4,TM7-5 and TM7-14 are prone to drug resistance,and it is recommended not to consider subsequent development.Preliminary structure-activity analysis showed that:1)the activity of PAS ethyl ester derivatives is better than that of methyl ester derivatives;2)The introduction of DHA improves the activity of compound（TM7）,which provides a reference for later derivatization and modification.86 target compounds and some intermediates were tested for the inhibitory activity of citrus canker pathogens Xanthomonas axonopodis.pv.citri strain Xac.Preliminary screening results show that the target compound has no gratifying inhibitory activity against citrus canker,which may have a certain relationship with the structure of the compound,proving that the compound we synthesized has a certain selectivity.In this project,a total of 15 intermediates and 86 target compounds were synthesized,all of which were new compounds;4 target compounds and 3 intermediates showed good anti-tuberculosis activity against wild-type M.smegmatis,some The activity of the molecule is stronger than that of some positive controls;some molecules show good antibacterial activity in vitro against Staphylococcus aureus;about half of the target molecules have the same inhibitory activity against Pichia pastoris strains as fluconazole;a few molecules have certain tumors Cells have good inhibitory activity;some molecules have the same inhibitory activity against plant fungi as prochloraz,but the activity against citrus pathogens is very poor.The physical parameters of all synthesized target compounds are calculated,so the highly active molecules obtained in this study can be further studied.This subject is the initial attempt of this laboratory to conduct molecular hybridization of multiple active fragments of anti-tuberculosis drugs based on the idea of drug hybridization,which lays a good foundation for subsequent design and research.