Study On The Design,Synthesis,Optimization And Biological Activity Of Clofazimine And Its Analogues

Clofazimine is a riminophenazine compound which has anti-mycobacterial,anti-protozoal and anti-inflammatory effects.In recent years,clofazimine has been effective in the treatment of drug-resistant tuberculosis,and can be applied to the prevention and treatment of other diseases such as COVID-19,which has aroused widespread concern all over the world.In this thesis,the synthesis and refining approach of clofazimine were first studied,and a new salt of clofazimine with p-toluenesulfonate was obtained,and its solubility and antibacterial activity were investigated.At the same time,a series of clofazimine analogues were designed and synthesized,and the compounds with the most potential for development were selected by investigating their antibacterial activity.(1)N-(4-chlorophenyl)-2-nitroaniline was synthesized through aromatic nucleophilic substitution reaction using o-fluoronitrobenzene and p-chloroaniline as starting materials.When the molar ratio of o-fluoronitrobenzene,p-chloroaniline and triethylamine was1:1.5:0.5,the yield of N-(4-chlorophenyl)-2-nitroaniline was 96%after solvent-free reaction at 140°C for 10 hours.Reduction reaction was conducted to prepare N-(4-chlorophenyl)benzene-1,2-diamine,the optimal conditions for the reduction approach were that the ratio of N-(4-chlorophenyl)-2-nitroaniline,thiourea dioxide and monoethanolamine was 1:5:9,and the reaction was carried out at 30°C in ethanol and water for 1.3 hours,N-(4-chlorophenyl)benzene-1,2-diamine was obtained in 88%yield.The reducing agent thiourea dioxide could solve the problems of complicated operation and environmental pollution in the original reduction approach.Two molecules of N-(4-chlorophenyl)-1,2-phenylenediamine were cyclized in the presence of ferric chloride,the optimal condition for the oxidative cyclization reaction was that the molar ratio of N-(4-chlorophenyl)benzene-1,2-diamine,hydrochloric acid and ferric chloride was 1:1.1:3,the hydrochloride salt of the cyclic compound was obtained after reacting for 10 hours at room temperature,which was then neutralized with aqueous ammonia to give2-(4-chloranilino)-3,5-dihydro-5-(4-chlorphenyl)-3-iminophenazine in 98%yield.The content of generated isomer is the least,and the phenomena and results of the experiment were basically consistent with the proposed reaction mechanism.2-(4-chloranilino)-3,5-dihydro-5-(4-chlorphenyl)-3-iminophenazine and excess isopropylamine could undergo addition-elimination reaction in the presence of glacial acetic acid,the crude clofazimine could be obtained in 91%yield after reacting at 110°C for 6 hours.(2)The refining process of clofazimine was studied,pure product could be obtained by volatilization and crystallization in a mixed solvent of dichloromethane and methanol(3:1,v/v).Three pure products were analysised by UV-Vis absorption spectra,infrared spectroscopy,thin layer chromatography,loss on drying,potentiometric titration and high performance liquid chromatography,the results showed that the quality of the refined product could meet the standards of the Chinese Pharmacopoeia and the European Pharmacopoeia,the total product yield reached 53%and the liquid phase purity reached more than 99.8%.Three crystal forms of clofazimine were prepared and characterized by PXRD,FTIR and MP.The results of the solubility and stability studies of the three crystal forms showed that three crystal forms all had good solubility in the acetic acid/ammonium acetate buffer solution(p H4.8),form?had the fastest dissolution rate and the largest percentage of dissolution in four buffers.In addition,the three crystal forms could exist stably for at least ten days under the conditions of high temperature(60°C),high humidity(25°C,relative humidity 90%±5%)and light(illuminance 4500lx)without crystal transformation,decomposition and hygroscopicity.Because the crystal form?has the best dissolution performance,it can be selected as the dominant medicinal crystal form for further in-depth research.(3)A new salt of clofazimine with p-toluenesulfonate was prepared.Its solubility was measured to be 2.7763 mg/m L after stirring in 60%ethanol for 24 hours,which was 69.06times that of clofazimine(0.0402 mg/m L).The solid dissolution experiment was carried out in 60%ethanol for 125 minutes.The curve was drawn with time as the abscissa and the dissolved amount as the ordinate.The salt of clofazimine with p-toluenesulfonate basically reached the plateau of the curve at about 80 minutes with the concentration of 0.45 mg/m L,which was 8.7 times that of clofazimine(0.05 mg/m L).Filter paper agar diffusion was used to qualitatively test the diameter of the inhibition zone,the results showed that clofazimine and its p-toluenesulfonate were ineffective against Gram-negative bacteria Escherichia coli,Pseudomonas aeruginosa and the fungus Candida albicans.It had good inhibitory effects on the gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis.When the test concentration is 25?g/m L,the diameters of inhibition zone of clofazimine against two bacteria were 8.39±0.08 mm and 11.15±0.65 mm,respectively;The diameter of inhibition zone of the p-toluenesulfonate of clofazimine against two bacteria were 6.97±0.27 mm and10.96±0.37 mm,respectively.The p-toluenesulfonate of clofazimine exhibited the minimal inhibitory concentrations(MIC)of 25?g/m L against Staphylococcus aureus by agar dilution method,which was slightly less active than clofazimine(MIC=3.1?g/m L);The p-toluenesulfonate of clofazimine exhibited potent activity against Bacillus subtilis with MIC value of 1.65?g/m L,which was equivalent to the activity of clofazimine(MIC=1.56?g/m L).(4)The synthesis of clofazimine analogs and their intermediate products was studied.Thirty-two clofazimine analogues 6-37 were designed and synthesized.The structures of these products were characterized by FTIR,¹H NMR,¹³C NMR and MS.The biological activity of clofazimine analogues 6-37 was studied.Compounds 6,13,14,19,21 and 22 had larger diameters of inhibitory zone,indicating good antibacterial activity.The results of MIC and Clog P showed that compounds 6,13,14,27,33 and 34 had better antibacterial activities,and their MICs against Staphylococcus aureus were 6.25,12.5,100,12.5,3.13 and 1.56?g/m L,respectively;The MICs against Bacillus subtilis were 3.13,6.25,1.56,6.25,3.13 and 25?g/m L,respectively.Five compounds except compound 34 had lower Clog P values than that of CFZ.