Study On The Synthesis Of Berberine Homologues And Their Pharmacological Activities

Berberine is an isoquinoline alkaloids isolated from Chinese herbs Coptidis Rhizoma or Cortex Phellodendri,was initially used as anti-microbial agent, anti-diarrhea, anti-inflammation, and anti-cardiovascular ipid-modulating, antihyperglycemic, antihypertension and antitumor diseases. To increase the pharmaceutica and antioxidant activity, some berberine derivative were synthesized, such as 8-alkyl-berberine,8-alkyl-13-bromo-berberine,13-halogenated-berberine,13-alkyl(or benzyl)-berberine,8,13-dialkyl-berberine,8-alkyl-13-benzyl-berberine, phenol- berberine and 8-alkyl- phenol-berberine. Some pharmacological effects of the berberine derivatives were studied including the antimicrobial activity, antihyperglycemic and antioxidant. The methods and results are as follows:1. Synthesis of berberine derivatives1.1 Synthesis of 8-alkyl-berberine,8-alkyl-13-bromo-berberine and 13-halogenated- berberine8-alkyl on the ring C were synthesized according to the pervious methods. All bromination reaction were performed in the similar way. The optimum conditions of synthesis of 8-alkyl-13-bromo-berberine are that the temperature is 30℃, the ratio of Br2 to 8-alkyl-berberine is 6:1,0.5000 g 8-alkyl-berberine is added in 60 mL glacial acetic acid.The order of reactants addition and the reaction temperature were the tow key factors to synthesize these bromo-substituted compounds. The yield of adding Br2 before adding berberine or alkyl-berberine was far better than reverse order. Adding Br2 before adding berberine or alkyl-berberine was far better than reverse order in terms of the yield.. Chlorination of berberine was comparably easy via chlorinating reagent chlorine. The high yield of chloro-berberine can be accomplished in shorter reaction time and at room temperature. However, in order to improve the yield, the following points must be obeyed. Firstly, all the reagent and solvent must be dried due to the stronger oxidizability of chlorine than the other two. Secondly, berberine must be quickly added after chlorine saturation. Lastly, small amount of FeCl3 must be added at the time when the experiment finished. FeCl3 is prone to cause production precipitation, so high-purity production would be easily obtained through recrystallization instead of column chromatography.Iodinations were more difficult to substitution compared to other halogen due to the lower electrophilicity of iodine.So the oxidant must be added for iodination.The substitute of iodination is different according to oxidant.C13 is iodinated in HNO3/I2/AcOH and C11 is iodinated in KI/KIO4/AcOH/H2O, maybe.1.2 Synthesis of 13-alkyl (or benzyl)-berberine,8-octyl-13-alkyl-berberine,8-octyl-13-benzyl-berberineA number of 13-substitued berberine derivatives were thus from berberine in 3steps via dihydroberberine or in 2 steps via 8-acetonyldiydroberberine derivatives.In the first synthetic approach, berberine was reduced with sodium borohydride in pyridine to afford dihydroberberine, followed by enamine alkylation with electrophiles, and subsequent oxidation with NCS(N-chlorosuccinimide) to give the 13-substitued berberine salt derivatives.The target compounds were also prepared with acetone and aqueous sodium hydroxide solution to afford 8-acetonydihydroberberine and subsequent enamine alkylation with electrophiles followed by elimination of acetone to afford the salt derivatives. The synthesis of 8-octyl-13-alkyl-berberine and 8-octyl -13-benzyl-berberine start from 8-octyldiydroberberine.The following steps are according to the first synthetic approach.1.3 Synthesis of phenol-berberine and alky- phenol-berberine derivativesBerberrubine derivatives,2,3-dihydroxyl-berberine derivatives,2,3,9-trihydroxyl-berberine derivatives and 2,3,9,10-tetrahydroxyl-berberine derivatives were prepared with dimethylform amide, phloroglucol/H2SO4, AlCl3/pyridine, BBr3/dichloromethane, respectively.2. Anti-microbial of berberine derivativesMICs of 8-alkyl-berberine,8-alkyl-13-bromo-berberine,13-halogenated-berberine,13-alkyl(or benzyl)-berberine,8,13-dialkyl-berberine,8-alkyl-13-benzyl-berberine homologues against 9 microorganisms were determined by turbidimetric method respectively.8-alkyl-berberine and 13-alkyl-berberine share the same result that the antimicrobial activity increased as the length of aliphatic chain elongated, but decreased gradually when the alkyl chain exceeded 8 carbon atoms. 8-octyl-berberine and 13-octyl-berberine showed the highest antimicrobial activity. But the antimicrobial activity of 8-alkyl-berberine was stronger than conrresponding 13-alkyl-berberine. Halogenation could further increase subtlely the antimicrobial activity of 8-alkyl-berberine and berberine,so the the antimicrobial activity of 8-alkyl-13-bromo-berberine and 13-halogenated-berberine were better than 8-alkyl-berberine and berberine. An interesting finding is that the 8,13-dioctyl-berberine did not showed the highest antimicrobial activity among all compounds, but 13-butyl-8-octyl-berberine.3. Effect of berberine derivatives on a-Amylase and ACEThe effect of 13-halogenated-berberine, berberine and phenol-berberine on a-Amylase were determined by iodine-starch colorimetry method. The result showed that berberine and its derivatives strongly inhibited a-Amylase.2,3,9-trihydroxyl-berberine and 2,3,9,10-tetrahydroxyl-berberine showed the best inhibiting a-Amylase activity among all the synthesized compounds, which was over 5-fold higher than its precursor (berberine). But the derivatives had little influence on ACE.4. Antihyperglycemic of berberine derivativesIn our study the glucose-lowering effect of berberine and its derivative was not due to an increment of cell number. On the contrary, they depressed the growth of HepG2 cells. Chloro-berberine and bromo-berberine were better than berberine in promoting glucose consumption. That did not indicate chloro-berberine and bromo-berberine carried out the mechanism different from berberine, but they cause less toxicity in HepG2 cells. The glucose-lowering effect of 8-octyl-berberine would abolished when its concentration increased from 0.5 to 4μg/ml, just because of its remarkably cytotoxicity. So the inhibition of cell proliferation increased by 8-octyl -berberine might has potentially beneficial anticancer effects because HepG2 cells are immortalized cell lines. So chloro-berberine which is less cytotoxicity might have more glucose-lowering effect in vitro than BBR. Therefore we infered that it might also be a good option in treating hyperglycemic patients with liver impairment.5. Antioxidant of berberine and its phenol-berberine derivativesAntioxidant of berberine and its phenol-berberine derivatives were tested by scavenging free radical, such as DPPH and·OH.2,3,9,10-tetrahydroxyl-berberine showed the best scavenging free radical capacity among all the other phenol-berberine derivatives.The IC50 of 2,3,9,10-tetrahydroxyl-berberine on DPPH was 0.13 mg/mL, but higher than that of BHA. Its IC50 was 0.27 mg/mL on·OH free radical, lower than Vc. These berberine derivatives had little influence on scavenging nitricol, but could block the formation of nitrosamines which was strong carcinogens. Restraining the oxidation of grease of 8-alkyl-2,3,9,10-tetrahydroxyl-berberine derivatives was investigated in the paper. The result showed that 8-alkyl-2,3,9,10-tetrahydroxyl-berberine derivatives could decrease POV and acid value of grease comparing with the placebo group. The activity increased as the length of aliphatic chain elongated and 8-dodecyl-2,3,9,10-tetrahydroxyl- berberine showed the best antioxidative activities on grease.