Design,Synthesis And Preliminary Antibacterial Activity And Anticancer Activity Of Berberine Derivatives

Berberine,a natural isoquinoline alkaloid,showed a wide range of biological activities.Berberine and its derivatives have been one of the research focuses in the pharmaceutical field.In this paper,novel berberine derivatives were designed,synthesized and their biological activities were studied.The following researches are carried out:Part One:Design,synthesis and antibacterial activity of 9,13-disubstituted berberine derivatives1.Based on the principle of bioisosterism,fifty-four new 9,13-disubstituted berberine derivatives were designed and synthesized for the first time.All the structures were characterized by NMR and HRMS spectra.2.The in vitro antibacterial activities of all berberine derivatives against S.aureus Newman and five multidrug-resistant strains(NRS-1、NRS-70、NRS-100、NRS-108、NRS-271)were evaluated.Most of the 9,13-disubstituted berberine derivatives showed good antibacterial activity against Newman and multidrug-resistant bacteria species,which was significantly better than berberine and better than the positive control kanamycin.Compound with the highest activity against Newman and NRS-1 was comparable to the positive control vancomycin.3.The preliminary structure-activity relationship was discussed:1)Compared with berberine derivatives with mono 9-substituted or 13-substituted,9-butylamino-13-benzyl substituted berberine derivatives showed satisfactory antibacterial activity against S.aureus Newman and five multidrug-resistant strains(NRS-1、NRS-70、NRS-100、NRS-108、NRS-271),which was better than berberine and kanamycin.2)Maintain the 13-position substitution of benzyl,alkylamino group with different chain length was introduced into the 9-position of berberine with different terminal groups such as methyl,phenyl and hydroxyl.The antibacterial activity showed that hydrophilic groups(hydroxyl)decreased the antibacterial potency and lipophilic groups increased the antibacterial potency.Also,antibacterial activity was relative to the length of alkylamino chain,and the best alkyl length is that consisting of five carbon atoms.3)Maintain the 9-position substitution of alkylamino,different substituted benzyl,straight-chain alkyl and heterocycle were introduced into the 13-position of berberine.The antibacterial activity showed that benzyl and straight-chain alkyl substitution were favorable for antibacterial potency,compounds with heterocycle at 13-position lose their inhibition on Staphylococcal growth.4)9-Butyl/pentyl amino-13-(4-methyl/fluoro)benzyl berberine derivatives showed nice inhibitory activity with MIC values of 0.78-3.13 μg/mL against Newman and five multidrug-resistant bacteria species,which was better than berberine and kanamycin.For Newman and NRS-1,9-pentylamino-13-(4-methyl)benzyl berberine showed the highest activity(MIC=0.78-1.56 μg/mL),which is comparable to the positive control vancomycin.Part Two:A series of novel phenylethylamine derivatives were designed and synthesized by splicing active groups.The structures of twenty-two new compounds were characterized by NMR and HRMS spectra;The in vitro antibacterial activities of all phenylethylamine derivatives against S.aureus Newman were evaluated.The unsaturated amide unit in C region and 5-nitrofuran in D region played an important role in improving antibacterial activity.That is,conjugated system was essential to maintain the antibacterial activity of these compounds.Compounds 2-98,2-100 and 2-103 exhibited good antibacterial activity(MIC<3.13 μg/mL).Part Three:Compounds with low MIC-values against Newman or multidrug-resistant strains were tested the cytotoxic activity on human fibroblast(HAF)cell line.Most 9,13-disubstituted berberine derivatives showed cytotoxicity with IC50 11-20 μg/mL,which is lower than positive eontrol totarol(IC50=8.46 μg/mL).Phenylethylamine derivatives 2-98,2-100 and 2-103 did not show obvious toxicity to HAF cells(ICso>50 μg/mL).In addition,compounds 2-23、2-32 and 2-37 were selected to investigate the cellular morphology of HAF.The ceytotoxicity assay demonstrated that these novel 9,13-disubstituted berberine derivatives bear good selectivity for bacterial over mammalian cells.Part Four:In consideration of some new compounds showed toxicity to HAF cells,fourteen representative compounds with low IC50 values against HAF were tested the inhibition proliferation activity against prostate cancer cell line(PC3,DU 145),breast cancer cell line(MDA-MB-2315 MCF-7),and colon cancer cell line(HCT-116,HT-29)using the SRB assay.The result showed that most of 9,13-disubstituted berberine derivatives exhibited moderate inhibitory activity against tumor cells,and the inhibitory activity against PC3 was superior to that of other cancer cell lines.The IC50 values of compounds 2-28 and 2-63 against PC3 were 0.64±0.03 μM and 0.87±0.23 μM,respectively.Part Five:In view of lipophilic substituents had a significant impact on the antitumor activity of 9,13-disubstituted berberine derivatives,seven new 9,13-disubstituted berberine derivatives were designed and synthesized by increasing the length of the alkyl chain in active compounds 2-28 and 2-63.The structures of new compounds were characterized by NMR and HRMS spectra.Their antiproliferative activities against PC3,DU145,MDA-MB-231,MCF-7,HCT-116 and HT-29 cancer cell lines were investigated.The results showed that prolonging the alkyl chain length of 9-substituted or 13-substituted could improve the inhibition ability of compounds against cancer cell lines.Among them,the IC50 values of 9-octylamino-13-(4-i-Pr)benzyl berberine(3-4)and 9-hexylamino-13-octyl berberine(3-8)against PC3 were 0.19±0.01 μM and 0.30±0.08 μM,three times higher than that of 2-28 and 2-63,respectively.Five compounds with low IC50 values against cancer cell lines were tested the cytotoxic activity on HAF cell line.The results showed that compounds with longer alkyl chain exhibited greater cytotoxicity.The selectivity index of the best compound 3-4 was 20.8.Part Six:Compound 3-4 was chosen as a representative compound to investigate the mechanism of PC3 cell death.The characteristics of PC3 death induced by 3-4 included cytoplasmic vacuolation,induction of endoplasmic reticulum stress,induced autophagy and the absence of inhibition by caspase inhibitors,fulfilling the criteria for paraptosis.It was the first time reported that berberine derivatives could induce cancer cell death by paraptosis,mechanism of inhibiting cell proliferation was different from the reported berberine derivatives.In addition,eompound 3-4 could inhibit tumor growth in mice in vivo.These findings would lay a foundation for the drug discovery for the treatment of bacterial,especially multidrag-resistant bacteria infections and cancer.