Design,synthesis And Anti-Hepatoma Activity Of Bruceine D Derivatives

Hepatocellular cancer（HCC）is the fifth most common malignancy worldwide.However,the mortality rate of HCC ranks the second among all the cancers in the world,due to lack of effective treatments.At the initial diagnosis,surgical resection and transplantation are considered as potentially curative modality for HCC.Patients with localized unresectable disease,chemotherapy and radiotherapy are often used.Until recently,the RAF inhibitor sorafenib is the only systemic treatment with statistically significant.However,several side effects were often observed concomitantly during sorafenib therapy,such as drug-resistance and tumor rebound and so on.Meanwhile,it brings a heavy burden for patients and their families because of high cost.Therefore,it is of urgent significance to develop novel drugs for the treatment of HCC.In previous study,a natural product Bruceine D was screened from the natural product library,which could specifically kill hepatoma cells.In vitro,Bruceine D could significantly inhibit Huh7 cells proliferation and induce cells apoptosis in a dose-and time-dependent manner.In vivo,xenograft tumor model in mice also proved that Bruceine D could significantly inhibit tumor growth.However,literature report and experimental study demonstrated that it had a strong toxicity and narrow therapeutic window,and this limited its further research.Research objective:In order to reduce the toxicity of Bruceine D,its derivatives were designed and synthesized.We hope to find analogues with lower toxicities and similar or stronger activities compared to Bruceine D.Since the targets of Bruceine D are not clear,we have carried out the structural modification and the anti-HCC research according to the classical methods of medicinal chemistry.Research contents:1.Firstly,the structure-activity relationships（SARs）of Bruceine D about anti-hepatoma activity were studied.To this end,we explored a related chemical reaction of the functional groups of Bruceine D,and the derivatives 2-1?2-14 were synthesized.On the other hand,we found analogues 2-15?2-25 from natural product library.Then,the total 25 analogues of Bruceine D were tested about proliferation inhibition activity against Huh7 and L-02 by CCK-8.Finally,the preliminary SARs were summarized as follows: 1)A ring: ○1E A α,β-unsaturated carbonyl is an essential group for anti-hepatoma activity.When it is reduced or replaced by other groups,the antiproliferative activity disappeared.A○2E A The free hydroxyl group of C-1 is beneficial to the activity,and the proliferation inhibition activity decreases when the ester is formed.2)B ring: the activity disappeared when introduction of acetyl group at C-6.3)C ring: A○1E A The free hydroxyl group of C-14 is essential to the activity,the activity decreased or disappeared when it is lost.A○2E A It lacks cytotoxic selectivity selectivity when introduction of hydroxyl group at C-20.4)D ring: A○1E A D-ring lactone is essential to the activity,and the activity disappeared when the lactone ring-opening or transferred.A○2E A The antiproliferative activity of derivatives of 15-position esterification is substantially comparable to Bruceine D,but the activity is significantly reduced or loss of selectivity when introduction of acetyl or hydroxyl.5)E ring: the furan ring is an active group,and the activity disappears when it opening or introduction of hydroxyl groups at C-19 position.2.Based on the study of SARs,we introduced the alkyl side chains and aryl side chains at the C-15 position of Bruceine D,and synthesized 33 derivatives（3-1?3-33）.At the concentration of 10 μM,Bruceine D and its derivatives were evaluated on the inhibition in Huh7 cells.The derivatives with inhibition rates more than 50% were further evaluated in different HCC cell lines（Huh7,SMMC-7721,Hep G-2,QGY-7703）for their antitumor activities,and evaluated their cytotoxicities in normal liver cells L-02.Finally,the alkyl derivative 3-7 was found better than Bruceine D about the cytotoxic selectivity.In addition,further study demonstrated that 3-7 could induce apoptosis of HCC cells in a dose-dependent manner.3.Based on hybridization principle,furazan NO-donating hybrids（4-13?4-30）were designed and synthesized.By evaluated in different HCC cell lines and normal liver cells in vitro,proliferation inhibition activity of hybrids 4-21 and 4-22 was similar to Bruceine D,but the cytotoxicity of the formers was greatly reduced（IC50 > 100 μM）.The NO releasing ability of the hybrids 4-13?4-30 was determined by Griess assay,which showed 4-21 and 4-22 had a strong ability to release NO.Based on the above results,the hybrids 4-21 and 4-22 were selected for further mechanism study in Huh7 cells.The results proved that 4-21 and 4-22 could induce apoptosis of HCC cells in a dose-dependent manner.4.According to the design principle of prodrugs,we selectively introduced the non-toxic benzoyl and cinnamoyl group into hydroxyl group at C-15 of Bruceine D,getting derivatives 3-20 and 3-25,which were carried out the research of hydrolysis stability and hydrolytic enzyme attribution in vitro.It was found that the two derivatives could selectively release Bruceine D by carboxylesterase 2,realizing the liver targeting function.In vitro on normal liver cells L-02,the toxicity of 3-25 is less than that of 3-20.So we choose 3-25 to do the following biological evaluation.In terms of LD₅₀ from ICR mice acute toxicity experiments,the toxicity of prodrug 3-25(LD₅₀ = 5.9 mg/kg/d)is less than that of Bruceine D(LD₅₀ = 2.8 mg/kg/d).Finally,we evaluated the anti-hepatocarcinoma efficacy of 3-25 and Bruceine D by orthotopic liver cancer transplantation model.At 1.5 mg/kg,they showed considerable efficacy.But due to the lower toxicity of 3-25,the dosage can be increased to 3.0 mg/kg,which is much better than the 1.5 mg/kg dose group.Besides,the results of HE staining and immunohistochemistry showed that 3-25 could effectively promote the apoptosis and inhibit the growth of HCC cells in vivo.ConclusionIn summary,a variety of structural modification strategies were used to reduce the toxicity of Bruceine D.By bio-evaluation,we finally found 4 derivatives（3-7,3-25,4-21 and 4-22）with higher cytotoxic selectivity than that of Bruceine D.As researching deeply,they hold great promise for the development of anti-liver cancer drugs in the future.Overall,the research of this paper provides an important reference value for searching for antihepatoma drugs.Meanwhile,it is practical and instructive for other natural products.