Anti-tumor Activity Of Gemcitabine Oral Derivative SL-01and Indazole Compound Based Diarylurea Derivatives

BackgroundsCancer remains a serious threat to mankind. Besides surgery, chemotherapy is still the major treatment for cancer. Currently, cytotoxic drugs are widely used chemotherapy drugs in clinic. In addition, targeted drugs such as small molecule inhibitors have been focused in recent years. This will lead the development trends of chemotherapy currently and in future for a long time. Nonetheless, toxicity and drug resistance are considered to be the main obstacles for successful chemotherapy. In order to overcome these disadvantages, we developed prodrugs for cytotoxic agent gemcitabine and made structure modification for small molecule targeted inhibitor sorafenib.Gemcitabine, showing effective against a variety of cancers, has played a prominent role in cancer treatment. However, gemcitabine is rapidly inactivated in plasma and liver. Serious toxcities and drug resistance also limited its use in clinic. The introduction of prodrug moieties at the N4-position of the cytidine ring to form a hydrolysable amide linkage would improve its oral bioavailability, decrease its susceptibility to deamination, increase metabolism stability, maintain the active concentration for a longer time, and reduce the toxicity. SL-01, dodecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) carbamoyl) pyrazine-2-carboxylate, was selected from these compounds. Our goal in this study in Part1was to evaluate the efficacy of SL-01on the growth of human cancers.Sorafenib, the first oral multi-kinase inhibitor, is a successful example of diarylurea derivatives. However, concomitant toxicities and drug resistance cannot be ignored in clinic. A new series of diarylurea compounds containing indazole or azaindazole moiety based on the structural features of sorafenib was synthesized in our group. These compounds showed good activiy in preliminary test. In Part2,15 diarylurea compounds were identified for their anticancer activity. The anticancer activity of compound1082-39(N-(4-(1-(4-bromoindazole))phenyl)-N’-(4-Chloro-3-trifluoromethylphenyl)urea), one of indazole diarylurea compounds, was evaluated in human melanoma cell M21and its mechanism underlying were also investigated.Part1Anti-tumor activity of SL-01, an oral derivative of gemcitabineObjective:Our goal in this study was to evaluate the efficacy of SL-01on the growth of human cancers and mechnasim underlying with gemcitabine as control.Methods:Experiments were performed on human non-small cell lung cancer NCI-H460and colon cancer HCT-116both in vitro and in vivo. In vitro assays, the anti-proliferative activity was determined by the MTT assay and the apoptosis induction was estimated with Hoechst33258staining and Annexin V-FITC and propidium iodide (PI) staining analysis by using flow cytometry. In vivo experiments, the anti-tumor effect of SL01was examined by NCI-H460and HCT-116xenografts bearing in mice. The TUNEL assay was used to evaluate the efficacy of apoptosis induction in tumor tissues. Further examination with western blotting analysis was employed to determine the levels of apoptosis related proteins including caspase-9, caspase3, PARP, Bax and Bcl-2.Results:After incubation for24,48and72h, SL-01(0.156-20μM) significantly inhibited the growth of NCI-H460and HCT-116cells in a dose-and time-dependent manner. The inhibitory potency is similar to that of gemcitabine. Both SL-01and gemcitabine (8μM) were found to potently induce NCI-H460and HCT-116cells to apoptosis, showing cell shrinkage and membrane integrity loss or deformation, nuclear fragmentation and chromatin compaction determined by Hochest33258staining. Annexin V-FITC/PI double staining showed that the apoptosis rates of NCI-H460and HCT-116were39.6%and41.4%after treatment with8μM SL-01for24h, which were higher than that of gemcitabine at the same dose (27.8%for NCI-H460and24.6%for HCT-116). In vivo, oral administration with SL-01once every three days for3weeks effectively delayed the growth of NCI-H460and HCT-116without significant loss of body weight in mice. At dosages of10,20and30μmol/kg of SL-01, the inhibition rates on growth of NCI-H460and HCT-116xenografts were31.8%,43.1%,58.0%and36.4%,45.8%,63.1%, respectively. In comparison, injection30μmol/kg gemcitabine once every three days inhibited the growth of NCI-H460and HCT-116xenografts by41.5%and46.9%, with a significant reduction in body weight. TUNEL staining showed that positive staining cells were increased in xenografts treated with SL-01. The percentages of apoptotic cells induced by10,20and30μmol/kg of SL-01were25.6%,41.7%,54.3%for NCI-H460and20.7%,33.6%.47.7%for HCT-116, respectively. While, gemcitabine (30μmol/kg) induced TUNEL staining positive cells in NCI-H460and HCT-116xenografts by37.2%and31.2%, respectively. Western blotting assay showed that SL-01induced activation of caspase-9, caspase-3and cleaved PARP, as well as increased Bax/Bcl-2ratio in cancer cells. These biological activities of SL-01were more potential than that of gemcitabine.Conclusion:SL-01showed similar in vitro but superior in vivo inhibitory effect to gemcitabine. The higher potency of SL-01in vivo was associated with its better ability in induction of caspase dependent apoptosis.Part2Anti-tumor activity study on indazole based diarylurea derivativesChapter1Identification of indazole based diarylurea derivatives for their in vitro anti-tumor activityObjective:15indazole diarylurea compounds were identified for their anticancer activity. The effects of the selected5diarylurea compounds on antiangiogenesis and anti-proliferation against human melanoma cell M21were further investigated.Methods:The antiproliferative activities of15indazole diarylurea compounds against tumor cells MDA-MB-231, PLC/PRF/5, HCT-116,786-0, NCI-H460, and compounds1121-39,1122-10,1122-38,1122-37,1082-39against M21cells and human umbilical vein endothelial cells HUVEC and EA.hy926cells, were determined by the MTT assay. The scratch assay was employed to investigate the effects of1121-39,1122-38and1082-39on the migaration ability of HUVECs. The effects of1121-39,1122-10,1122-38,1122-37and1082-39on angiogenesis were evaluated by using the model of zebrafish embryo.Results:MTT assay showed that most of these diarylureas derivatives, except1121-43,1122-26and1121-40, showed lower IC50s than sorafenib in MDA-MB-231, PLC/PRF/5, HCT-116, and NCI-H460cells. But in786-0cells, only IC50s for1122-38,1082-39,1122-37,1104-40and1106-78were lower than that of sorafenib. IC50s for these5selected compounds1121-39,1122-10,1122-38,1122-37and1082-39against EA.hy926cells were lower than that of sorafenib. Only1121-39significantly inhibited the proliferation of HUVEC cells with IC50at4.81±1.97μM. In M21cell, IC50s forl122-38and1082-39were lower than that of sorafenib.1122-10and1122-37exihibited similar IC50S to sorafenib.1121-39showed no effect on proliferation of M21. Exposure at5μM for24h,1121-39,1122-38and1082-39inhibited the migration of HUVEC by23.16%,41.32%and45.63%, respectively, as estimated by scratch assay. Their effects were much lower than sorafenib with the rate of71.85%. Results from the model of Zebrafish embryo showed that these compounds markedly inhibited the formation of intersegmental blood vessel in Zebrafish embryo, leading to blakage of blood flow and pericardium edema. However, compounds1121-39,1122-10,1122-38,1122-37and1082-39showed no effect on the formation of intersegmental blood vessel in zebrafish embryo.Conclusion:Indazole diarylurea compounds1121-39,1122-10,1122-38,1122-37and1082-39showed more potential anti-proliferative activity than sorafenib against most tumor cell lines in vitro. These compounds exhibited no effect on the angiogenesis of Zebrafish embryo.Chapter2Anti-tumor activity in vitro and mechanism study of1082-39Objective:To evaluate the efficacy of1082-39on proliferation of human melanoma cell M21and mechnasim underlying with sorafenib as control.Methods:After treatment with1082-39or sorafenib for24,48,72h, the IC50s were determined by MTT assay. Annexin V-FITC and PI staining was exploited to assess the apoptosis rates induced by1082-39in M21cell. After JC-1staining, the change of M21cell mitochondria membrane potential was observed under fluorescence microscope. The expression of c-Kit in M21cells; the effect of1082-39on the expressions of proliferating cell nuclear antigen PCNA; apoptosis related proteins including caspase-9, caspase3, PARP, Bax, Bcl-2, Mcl-1; Akt and Akt signaling pathway related proteins such as NF-κB, mTOR, GSK3β, c-Myc, surviving, PI3K, EGFR, Wnt2, as well as the redistribution of cytochrome c and the nucleal translocation of β-catenin were evaluated by using western blotting assay.Results:MTT showed that IC50S for1082-39against M21cells were lower than that of sorafenib at indicated exposure time, especially72h. Annexin V-FITC and PI staining indicated that1082-39effectively induced M21cells to apoptosis. The rate of apoptosis caused by48h treatment with5μM of1082-39was40.7%, which was higher than that of the same dose of sorafenib with the rate at25.3%. After treatment with0,0.625,1.25,2.5,5μM of1082-39for48h, the red fluorescence of JC-1was gradually decreased and the green fluorescence was correspondingly increased. The ratios of green to red fluorescence were increased in a dose-dependent manner to the maximum at2.71±0.2, which was a little higher than that of sorafenib (2.13±0.6).1082-39was found to down-regulate the expression of PCNA in M21cells more potential than sorafenib as determine by western blotting assay.1082-39significantly increased the expression of Bax and slightly decreased the expression of Mcl-1. Sorafenib markedly decreased the expression of Mcl-1with weak effect on Bax and Bcl-2. Both1082-39and sorafenib could increase the ratio of Bax/Bcl-2and Bax/Mcl-1, leading to redistribution of cytochrome c.1082-39significantly induced the activation of caspase-9, caspase-3and cleaved PARR However, sorafenib showed less effct on these proteins. Western blotting results also showed that there was no expression of c-Kit in M21cells. But the phosphorylation of Akt, and the expression or activation of its upstream proteins PI3K, EGFR, Wnt2. as well as downstream proteins NF-κB, mTOR, GSK3β,c-Myc, survivin, were inhibited by1082-39and sorafenib. These biological activities of1082-39were more potential than that of sorafenib.Conclusion:1082-39showed better anti-proliferative and pro-apoptotic activiy than sorafenib in M21cells. The mechnasim underlying was associated with its activity in regulation of Akt signaling pathway.