Anticancer Bioactivity And Mechanistic Study Of Novel Sulfur-Nitrogen Heterocyclic Compounds

Cancer, also called malignant tumors or neoplasm, is such a disease that can arise from anywhere of human bodies. One hallmark of cancer is that the abnormal cells divide fast and exceed their normal growth limit to metastasize to other organs. Cancer is an important cause of human death. Among the dead 7.6 million people in 2008, about 13% of them died of cancer. Meantime, about 70% cancer-caused death occurred in undeveloped countries like China (data from IARC). Based on the reports of WTO, the human death number of cancer will continue increasing and achieve 13.10 million in the year of 2030.At present, there are mainly three ways of cancer therapy:operative treatment, radiotherapy and chemical treatment. Among them, the chemical treatment (chemotherapy) can arrive into almost anywhere of human following the blood, which is essential and plays import role in cancer therapy. The existing mechanism of chemotherapy is mainly as following:inhibiting the formation of nucleic acid, for example, fluorouracil; disrupting the DNA, for example, alkylating agents and daunorubicin et.al.; disrupting the microtubule, for example, vincristine and paclitaxel; inhibiting the cell enzyme, for example, etoposide and cytarabine; and hormone drugs, for example, estrogen and androgen et. al.However, there are serious bottlenecks of present chemotherapy drugs:one is that most chemotherapy drugs can cause serious adverse reactions, toxic and side effects, including gastrointestinal reaction, myelosuppression and neurotoxicity and so on. For example, vincristine caused lymphopenia, vomit, numbness and so on. Another one is that the potent chemotherapy drugs are still limited and more and more resisted by multidrug resistant cancers. Therefore, it is urgent to develop novel potent and safe anticancer drugs.According to the literatures, tricyclic compounds and thiazolidinone derivatives are bioactive at several areas, of which many have been applied in clinical and basic research. So we designed and synthesized a series of tricyclic thiazepine derivatives (27 compounds) and a 4-thiazolidinone library (372 compounds), using of active scaffold redevelopment design and combinatorial chemistry two methods, respectively. The detailed contents are described as followed:Tricyclic compounds mainly refer to the drugs with tricyclic frame used as antipsychotic drugs, like tianeptine and quetiapine. There are also several reports demonstrating that tricyclic derivatives are bioactive as anti-HIV agents. A recent report identified tricyclic antidepressants and related molecules as being anticancer reagents. To our knowledge, there is no report on the anti-cancer activity and molecular mechanism of tricyclic thiazepine derivatives so far. Lung cancer is one of diseases with the most morbidity and mortality. About 80% of lung cancer is non-small cell lung cancer. The most important reason of failure of chemotherapy to cancer is the drug resistance, especially in cases of paclitaxel and vincristine these natural chemotherapy drugs. The origin of drug resistance is directly related to one kind of trans-membrane protein-p-glycoprotein (P-gp), which is a 170-kDa protein, pumping out many drugs specifically. So P-gp was also called "drug pump" vividly. Therefore, it is attractive to develop the potent anti-NSCLC drugs without transport by the drug pump, P-gp.Therefore, with the help of Prof. Xu Bai in Jilin University, using active scaffold redevelopment design method, a series of 27 novel tricyclic thiazepine derivatives were designed, synthesized and characterized. After screening their anti-non-small cell lung cancer activity, we found that six hits (Compounds 4,9-12, and 14) were potent and selective against the non-small cell lung cancer cell lines H460 and drug-resistant H460iaxR with both EC50 of<1μM. Then, by in vitro ADME assays, we found that these 6 hits also exhibited suitable drug-like properties for further in vivo experiment. We selected the most active compound 4, named 6-(p-tolyl)benzo[f]pyrido[2,3-b][1,4]thiazepine 11,11-dioxide (TBPT) for further investigation in vivo. The following in vivo experiments indicated that TBPT significantly inhibited the growth of H460 and H460TaxR tumors without obvious side-effects. However, paclitaxel is weakened a lot in H460TaxR in vitro and in vivo, compared with H460, because of the transport of P-gp. TBPT was found that it could avoid the transport of P-gp to exert its bioactivity in resistant lung cancer cells, because it was not a substrate of P-gp. By DNA array, we determined the affected genes and cell processes in TBPT-treated cells. We found that microtubule activity and nuclear metabolism were significantly regulated. By the further experiments, it was proved that TBPT acted as a novel microtubule depolymerizing agent inducing cell cycle arrest and apoptosis. Meantime, TBPT did not disrupt the normal cell cycle and apoptosis state of human fibroblast cells NHFB and mice hippocampus cells. By kinase screening assay, we found TBPT did not inhibit the kinases from a panel of 442 kinases kinome. Therefore, TBPT may not rudely disrupt the normal cell signal pathways mediated by kinases. This may be one of key aspects of the low toxicity of TBPT.Thiazolidone derivatives belong to a kind of alkane of which the frame is a five-membered ring containing sulfur and nitrogen. As previously reported, thiazolidone derivatives is bioactive in many areas, such as antibacterial, anti-virus, anti-diabetic, and anti-inflammation et.al. The clinical drugs of thiazolidone have been compounds used as oral anti-diabetic drugs that contain two carbanyl groups. Latest study reported thiazolidinone is also bioactive against cancers. At present, the anticancer activity of thiazolidinone is more and more attractive. Rhabdomyosarcoma is one kind of malignant tumors, it arises from striated muscle or mesenchyme cells, and it is the most frequent cancer of children soft tissue sarcoma. There are two main subtypes of rhabdomyosarcoma:embryo (ERMS) and alveolar (ARMS). The standard chemotherapy adopted by Children’s Oncology Group and the Intergroup RMS Study Group (IRSG) is the three-drug combination of vincristine, dactinomycin and cyclophosphamide. However, these traditional chemotherapy drugs can lead to serious toxic side effects even cause death, due to their severe toxicity, which limited their clinical application.Therefore, by combinatorial chemistry we designed and synthesized a thiazolidinone library containing 372 compounds with novel structures, previously. Then, by high throughput screening of these compounds for anticancer activity, compound 2-((4-hydroxyphenyl)imino)-5-(3-methoxybenzylidene)thiazolidin-4-one (MHPT), was found bioactive against rhabdomyosarcoma cell lines RD (ERMS) and SJ-RH30 (ARMS) with EC50 of about 0.4μM and 1.4μM, respectively. Meantime, MHPT did not exhibit obvious toxicity to normal human fibroblast cells NHFB with EC50 of over 100μM. Further investigation demonstrated that MHPT inhibited the polymerization of tubulin to disrupt the normal formation of microtubule, leading to G2/M cell cycle arrest and cell apoptosis. The following in vivo experiments indicated that MHPT significantly inhibited the growth of RD tumors at 40 mg/kg and no detectable toxicity was found to nude mice at our experiment conditions. However, the clinical drug vincristine caused serious toxicities, such as body weight loss, blood toxicity, even death when achieving the similar tumor inhibition level of MHPT. Then, we discussed the mechanism of MHPT. We speculate that, as a moderate tubulin polymerization inhibitor, MHPT requires a more dynamic tubulin turn-over to inhibit the cell cycle, and this scenario only exists in cancer cells. Therefore, the cell cycle inhibitory effect of MHPT may be adequate to cause cytotoxicity in cancer cells but not robust enough to induce similar cytotoxicity in normal cells. Taken together, MHPT was found as a potent and selective microtubule inhibitor against rhabdomyosarcoma.In summary, using active scaffold redevelopment design and combinatorial chemistry, we previously synthesized two kinds of sulfur-nitrogen heterocyclic derivatives (tricyclic thiazepine and 4-thiazolidinone derivatives). Then, after screening their anticancer activity, we discovered two potent anticancer agents, the anti-non-small cell lung cancer compound TBPT and anti-rhabdomyosarcoma compound MHPT. Meanwhile, both their toxicity towards normal cells or mice organs are less than the clinical drugs paclitaxel and vincristine. The further mechanism investigation of TBPT and MHPT indicated that they both can inhibit the polymerization of tubulin, cause cancer cell cycle arrest and induce apoptosis.