Design, Synthesis, And Evaluation Of Novel Gemcitabine Derivatives As Anti-tumor Agents

Cancer is one of the most serious diseases threatening human health in the world.Various nucleoside analogue drugs are widely used for the treatment of cancer.Gemcitabine(2′, 2′-difluorodeoxycytidine, d Fd C), a metabolic inhibition of nucleoside analogue, has been approved for the treatment of various solid tumors, such as pancreatic cancer, non-small-cell lung cancer(NSCLC), bladder cancer, and breast cancer. It is mainly through inhibit DNA synthesis related enzymes and prevent cells division and reproduction and induce tumor cells apoptosis. However, there are some drawbacks of gemcitabine in the clinical use, including poor fat-soluble, poor transmembrane, degraded deaminase inactivation, short half-life, low bioavailability,and drug resistance. Hence, based on the above deficiencies in clinical application, it is urgent for researchers to further modify and design novel gemcitabine derivatives.Farnesylthiosalicylic acid(FTA), as a novel Ras inhibitor in clinical trial, which is known that combined medication of FTA and gemcitabinecould increase the tumor cell’s sensitivity to medicine and inhibit the growth of cancer cells. In addition, Ras mutation of tumor cells could be FTA selective recognition, and K-ras oncogene by inhibition of FTA could help improve the effect of gemcitabine in the treatment of cancer diseases with synergistic effect. Furthermore, Nitric oxide(NO) as a signaling and/or effector molecule plays a pivotal role in the numerous physiologic and pathologic processes. It is well-known that high levels of NO generated from NO-donors can not only induce apoptosis and inhibit metastasis of tumor cells, but also sensitize tumor cells to chemotherapy, radiation, and immunotherapy in vitro and in vivo. Based on the above investigations, two different series of gemcitabine derivatives through coupling gemcitabine with FTA or phenylsulfonyl furoxans were designed and synthesized with the promise to enhance anticancer activities, selectively inhibit tumor cell proliferation and induced tumor cell apoptosis with a synergistic effect. The 27 target compounds were purified by column chromatography, and their structures were characterized by IR,mp, 1H NMR, 13 C NMR, and MS.In this paper, the N4-position amino of gemcitabine with other compounds through a hydrolysable amide linkage is one of the most critical. Comparing different experimental methods and the method of active ester is the best reaction. The active ester of 6a-m are prepared from 6a-m and NHS, and then reacted with 8. Not only reaction condition is mild, strong specificity, but also final treatment is relatively simple.In addition, the synthesis of 18 need under protection from dark and N2 are the most critical, in order to avoid the formation of by-products and improve the yield.For gemcitabine/phenylsulfonyl furoxans hybrids 10a-m, their in vitro growth inhibitory assays showed that most compounds displayed strong anti-proliferation activity against five cancer cells, and compound 10e(IC50=1.02-3.27 μmol/L) exhibited comparable to or superior to gemcitabine. The maximum amount of NO released by the most active compound 10 e was about 32.0 μmol/L at 300 min, and treatment with hemoglobin partially reduced cytotoxicities of 10 e in Hep G2 cells. These results suggested that the high concentration of NO release by NO donor moieties of 10 e and the bioactivity of gemcitabine segment may synergistically contribute to the antitumor activity of 10 e in vitro. Furthermore, inhibition of nucleoside transport significantly reduced the inhibitory activity for gemcitabine, but not for 10 e, underlining the independence of a nucleoside transporter. In addition, 10 e could apparently induce cancer cells apoptosis by reducing the levels of Bcl-2 and Parp-1 and up-regulating the expression of Bax and caspase-3.Analysis of SAR revealed that most of 10a-m exhibited remarkable anti-tumor activities against five human cancer cells. Compounds 10a-h with diol linker chain showed relatively higher anti-perliferative activities compared to compounds 10i-m with alcohol amine chain. As for the length of the linker, hybrids(10b-e, 10 j, and 10k)with the linker comprised of three or four carbon units displayed obviously stronger growth inhibitory effects than the other hybrids. In sharp contrast, among tested compounds, the hybrid 10 e with 1, 4-butyl acetylene glycol group had the strongestinhibitory activity, which may come from higher levels of NO release and gemcitabine fragment.