Design, Synthesis And Anti-tumor Activities Of Novel 3-Aryl Flavone Acetic Acid Derivatives

Tumor vascular disrupting agents (VDAs) have been shown to selectively affect endothelial cells of established tumor blood vessels, resulting in ischemia in the central component of tumor masses with the persistence of a viable layer of cancer cells in the periphery. Flavonoids are one part of tumor vascular-disrupting agents, function by activating the immune system and inducing the release of tumor necrosis factor (TNF-a) to destruct tumor vasculars. A notable example among the flavono id-based VDAs developed to date is 5,6-dimethylxanthenone-4-acetic acid (DMXAA), where the xanthenone moiety can be viewed as a fused flavone. Although phase III trials of DMXAA against lung cancers discontinued because of no survival benefit of DMXAA combination therapy for patients in the late stage of lung cancer, the outcomes have clearly demonstrated that DMXAA is safe and well-tolerated in humans.In previous study on the creation of potent flavono id-based VDAs, a series of 7-methoxy-3-arylflavone-8-acetic acids (AFAA) bearing a methoxy group at the 7-position and diverse subsitiuents on the benzene ring at the 2- and 3-positions of flavone nucleus has been designed and synthesized, using DMXAA and FAA (flavone-8-acetic acid) as lead compounds. Their direct and indirect antiproliferative activities were evaluated. However, the results showed that most compounds showed lower levels of indirect cytotoxicity than DMXAA. The result urged us to investigate the reason. A proposition has been reported that pyrylium-type salts derived from DMXAA and FAA are proposed to play an important mechanistic role in anticancer action. In addition,7-methoxy-3-arylflavone-8-acetic acids may exist in two intramolecular hydrogen bonded forms. Thus, we speculated that the intramolecular hydrogen bond formed between the acetic acid group at the 8-position of flavone nucleus and the oxygen atom of pyrylium ring may be propitious for the formation of pyrylium-type salts, whereas the intramolecular hydrogen bond formed between the methoxy group at the 7-position and the acetic acid group at the 8-position of flavone nucleus may obstruct the formation of pyrylium-type salts.In order to verify our hypothesis and obtain potent flavono id-based VDAs, eighteen 3-arylflavone-8-acetic acids (YFAA01-18) without a methoxy group at the 7-position of flavone nucleus were synthesized in five steps from 3-methyl-2-hydroxy-deoxybenzoins with good yields. Their direct cytotoxicities against human colon adenocarcinoma (HT-29) and non-small-lung adenocarcinoma (A549) cell lines, and indirect antiproliferative activities against A549 cell line in the transwell co-culture system were evaluated, using DMXAA as a control. Meanwhile, the cytotoxicities of 3-arylflavone-8-acetic acids and 7-methoxy-3-arylflavone-8-acetic acids against human immortalised myelogenous leukemia (K-562) and human acute myelocytic leukemia cell lines (Kasumi-1) were also evaluated.The results indicated that most of these compounds bearing electron-withdrawing groups on the benzene rings at the 2- and 3-positions of flavone nucleus, showed comparable direct antiproliferative activities against HT-29 and A549 cells with DMXAA, except for compounds YFAA02 and YFAA05. Nevertheless, all the compounds bearing methoxy groups on the benzene ring at the 2- and 3-positions of flavone nucleus, showed very weak cytotoxicities against HT-29 and A549 cells.In contrast with their direct antiproliferative activities, most of these compounds bearing electron-withdrawing groups on the benzene rings at the 2- or 3-position of flavone nucleus, exhibited lower indirect inhibitory activities against A549 cells than DMXAA at the concentration of 50 μM. Only compounds YFAA05 and YFAA09 showed comparable indirect cytotoxicity with DMXAA. On the contrary, all the compounds bearing methoxy groups on the benzene rings at the 2-or 3-position of flavone-8-acetic acid exhibited higher indirect inhibitory activity against A549 cells than DMXAA at the concentration of 50μM, such as compounds YFAA14-18.To further determine the pharmacological activities of compounds YFAA05, YFAA09 and YFAA14-18,50% inhibiting concentrations (IC50) of these compounds were measured on A549 cells co-cultured with human PBMCs. The results showed that the indirect cytotoxicity of these compounds induced in human PBMCs is substantially higher than their direct cytotoxicity. Moreover, the indirect cytotoxicity of compounds YFAA16 (IC50=38.5μM), YFAA17 (IC50=44.5 μM) and YFAA18 (IC50=35.7 μM) were 2.2,1.9 and 2.4-fold higher than that of DMXAA (IC50=84.4 μM) respectively. In addition, compounds YFAA16, YFAA17 and YFAA18 were found to be able to induce TNF-a production in human peripheral blood mononuclear cells. All 3-arylflavone-8-acetic acids showed higher indirect cytotoxicity than 7-methoxy-3-arylflavone-8-acetic acids (AFAA) bearing the same subsitiuents on the benzene ring at the 2- and 3-positions of flavone nucleus at the concentration of 50 μM. This result indicated that the methoxy group at the 7-position of flavone nucleus decrease the indirect cytotoxicities of 3-arylflavone-8-acetic acids.In addition, the cytotoxicities of 3-arylflavone-8-acetic acids and 7-methoxy-3-aryl-flavone-8-acetic acids against K-562 and Kasumi-1 cells were also evaluated in the meantime, and using DMXAA, FAA, Chrysin and Cytosine arabinoside (Ara-C, Clinical drug) as controls. The results showed that the cytotoxicities of FAA and DMXAA against K-562 and Kasumi-1 cells are very weak. However, compounds YFAA12, YFAA13 and AFAA09 exhibited higher inhibitory activities against K-562 and Kasumi-1 cells than Chrysin. Moreover, compounds YFAA12 and AFAA09 showed comparable cytotoxicities against Kasumi-1, HL-60 and HL-60/ADM cells with Ara-C.Furthermore, we developed a mild and practical approach for the synthesis of 2-hydroxydeoxybenzoins bearing diverse substituents from the microwave-assisted alkali degradation of 3-aryl-4-hydroxycoumarins in water. This approach is characterized by excellent yield, high efficacy and easy manipulation, and thus applicable for the large-scale synthesis of 2-hydroxydeoxybenzoins. Moreover, it reduces the use of organic solvents, minimizes the formation of waste, and improves energy consumption. In addition, we have also revisited the intramolecular claisen condensation reaction of methyl 2-(2-arylacetoxy) benzoates as key starting materials for the synthesis of 2-hydroxydeoxybenzoins, and found that cesium carbonate/acetone at room temperature is an optimized reaction condition with excellent yields, little hydrolysis and simple workup procedures.