**2. Results**

#### *2.1. Anti-Proliferative Activity of* **1**–**4** *In Vitro*

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [20,21] was used to evaluated the anti-proliferative activities of **1**–**4** against the human epidermoid carcinoma cell lines A431 and human NSCLC cell lines A549. Cells were cultured with indicated concentrations (250, 200, 100, and 25 μM) of **1**–**4** or the reference compound gefitinib (an epidermal growth factor receptor inhibitor) for 72 h, and living cells were detected by MTT assay. The results are shown in Figure 2. When against A549 cells, compared with the control cells, significant growth inhibitor effect could be observed when cells were treated with 200 μM of **1**, 200, 100, and 50 μM of **3**, 250 μM of **4**, and 250, 200, 100, and 50 μM of gefitinib (Figure 2A). When against A431 cells, compared with the control cells, significant growth inhibitor effect could be observed when cells were treated by 250 μM of **1**, 200 μM of **2**, and 250, 200, 100, and 50 μM of **4** and gefitinib (Figure 2B). The results are further shown in Figure 2C and D, when A549 cells were treated with 250 μM of **4** (multifloroside) or 25 μM of gefitinib, cell viabilities decreased markedly to 30.30% and 70.85% compared with the control group, respectively (*p* < 0.001), when A431 cells were treated with 250, 200, 100, 50, and 25 μM of **4** (multifloroside) or 25 μM of gefitinib, cell viabilities decreased markedly to 7.21%, 12.44%, 70.29%, 75.87%, 84.62%, and 34.02% compared with the control group, respectively (*p* < 0.001), and the inhibitory effect was concentration-dependent. The above results sugges<sup>t</sup> that **1**–**4** possess different anti-proliferative activities against A549 and A431 cells, and **4** (multifloroside) is the most potent agen<sup>t</sup> against A431 cells.

**Figure 2.** Anti-proliferative activity of compounds in two human cancer cell lines (A549 and A431) as determined by the MTT assay. (**A**) **1**–**4** against A549 cells, (**B**) **1**–**4** against A431 cells, (**C**) Multifloroside (**4**) against A549 cells, (**D**) Multifloroside (**4**) against A431 cells. All results are shown as the mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 indicate significant differences compared with the control.
