*2.2. Biological Evaluation*

2.2.1. The Effect of Compounds **1**, **5**, and **6** on Cancer Cells' Viability and Proliferation of Normal and Cancer Cells

In the first step of bioactivity investigations, the cytotoxicity of compounds **1**, **5**, and **6** was determined by measuring the metabolic activity of normal mouse epidermal JB6 Cl41, human colorectal carcinoma HT-29, and breast cancer MDA-MB-231 cells using MTS reagent. None of the tested compounds inhibited the viability of JB6 Cl41, HT-29, and MDA-MB-231 cells by 50% at concentrations up to 100 μM. The compounds **1**, **5**, and **6** decreased the cell viability by less than 20% at 100 μM (data not shown).

Next we determined the ability of the investigated compounds to affect cell proliferation of the tested cell lines. JB6 Cl41, HT-29, and MDA-MB-231 cells were treated with compounds **1**, **5**, and **6** at a non-toxic concentration of 20 μM for 24, 48, and 72 h. All tested compounds inhibited cell growth to a comparable degree (Figure 4). Compounds **1**, **5**, and **6** decreased proliferation of JB6 Cl41 cells by 20%, 22%, and 24%, respectively; HT-29 cells by 20%, 22%, and 26%, respectively; and MDA-MB-231 cells by 24%, 27%, and 29%, respectively, after 72 h of treatment.

**Figure 4.** The effect of compounds **1**, **5**, and **6** on cell proliferation**.** (**A**) JB6 Cl41; (**B**) HT-29; or (**C**) MDA-MB-231 cells were treated with compounds **1**, **5**, and **6** at concentration of 20 μM for 24 h, 48 h, and 72 h. Cell viability was estimated using the MTS assay. Data are represented as the mean ± SD as determined from triplicate experiments. A Student's t-test was used to evaluate the data with the following significance levels: \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001.
