2.2.2. The Effect of Compounds **1**, **5**, and **6** on Colony Formation of Cancer Cells

The effect of compounds **1**, **5**, and **6** on the colony formation of human cancer HT-29 and MDA-MB-231 cells was investigated using the soft agar assay. Compounds **1**, **5**, and **6** (20 μM) were demonstrated to possess comparable inhibiting activity on colony formation of cancer cells and the decrease in the colony number of HT-29 cells was by 18%, 12%, and 18%, respectively, while MDA-MB-231 cells was by 35%, 30%, and 31%, respectively as compared with non-treated cells (control) (Figure 5). In the present study it was demonstrated that triple negative human breast cancer cells MDA-MB-231 were more sensitive to the treatment of compounds **1**, **5**, and **6** than colorectal carcinoma cells HT-29.

**Figure 5.** The effect of compounds **1**, **5**, and **6** on colony formation in human cancer cells. (**A**) HT-29; or (**B**) MDA-MB-231 cells (2.4 <sup>×</sup> 104) with or without investigated compounds (20 <sup>μ</sup>M) treatment were subcultured onto 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 μg/mL gentamicin. After 14 days of incubation, the number of colonies was evaluated under a microscope with the aid of the ImageJ software program. Results are expressed as the mean ± standard deviation (SD). The asterisk (\*) indicates a significant decrease in colony number of cancer cells treated by compounds as compared with the control (\**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001).

In summary, compounds **1**, **5**, and **6** are non-cytototoxic against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cell lines at concentrations up to 100 μM, however, they are able to suppress cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

#### **3. Experimental Section**

#### *3.1. General Procedures*

The 1H- and 13C- NMR spectra were recorded on a Bruker Avance III 500 HD (Bruker, Germany) spectrometer at 500.13 and 125.76 MHz and a Bruker Avance III 700 spectrometer (Bruker, Germany) at 700.13 and 176.04 MHz, respectively. Chemical shifts (ppm) were internally referenced to the corresponding residual solvent signals δ<sup>H</sup> 3.30/δ<sup>C</sup> 49.0 for CD3OD. HRESIMS mass spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); the samples were dissolved in MeOH (c 0.001 mg/mL). Optical rotation was measured using the Perkin Elmer 343 polarimeter (Waltham, MA, USA). IR spectra were recorded on a Bruker OPUS Vector-22 infrared spectrophotometer. HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara,.CA, USA) equipped with a differential refractometer; Discovery C18 (5 μm, 250 × 10 mm, Supelco, Bellefonte, PA, USA) and YMC-Pack Pro C18 (5 μm, 250 × 4.6 mm, YMC CO., LTD, Kyoto, Japan) columns were used. Low pressure column liquid chromatography was performed using Amberlite XAD-2 (20 to 60 mesh, Supelco, Bellefonte, PA., USA), and silica gel KSK (50 to 160 μm, Sorbpolimer, Krasnodar, Russia). Sorbfil silica gel plates (4.5 × 6.0 cm, 5 to 17 μm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography.
