*3.5. Preparation of the MTPA Esters of Compound 1*

Aliquots (0.7 mg each) of compound **1** were treated with *S*-(+)- and *R*-(–)-α-methoxyα-(trifluoromethyl)-phenylacetyl (MTPA) chloride (0.7 μL) in dry pyridine (180 μL) for 3 h at room temperature. After removal of the solvent, the products were purified on a Si gel column (0.8 × 5 cm) using CHCl3-EtOH (50:1, *v*/*v*) to obtain the corresponding (*R*)- and (*S*)-MTPA esters of **1**.

3,15,26-tri-(*R*)-MTPA ester of **1**: Selected 1H-NMR (500.13 MHz, CD3OD): δ<sup>H</sup> 0.75 (1H, m, H-7), 0.87 (3H, d, *J* = 6.7 Hz, H3-21), 0.91 (3H, d, *J* = 6.8 Hz, H3-27), 1.07 (3H, s, H3-18), 1.12 (3H, s, H3-19), 1.49 (1H, m, H- -7), 1.83 (1H, m, H-25), 2.10 (1H, m, H-17), 3.05 (1H, dd, *J* = 3.6, 2.5 Hz, H-6), 4.15 (1H, m, H-16), 4.15 (1H, dd, *J* = 10.7, 5.7 Hz, H-26), 4.17 (1H, dd, *J* = 10.7, 6.0 Hz, H- -26), 5.03 (1H, d, *J* = 3.1 Hz, H-15), 5.39 (1H, m, H-3).

3,6,15,26-tetra-(*S*)-MTPA ester of **1**: Selected 1H-NMR (500.13 MHz, CD3OD): δ<sup>H</sup> 0.62 (3H, s, H3-19), 0.87 (3H, d, *J* = 6.7 Hz, H3-21), 0.90 (3H, d, *J* = 6.8 Hz, H3-27), 1.02 (3H, s, H3-18), 1.33 (1H, m, H-7), 1.82 (1H, m, H-25), 2.02 (1H, dd, *J* = 11.2, 8.2 Hz, H-17), 2.09 (1H, m, H- -7), 4.08 (2H, m, H-16, H-26), 4.09 (1H, m, H-16), 4.24 (1H, dd, *J* = 10.7, 5.5 Hz, H- -26), 4.83 (1H, t, *J* = 3.0 Hz, H-6), 5.19 (1H, d, *J* = 3.4 Hz, H-15), 5.32 (1H, m, H-3).

#### *3.6. Bioactivity Assay*

#### 3.6.1. Reagents

Phosphate buffered saline (PBS), L-glutamine, penicillin-streptomycin solution (10,000 U/mL, 10 μg/mL) were from Sigma-Aldrich company (St. Louis, MO, USA). MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Madison, Wisconsin, USA). The Basal Medium Eagle (BME), Dulbecco's modified Eagle's medium (DMEM), and McCoy's 5A modified medium (McCoy's 5A), trypsin, fetal bovine serum (FBS), and agar were purchased from ThermoFisher Scientific (Waltham, MA, USA).

#### 3.6.2. Cell Lines and Culture Conditions

Mouse epidermal cells JB6 Cl41 (ATCC® no. CRL-2010™), human colorectal carcinoma HT-29 (ATCC® no. HTB-38™), and human breast adenocarcinoma MDA-MB-231 (ATCC® HTB-26™) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

JB6 Cl41 cells grown in MEM supplemented with 5% fetal bovine serum (FBS), HT-29 cells were cultured in McCoy's 5A with 10% FBS, and MDA-MB-231 cells were grown in DMEM with 10% FBS. The cell cultures were maintained at 37 ◦C in humidified atmosphere containing 5% CO2.

#### 3.6.3. MTS Assay

To determine the cytotoxic activity of compounds **1**, **5**, and **6**, JB6 Cl41, HT-29, and MDA-MB-231 cells were seeded at a density of 1.0 <sup>×</sup> 104cells/200 <sup>μ</sup>L of complete MEM/5% FBS, McCoy's 5A/10% FBS, and DMEM/10% FBS media, respectively, in 96-well plates. After incubation for 24 h, the cells were treated with tested compounds in the range of concentration 5–100 μM, while the control was treated with the complete medium only. Cells were cultured for additional 24 h at 37 ◦C in 5% CO2 atmosphere. Subsequently, MTS reagent (20 μL) was added to each well, and the cells were incubated for an additional 3 h at 37 ◦C in 5% CO2. Absorbance was measured at 490/630 nm by a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). All tested samples were carried out in triplicates. Compound's concentration causing 50% of cell viability inhibition (IC50) were calculated.

To analyze the anti-proliferative activity of compounds **<sup>1</sup>**, **<sup>5</sup>**, and **<sup>6</sup>**, the cells (1.0 <sup>×</sup> <sup>10</sup>4cells/<sup>200</sup> <sup>μ</sup>L) were treated with tested compounds at concentration of 20 μM and incubated for an additional 24, 48, and 72 h at 37 ◦C in 5% CO2. MTS reagent (20 μL) was added to each well, and the cells were incubated for an additional 3 h at 37 ◦C in 5% CO2. Absorbance was measured at 490/630 nm using a microplate reader. All tested samples were analyzed in triplicates.

### 3.6.4. Soft Agar Assay

Cells (2.4 <sup>×</sup> 104/mL) were grown in 1 mL of 0.3% Basal Medium Eagle's agar containing 10% FBS. The cells were treated by compounds **1**, **5**, and **6** at non-toxic concentration of 5, 10, and 20 μM. The cultures were maintained at 37 ◦C in 5% CO2 incubator for 2 weeks and the number and size of the colonies were determined using a Motic microscope AE 20 (XiangAn, Xiamen, China) and ImageJ software bundled with 64-bit Java 1.8.0\_112 (NIH, Bethesda, Maryland, USA).

#### 3.6.5. Statistical Analysis

Results are expressed as the mean ± standard deviation (SD). Student's T test was used to evaluate the data with the following significance levels: \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001. All assays were performed in at least three independent experiments.
