*{4-[{(2E)-2-[(2S,3aS,3bS,6S,7R,9aS,10R,11aR)-2-(Acetyloxy)-7,10-dihydroxy-3a,3b,6,9atetramethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-ylidene]-6-methylhept-5-enoyl} (carboxymethyl)amino]-2,2,6,6-tetramethylpiperidin-1-yl}oxidanyl* (**17**)

Obtained using the general method **C2**, the reaction crude reaction product was purified by silica gel column chromatography (DCM/MeOH 8:2) to yield compound **17** (12 mg, 0.016 mmol, 66%) as orange oil. *R<sup>F</sup>* 0.15 (DCM/MeOH 8:2). NMR of the corresponding hydroxylamine after the addition of hydrazobenzene. <sup>1</sup>H-NMR (400 MHz, DMSO-*d6*) δ 8.89 (s, 1H), 5.44 (d, *J* = 8.5 Hz, 1H), 5.10 (t, *J* = 7.0 Hz, 1H), 4.18–4.13 (m, 1H), 3.98 (d, *J* = 3.7 Hz, 1H), 3.95 (d, 1H), 3.87 (d, *J* = 17.0 Hz, 2H), 3.53–3.49 (m, 1H), 2.97–2.87 (m, 1H), 2.73–2.66 (m, 1H, TEMPO), 2.26–2.17 (m, 3H), 2.10–1.97 (m, 5H), 1.94–1.82 (m, 5H), 1.81–1.70 (m, 2H, TEMPO), 1.64 (s, 3H), 1.57 (s, 3H), 1.53–1.29 (m, 6H), 1.27 (s, 3H), 1.26–1.12 (m, 3H), 1.10–0.95 (m, 14H, TEMPO), 0.88 (s, 3H), 0.84 (s, 3H), 0.79 (d, *J* = 6.7 Hz, 3H). <sup>13</sup>C-NMR (101 MHz, DMSO-*d6*) δ 175.94, 171.54, 170.02, 154.96, 147.92, 133.53, 123.91,

74.27, 69.74, 66.27, 58.63, 58.47, 54.20, 49.43, 49.10, 48.97, 43.02, 39.25, 38.90, 36.94, 36.69, 36.09, 35.71, 31.77, 29.78, 27.52, 26.07, 23.86, 23.22, 21.37, 20.72, 20.18, 18.21, 18.15, 16.78, 14.43. HRMS (ESI) *m/z*: 726.4807 [M − H]−, calcd. for [C42H66N2O8] − 726.4819.

*{4-[{(2E)-2-[(2S,3aS,3bS,6S,7R,9aS,10R,11aR)-2-(Acetyloxy)-7,10-dihydroxy-3a,3b,6,9atetramethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-ylidene]-6-methylhept-5-enoyl}(2-{[2- ({2-[6-(dimethylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl]ethyl}amino)- 2-oxoethyl][3-(triphenylphosphaniumyl)propyl]amino}-2-oxoethyl)amino]-2,2,6,6 tetramethylpiperidin-1-yl}oxidanyl bromide* (**18**)

Obtained using the general method **A** in 0.013 mmol scale (3-aminopropyl) triphenylphosphonium bromide and paraformaldehyde were used, then fusidic acid derivative **17** and Yudin's isonitrile were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 8:2) to yield compound **18** (6 mg, 0.004 mmol, 34%) as yellow powder. *R<sup>F</sup>* 0.12 (DCM/MeOH 8:2). NMR of the corresponding hydroxylamine after the additon of hydrazobenzene. <sup>1</sup>H-NMR (400 MHz, DMSO-*d6*) δ 8.57 (d, *J* = 8.8 Hz, 1H), 8.54 (d, *J* = 7.5 Hz, 1H), 8.51 (d, *J* = 8.3 Hz, 1H), 7.87–7.72 (m, 15H), 7.18–7.15 (m, 1H), 5.44 (q, *J* = 15.3, 12.0 Hz, 1H), 5.10 (d, *J* = 10.6 Hz, 1H), 4.62 (t, *J* = 7.3 Hz, 2H), 4.20–4.14 (m, 1H), 4.08–3.97 (m, 6H), 3.62–3.52 (m, 4H), 3.37 (d, *J* = 7.9 Hz, 2H), 3.16 (s, 4H), 3.10 (s, 6H), 2.95–2.88 (m, 1H), 2.69–2.62 (m, 1H), 2.46–2.11 (m, 8H), 2.11–1.92 (m, 7H), 1.50 (s, 3H), 1.47–1.32 (m, 6H), 1.30 (s, 3H), 1.24 (s, 3H), 1.15–0.95 (m, 17H), 0.89 (s, 3H), 0.85 (s, 3H), 0.80 (d, *J* = 6.7 Hz, 3H). <sup>13</sup>C-NMR (101 MHz, DMSO-*d6*) δ 171.12, 169.83, 169.46, 168.61, 163.83, 163.15, 156.51, 148.71, 135.13, 135.10, 133.60, 133.49, 132.17, 131.82, 131.51, 130.42, 130.29, 129.73, 128.76, 128.46, 124.98, 123.47, 122.99, 118.40, 117.82, 113.56, 112.99, 73.58, 69.19, 65.74, 58.08, 57.89, 54.62, 51.14, 48.94, 48.58, 48.16, 44.36, 43.54, 42.08, 38.59, 36.66, 36.42, 36.33, 36.26, 36.16, 35.84, 31.78, 30.21, 27.01, 25.55, 23.46, 23.33, 21.07, 20.80, 20.67, 20.08, 19.64, 18.37, 17.84, 17.71, 17.49, 16.25. HRMS (ESI) *m/z*: 1352.7621 [M]<sup>+</sup> , calcd. for [C81H105N6O10P]<sup>+</sup> 1352.7624.

#### *4.2. EPR Spectroscopy and Sample Preparation*

X-Band (~9.43 GHz) room temperature CW-EPR measurements were performed on a Magnettech MiniScope MS400 benchtop spectrometer (Magnettech, Berlin, Germany). Spectra were recorded with a microwave power of 3.16 mW, 100 kHz modulation frequency, modulation amplitude of 0.1mT and 4096 points. The final spectra were accumulations of 10 scans, each took 60 s. The samples were dissolved in methanol. Therefore, to reduce the line broadening effect due to the dissolved oxygen in the solvent, all samples were flushed with argon before EPR measurements.

#### *4.3. Biology*

#### 4.3.1. Cell Lines and Cultivation

PC3 and HT29 cell lines were supplied by the Leibniz Institute of Plant Biochemistry. The cells were grown in RPMI 1640 completed medium (supplemented with 10% FCS, 1% glutamine, and 1% penicillin/streptomycin) at 37 ◦C and 5% CO2. Cells were seeded at 5 × 103 cells/well in 96-well plates for viability determination and 1.5 × 105 cells/well in 6-well plates for flow cytometry and western blotting.

#### 4.3.2. MTT and CV Assays

For the fast screening the two cell lines were treated with 0.1 and 10 µM of the synthesized compounds 6-12, and 18 for 48 h. The compounds which showed anticancer activity was further analyzed to determine their IC50, in which, each compound was tested in 7 different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.56 µM) for 48 h. Afterward, for the CV assay, the cells were fixed by 4% paraformaldehyde for 15 min at RT and then the cells were stained with 0.1% CV solution for 15 min. Subsequently, the cells were washed with dd H2O, dried overnight and the dye was dissolved using 33% acetic acid. For MTT assay, the cells were incubated with MTT (0.5 mg/mL) for 20 min. Then, the

MTT solution was removed and the dye was dissolved using DMSO. The dissolved dyes were measured using an automated microplate reader (Spectramax, Molecular Devices, San José, CA, USA) at 570 nm with a background wavelength of 670 nm. The IC<sup>50</sup> values were calculated using the four-parameter logistic function and presented as the mean and all assays were performed in three biological replicates. The cell viability was expressed as a percentage compared to a negative control which was cells treated with complete medium and a positive control which was cells treated with digitonin (125 µM) [41,42].

#### 4.3.3. Apoptosis Analysis

The PC3 cells were prepared in a 6 well plate, treated with IC<sup>50</sup> and 2 × IC<sup>50</sup> of compound **8** (7.4 and 14.9 µM), and incubated for 48 h at 37 ◦C and 5% CO2. After the incubation, cells were stained by AnnV and PI (5 µL of AnnV, 2 µL of PI in 100 µL PBS) to determine apoptosis using flow cytometry (FACSAria III, BD Biosciences, Franklin Lakes, NJ, USA). The procedure was carried out according to the manufacturer's supplied instructions [42].

#### 4.3.4. Cell Cycle Analysis

The PC3 cells were prepared in a 6 well-plate and treated with IC<sup>50</sup> and 2 × IC<sup>50</sup> of compound **8** (7.4 and 14.9 µM) and incubated for 48 h at 37 ◦C and 5% CO2. Afterward, the cells were fixed in 70% ethanol overnight at 2 ◦C and then, stained with 1 µg/mL of DAPI at room temperature for 10 min. At last, the cells were analyzed by flow cytometry (FACSAria III) [42].

#### 4.3.5. Western Blot Analysis

PC3 cells were cultivated with an IC<sup>50</sup> dose of **8** for 2 h, 6 h, 12 h, 24 h, and 48 h. The cell lysis was performed using protein lysis buffer (62.5 mMTris–HCl (pH 6.8), 2% (*w*/*v*) SDS, 10% glycerol, and 50 mM dithiothreitol). The proteins were electrically separated using 12% SDS-polyacrylamide gels where a PageRuler prestained ladder was used as a protein molecular weight marker. The proteins were electrically transferred to nitrocellulose membranes by western blot system (Owl HEP-1, ThermoFisher Scientific, Schwerte, Germany). The membranes were blocked by 5% (*w*/*v*) BSA in PBS with 0.1% Tween 20 for 1 h at RT. Afterwards, blots were incubated overnight at 4 ◦C with α/β-Tubulin rabbit Ab, Caspase-3 rabbit Ab, β-actin rabbit Ab, and Bcl-XL rabbit Ab. As a secondary antibody Anti-rabbit IgG, HRP-linked Antibody was used. Bands were visualized using an ECL Prime Western Blotting System.

#### 4.3.6. Investigation of ROS Production

For the detection of reactive oxygen and nitrogen species, PC3 cells were stained with 1 µM of DHR solution in 0.1% PBS for 10 min. Afterwards, the cells were treated with IC<sup>50</sup> and 2 × IC<sup>50</sup> of compound **8** for 48 h. After 48 h, cells were trypsinized, washed with PBS, and then analyzed with flow cytometry [43,44].

#### *4.4. Microscopy*

#### Fluorescent Microscopy

PC3 cells were seeded in a 6-well plate for 24 h at 37 ◦C and 5% CO2. Afterward, cells were stained with 0.1 µM of MitoTrackerTM Deep Red in a complete medium for 15 min (based on the manufacturer's protocol). The cells were washed twice with PBS. After washing, cells were treated with the IC<sup>50</sup> of the tested compound for 24 h. The cells were washed twice with PBS, upon which 1 mL of medium was added. Finally, the cells were observed using GFP and Texas Red channels using LSM700 (Carl Zeiss, Jena, Germany) and EVOS FL AUTO (ThermoFisher, Schwerte, Germany).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22137125/s1. Table S1. EPR charactersitics for nitroxides 6-12, 14, 16-18; <sup>1</sup>H-, <sup>13</sup>C-NMR spectra and HPLC-profiles of products 6-12, 14, 16-18.

**Author Contributions:** H.N.S. synthesized and analyzed the compounds, wrote the initial manuscript, I.M. carried out the biological experiments and assisted in the biological data compilation, H.H. co-analyzed the spectroscopic data and assisted in the initial manuscript, A.H.R. and H.H.H. measured and analyzed the EPR-spectra, G.N.K. oversaw the biological assay data and assisted in the final manuscript, D.H. oversaw the EPR-analysis and co-designed the project, B.W. designed and coordinated the project, finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Hidayat Hussain gratefully acknowledges support by the AvH-foundation.

**Conflicts of Interest:** The authors declare no competing financial interest.

#### **References**


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