*[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-(tertbutylamino)-2-oxoethyl]amino)-2,2,6,6-tetramethylpiperidin-1-yl]oxidanyl* (**8**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then fusidic acid and *t*-butyl isonitrile were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 9:1) to yield compound **8** (51 mg, 0.057 mmol, 57%) as red solid. *R<sup>F</sup>* 0.65 (DCM/MeOH 9:1). NMR of the corresponding hydroxylamine after phenylhydrazine reduction. <sup>1</sup>H-NMR (600 MHz, CDCl3) δ 5.69 (d, *J* = 8.6 Hz, 1H), 5.07 (m, 1H), 4.33–4.22 (m, 3H), 3.71 (s, 2H), 3.26 (d, *J* = 14.6 Hz, 1H),

3.05–3.02 (m, 1H), 2.80–2.75 (m, 2H), 2.32–2.28 (m, 1H), 2.22–2.02 (m, 5H), 1.89 (s, 3H), 1.86–1.82 (m, 2H), 1.76–1.71 (m, 4H), 1.68 (s, 3H), 1.61 (s, 3H), 1.60–1.53 (m, 4H), 1.34 (d, *J* = 7.0 Hz, 3H), 1.28 (s, 9H), 1.26–1.14 (m, 15H), 1.13–1.08 (m, 2H), 0.96 (s, 3H), 0.91–0.88 (m, 6H). <sup>13</sup>C-NMR (151 MHz, CDCl3) δ 173.21, 169.87, 169.06, 133.15, 132.45, 122.56, 74.81, 73.10, 71.29, 68.10, 50.95, 50.84, 49.76, 49.63, 49.42, 44.51, 41.63, 39.45, 39.26, 37.09, 36.22, 36.09, 35.22, 32.28, 32.28, 30.28, 30.24, 28.69, 28.57 (4 x CH<sup>3</sup> (TEMPO)), 28.52, 25.86, 22.97, 20.86, 20.51, 20.10, 18.05, 17.88, 16.03. HRMS (ESI) *m/z*: 783.5744 [M + H]<sup>+</sup> , calcd. for [C46H77N3O7] <sup>+</sup> 783.5762.

*[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-oxo-2-[(2,4,4-trimethoxybutyl)amino]ethyl}amino)-2,2,6,6-tetramethylpiperidin-1-yl]oxidanyl* (**9**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then fusidic acid and IPB isonitrile **5** were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 9:1) to yield compound **9** (60 mg, 0.068 mmol, 69%) as red solid. *R<sup>F</sup>* 0.72 (DCM/MeOH 9:1). NMR of the corresponding hydroxylamine after phenylhydrazine reduction. <sup>1</sup>H-NMR (600 MHz, CDCl3) δ 5.69 (d, *J* = 8.4 Hz, 1H), 5.08 (m, 1H), 4.51 (m, 1H), 4.34–4.31 (m, 1H), 3.76–3.73 (m, 1H), 3.65 (s, 2H), 3.39–3.29 (m, 12H), 3.14 (m, 1H), 3.07–3.02 (m, 1H), 2.75 (m, 1H), 2.37–2.27 (m, 1H), 2.23–2.08 (m, 5H), 2.04 (s, 3H), 1.92 (s, 3H), 1.88–1.80 (m, 4H), 1.76–1.71 (m, 4H), 1.68 (s, 3H), 1.62 (s, 3H), 1.60–1.48 (m, 4H), 1.36 (s, 3H), 1.31–1.17 (m, 15H), 1.15–1.10 (m, 2H), 0.97 (s, 3H), 0.93–0.88 (m, 6H). <sup>13</sup>C-NMR (151 MHz, CDCl3) δ 173.21, 169.87, 169.06, 133.15, 132.45, 122.56, 74.81, 71.29, 68.10, 50.95, 49.63, 49.42, 44.51, 42.68, 39.45, 39.26, 37.09, 36.37, 36.22, 36.09, 35.22, 32.28, 30.28, 30.24, 30.07, 28.57 (4 x CH<sup>3</sup> (TEMPO)), 28.52, 27.81, 27.73, 25.86, 23.85, 22.97, 21.43, 21.21, 20.86, 20.51, 20.10, 18.19, 18.05, 17.88, 16.03.15.68. HRMS (ESI) *m/z*: 873.6062 [M + H]<sup>+</sup> , calcd. for [C49H83N3O10] <sup>+</sup> 873.6078.

*[4-([2-(tert-Butylamino)-2-oxoethyl]{(4R)-4-[(1R,3aS,4R,7R,9aS,9bS,11S,11aR)-4,7,11-trihydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]pentanoyl}amino)-2,2,6,6 tetramethylpiperidin-1-yl]oxidanyl* (**10**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then cholic acid and *t*-butyl isonitrile were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 9:1) to yield compound **10** (55 mg, 0.081 mmol, 81%) as red solid. *R<sup>F</sup>* 0.62 (DCM/MeOH 9:1). NMR of the corresponding hydroxylamine after phenylhydrazine reduction. <sup>1</sup>H-NMR (600 MHz, CDCl3) δ 4.14–4.08 (m, 1H), 4.06–3.98 (m, 2H), 3.97–3.91 (m, 1H), 3.85–3.75 (m, 1H), 2.51–2.42 (m, 1H), 2.36–2.28 (m, 1H), 2.27–2.13 (m, 4H), 1.94–1.80 (m, 5H), 1.79–1.60 (m, 7H), 1.61–1.46 (m, 7H), 1.37–1.15 (m, 25H), 1.13–1.00 (m, 3H), 0.97 (s, 3H), 0.87 (s, 3H), 0.67 (d, *J* = 7.5 Hz, 3H). <sup>13</sup>C-NMR (151 MHz, CDCl3) δ 173.21, 169.87, 169.06, 133.15, 132.45, 122.56, 74.81, 73.10, 71.29, 68.10, 50.95, 50.84, 49.76, 49.63, 49.42, 44.51, 41.63, 39.45, 39.26, 37.09, 36.22, 36.09, 35.22, 32.28, 32.28, 30.28, 30.24, 28.57, 28.52, 28.52, 25.86, 22.97, 20.86, 20.51, 20.10, 18.05, 17.88, 16.03. HRMS (ESI) *m/z*: 675.5164 [M + H]<sup>+</sup> , calcd. for [C39H69N3O6] <sup>+</sup> 675.5186.

*[2,2,6,6-Tetramethyl-4-({2-oxo-2-[(2,4,4-trimethoxybutyl)amino]ethyl}{(4R)-4-[(1R,3aS,4R,7R,9aS, 9bS,11S,11aR)-4,7,11-trihydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1 yl]pentanoyl}amino)piperidin-1-yl]oxidanyl* (**11**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then cholic acid and IPB isonitrile **5** were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 9:1) to yield compound **11** (47 mg, 0.061 mmol, 61%) as red solid. *R<sup>F</sup>* 0.55 (DCM/MeOH 9:1). NMR of the corresponding hydroxylamine after phenylhydrazine reduction. <sup>1</sup>H-NMR (600 MHz, CDCl3) δ 4.55–4.48 (m, 1H), 4.12–4.00 (m, 1H), 3.95–3.81 (m, 2H), 3.45 (s, 3H), 3.37–3.28 (m, 12H), 3.28–3.22 (m, 1H), 2.48 (m, 2H), 2.36–2.11 (m, 5H), 1.98–1.80 (m, 6H), 1.80–1.69 (m, 5H), 1.69–1.39 (m, 7H), 1.40–1.16 (m, 15H), 1.15–1.00 (m, 4H), 0.96 (s, 3H), 0.88 (s, 3H), 0.68 (s, 3H). <sup>13</sup>C-NMR (151 MHz, CDCl3) δ 174.58, 170.07, 151.16, 101.75, 76.18, 72.86, 71.70, 68.28,

59.33, 59.28, 57.03, 53.33, 53.06, 52.96, 50.55, 46.99, 46.39, 45.61, 41.80, 41.38, 41.28, 35.57, 35.20, 35.09, 34.64, 32.16, 31.54, 30.85, 30.39, 28.20, 27.46, 26.51, 23.12, 22.42, 19.88, 17.44, 14.62, 12.48. HRMS (ESI) *m/z*: 764.5410 [M]<sup>+</sup> , calcd. for [C42H74N3O9] <sup>+</sup> 764.5425.

*[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-[6-(dimethylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl]ethyl}amino)-2 oxoethyl]amino)-2,2,6,6-tetramethylpiperidin-1-yl]oxidanyl* (**12**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then fusidic acid and Yudin's isonitrile were added. The crude reaction product was purified by silica gel column chromatography (DCM/MeOH 9:1) to yield compound **12** (35 mg, 0.035 mmol, 35%) as yellow powder. *R<sup>F</sup>* 0.1 (DCM/MeOH 9:1). NMR of the corresponding hydroxylamine after the addition of hydrazobenzene. <sup>1</sup>H-NMR (400 MHz, DMSO-*d*6) δ 8.49 (dd, *J* = 8.5, 2.1 Hz, 1H), 8.45 (d, *J* = 7.5 Hz, 1H), 8.34 (dd, *J* = 8.2, 1.9 Hz, 1H), 7.78–7.72 (m, 1H), 7.20 (d, *J* = 8.6 Hz, 1H), 5.65 (d, *J* = 8.5 Hz, 1H), 5.09–5.01 (m, 1H), 4.91–4.76 (m, 2H), 4.18–4.11 (m, 1H), 4.07 (d, *J* = 3.9 Hz, 1H), 3.99 (d, *J* = 3.9 Hz, 1H), 3.90–3.81 (m, 2H), 3.53 (s, 2H), 3.50–3.47 (m, 1H), 3.07 (s, 6H), 2.99–2.85 (m, 1H), 2.70–2.60 (m, 1H), 2.40–2.17 (m, 3H), 2.16–1.93 (m, 5H), 1.85 (d, *J* = 13.3 Hz, 2H), 1.62 (dd, *J* = 9.8, 5.2 Hz, 7H, (Fusidic acid; 5H+TEMPO; 2H)), 1.57–1.50 (m, 6H), 1.49–1.28 (m, 6H), 1.25 (s, 3H), 1.08–0.95 (m, 17H, (fusidic acid 3H, TEMPO 14H)), 0.86 (s, 3H), 0.81–0.75 (m, 6H). <sup>13</sup>C-NMR (101 MHz, DMSO-*d6*) δ 171.61, 169.91, 169.10, 164.32, 163.64, 147.18, 133.72, 132.31, 131.95, 131.88, 130.94, 130.21, 125.46, 124.76, 123.96, 123.48, 114.04, 113.47, 74.34, 69.68, 66.23, 58.57, 58.38, 51.63, 49.42, 49.07, 48.98, 48.65, 44.84, 43.22, 39.05, 36.90, 36.81, 35.67, 35.55, 33.31, 33.05, 32.37, 32.25, 30.69, 29.93, 28.82, 28.49, 25.92, 23.94, 23.18, 20.67, 20.13, 18.01, 17.97, 16.74, 16.71. HRMS (ESI) *m/z*: 993.6145 [M + H]<sup>+</sup> , calcd. for [C58H83N5O9] <sup>+</sup> 993.6191.

4.1.3. General Procedure **B** for the Conversion of Ugi Products 7 and 9 to Corresponding Spin-Labelled N-Acylpyrroles **13** and **15**

To a solution of Ugi products **7** and **9** (0.05 mmol) in toluene (10 mL) was added 10-camphorsulfonic acid (10 mol%) and quinoline (10 mol%). The mixture was stirred for 1 min at room temperature and then refluxed for at least 30 min until TLC showed complete conversion. The mixture was cooled to room temperature, transferred to a separatory funnel and washed with 1M aqueous HCl (2 × 30 mL). The acidic aqueous phase was further extracted with ethyl acetate (1 × 20 mL). The organic layers were combined, washed with NaHCO<sup>3</sup> and brine (2 × 20 mL), dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to obtain the *N*-acyl pyrrole derivatives **13** and **15**, which were used in the next step without further purification.

4.1.4. General Procedure **C** for the Conversion of the N-Acylpyrroles **13** and **15** into Their Corresponding Carboxylic Acids **14** and **16** Method **C1**

To a solution of intermediates **13** and **15** (0.025 mmol) in a mixture of THF (2 mL), methanol (2 mL), water (2 mL), potassium hydroxide (0.5 mmol) was added. This mixture was heated to 110 ◦C for 30 min in a microwave (90 W heating, 6 W keeping temperature). The reaction mixture was diluted with methanol (10 mL) and the pH value was set to pH = 2 by the addition of saturated aqueous NaHSO<sup>4</sup> solution. The aqueous phase was extracted with ethyl acetate (3 × 100 mL) and it was dried over Na2SO4. After filtration and evaporation of the solvent, the crude residue was purified by column chromatography (DCM/MeOH 8:2). By this method compounds, **14** and **16** were obtained.

*(4-{(Carboxymethyl)[(1R,3aS,5aR,5bR,9S,11aR)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1 en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carbonyl]amino}-2,2,6,6-tetramethylpiperidin-1 yl)oxidanyl* (**14**)

Obtained using the general method **C1**, the reaction crude reaction product was purified by silica gel column chromatography (DCM/MeOH 8:2) to yield compound **14** (11.8 mg, 0.014 mmol, 70%) as orange powder. *R<sup>F</sup>* 0.12 (DCM/MeOH 8:2). NMR of the corresponding hydroxylamine after the addition of hydrazobenzene. <sup>1</sup>H-NMR (400 MHz, DMSO-*d6*) δ 11.92 (s, 1H), 4.64 (d, *J* = 2.7 Hz, 1H), 4.57–4.51 (m, 1H), 4.26 (d, *J* = 5.1 Hz, 1H), 3.80–3.62 (m, 1H), 3.52 (s, 2H), 2.97 (m, 2H), 2.80 (m, 1H), 2.68 (m, 1H), 2.36–2.31 (m, 1H), 1.96 (m, 2H), 1.78 (dd, *J* = 13.0, 9.0 Hz, 2H), 1.64 (s, 3H), 1.55 (t, *J* = 12.7 Hz, 5H), 1.51–1.38 (m, 6H), 1.38–1.21 (m, 8H), 1.08 (d, *J* = 3.1 Hz, 14H), 0.92 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.77 (s, 3H), 0.66 (s, 3H). <sup>13</sup>C-NMR (101 MHz, DMSO-*d6*) δ 173.71, 171.03, 151.04, 109.22, 76.75, 58.20, 54.97, 53.73, 52.04, 50.17, 48.12, 45.40, 43.82, 41.50, 38.48, 38.28, 38.23, 36.73, 35.18, 33.92, 32.46, 30.63, 29.07, 27.15, 27.12, 25.15, 20.65, 19.78, 19.63, 19.07, 17.93, 15.97, 15.85, 15.78, 14.31. HRMS (ESI) *m/z*: 666.4996 [M − H]−, calcd. for [C41H66N2O5] − 666.4972.

*{4-[(Carboxymethyl){(2E)-6-methyl-2-[(2S,3aS,3bS,6S,7R,9aS,10R,11aR)-2,7,10-trihydroxy-3a, 3b,6,9a-tetramethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-ylidene]hept-5-enoyl}-amino]- 2,2,6,6-tetramethylpiperidin-1-yl}oxidanyl* (**16**)

Obtained using the general method **C1**, the reaction crude reaction product was purified by silica gel column chromatography (DCM/MeOH 8:2) to yield compound **16** (10 mg, 0.014 mmol, 58%) as orange oil. *R<sup>F</sup>* 0.10 (DCM/MeOH 8:2). NMR of the corresponding hydroxylamine after the additon of hydrazobenzene. <sup>1</sup>H-NMR (400 MHz, DMSO-*d6*) δ 10.22 (s, 1H), 5.46 (d, *J* = 8.6 Hz, 1H), 5.14–5.05 (m, 1H), 4.48 (d, *J* = 8.3 Hz, 1H), 4.17–4.07 (m, 1H), 4.00 (d, *J* = 3.6 Hz, 1H), 3.94 (d, *J* = 3.9 Hz, 1H), 3.51 (t, *J* = 3.2 Hz, 1H), 3.17 (dd, *J* = 15.1, 9.9 Hz, 1H), 2.99–2.75 (m, 1H), 2.71–2.55 (m, 1H), 2.40–1.93 (m, 8H), 1.93–1.66 (m, 4H), 1.63 (s, 3H), 1.55 (t, *J* = 2.2 Hz, 3H), 1.52–1.29 (m, 6H), 1.26 (s, 3H), 1.24–1.07 (m, 3H), 1.07–0.91 (m, 14H), 0.89 (d, *J* = 6.2 Hz, 6H), 0.79 (d, *J* = 6.7 Hz, 3H). <sup>13</sup>C-NMR (101 MHz, DMSO-*d6*) δ 174.42, 171.52, 147.95, 147.48, 135.99, 124.70, 69.98, 69.76, 66.50, 58.62, 58.55, 56.47, 49.67, 49.07, 48.89, 43.28, 41.71, 39.22, 36.97, 35.86, 35.67, 33.37, 33.13, 30.75, 29.57, 27.60, 25.89, 23.95, 23.06, 21.21, 19.01, 18.17, 16.77, 16.00. HRMS (ESI) *m/z*: 684.4732 [M − H]−, calcd. for [C40H64N2O7] − 684.4714.

#### Method **C2**

Method **C2** was established to keep the C-16 acetyl group intact on position 16 of the fusidic acid skeleton. *N*-acylpyrrole **15** (0.025 mmol) was dissolved in a mixture of *t*-BuOH (10 mL) and H2O (5 mL). Then, DMAP (0.015 mmol) was added and the reaction mixture was heated at reflux for 5 h, after which TLC (DCM/MeOH 8:2) indicated the saponification into the carboxylic acid **17**. The reaction mixture was concentrated to a volume of 10 mL in a rotary evaporator. Saturated NaHCO<sup>3</sup> solution (10 mL) and CH2Cl<sup>2</sup> (20 mL) were added. After the separation of the organic layer, the water layer was extracted with CH2Cl<sup>2</sup> (2 × 30 mL). Then the water layer was acidified with NaHSO<sup>4</sup> (2 M) and extracted with EtOAc (3 × 20 mL). The combined organic solutions of the acidic extraction were dried over Na2SO4, filtered, and evaporated to give carboxylic acid derivative, which was further purified by column chromatography (DCM/MeOH 8:2).
