Análisis bioquímicos del agua

For general methods, see the materials and methods Section of Chapter 2 (paragraph 2.4).

2,5-Dioxopyrrolidin-1-yl 2-azidoacetate (3). To a solution of 2-azido acetic acid (200 mg, 2.0

mmol, 1.0 equiv.) in dry DCM (10 mL), N-hydroxysuccinamide (273 mg, 2.4 mmol, 1.2 equiv.) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (455 mg, 2.4 mmol, 1.2 equiv.) were added and the reaction mixture was stirred at r.t. for 17 h. The reaction mixture was washed with H2O and brine, and dried over Na2SO4. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography (0-25% EtOAc in heptane) to yield title compound 3 as a white solid (274 mg, 1.4 mmol, 70%). Rf = 0.25 (EtOAc/heptane 1:1); IR (film)

2113, 1818, 1786, 1736, 1426, 1366, 1283, 1202, 1095, 1066, 811, 647 cm-1_; 1_{H NMR (400 MHz,} chloroform-d) δ 4.24 (s, 2H), 2.88 (s, 4H). 13_{C NMR (126 MHz, chloroform-d) δ 168.40, 164.14,} 47.99, 25.56. Mass could not be found.

2,5-Dioxopyrrolidin-1-yl 14-azido-3,6,9,12-tetraoxatetradecanoate (4). A solution of 14-

Azido-3,6,9,12-tetraoxatetradecanoic acid (50 mg, 0.18 mmol, 361 µl, 1.0 equiv.) in tBME was added to dry DCM (2.5 mL). N-hydroxysuccinamide (25 mg, 0.22 mmol, 1.2 equiv.) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (42 mg, 0.22 mmol, 1.2 equiv.) were added and the reaction mixture was stirred at r.t. for 17 h. The reaction mixture was washed with H2O and brine, and dried over Na2SO4. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography (50-60% EtOAc in heptane) to yield title compound 4 as a white solid (44 mg, 0.12 mmol, 65%). Rf = 0.29 (60%

EtOAc in heptane); IR (film) 2873, 2103, 1819, 1786, 1736, 1431, 1352, 1301, 1203, 1070, 814, 648, 558 cm-1_; 1_{H NMR (400 MHz, chloroform-d) δ 4.51 (s, 2H), 3.81 – 3.76 (m, 2H), 3.69 (dd, J} = 4.0, 1.8 Hz, 2H), 3.68 – 3.66 (m, 2H), 3.65 (s, 8H), 3.40 – 3.35 (m, 2H), 2.84 (s, 4H). 13_{C NMR} (101 MHz, chloroform-d) δ 171.95, 168.96, 166.13, 71.42, 70.75, 70.71, 70.63, 70.10, 66.62, 50.78, 25.69, 25.48. Mass could not be found.

6-(Hydroxymethyl)picolinaldehyde (5). 2,6-Pyridinedimethanol (500 mg, 3.6 mmol, 1.0 equiv.)

and SeO2 (200 mg, 1.8 mmol, 0.5 equiv.) were added to a Schlenk tube and put under N2. 1,4-Dioxane (10 mL, not degassed or dried) was added, the mixture was sonicated for 2 min and then stirred at 65 °C for 24 h. After the mixture was cooled to r.t., it was diluted with DCM and filtered over celite. Then, the volatiles were removed under reduced pressure and the product was purified with column

chromatography (2.5% MeOH in DCM) yielding aldehyde 5 (405 mg, 82%) as a slightly yellow oil.

Rf = 0.17 (2.5% MeOH in DCM). 1H NMR (400 MHz, chloroform-d) δ 10.09 (s, 1H), 7.93 – 7.83

(m, 2H), 7.56 – 7.47 (m, 1H), 4.88 (s, 2H). 13_{C NMR (100 MHz, chloroform-d) δ 193.2, 160.2,} 151.8, 137.9, 124.9, 120.6, 64.2. LRMS (ESI+) m/z calcd. for C7H7NNaO2+ _[M+Na]+ _{138.1, found:} 138.0. The obtained data agrees with literature.4

(6-Formylpyridin-2-yl)methyl methanesulfonate (6). Alcohol 5 (350 mg 2.6 mmol, 1.0 equiv.)

was dissolved in dry DCM, the solution was cooled to 0 °C and Et3N (1.1 mL, 7.7 mmol, 3.0 equiv.) was added. Then, MsCl (237 µL, 3.1 mmol, 1.2 equiv.) was added dropwise and the mixture was stirred at r.t. for 30 min. The reaction was quenched with sat. NaHCO3, the layers were separated and the aqueous layer was extracted with DCM (3 x). The combined organic layers were dried over Na2SO4 and the volatiles were removed under reduced pressure. The brown oil was purified with column chromatography (60% EtOAc in heptane) yielding methanesulfonate 6 (399 mg, 73%) as a slightly

yellow solid. Rf = 0.35 (EtOAc/heptane, 3:2).1H NMR (400 MHz, chloroform-d) δ 10.05 (s, 1H),

7.98 – 7.92 (m, 2H), 7.74 – 7.69 (m, 1H), 5.42 (s, 2H), 3.14 (s, 3H). 13_{C NMR (100 MHz,} chloroform-d) δ 192.8, 154.8, 152.6, 138.5, 126.5, 121.7, 70.8, 38.2. LRMS (ESI+) m/z calcd. for C8H10NO4S+ _[M+H]+ _{216.0, found: 216.1. The obtained data agrees with literature.}4

tert-Butyl 4-((6-formylpyridin-2-yl)methyl)piperazine-1-carboxylate (7). Methanesulfonate 6

(370 mg, 1.7 mmol, 1.0 equiv.) was dissolved in dry MeCN (10 mL) and 1-Boc-piperazine (384 mg, 2.0 mmol, 1.2 equiv.) and K2CO3 (475 mg, 3.4 mmol, 2.0 equiv.) were added. The mixture was stirred at 60 °C for 16 h, whereupon the solvent was evaporated. DCM and sat. NaHCO3 were added, the layers were separated and the aqueous layer was extracted with DCM (3 x). The combined organic layers were dried over Na2SO4 and the volatiles were removed using a rotary evaporator. Then, the product was purified using column chromatography (80% EtOAc in heptane) yielding tertiary amine 7 (456 mg, 87%) as a white solid. Rf = 0.26 (EtOAc/heptane, 4:1).

1_{H NMR (400 MHz, chloroform-d) δ 10.07 (s, 1H), 7.88 – 7.82 (m, 2H), 7.73 – 7.66 (m, 1H), 3.77} (s, 2H), 3.52 – 3.41 (m, 4H), 2.52 – 2.42 (m, 4H), 1.46 (s, 9H). 13_{C NMR (100 MHz, chloroform-} d) δ 193.7, 159.6, 154.9, 152.5, 137.6, 127.5, 120.4, 79.9, 64.3, 53.3, 28.6. LRMS (ESI+) m/z calcd. for C16H24N3O3+ _[M+H]+ _{306.2, found: 305.9. The obtained data agrees with literature.}4

6-((4-(2-Azidoacetyl)piperazin-1-yl)methyl)picolinaldehyde (8). tert-Butyl 4-((6- formylpyridin-2-yl)methyl)piperazine-1-carboxylate 7 (25 mg, 82

µmol, 1.0 equiv.) was dissolved in dry DCM (1 mL) under N2 and 4M HCl in dioxane (205 µL, 820 µmol, 10.0 equiv.) was added. The mixture was stirred for 2 h, whereupon the volatiles were evaporated. The solid was dissolved in DMF (1 mL) and 2,5-dioxopyrrolidin-1-yl 2-azidoacetate (3) (19 mg, 98 µmol, 1.2 equiv.) was added. Then, Et3N (34 µL, 250 µmol, 3.0 equiv.) was added and the solution was stirred for 2 h. The volatiles were evaporated and the product was purified using column chromatography (50 to 100% EtOAc in heptane) yielding 2-PCA-N3 8 (19

mg, 80%) as a white solid. Rf = 0.49 (5% MeOH in EtOAc). IR (film) 2107, 1711, 1657, 1592,

1457, 1220, 1076, 1000, 788, 655 cm-1_. 1_{H NMR (500 MHz, chloroform-d) δ 10.06 (s, 1H), 7.87 (d,} 160

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7.4 Acknowledgements

Camille Le Gall and dr. Martijn Verdoes (Radboud Institute for Molecular Life Sciences) are gratefully acknowledged for providing us with the anti-CD20 Fab fragments and their collaboration in this project. Mike Smeenk is thanked for the synthesis of iminosydnones 10-12, 14-18. We thank dr. Selma Eising for the synthesis of compound 5-7.

7.5 Materials and methods

For general methods, see the materials and methods Section of Chapter 2 (paragraph 2.4).

2,5-Dioxopyrrolidin-1-yl 2-azidoacetate (3). To a solution of 2-azido acetic acid (200 mg, 2.0

mmol, 1.0 equiv.) in dry DCM (10 mL), N-hydroxysuccinamide (273 mg, 2.4 mmol, 1.2 equiv.) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (455 mg, 2.4 mmol, 1.2 equiv.) were added and the reaction mixture was stirred at r.t. for 17 h. The reaction mixture was washed with H2O and brine, and dried over Na2SO4. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography (0-25% EtOAc in heptane) to yield title compound 3 as a white solid (274 mg, 1.4 mmol, 70%). Rf = 0.25 (EtOAc/heptane 1:1); IR (film)

2113, 1818, 1786, 1736, 1426, 1366, 1283, 1202, 1095, 1066, 811, 647 cm-1_; 1_{H NMR (400 MHz,} chloroform-d) δ 4.24 (s, 2H), 2.88 (s, 4H). 13_{C NMR (126 MHz, chloroform-d) δ 168.40, 164.14,} 47.99, 25.56. Mass could not be found.

2,5-Dioxopyrrolidin-1-yl 14-azido-3,6,9,12-tetraoxatetradecanoate (4). A solution of 14-

Azido-3,6,9,12-tetraoxatetradecanoic acid (50 mg, 0.18 mmol, 361 µl, 1.0 equiv.) in tBME was added to dry DCM (2.5 mL). N-hydroxysuccinamide (25 mg, 0.22 mmol, 1.2 equiv.) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (42 mg, 0.22 mmol, 1.2 equiv.) were added and the reaction mixture was stirred at r.t. for 17 h. The reaction mixture was washed with H2O and brine, and dried over Na2SO4. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography (50-60% EtOAc in heptane) to yield title compound 4 as a white solid (44 mg, 0.12 mmol, 65%). Rf = 0.29 (60%

EtOAc in heptane); IR (film) 2873, 2103, 1819, 1786, 1736, 1431, 1352, 1301, 1203, 1070, 814, 648, 558 cm-1_; 1_{H NMR (400 MHz, chloroform-d) δ 4.51 (s, 2H), 3.81 – 3.76 (m, 2H), 3.69 (dd, J} = 4.0, 1.8 Hz, 2H), 3.68 – 3.66 (m, 2H), 3.65 (s, 8H), 3.40 – 3.35 (m, 2H), 2.84 (s, 4H). 13_{C NMR} (101 MHz, chloroform-d) δ 171.95, 168.96, 166.13, 71.42, 70.75, 70.71, 70.63, 70.10, 66.62, 50.78, 25.69, 25.48. Mass could not be found.

6-(Hydroxymethyl)picolinaldehyde (5). 2,6-Pyridinedimethanol (500 mg, 3.6 mmol, 1.0 equiv.)

and SeO2 (200 mg, 1.8 mmol, 0.5 equiv.) were added to a Schlenk tube and put under N2. 1,4-Dioxane (10 mL, not degassed or dried) was added, the mixture was sonicated for 2 min and then stirred at 65 °C for 24 h. After the mixture was cooled to r.t., it was diluted with DCM and filtered over celite. Then, the volatiles were removed under reduced pressure and the product was purified with column

chromatography (2.5% MeOH in DCM) yielding aldehyde 5 (405 mg, 82%) as a slightly yellow oil.

Rf = 0.17 (2.5% MeOH in DCM). 1H NMR (400 MHz, chloroform-d) δ 10.09 (s, 1H), 7.93 – 7.83

(m, 2H), 7.56 – 7.47 (m, 1H), 4.88 (s, 2H). 13_{C NMR (100 MHz, chloroform-d) δ 193.2, 160.2,} 151.8, 137.9, 124.9, 120.6, 64.2. LRMS (ESI+) m/z calcd. for C7H7NNaO2+ _[M+Na]+ _{138.1, found:} 138.0. The obtained data agrees with literature.4

(6-Formylpyridin-2-yl)methyl methanesulfonate (6). Alcohol 5 (350 mg 2.6 mmol, 1.0 equiv.)

was dissolved in dry DCM, the solution was cooled to 0 °C and Et3N (1.1 mL, 7.7 mmol, 3.0 equiv.) was added. Then, MsCl (237 µL, 3.1 mmol, 1.2 equiv.) was added dropwise and the mixture was stirred at r.t. for 30 min. The reaction was quenched with sat. NaHCO3, the layers were separated and the aqueous layer was extracted with DCM (3 x). The combined organic layers were dried over Na2SO4 and the volatiles were removed under reduced pressure. The brown oil was purified with column chromatography (60% EtOAc in heptane) yielding methanesulfonate 6 (399 mg, 73%) as a slightly

yellow solid. Rf = 0.35 (EtOAc/heptane, 3:2).1H NMR (400 MHz, chloroform-d) δ 10.05 (s, 1H),

7.98 – 7.92 (m, 2H), 7.74 – 7.69 (m, 1H), 5.42 (s, 2H), 3.14 (s, 3H). 13_{C NMR (100 MHz,} chloroform-d) δ 192.8, 154.8, 152.6, 138.5, 126.5, 121.7, 70.8, 38.2. LRMS (ESI+) m/z calcd. for C8H10NO4S+ _[M+H]+ _{216.0, found: 216.1. The obtained data agrees with literature.}4

tert-Butyl 4-((6-formylpyridin-2-yl)methyl)piperazine-1-carboxylate (7). Methanesulfonate 6

(370 mg, 1.7 mmol, 1.0 equiv.) was dissolved in dry MeCN (10 mL) and 1-Boc-piperazine (384 mg, 2.0 mmol, 1.2 equiv.) and K2CO3 (475 mg, 3.4 mmol, 2.0 equiv.) were added. The mixture was stirred at 60 °C for 16 h, whereupon the solvent was evaporated. DCM and sat. NaHCO3 were added, the layers were separated and the aqueous layer was extracted with DCM (3 x). The combined organic layers were dried over Na2SO4 and the volatiles were removed using a rotary evaporator. Then, the product was purified using column chromatography (80% EtOAc in heptane) yielding tertiary amine 7 (456 mg, 87%) as a white solid. Rf = 0.26 (EtOAc/heptane, 4:1).

1_{H NMR (400 MHz, chloroform-d) δ 10.07 (s, 1H), 7.88 – 7.82 (m, 2H), 7.73 – 7.66 (m, 1H), 3.77} (s, 2H), 3.52 – 3.41 (m, 4H), 2.52 – 2.42 (m, 4H), 1.46 (s, 9H). 13_{C NMR (100 MHz, chloroform-} d) δ 193.7, 159.6, 154.9, 152.5, 137.6, 127.5, 120.4, 79.9, 64.3, 53.3, 28.6. LRMS (ESI+) m/z calcd. for C16H24N3O3+ _[M+H]+ _{306.2, found: 305.9. The obtained data agrees with literature.}4

6-((4-(2-Azidoacetyl)piperazin-1-yl)methyl)picolinaldehyde (8). tert-Butyl 4-((6- formylpyridin-2-yl)methyl)piperazine-1-carboxylate 7 (25 mg, 82

µmol, 1.0 equiv.) was dissolved in dry DCM (1 mL) under N2 and 4M HCl in dioxane (205 µL, 820 µmol, 10.0 equiv.) was added. The mixture was stirred for 2 h, whereupon the volatiles were evaporated. The solid was dissolved in DMF (1 mL) and 2,5-dioxopyrrolidin-1-yl 2-azidoacetate (3) (19 mg, 98 µmol, 1.2 equiv.) was added. Then, Et3N (34 µL, 250 µmol, 3.0 equiv.) was added and the solution was stirred for 2 h. The volatiles were evaporated and the product was purified using column chromatography (50 to 100% EtOAc in heptane) yielding 2-PCA-N3 8 (19

mg, 80%) as a white solid. Rf = 0.49 (5% MeOH in EtOAc). IR (film) 2107, 1711, 1657, 1592,

1457, 1220, 1076, 1000, 788, 655 cm-1_. 1_{H NMR (500 MHz, chloroform-d) δ 10.06 (s, 1H), 7.87 (d,} 161 Summary, Future Perspectives and Concluding Remarks

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J = 3.9 Hz, 2H), 7.70 – 7.65 (m, 1H), 3.93 (s, 2H), 3.82 (s, 2H), 3.73 – 3.66 (m, 2H), 3.50 – 3.39 (m, 2H), 2.60 (q, J = 5.9 Hz, 4H). 13_{C NMR (126 MHz, chloroform-d) δ 193.30, 172.07, 165.66,} 158.46, 152.43, 137.69, 127.57, 120.65, 76.78, 63.60, 52.71, 50.68, 44.93, 41.88, 25.41. HRMS (ESI+) m/z calcd. for C13H17N6O2 [M+H]+ _{289.14075, found: 289.14130. m/z calcd. for} C13H16N6NaO2 [M+Na]+ _{311.12269, found 311.12324}

6-((4-(14-Azido-3,6,9,12-tetraoxatetradecanoyl)piperazin-1-yl)methyl)picolinaldehyde (9).

tert-Butyl 4-((6-formylpyridin-2-yl)methyl) piperazine-1-carboxylate 7 (50 mg, 0.16

mmol, 1.0 equiv.) was dissolved in dry DCM (2 mL) under N2 and 4M HCl in dioxane (410 µL, 1.6 mmol, 10.0 equiv.) was added. The mixture was stirred for 2 h, whereupon the volatiles were evaporated. The solid was dissolved in DMF (1 mL) and 2,5-dioxopyrrolidin-1-yl 14-azido-3,6,9,12-tetraoxatetradecanoate (4) (74 mg, 0.20 mmol, 1.2 equiv.) was added. Then, Et3N (69 µL, 0.49 mmol, 3.0 equiv.) was added and the solution was stirred for 2 h. The volatiles were evaporated and the product was purified using preparative HPLC (gradient: 5 to 100% MeCN in H2O in 55 minutes) yielding 2- PCA-PEG4-N3 9 (8 mg, 11%) as a yellow oil. Rf = 0.45 (75% EtOAc in heptane). IR (film) 2104,

1711, 1648, 1592, 1458, 1214, 1094, 799, 653 cm-1_. 1_{H NMR (500 MHz, chloroform-d) δ 10.03 (s,} 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.83 (d, J = 6.4 Hz, 1H), 4.41 (s, 2H), 4.21 (s, 2H), 3.98 – 3.87 (m, 4H), 3.69 – 3.60 (m, 14H), 3.39 (q, J = 7.4, 4.9 Hz, 4H), 3.27 (s, 2H). 13_{C NMR (126 MHz,} chloroform-d) δ 192.28, 167.85, 152.74, 150.53, 138.94, 129.88, 122.27, 70.63, 70.53, 70.52, 70.38, 70.30, 70.28, 70.10, 69.94, 61.04, 52.29, 51.82, 50.59, 42.49, 39.05. HRMS (ESI+) m/z calcd. for C21H33N6O6 [M+H]+ _{465.24561, found: 465.24616 m/z calcd. C21H32N6NaO6 [M+Na]}+ _487.22755, found: 487.22607.

2-((4-Iodophenyl)amino)acetonitrile (10). Iodoaniline (5.00 g, 23 mmol, 1 equiv.) was dissolved

in dry acetonitrile (60 mL). To this solution was subsequently added K2CO3 (3.80 g, 28 mmol, 1.2 equiv.) and NaI (3.40 g, 23 mmol, 1 equiv.). Chloroacetonitrile (2.89 mL, 46 mmol, 2 equiv.) was added dropwise and the mixture was then refluxed under argon for 3 days. Afterwards, the reaction was cooled down to room temperature and filtered over celite and rinsed with EtOAc. The organic layer was washed once with brine and dried over MgSO4. The volatiles were evaporated and the product (4.82 g, 82% yield) was obtained after purifying on a dry loaded column (20-50% EtOAc in heptane). Rf = 0.52 (50% EtOAc in heptane). 1_{H NMR (400 MHz,} chloroform-d) δ 7.56 – 7.51 (m, 2H), 6.52 – 6.47 (m, 2H), 4.09 (d, J = 1.0 Hz, 1H), 4.07 (s, 2H). 13_{C-NMR (100 MHz, chloroform-d): δ 144.76, 138.38 (2C), 116.55, 115.87 (2C), 81.76, 32.61.} LRMS (ESI+): m/z calcd for C8H7IN2+ _[M+H]+ _{258.8, found 258.9.}

N-(Cyanomethyl)-N-(4-iodophenyl)nitrous amide (11). Compound 10 (740 mg, 2.9 mmol, 1

equiv.) was dissolved in dry THF (3.8 mL) and isoamyl nitrite (1.19 mL, 8.6 mmol, 3 equiv.) was added dropwise to the mixture. This was left to stir for 2 hours at room temperature. Afterwards, the volatiles were removed yielding a crude product (821 mg, 2.9 mmol) with a high purity (>95%), so it was used in the next step without further purification. Rf = 0.62 (50% EtOAc in heptane). 1_H-

NMR (400 MHz, chloroform-d): δ 7.89 (d, 2H, J = 8.8 Hz), 7.33 (d, 2H, J = 8.8 Hz), 4.77 (s, 2H). 13_{C-NMR (100 MHz, chloroform-d): δ 139.59, 139.20 (2C), 121.42, 112.12 (2C), 93.74, 30.34.} LRMS (ESI+): m/z calcd for C8H7IN3O+ _[M+H]+ _{288.0, found 288.1.}

5-Amino-3-(4-iodophenyl)-1,2,3-oxadiazol-3-ium chloride (12). Compound 11 was dissolved

in 6 mL 4M HCl in dioxane and was stirred at room temperature overnight. The product was obtained by trituration in diethyl ether (95% yield over two steps). 1_{H-NMR (400 MHz, DMSO-d6): δ 9.97 (s, 2H), 8.66} (s, 1H), 8.16 (d, 2H, J = 8.92 Hz), 7.84 (d, 2H, J = 8.93). 13_{C-NMR (100} MHz, DMSO-d6): δ 169.45, 139.19 (2C), 132.44, 124.36 (2C), 101.43 (1C missing). LRMS (ESI+):

m/z calcd for C8H7IN3O+ _[M-Cl]+ _{288.0, found 288.0}

(tert-Butoxycarbonyl)(3-(4-iodophenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (13).

Compound 12 (6.50 g, 20 mmol, 1 equiv.), NaHCO3 (1.90 g, 34 mmol, 1.7 equiv.) and DMAP (276 mg, 2.3 mmol, 10 mol%) were dissolved in dry THF (100 mL). The mixture was cooled down to 0 °C. To this mixture, Boc2O (7.40 g, 34 mmol, 1.7 equiv.) in THF (65 mL) was added dropwise and the reaction was stirred for 70 hours. The reaction was then quenched with 165 mL of aq. sat. NH4Cl solution, THF was removed under reduced pressure and the product was extracted with EtOAC (3 x 80 mL). The organic layer was then dried and the product was obtained as an orange solid (4.79 g, 62% yield) after flash purification (0-50% EtOAc in heptane). Rf = 0.58 (50% EtOAc in heptane). 1_{H-NMR (400 MHz, chloroform-d): δ 8.09 (s,} 1H), 8.00 (d, 2H, J = 8.94), 7.54 (d, 2H, J = 8.94), 1.52 (s, 9H). 13_{C-NMR (100 MHz, chloroform-} d): δ 174.64, 160.72, 139.78, 139.75 (2C), 122.76, 122.74 (2C), 99.45, 79.24, 28.23 (3C). LRMS (ESI+): m/z calcd for C13H14IN3O3+ _[M+H]+ _{387.01, found 387.9}

(tert-Butoxycarbonyl)(3-(4-(ethoxycarbonyl)phenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (14). Compound 13 (1.20 g, 3.1 mmol, 1 equiv.) was dissolved

in dry ethanol (10 mL) and NEt3 (10 mL). To this, PdCl2(PPh3)2 (324 mg, 0.46 mmol, 15 mol%) was added. The compound was allowed to react in a CO atmosphere at 60 °C for 2.5 hours. The reaction mixture was then allowed to cool down and was filtered over celite. The mixture was then concentrated and purified using flash chromatography (0-50% EtOAc in heptane) to obtain an orange solid (668 mg, 65% yield). Rf = 0.35 (50% EtOAc in heptane). 1_{H-NMR (400 MHz, chloroform-d): δ 8.32 (d, 2H, J = 8.87), 8.16} (s, 1H), 7.89 (d, 2H, J = 8.87), 4.45 (q, 2H, J = 7.14), 1.52 (s, 9H), 1.43 (t, 3H, J = 7.14). 13_C-NMR (100 MHz, chloroform-d): δ 174.88, 164.53, 160.95, 136.82, 134.93, 131.86 (2C), 121.63 (2C), 102.62, 79.36, 62.20, 28.36 (3C), 14.39. LRMS (ESI+): m/z calcd for C16H20N3O5+ _[M+H]+ _334.1, found 334.4.

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J = 3.9 Hz, 2H), 7.70 – 7.65 (m, 1H), 3.93 (s, 2H), 3.82 (s, 2H), 3.73 – 3.66 (m, 2H), 3.50 – 3.39 (m, 2H), 2.60 (q, J = 5.9 Hz, 4H). 13_{C NMR (126 MHz, chloroform-d) δ 193.30, 172.07, 165.66,} 158.46, 152.43, 137.69, 127.57, 120.65, 76.78, 63.60, 52.71, 50.68, 44.93, 41.88, 25.41. HRMS (ESI+) m/z calcd. for C13H17N6O2 [M+H]+ _{289.14075, found: 289.14130. m/z calcd. for} C13H16N6NaO2 [M+Na]+ _{311.12269, found 311.12324}

6-((4-(14-Azido-3,6,9,12-tetraoxatetradecanoyl)piperazin-1-yl)methyl)picolinaldehyde (9).

tert-Butyl 4-((6-formylpyridin-2-yl)methyl) piperazine-1-carboxylate 7 (50 mg, 0.16

mmol, 1.0 equiv.) was dissolved in dry DCM (2 mL) under N2 and 4M HCl in dioxane (410 µL, 1.6 mmol, 10.0 equiv.) was added. The mixture was stirred for 2 h, whereupon the volatiles were evaporated. The solid was dissolved in DMF (1 mL) and 2,5-dioxopyrrolidin-1-yl 14-azido-3,6,9,12-tetraoxatetradecanoate (4) (74 mg, 0.20 mmol, 1.2 equiv.) was added. Then, Et3N (69 µL, 0.49 mmol, 3.0 equiv.) was added and the solution was stirred for 2 h. The volatiles were evaporated and the product was purified using preparative HPLC (gradient: 5 to 100% MeCN in H2O in 55 minutes) yielding 2- PCA-PEG4-N3 9 (8 mg, 11%) as a yellow oil. Rf = 0.45 (75% EtOAc in heptane). IR (film) 2104,

1711, 1648, 1592, 1458, 1214, 1094, 799, 653 cm-1_. 1_{H NMR (500 MHz, chloroform-d) δ 10.03 (s,} 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.83 (d, J = 6.4 Hz, 1H), 4.41 (s, 2H), 4.21 (s, 2H), 3.98 – 3.87 (m, 4H), 3.69 – 3.60 (m, 14H), 3.39 (q, J = 7.4, 4.9 Hz, 4H), 3.27 (s, 2H). 13_{C NMR (126 MHz,} chloroform-d) δ 192.28, 167.85, 152.74, 150.53, 138.94, 129.88, 122.27, 70.63, 70.53, 70.52, 70.38, 70.30, 70.28, 70.10, 69.94, 61.04, 52.29, 51.82, 50.59, 42.49, 39.05. HRMS (ESI+) m/z calcd. for C21H33N6O6 [M+H]+ _{465.24561, found: 465.24616 m/z calcd. C21H32N6NaO6 [M+Na]}+ _487.22755, found: 487.22607.

2-((4-Iodophenyl)amino)acetonitrile (10). Iodoaniline (5.00 g, 23 mmol, 1 equiv.) was dissolved

in dry acetonitrile (60 mL). To this solution was subsequently added K2CO3 (3.80 g, 28 mmol, 1.2 equiv.) and NaI (3.40 g, 23 mmol, 1 equiv.). Chloroacetonitrile (2.89 mL, 46 mmol, 2 equiv.) was added dropwise and the mixture was then refluxed under argon for 3 days. Afterwards, the reaction was cooled down to room temperature and filtered over celite and rinsed with EtOAc. The organic layer was washed once with brine and dried over MgSO4. The volatiles were evaporated and the product (4.82 g, 82% yield) was obtained after purifying on a dry loaded column (20-50% EtOAc in heptane). Rf = 0.52 (50% EtOAc in heptane). 1_{H NMR (400 MHz,} chloroform-d) δ 7.56 – 7.51 (m, 2H), 6.52 – 6.47 (m, 2H), 4.09 (d, J = 1.0 Hz, 1H), 4.07 (s, 2H). 13_{C-NMR (100 MHz, chloroform-d): δ 144.76, 138.38 (2C), 116.55, 115.87 (2C), 81.76, 32.61.} LRMS (ESI+): m/z calcd for C8H7IN2+ _[M+H]+ _{258.8, found 258.9.}

N-(Cyanomethyl)-N-(4-iodophenyl)nitrous amide (11). Compound 10 (740 mg, 2.9 mmol, 1

equiv.) was dissolved in dry THF (3.8 mL) and isoamyl nitrite (1.19 mL, 8.6 mmol, 3 equiv.) was added dropwise to the mixture. This was left to stir for 2 hours at room temperature. Afterwards, the volatiles were removed yielding a crude product (821 mg, 2.9 mmol) with a high purity (>95%), so it was used in the next step without further purification. Rf = 0.62 (50% EtOAc in heptane). 1_H-

NMR (400 MHz, chloroform-d): δ 7.89 (d, 2H, J = 8.8 Hz), 7.33 (d, 2H, J = 8.8 Hz), 4.77 (s, 2H). 13_{C-NMR (100 MHz, chloroform-d): δ 139.59, 139.20 (2C), 121.42, 112.12 (2C), 93.74, 30.34.} LRMS (ESI+): m/z calcd for C8H7IN3O+ _[M+H]+ _{288.0, found 288.1.}

5-Amino-3-(4-iodophenyl)-1,2,3-oxadiazol-3-ium chloride (12). Compound 11 was dissolved

in 6 mL 4M HCl in dioxane and was stirred at room temperature overnight. The product was obtained by trituration in diethyl ether (95% yield over two steps). 1_{H-NMR (400 MHz, DMSO-d6): δ 9.97 (s, 2H), 8.66} (s, 1H), 8.16 (d, 2H, J = 8.92 Hz), 7.84 (d, 2H, J = 8.93). 13_{C-NMR (100} MHz, DMSO-d6): δ 169.45, 139.19 (2C), 132.44, 124.36 (2C), 101.43 (1C missing). LRMS (ESI+):

m/z calcd for C8H7IN3O+ _[M-Cl]+ _{288.0, found 288.0}

(tert-Butoxycarbonyl)(3-(4-iodophenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (13).

Compound 12 (6.50 g, 20 mmol, 1 equiv.), NaHCO3 (1.90 g, 34 mmol, 1.7 equiv.) and DMAP (276 mg, 2.3 mmol, 10 mol%) were dissolved in dry THF (100 mL). The mixture was cooled down to 0 °C. To this mixture, Boc2O (7.40 g, 34 mmol, 1.7 equiv.) in THF (65 mL) was added dropwise and the reaction was stirred for 70 hours. The reaction was then quenched with 165 mL of aq. sat. NH4Cl solution, THF was removed under reduced pressure and the product was extracted with EtOAC (3 x 80 mL). The organic layer was then dried and the product was obtained as an orange solid (4.79 g, 62% yield) after flash purification (0-50% EtOAc in heptane). Rf = 0.58 (50% EtOAc in heptane). 1_{H-NMR (400 MHz, chloroform-d): δ 8.09 (s,} 1H), 8.00 (d, 2H, J = 8.94), 7.54 (d, 2H, J = 8.94), 1.52 (s, 9H). 13_{C-NMR (100 MHz, chloroform-} d): δ 174.64, 160.72, 139.78, 139.75 (2C), 122.76, 122.74 (2C), 99.45, 79.24, 28.23 (3C). LRMS (ESI+): m/z calcd for C13H14IN3O3+ _[M+H]+ _{387.01, found 387.9}

(tert-Butoxycarbonyl)(3-(4-(ethoxycarbonyl)phenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (14). Compound 13 (1.20 g, 3.1 mmol, 1 equiv.) was dissolved

in dry ethanol (10 mL) and NEt3 (10 mL). To this, PdCl2(PPh3)2 (324 mg, 0.46 mmol, 15 mol%) was added. The compound was allowed to react in a CO atmosphere at 60 °C for 2.5 hours. The reaction mixture was then allowed to cool down and was filtered over celite. The mixture was then concentrated and purified using flash chromatography (0-50% EtOAc in heptane) to obtain an orange solid (668 mg, 65% yield). Rf = 0.35 (50% EtOAc in heptane). 1_{H-NMR (400 MHz, chloroform-d): δ 8.32 (d, 2H, J = 8.87), 8.16} (s, 1H), 7.89 (d, 2H, J = 8.87), 4.45 (q, 2H, J = 7.14), 1.52 (s, 9H), 1.43 (t, 3H, J = 7.14). 13_C-NMR (100 MHz, chloroform-d): δ 174.88, 164.53, 160.95, 136.82, 134.93, 131.86 (2C), 121.63 (2C), 102.62, 79.36, 62.20, 28.36 (3C), 14.39. LRMS (ESI+): m/z calcd for C16H20N3O5+ _[M+H]+ _334.1, found 334.4.

163 Summary, Future Perspectives and Concluding Remarks

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(tert-Butoxycarbonyl)(3-(4-carboxyphenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (15).

Compound 14 (650 mg, 2.0 mmol, 1 equiv.) was dissolved in

THF (15 mL) and LiOH·H2O (246 mg, 5.9 mmol, 3 equiv.) was added to water (15 mL). The two solutions were mixed for 2.5 hours at room temperature. The reaction mixture was then diluted with water and extracted with EtOAc (3 x 30 mL). The water layer was then acidified to pH 2 with 1M HCl and again extracted with EtOAc (3 x 30 mL). The organic layers were combined and dried with MgSO4. The solvents were evaporated to yield a pure yellow compound (543 mg, 92% yield). Rf = 0.30 (10% MeOH, 0.5% AcOH in DCM). 1_{H-NMR (400 MHz, MeOH-d4): δ 8.46 (s, 1H), δ 8.32 (d, 2H, J =} 8.92), 8.11 (d, 2H, J = 8.92), 1.51 (s, 9H). 13_{C-NMR (100 MHz, MeOH-d4): δ 166.32, 159.21, 136.65,} 135.40, 131.29 (2C), 122.13 (2C), 108.99, 79.14, 27.11 (3C). HRMS (ESI+): m/z calcd for C14H15N3O5+ _[M+H]+ _{306.10900, found 306.10957}

(tert-Butoxycarbonyl)(3-(4-(prop-2-yn-1-ylcarbamoyl)phenyl)-1,2,3-oxadiazol-3-ium-5- yl)amide (16). Compound 15 (540 mg, 1.8 mmol, 1 equiv.)

was dissolved in DMF (20 mL). To this mixture was added HATU (738 mg, 2.2 mmol, 1.1 equiv.) and DiPEA (620 µL, 3.6 mmol, 2 equiv.) and was left to stir at room temperature for 5 minutes. Propargylamine (227 µL, 3.6 mmol, 2 equiv.) was added and stirred for 2 hours at room temperature. DMF was removed under reduced pressure and the product was obtained after flash chromatography (5% MeOH in DCM) to yield a yellow solid (349 mg, 58% yield). Rf = 0.24 (5% MeOH in DCM). 1_{H-NMR (400 MHz, MeOH-d4): δ 9.26 (t, 1H, J = 5.45), 8.60 (s, 1H), 8.21 (d,} 2H, J = 8.82), 8.14 (d, 2H, J = 8.82), 4.11 (dd, 2H, J = 5.45, 2.49), 3.18 (t, 1H, J = 2.49), 1.44 (s, 9H). 13_{C-NMR (100 MHz, MeOH-d4): δ 174.72, 164.83, 160.13, 137.82, 136.01, 129.53 (2C), 122.90} (2C), 81.34, 77.81, 73.67, 29.19, 28.48 (3C) (1C missing). HRMS (ESI+): m/z calcd for C17H19N4O4+ _{[M+H] 343.14008, found 343.14078. C17H18N4NaO4}+ _[M+Na]+ _{365.12203, found,} 365.12403.

5-Amino-3-(4-(prop-2-yn-1-ylcarbamoyl)phenyl)-1,2,3-oxadiazol-3-ium 2,2,2-trifluoro acetate (17). Compound 16 (349 mg, 1.1 mmol, 1 equiv.) was

dissolved in DCM (20 mL). TFA (2 mL) was added dropwise. The mixture was left to stir for 4 hours at room temperature. The DCM was evaporated and the TFA was fully removed with co-evaporation with DCM (3 x) to yield a red/brown powder (259 mg, 80% yield, contaminated with a HATU remainder). 1_{H-NMR (400 MHz, MeOH-d4): δ} 8.41 (s, 1H), 8.17 (d, 2H, J = 8.99), 8.12 (d, 2H, J = 8.99), 4.20 (d, 2H, J = 2.54), 2.65 (t, 1H, J = 2.54). 13_{C-NMR (100 MHz, MeOH-d4): δ 165.89, 140.19, 138.85, 129.28 (2C), 122.51 (2C), 78.91,} 71.02, 37.48, 28.77 (1C missing). HRMS (ESI+): m/z calcd for C12H11N4O2+ _[M-TFA]+ _243.08820, found 243.08871.

Fmoc-Orn(Cbz)-OtBu. Fmoc-Orn(Cbz)-OH (1.73 g, 3.7 mmol, 1.0 equiv.) was dissolved in

THF/Hexane (30 mL, 1:1). This was cooled down to 0 °C and afterwards tert-butyl 2,2,2-trichloroacetimidate (1.32 mL, 7.4 mmol, 2.0 equiv.) was added. This was stirred for 15 min and then BF3 etherate (228 uL, 1.8 mmol, 0.5 equiv.) was added to the mixture and the reaction was stirred overnight at room temperature. The product was obtained via column chromatography in a gradient of EtOAc in heptane (0% to 40%). Rf = 0.30 (30% EtOAc in heptane). 1_{H NMR (500} MHz, MeOH-d4) δ 7.79 (d, J = 7.5 Hz, 2H), 7.67 (t, J = 7.0 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.37 – 7.26 (m, 7H), 5.49 (s, 2H), 5.08 (s, 2H), 4.37 (qd, J = 10.6, 7.1 Hz, 2H), 4.22 (t, J = 6.8 Hz, 1H), 4.06 (dd, J = 8.6, 5.0 Hz, 1H), 3.15 (t, J = 6.7 Hz, 2H), 1.89 – 1.61 (m, 2H), 1.59 (d, J = 6.5 Hz, 2H), 1.46 (s, 9H). 13_{C NMR (126 MHz, MeOH-d4) δ 171.88, 157.50, 157.19, 143.92, 143.76, 141.18} (2C), 137.01, 128.06 (2C), 127.54 (2C), 127.38 (3C), 126.77 (2C), 124.86 (2C), 119.53 (2C), 81.36, 66.49, 65.96, 54.57, 47.04, 39.92, 28.55, 26.90 (3C), 26.00. HRMS (ESI+) m/z calcd for C32H36N2NaO6+ _[M+Na]+ _{567.24711, found 567.24580.}

Fmoc-Orn-OtBu (18). Fmoc-Orn(Cbz)-OtBu was dissolved in methanol (40 mL). To this

palladium on activated carbon (40 mg, 0.14 mmol) was added. The flask was then closed and H2 was bubbled through while stirring till completion. The product was filtrated over celite and obtained via column chromatography in 10% methanol in DCM (1.20 g, 80% yield over two steps). Rf = 0.23 (10% MeOH in DCM). 1_{H NMR (500 MHz, MeOH-d4) δ 7.78 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.8} Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 6.25 (s, 1H), 4.89 (s, 2H), 4.42 (dd, J = 10.6, 6.8 Hz, 1H), 4.31 (dd, J = 10.6, 6.8 Hz, 10H), 4.20 (t, J = 6.9 Hz, 1H), 4.06 (t, J = 8.8 Hz, 1H), 2.95 (t, J = 7.1 Hz, 1H), 1.95 – 1.83 (m, 1H), 1.74 (dt, J = 16.6, 8.9 Hz, 3H), 1.46 (s, 9H). 13_C NMR (126 MHz, MeOH-d4) δ 171.36, 157.29, 143.75 (2C), 141.20 (2C), 127.42 (2C), 126.79 (2C), 124.80 (2C), 119.57 (2C), 81.66, 66.59, 54.28, 46.99, 38.92, 28.15, 26.89 (3C), 23.85. HRMS (ESI+) m/z calcd for C24H31N2O4+ _[M+H]+ _{411.228383, found 411.22757. C24H30N2NaO4}+ _[M+Na]+ 433.21033, found 433.20943.

Fmoc-Orn(Im-alkyne)-OtBu (19). Compound 18 (410 mg, 1.0 mmol, 1 equiv.) was dissolved in

a mixture of DCM/sat. NaHCO3 (1:1, 20 mL) and cooled down to 0 °C. While stirring vigorously, triphosgene (99 mg, 0.33 mmol, 0.3 equiv.) dissolved in DCM (1 mL) was added dropwise. This was left to react for 10 min. Afterwards, 17 (356

mg, 1.0 mmol, 1 equiv.) dissolved in DCM (1 mL) was added dropwise at 0 °C and stirred for 1 hour. The reaction was then quenched with brine (20 mL) and extracted with EtOAc (3 x 30 mL). The product was obtained after purification with flash chromatography (2.5-5% MeOH in DCM) to yield an orange solid (122 mg, 18% yield). Rf = 0.22 (5% MeOH in DCM). 1_{H-NMR (400 MHz,} DMSO-d6): δ 8.26 (s, 1H), 8.12 (d, 2H, J = 8.88), 8.05 (d, 2H, J = 8.88), 7.78 (d, 2H, J = 7.53), 7.71 – 7.66 (m, 2H), 7.39 (t, 2H, J = 7.50), 7.32 (t, 2H, J = 7.39), 4.41 – 4.34 (m, 2H), 4.25 (d, J = 7.1 Hz, 1H), 4.22 (d, J = 2.5 Hz, 2H), 4.10 (dd, J = 8.7, 4.9 Hz, 1H), 3.25 (t, J = 6.2 Hz, 2H), 2.67 (t, J = 2.5 Hz, 1H), 1.93 – 1.81 (m, 1H), 1.78 – 1.69 (m, 1H), 1.68 – 1.60 (m, 2H), 1.48 (s, 9H). HRMS 164 Chapter 7

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7

(tert-Butoxycarbonyl)(3-(4-carboxyphenyl)-1,2,3-oxadiazol-3-ium-5-yl)amide (15).

Compound 14 (650 mg, 2.0 mmol, 1 equiv.) was dissolved in

THF (15 mL) and LiOH·H2O (246 mg, 5.9 mmol, 3 equiv.) was added to water (15 mL). The two solutions were mixed for 2.5 hours at room temperature. The reaction mixture was then diluted with water and extracted with EtOAc (3 x 30 mL). The water layer was then acidified to pH 2 with 1M HCl and again extracted with EtOAc (3 x 30 mL). The organic layers were combined and dried with MgSO4. The solvents were evaporated to yield a pure yellow compound (543 mg, 92% yield). Rf = 0.30 (10% MeOH, 0.5% AcOH in DCM). 1_{H-NMR (400 MHz, MeOH-d4): δ 8.46 (s, 1H), δ 8.32 (d, 2H, J =} 8.92), 8.11 (d, 2H, J = 8.92), 1.51 (s, 9H). 13_{C-NMR (100 MHz, MeOH-d4): δ 166.32, 159.21, 136.65,} 135.40, 131.29 (2C), 122.13 (2C), 108.99, 79.14, 27.11 (3C). HRMS (ESI+): m/z calcd for C14H15N3O5+ _[M+H]+ _{306.10900, found 306.10957}

(tert-Butoxycarbonyl)(3-(4-(prop-2-yn-1-ylcarbamoyl)phenyl)-1,2,3-oxadiazol-3-ium-5- yl)amide (16). Compound 15 (540 mg, 1.8 mmol, 1 equiv.)

was dissolved in DMF (20 mL). To this mixture was added HATU (738 mg, 2.2 mmol, 1.1 equiv.) and DiPEA (620 µL, 3.6 mmol, 2 equiv.) and was left to stir at room temperature for 5 minutes. Propargylamine (227 µL, 3.6 mmol, 2 equiv.) was added and stirred for 2 hours at room temperature. DMF was removed under reduced pressure and the product was obtained after flash chromatography (5% MeOH in DCM) to yield a yellow solid (349 mg, 58% yield). Rf = 0.24 (5% MeOH in DCM). 1_{H-NMR (400 MHz, MeOH-d4): δ 9.26 (t, 1H, J = 5.45), 8.60 (s, 1H), 8.21 (d,} 2H, J = 8.82), 8.14 (d, 2H, J = 8.82), 4.11 (dd, 2H, J = 5.45, 2.49), 3.18 (t, 1H, J = 2.49), 1.44 (s, 9H). 13_{C-NMR (100 MHz, MeOH-d4): δ 174.72, 164.83, 160.13, 137.82, 136.01, 129.53 (2C), 122.90} (2C), 81.34, 77.81, 73.67, 29.19, 28.48 (3C) (1C missing). HRMS (ESI+): m/z calcd for C17H19N4O4+ _{[M+H] 343.14008, found 343.14078. C17H18N4NaO4}+ _[M+Na]+ _{365.12203, found,} 365.12403.

5-Amino-3-(4-(prop-2-yn-1-ylcarbamoyl)phenyl)-1,2,3-oxadiazol-3-ium 2,2,2-trifluoro acetate (17). Compound 16 (349 mg, 1.1 mmol, 1 equiv.) was

dissolved in DCM (20 mL). TFA (2 mL) was added dropwise. The mixture was left to stir for 4 hours at room temperature. The DCM was evaporated and the TFA was fully removed with co-evaporation with DCM (3 x) to yield a red/brown powder (259 mg, 80% yield, contaminated with a HATU remainder). 1_{H-NMR (400 MHz, MeOH-d4): δ} 8.41 (s, 1H), 8.17 (d, 2H, J = 8.99), 8.12 (d, 2H, J = 8.99), 4.20 (d, 2H, J = 2.54), 2.65 (t, 1H, J = 2.54). 13_{C-NMR (100 MHz, MeOH-d4): δ 165.89, 140.19, 138.85, 129.28 (2C), 122.51 (2C), 78.91,} 71.02, 37.48, 28.77 (1C missing). HRMS (ESI+): m/z calcd for C12H11N4O2+ _[M-TFA]+ _243.08820, found 243.08871.

Fmoc-Orn(Cbz)-OtBu. Fmoc-Orn(Cbz)-OH (1.73 g, 3.7 mmol, 1.0 equiv.) was dissolved in

THF/Hexane (30 mL, 1:1). This was cooled down to 0 °C and afterwards tert-butyl 2,2,2-trichloroacetimidate (1.32 mL, 7.4 mmol, 2.0 equiv.) was added. This was stirred for 15 min and then BF3 etherate (228 uL, 1.8 mmol, 0.5 equiv.) was added to the mixture and the reaction was stirred overnight at room temperature. The product was obtained via column chromatography in a gradient of EtOAc in heptane (0% to 40%). Rf = 0.30 (30% EtOAc in heptane). 1_{H NMR (500} MHz, MeOH-d4) δ 7.79 (d, J = 7.5 Hz, 2H), 7.67 (t, J = 7.0 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.37 – 7.26 (m, 7H), 5.49 (s, 2H), 5.08 (s, 2H), 4.37 (qd, J = 10.6, 7.1 Hz, 2H), 4.22 (t, J = 6.8 Hz, 1H),

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