\\n Experimental Section\\n \\n Chemistry. Synthesis\\n Chemicals,\\ncommercially available,\\nwere purchased from Apollo Scientific, Sigma-Aldrich, Fluorochem,\\nor Manchester Organics and used without any further purification.\\nAll reactions were carried out using anhydrous solvents. Analytical\\nthin-layer chromatography (TLC) was performed on precoated TLC plates\\n(layer 0.20 mm silica gel 60 with fluorescent indicator (UV 254: Merck)).\\nThe TLC plates were air-dried and revealed under UV lamp (254/365\\nnm). Flash column chromatography was performed using prepacked silica\\ngel cartridges (230–400 mesh, 40–63 mm; SiliCycle) using\\na Teledyne ISCO Combiflash Companion or Combiflash Retrieve using\\nthe solvent mixtures stated for each synthesis as mobile phase. Liquid\\nchromatography–mass spectrometry (LC–MS) analyses were\\nperformed with either an Agilent HPLC 1100 series connected to a Bruker\\nDaltonics MicroTOF or an Agilent Technologies 1200 series HPLC connected\\nto an Agilent Technologies 6130 quadrupole spectrometer or a Waters\\n2795 connected to a Waters ZQ Micromass spectrometer; all instruments\\nwere connected to a diode array detector. All of the final compounds\\nused in all of the experiments were evaluated after preparative LC–MS\\nseparations with a Waters X-bridge C18 column (50 mm × 2.1 mm\\n× 3.5 mm particle size); flow rate, 0.5 mL/min with a mobile\\nphase of water/MeCN + 0.1% NH4OH or water/MeCN + 0.1% CHOOH;\\n95/5 water/MeCN was initially held for 0.5 min followed by a linear\\ngradient from 95/5 to 5/95 water/MeCN over 3.5 min which was then\\nheld for 2 min. The purity of all the compounds was evaluated using\\nthe analytical LC–MS system described before, and purity was\\n>95%. 1H NMR and 13C NMR spectra were recorded\\non a Bruker Avance II 500 spectrometer (1H at 500.1 MHz, 13C at 125.8 MHz) or on a Bruker DPX-400 spectrometer (1H at 400.1 MHz, 13C at 101 MHz). Chemical shifts\\n(δ) are expressed in ppm reported using residual solvent as\\nthe internal reference in all cases. Signal splitting patterns are\\ndescribed as singlet (s), doublet (d), triplet (t), multiplet (m),\\nor a combination thereof. Coupling constants (J)\\nare quoted to the nearest 0.1 Hz.
\\n \\n \\n General Method A\\n To a mixture of aryl bromide (1 equiv)\\nin dioxane (0.2 M), Pd(dppf)Cl2 (0.1 equiv), bis(pinacolato)diboron\\n(1.2 equiv), and KOAc (3 equiv) were added. The mixture was heated\\nunder microwave conditions at 140 °C for 40 min. Then aryl iodide 14 (1 equiv) and a degassed solution of K2CO3 2 M (2 equiv) were added. The reaction mixture was heated\\nin microwave at 120 °C for 30 min. The resulting mixture was\\nfiltered through Celite and washed several times with DCM. The organic\\nphase was washed with H2O and brine, dried over MgSO4, filtered, and evaporated in vacuum. The crude was purified\\nby flash column chromatography using a gradient from 0% to 20% of\\nMeOH in DCM to obtain the desired compound. Then the intermediate\\nwas taken up in a mixture 1:1 HCl 4 N in dioxane/DCM and stirred at\\nrt for 3 h. The solvents were evaporated to dryness to obtain the\\ndesired compound as hydrochloride salt.
\\n \\n \\n General Method B\\n To a solution of acid (1 equiv) in\\nDMF (0.2 M), HATU (1 equiv), HOAt (1 equiv), amine (1 equiv), and\\nDIPEA (5 equiv) were added. The reaction mixture was stirred at rt\\nfor 2 h. The solvent was evaporated under reduced pressure to give\\nthe corresponding crude, which was chromatographically purified to\\nyield the final compound.
\\n \\n \\n General Method C\\n A mixture of aldehyde (1 equiv), amine\\n(HCl salt, 1.1 equiv), and TEA (1.1 equiv) in DMF (0.2 M) was stirred\\nat rt. After 15 min, NaBH(OAc)3 (1.5 equiv) was added,\\nand the reaction was stirred overnight at rt under nitrogen. The solvent\\nwas evaporated under reduced pressure to give the corresponding crude,\\nwhich was purified accordingly to yield the desired compound.
\\n \\n \\n General\\nMethod D\\n To a stirred solution of oxalyl chloride\\n(1.5 equiv) in DCM (0.3 M), DMSO (2 equiv) was added dropwise at −78\\n°C. After 10 min at −78 °C, the alcohol starting\\nmaterial (1 equiv) in DCM (0.3 M) was added. The reaction was stirred\\nat −78 °C for 1 h, TEA (10 equiv) was added dropwise,\\nand temperature was increased to rt. After 1 h, the reaction mixture\\nwas diluted with DCM, washed with a saturated solution of ammonium\\nchloride. The organic phases were combined, washed with brine, dried\\nover MgSO4, filtered, and evaporated to dryness. Then the\\ncrude was dissolved in DMF (0.05 M); 1c (1 equiv) and\\nTEA (1.1 equiv) were added. After 15 min, NaBH(OAc)3 was\\nadded and the reaction mixture was stirred overnight at rt under nitrogen.\\nThe solvent was evaporated under reduced pressure to give the corresponding\\ncrude, which was purified by preparative HPLC to yield the desired\\ncompound.
\\n \\n \\n General Method E\\n To a solution of\\nthe alcohol starting\\nmaterial (1 equiv) and trimethylamine (1.5 equiv) in DCM (0.2 M),\\nmethansulfonyl chloride (1.2 equiv) was added at 0 °C. The reaction\\nmixture was stirred at 0 °C for 3 h. The reaction was quenched\\nwith a saturated solution of ammonium chloride and extracted with\\nDCM. The organic phases were combined, dried over MgSO4, filtered, and evaporated to dryness. The crude (1.5 equiv) was\\nsuspended in DMF (0.2 M), and 2b–d inhibitor derivative (1 equiv) and K2CO3 (3\\nequiv) were added. The mixture was heated overnight at 70 °C.\\nThe reaction mixture was diluted with water and extracted with DCM.\\nThe organic phases were combined, dried over MgSO4, filtered,\\nand evaporated to dryness. The crude was purified by flash column\\nchromatography using a gradient from 0% to 20% of MeOH in DCM to yield\\nthe desired compound.
\\n \\n \\n General Method F\\n A 1 M aqueous solution\\nof HCl (1 mL)\\nwas added to the VHL-acetal linker (0.06 mmol) in THF (1 mL). The\\nreaction mixture was heated at 50 °C for 2 h. The solvent was\\nevaporated under reduced pressure, and the product was extracted with\\nDCM/MeOH (9:1). The organic phases were combined, dried over MgSO4, filtered, and evaporated to dryness. The crude was used\\ndirectly without any further purification.
\\n \\n \\n General method G\\n Aldehyde (1 equiv) was taken up in t-BuOH and water\\n(4:1) (0.3 M). Then 2-methyl-2-butene 2\\nM in THF (4 equiv) was added, followed by sodium phosphate dibasic\\n(1 equiv) and sodium chlorite (2 equiv). The reaction mixture was\\nstirred at rt for 4 h. A 1 M aqueous solution of HCl was added. The\\nproduct was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThe crude was used directly without any further purification.
\\n \\n \\n 4-(3,5-Dimethoxy-4-(piperazin-1-ylmethyl)phenyl)-2-methyl-2,7-naphthyridin-1(2<italic>H</italic>)-one (<bold>1c</bold>)\\n Following general method\\nA, compound 1c was obtained from compound 12 and 14 as brown powder. Yield: 50 mg, 55%. 1H NMR (400 MHz, MeOD) δ: 9.67 (s, 1H), 8.76 (d, J = 6.9 Hz, 1H), 8.27 (s, 1H), 8.21 (d, J = 6.9 Hz,\\n1H), 6.95 (s, 2H), 4.62 (s, 2H), 4.03 (s, 6H), 3.81 (s, 3H), 3.73\\n(s, 8H). 13C NMR (101 MHz, CDCl3) δ: 161.5,\\n161.4, 148.6, 146.3, 145.5, 141.9, 139.7, 122.9, 117.9, 107.1, 105.8,\\n57.2, 50.8, 49.7, 41.9, 37.9. MS m/z calcd for C22H26N4O3 394.2, found 395.3 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((S)-2-(2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide\\n(<bold>5</bold>)\\n Following general method B, compound 5 was obtained from 19 and 2a (synthesized\\naccordingly to literature61). The crude\\nwas purified by HPLC using a gradient of 5% to 95% v/v acetonitrile\\nin 0.1% aqueous solution of formic acid to obtain 5 as\\nwhite powder. Yield: 6.3 mg, 44%. 1H NMR (400 MHz, MeOD)\\nδ: 9.44 (s, 1H), 8.80 (s, 1H), 8.59 (d, J =\\n5.5 Hz, 1H), 7.66 (s, 1H), 7.54 (d, J = 5.9 Hz, 1H),\\n7.37 (q, J = 7.8 Hz, 4H), 6.73 (s, 2H), 4.66 (s,\\n1H), 4.52–4.26 (m, 4H), 4.05–3.95 (m, 4H), 3.86–3.72\\n(m, 8H), 3.69–3.59 (m, 9H), 2.96–2.73 (m, 10H), 2.40\\n(s, 3H), 2.21–2.14 (m, 1H), 2.06–1.97 (m, 1H), 0.99\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 174.3, 172.0,\\n171.7, 163.0, 161.0, 152.8, 151.7, 151.2, 149.1, 143.5, 140.3, 139.0,\\n133.3, 131.6, 130.5, 130.4, 129.5, 129.0, 121.7, 119.2, 119.0, 106.6,\\n72.2, 71.3, 71.1, 71.0, 69.1, 60.9, 58.2, 58.1, 57.9, 56.6, 52.8,\\n52.4, 43.7, 39.0, 37.4, 37.2, 27.0, 15.8. HRMS m/z calcd for C50H64N8O9S 952.45, found 953.4728 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(<italic>tert</italic>-Butyl)-17-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide\\n(<bold>6</bold>)\\n Following general method B, compound 6 was obtained from 20 and 2a (synthesized\\naccordingly to literature61). The crude\\nwas purified by HPLC using a gradient of 5% to 95% v/v acetonitrile\\nin 0.1% aqueous solution of formic acid to obtain 6 as\\nwhite powder. Yield: 4.9 mg, 30%. 1H NMR (400 MHz, MeOD)\\nδ: 9.44 (s, 1H), 8.81 (s, 1H), 8.60 (d, J =\\n5.6 Hz, 1H), 7.67 (s, 1H), 7.54 (d, J = 5.6 Hz, 1H),\\n7.37 (q, J = 9.0 Hz, 4H), 6.73 (s, 2H), 4.63 (s,\\n1H), 4.53–4.26 (m, 4H), 4.02–3.94 (m, 4H), 3.84–3.72\\n(m, 8H), 3.66–3.50 (m, 17H), 2.89–2.67 (m, 10H), 2.41\\n(s, 3H), 2.20–2.14 (m, 1H), 2.06–1.97 (m, 1H), 0.98\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 174.4, 172.0,\\n171.7, 163.0, 161.0, 152.9, 151.7, 151.2, 149.1, 143.5, 140.3, 139.0,\\n138.6, 133.4, 131.5, 130.5, 130.4, 129.5, 129.0, 121.7, 119.2, 119.1,\\n106.6, 72.3, 71.7, 71.6, 71.5, 71.4, 71.1, 71.0, 68.9, 60.9, 58.2,\\n58.0, 56.6, 52.8, 43.7, 39.0, 37.4, 37.1, 27.0, 15.9. HRMS m/z calcd for C54H72N8O11S 1040.50, found 1041.5287 [M + H+].
\\n \\n \\n 2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)-<italic>N</italic>-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide\\n(<bold>7</bold>)\\n Following general method B, compound 7 was obtained from compound 19 and 3a (synthesized accordingly to literature73). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid to obtain compound 7 as yellow powder. Yield: 5.6 mg, 33%. 1H NMR\\n(400 MHz, MeOD) δ: 9.54 (d, J = 0.8 Hz, 1H),\\n8.70 (d, J = 6.0 Hz, 1H), 8.11 (s, 1NH), 7.84 (s,\\n1H), 7.73 (dd, J = 0.6, 6.0 Hz, 1H), 7.55–7.49\\n(m, 1H), 7.10–7.03 (m, 2H), 6.87 (s, 2H), 5.08 (dd, J = 5.5, 12.7 Hz, 1H), 4.38 (s, 2H), 4.05 (s, 2H), 3.96\\n(s, 6H), 3.77–3.67 (m, 9H), 3.51–3.46 (m, 4H), 3.39\\n(s, 4H), 3.20 (s, 4H), 3.01 (t, J = 4.8 Hz, 2H),\\n2.94–2.67 (m, 4H). 13C NMR (101 MHz, MeOD) δ:\\n174.6, 173.4, 171.6, 170.6, 169.1, 162.7, 161.1, 150.9, 149.8, 147.9,\\n144.0, 140.1, 137.2, 133.9, 121.8, 119.6, 118.5, 117.9, 112.2, 111.4,\\n106.7, 71.7, 71.3, 71.0, 67.7, 57.5, 56.8, 51.8, 51.1, 50.2, 50.0,\\n42.6, 39.4, 37.5, 32.2, 23.8. HRMS m/z calcd for C43H50N8O10 838.36, found 839.3796 [M + H+].
\\n \\n \\n 14-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-<italic>N</italic>-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)-3,6,9,12-tetraoxatetradecanamide\\n(<bold>8</bold>)\\n Following general method B, compound 8 was obtained from compound 20 and 3a (synthesized accordingly to literature73). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid to obtain compound 8 as yellow powder. Yield: 10 mg, 54%. 1H NMR (400\\nMHz, MeOD) δ: 9.53 (s, 1H), 8.70 (d, J = 5.7\\nHz, 1H), 8.15 (s, 1NH), 7.77 (s, 1H), 7.64 (d, J =\\n5.7 Hz, 1H), 7.59–7.53 (m, 1H), 7.17–7.13 (m, 1H), 7.08–7.04\\n(m, 1H), 6.86 (s, 2H), 5.07 (dd, J = 5.5, 12.5 Hz,\\n1H), 4.29 (s, 2H), 4.01 (s, 2H), 3.96 (s, 6H), 3.72–3.63 (m,\\n17H), 3.54 (s, 4H), 3.25 (s, 4H), 3.02 (s, 4H), 2.94–2.67 (m,\\n6H). 13C NMR (101 MHz, MeOD) δ: 174.6, 173.5, 171.5,\\n170.6, 169.2, 163.0, 161.0, 151.7, 151.2, 148.0, 143.4, 139.8, 139.2,\\n137.3, 133.9, 121.7, 119.1, 118.7, 118.1, 112.1, 111.4, 108.1, 106.7,\\n72.0, 71.4, 71.1, 68.5, 57.6, 56.8, 52.4, 51.6, 50.2, 49.9, 42.5,\\n39.4, 37.5, 32.2, 23.8. HRMS m/z calcd for C47H58N8O12 926.42, found 927.4396 [M + H+].
\\n \\n \\n <italic>N</italic>-(4-(<italic>N</italic>-(3-Chloro-1<italic>H</italic>-indol-7-yl)sulfamoyl)benzyl)-2-(2-(2-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)acetamide\\n(<bold>9</bold>)\\n Following general method B, compound 9 was obtained from compound 19 and 4a (synthesis detailed in the Supporting Information). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid to obtain compound 9 as white powder. Yield: 10.8 mg, 34%. 1H NMR\\n(400 MHz, MeOD) δ: 9.54 (s, 1H), 8.69 (d, J = 5.8 Hz, 1H), 8.27 (s, 1NH), 7.75 (s, 1H), 7.68–7.62 (m,\\n3H), 7.39 (d, J = 8.3 Hz, 2H), 7.33 (dd, J = 0.9, 8.1 Hz, 1H), 7.26 (s, 1H), 6.91 (t, J = 7.8 Hz, 1H), 6.85 (s, 2H), 6.70 (d, J = 7.7 Hz,\\n1H), 4.49 (s, 2H), 4.28 (s, 2H), 4.11 (s, 2H), 3.94 (s, 6H), 3.76–3.66\\n(m, 7H), 3.63 (t, J = 5.3 Hz, 2H), 3.22 (s, 4H),\\n2.88 (s, 4H), 2.73 (t, J = 5.1 Hz, 2H). 13C NMR (101 MHz, MeOD) δ: 173.0, 166.4, 163.0, 161.0, 151.7,\\n151.2, 145.5, 143.3, 140.0, 139.4, 139.1, 132.0, 128.8, 128.7, 128.2,\\n123.4, 122.7, 121.6, 120.9, 119.4, 119.1, 118.8, 117.0, 107.5, 106.7,\\n106.2, 71.9, 71.4, 71.3, 68.7, 57.6, 56.8, 52.5, 51.3, 49.9, 43.0,\\n37.5. HRMS m/z calcd for C43H48ClN7O8S 857.30, found 858.3111\\n[M + H+].
\\n \\n \\n <italic>N</italic>-(4-(<italic>N</italic>-(3-Chloro-1<italic>H</italic>-indol-7-yl)sulfamoyl)benzyl)-14-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecanamide\\n(<bold>10</bold>)\\n Following general method B, compound 10 was obtained from compound 20 and 4a (synthesis detailed in the Supporting Information). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid to obtain compound 10 as white powder. Yield: 11.1 mg, 37%. 1H NMR\\n(400 MHz, MeOD) δ: 9.54 (d, J = 0.6 Hz, 1H),\\n8.69 (d, J = 5.7 Hz, 1H), 8.30 (s, 1NH), 7.75 (s,\\n1H), 7.68–7.62 (m, 3H), 7.41 (d, J = 8.2 Hz,\\n2H), 7.34 (dd, J = 0.8, 8.1 Hz, 1H), 7.27 (s, 1H),\\n6.92 (t, J = 7.8 Hz, 1H), 6.84 (s, 2H), 6.72 (dd, J = 0.9, 7.5 Hz, 1H), 4.50 (s, 2H), 4.26 (s, 2H), 4.09 (s,\\n2H), 3.94 (s, 6H), 3.73–3.67 (m, 7H), 3.65–3.59 (m,\\n4H), 3.58–3.53 (m, 6H), 3.22 (s, 4H), 2.96 (s, 4H), 2.82 (t, J = 5.3 Hz, 2H). 13C NMR (101 MHz, MeOD) δ:\\n173.1, 166.7, 163.1, 161.0, 151.7, 151.2, 145.5, 143.4, 139.7, 139.2,\\n139.0, 131.9, 128.9, 128.6, 128.2, 123.4, 122.7, 121.6, 121.0, 119.3,\\n119.2, 118.9, 117.0, 108.0, 106.7, 106.2, 72.0, 71.3, 71.3, 71.2,\\n68.3, 57.5, 56.8, 52.3, 51.5, 49.9, 42.9, 37.6. HRMS m/z calcd for C47H56ClN7O10S 945.35, found 946.3676 [M + H+].
\\n \\n \\n <italic>tert</italic>-Butyl 4-(4-Bromo-2,6-dimethoxybenzyl)piperazine-1-carboxylate\\n(<bold>12</bold>)\\n Following general method C, compound 12 was obtained from 4-bromo-2,6-dimethoxybenzaldehyde\\n(11) and boc-piperazine (both commercially available).\\nThe reaction was quenched with saturated solution of NaHCO3, extracted with DCM, washed with water and brine. The organic phases\\nwere combined, dried over MgSO4, filtered, and evaporated\\nto dryness to give the desired compound without any further purification\\nas sticky oil. Yield: 495 mg, 97%. 1H NMR (500 MHz, CDCl3) δ: 6.69 (s, 2H), 3.78 (s, 6H), 3.62 (s, 2H), 3.38\\n(t, J = 5.0 Hz, 4H), 2.41 (t, J =\\n4.4 Hz, 4H), 1.43 (s, 9H). 13C NMR (125 MHz, CDCl3) δ: 159.9, 154.7, 122.8, 107.7, 79.8, 56.1, 51.8, 48.5, 31.0,\\n28.5. MS m/z calcd for C18H27BrN2O4 414.12, found 415.2 [M\\n+ H+].
\\n \\n \\n 4-Iodo-2-methyl-2,7-naphthyridin-1(2<italic>H</italic>)-one\\n(<bold>14</bold>)\\n A mixture of 4-iodo-2,7-naphthyridin-1(2H)-one 13 (synthesized accordingly to literature74) (1 equiv) and NaH (2 equiv) was stirred 30\\nmin at 0 °C in DMF (0.2 M). Then CH3I (1.6 equiv)\\nwas added and the reaction mixture was stirred at 0 °C for 5\\nh. Water was added, and the precipitate formed was filtered and dried\\nin vacuum. Yield: 300 mg, 95%. 1H NMR (400 MHz, DMSO) δ:\\n9.27 (s, 1H), 8.82 (d, J = 5.6 Hz, 1H), 8.26 (s,\\n1H), 7.47 (d, J = 5.5 Hz, 1H), 3.52 (s,3H). 13C NMR (101 MHz, DMSO) δ: 160.3, 152.0, 150.3, 144.8,\\n143.0, 122.4, 120.7, 67.9, 36.2. MS m/z calcd for C9H7IN2O 285.96, found\\n287.1 [M + H+].
\\n \\n \\n <italic>tert</italic>-Butyl\\n2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)acetate\\n(<bold>17</bold>)\\n Following general method D, compound 17 was obtained from tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate 15 and 1c after purification by HPLC using a\\ngradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of\\nammonia as white powder. Yield: 8.8 mg, 32%. 1H NMR (500\\nMHz, CDCl3) δ: 9.69 (d, J = 0.8\\nHz, 1H), 8.70 (d, J = 5.7 Hz, 1H), 7.43 (dd, J = 0.8, 5.6 Hz, 1H), 7.26 (s, 1H), 6.52 (s, 2H), 4.00 (s,\\n2H), 3.81 (s, 6H), 3.70–3.60 (m, 11H), 2.65–2.56 (m,\\n10H), 1.46 (s, 9H). 13C NMR (125 MHz, CDCl3)\\nδ: 170.0, 161.8, 160.0, 152.0, 151.3, 142.1, 136.1, 135.5, 120.8,\\n118.4, 117.8, 113.9, 105.7, 81.9, 71.0, 70.7, 69.3, 58.1, 56.2, 53.7,\\n52.6, 48.9, 37.4, 28.4. MS m/z calcd\\nfor C32H44N4O7 596.32,\\nfound 597.34 [M + H+].
\\n \\n \\n <italic>tert</italic>-Butyl\\n14-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecanoate\\n(<bold>18</bold>)\\n Following general method D, compound 18 was obtained from tert-butyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate 16 and 1c after purification by HPLC using a\\ngradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of\\nammonia as white powder. Yield: 10.7 mg, 34%. 1H NMR (500\\nMHz, CDCl3) δ: 9.69 (d, J = 0.8\\nHz, 1H), 8.70 (d, J = 5.6 Hz, 1H), 7.43 (d, J = 5.6 Hz, 1H), 7.26 (s, 1H), 6.52 (s, 2H), 4.00 (s, 2H),\\n3.81 (s, 6H), 3.73–3.57 (m, 19H), 2.67–2.55 (m, 10H),\\n1.46 (s, 9H). 13C NMR (125 MHz, CDCl3) δ:\\n170.0, 161.8, 160.0, 152.0, 151.3, 142.1, 136.2, 120.8, 118.4, 117.8,\\n105.7, 81.9, 71.1, 70.9, 70.7, 69.4, 69.2, 58.0, 56.3, 53.8, 52.4,\\n48.9, 37.5, 28.5. MS m/z calcd for\\nC36H52N4O9 684.37, found\\n685.39 [M + H+].
\\n \\n \\n 2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)acetic\\nAcid (<bold>19</bold>)\\n A mixture of compound 17 (0.015 mmol), TFA (0.5 mL), and DCM (0.5 mL) was stirred at rt for\\n3 h. Then the solvent was evaporated; the crude was dried under high\\npressure overnight and used directly in the next step without any\\nfurther purification. Quantitative yield. MS m/z calcd for C28H36N4O7 540.26, found 541.4 [M + H+].
\\n \\n \\n 17-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-3,6,9,12,15-pentaoxaheptadecanoic\\nAcid (<bold>20</bold>)\\n A mixture of compound 18 (0.034 mmol), TFA (0.5 mL), and DCM (0.5 mL) was stirred at rt for\\n3 h. Then the solvent was evaporated; the crude was dried under high\\npressure overnight and used directly without any further purification.\\nQuantitative yield. MS m/z calcd\\nfor C34H48N4O10 672.34,\\nfound 673.36 [M + H+].
\\n \\n \\n (2<italic>R</italic>,4<italic>S</italic>)-1-((<italic>R</italic>)-2-(2-(2-((5-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)pentyl)oxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide\\n(<bold>23</bold>)\\n A mixture of 35 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThe crude was dissolved in DCE, and a mixture of 1c (1\\nequiv) and TEA (1.1 equiv) in DCE (0.02 M) was added. After 15 min,\\nNaBH(OAc)3 was added and the reaction mixture was stirred\\nat rt for 4 h under nitrogen. The solvent was evaporated under reduced\\npressure to give the corresponding crude, which was purified by HPLC\\nusing a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution\\nof ammonia. Compound 23 was obtained as a white powder.\\nYield: 6.9 mg, 54%. 1H NMR (400 MHz, CDCl3)\\nδ: 9.68 (s, 1H), 8.69 (d, J = 5.6 Hz, 1H),\\n8.66 (s, 1H), 7.41 (dd, J = 0.6, 5.6 Hz, 1H), 7.37–7.32\\n(m, 4H), 7.27 (s, 1H), 6.53 (s, 2H), 4.72 (t, J =\\n8.0 Hz, 1H), 4.59–4.49 (m, 3H), 4.35–4.29 (m, 1H), 4.07–3.95\\n(m, 3H), 3.85–3.80 (m, 8H), 3.67–3.55 (m, 8H), 3.43\\n(t, J = 6.4 Hz, 2H), 2.74–2.37 (m, 14H), 2.15–2.09\\n(m, 1H), 1.60–1.47 (m, 4H), 1.36–1.27 (m, 2H), 0.94\\n(s, 9H). 13C NMR (101 MHz, CDCl3) δ: 171.5,\\n170.9, 170.5, 168.6, 161.6, 159.8, 151.8, 151.1, 150.4, 148.6, 141.9,\\n138.3, 136.0, 131.7, 131.1, 129.6, 128.3, 120.6, 118.1, 117.6, 105.5,\\n71.4, 71.3, 70.6, 70.1, 58.6, 58.2, 57.2, 56.9, 56.1, 52.5, 51.7,\\n48.6, 43.4, 37.3, 36.1, 35.2, 29.4, 26.6, 26.0, 24.2, 16.2. HRMS m/z calcd for C53H70N8O9S 994.50, found 995.5178 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>26</bold>)\\n A mixture of 40 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, compound 26 was synthesized\\nfrom the aldehyde derivative and 1c. The solvent was\\nremoved in vacuum to give the corresponding crude, which was purified\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of formic acid to yield compound 26 as white\\npowder. Yield: 7 mg, 45%. 1H NMR (400 MHz, MeOD) δ:\\n9.54 (s, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.8 Hz,\\n1H), 8.58 (s, 1NH), 7.75 (s, 1H), 7.63 (dd, J = 0.6,\\n5.7 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.10–7.04\\n(m, 2H), 6.79 (s, 2H), 4.78 (s, 1H), 4.66 (t, J =\\n8.4 Hz, 1H), 4.55–4.42 (m, 3H), 4.32–4.23 (m, 2H), 3.97–3.77\\n(m, 14H), 3.73–3.66 (m, 7H), 2.85–2.68 (m, 10H), 2.52\\n(s, 3H), 2.30–2.24 (m, 1H), 2.16–2.09 (m, 1H), 1.46–1.27\\n(m, 4H), 1.07 (s, 9H). 13C NMR (101 MHz, MeOD) δ:\\n174.2, 171.7, 171.5, 171.3, 170.2, 163.0, 161.0, 158.0, 152.8, 151.7,\\n151.2, 149.1, 143.6, 138.9, 138.1, 133.5, 132.9, 130.0, 128.4, 122.9,\\n121.7, 119.2, 113.8, 106.5, 80.3, 78.0, 71.8, 71.4, 71.0, 70.8, 69.4,\\n69.3, 60.8, 58.7, 58.3, 58.2, 56.5, 53.3, 53.0, 39.5, 39.0, 37.4,\\n37.3, 26.9, 16.0, 14.1, 14.0, 13.9. MS m/z calcd for C54H69FN8O10S 1040.48, found 1041.4430 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-<italic>N</italic>-(2-(2-(2-(2-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>27</bold>)\\n A mixture of 42 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, from the aldehyde derivative and 1c compound 27 was obtained after purification\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of formic acid as white powder. Yield: 11 mg, 44%. 1H NMR (400 MHz, MeOD) δ: 9.55 (s, 1H), 8.90 (s, 1H), 8.69 (d, J = 5.8 Hz, 1H), 8.58 (s, 1NH), 7.75 (s, 1H), 7.63 (d, J = 5.6 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H),\\n7.10–7.03 (m, 2H), 6.79 (s, 2H), 4.71–4.63 (m, 2H),\\n4.56–4.42 (m, 3H), 4.33–4.23 (m, 2H), 3.98–3.66\\n(m, 21H), 2.86–2.69 (m, 10H), 2.52 (s, 3H), 2.30–2.23\\n(m, 1H), 2.16–2.08 (m, 1H), 1.70–1.55 (m, 4H), 1.06\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 174.1, 171.5,\\n170.1, 167.1, 163.0, 161.0, 158.1, 152.8, 151.7, 151.2, 149.1, 143.5,\\n138.9, 138.1, 133.4, 132.9, 130.1, 128.4, 122.8, 121.7, 120.8, 119.2,\\n119.2, 113.8, 106.5, 71.8, 71.4, 71.0, 70.8, 69.4, 69.2, 60.8, 59.5,\\n58.3, 58.1, 56.5, 53.2, 53.0, 39.5, 38.9, 37.7, 37.4, 26.8, 18.1,\\n18.0, 16.0, 14.8. HRMS m/z calcd\\nfor C55H69N9O10S 1047.49,\\nfound 1048.4493 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((S)-2-Acetamido-3,3-dimethylbutanoyl)-<italic>N</italic>-(2-(2-(2-(2-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>28</bold>)\\n A mixture of 44 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, from the aldehyde derivative and 1c compound 28 was obtained after purification\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of formic acid as white powder. Yield: 3.3 mg, 17%. 1H NMR (400 MHz, MeOD) δ: 9.55 (d, J = 0.7 Hz, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.9 Hz,\\n1H), 7.76 (s, 1H), 7.64 (dd, J = 0.7, 5.9 Hz, 1H),\\n7.52 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 1.7, 7.7 Hz, 1H),\\n6.79 (s, 2H), 4.65–4.60 (m, 2H), 4.54–4.42 (m, 3H),\\n4.28–4.25 (m, 2H), 3.96–3.65 (m, 21H), 2.86–2.67\\n(m, 10H), 2.52 (s, 3H), 2.27–2.21 (m, 1H), 2.15–2.08\\n(m, 1H), 2.04 (s, 3H), 1.05 (s, 9H). 13C NMR (101 MHz,\\nMeOD) δ: 174.4, 173.1, 172.3, 163.1, 161.0, 158.0, 152.8, 151.7,\\n151.2, 149.1, 143.6, 138.9, 133.5, 132.9, 130.0, 128.4, 122.8, 121.7,\\n119.2, 113.7, 106.5, 71.8, 71.4, 71.1, 70.8, 69.4, 69.2, 60.7, 59.2,\\n58.2, 58.0, 56.5, 53.2, 53.0, 49.8, 39.4, 38.9, 37.4, 36.5, 27.0,\\n22.3, 16.0. HRMS m/z calcd for C52H68N8O10S 996.48, found\\n997.5008 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((14-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>29</bold>)\\n A mixture of 41 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, from the aldehyde derivative and 1c compound 29 was obtained after purification\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of ammonia as white powder. Yield: 9.5 mg, 46%. 1H NMR (400 MHz, MeOD) δ: 9.54 (s, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.64 (dd, J = 0.6, 5.9 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.10–7.03\\n(m, 2H), 6.77 (s, 2H), 4.78 (s, 1H), 4.69–4.63 (m, 1H), 4.55–4.42\\n(m, 3H), 4.31–4.23 (m, 2H), 3.97–3.57 (m, 29H), 2.71–2.60\\n(m, 10H), 2.52 (s, 3H), 2.30–2.24 (m, 1H), 2.17–2.08\\n(m, 1H), 1.43–1.29 (m, 4H), 1.07 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 174.2, 171.6, 171.3, 163.0, 161.0, 158.0,\\n152.8, 151.7, 151.2, 149.1, 143.6, 138.8, 137.3, 133.5, 132.9, 130.0,\\n128.4, 122.8, 121.7, 119.4, 119.3, 113.8, 113.2, 106.5, 80.3, 78.0,\\n73.7, 71.8, 71.6, 71.5, 71.4, 71.0, 70.8, 69.6, 69.4, 62.2, 60.8,\\n58.7, 58.6, 58.2, 56.3, 54.0, 53.3, 39.5, 38.9, 37.4, 37.3, 26.9,\\n16.0, 14.1, 14.0, 13.9. HRMS m/z calcd for C58H77FN8O12S 1128.54, found 1129.5445 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-<italic>N</italic>-(2-((14-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>30</bold>)\\n A mixture of 43 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, from the aldehyde derivative and 1c compound 30 was obtained after purification\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of ammonia as white powder. Yield: 9 mg, 39%. 1H NMR (400 MHz, MeOD) δ: 9.55 (d, J = 0.7\\nHz, 1H), 8.89 (s, 1H), 8.70 (d, J = 5.9 Hz, 1H),\\n7.74 (s, 1H), 7.64 (dd, J = 0.7, 5.8 Hz, 1H), 7.49\\n(d, J = 7.7 Hz, 1H), 7.09–7.02 (m, 2H), 6.76\\n(s, 2H), 4.69–4.63 (m, 2H), 4.55–4.42 (m, 3H), 4.34–4.23\\n(m, 2H), 3.95 (t, J = 4.5 Hz, 2H), 3.89 (s, 6H),\\n3.84–3.75 (m, 6H), 3.73–3.70 (m, 5H), 3.69–3.58\\n(m, 9H), 2.68–2.56 (m, 10H), 2.52 (s, 3H), 2.32–2.22\\n(m, 1H), 2.17–2.09 (m, 1H), 1.71–1.55 (m, 4H), 1.06\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 174.1, 171.5,\\n167.2, 163.1, 161.0, 158.1, 152.8, 151.7, 151.2, 149.2, 143.7, 138.8,\\n137.2, 133.5, 133.0, 130.2, 128.4, 122.8, 121.7, 120.7, 119.5, 119.3,\\n113.9, 113.7, 106.5, 71.8, 71.6, 71.6, 71.4, 71.1, 70.9, 69.7, 69.4,\\n60.8, 59.5, 58.7, 58.1, 56.3, 54.2, 53.4, 39.6, 38.9, 37.4, 37.3,\\n26.8, 18.1, 18.0, 16.0, 14.9. HRMS m/z calcd for C59H77N9O12S 1035.54, found 1036.5169 [M + H+].
\\n \\n \\n 14-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)-<italic>N</italic>-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)-3,6,9,12-tetraoxatetradecanamide\\n(<bold>31</bold>)\\n A mixture of 45 (1 equiv),\\nosmium tetroxide 4% in H2O (0.2 equiv), sodium periodate\\n(4 equiv), pyridine (2 equiv) in dioxane/H2O (3:1) was\\nstirred at rt for 48 h. Then H2O was added to the reaction\\nand the product was extracted with DCM. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThen, following general method A, from the aldehyde derivative and 1c compound 31 was obtained after purification\\nby HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous\\nsolution of formic acid as white powder. Yield: 3.7 mg, 17%. 1H NMR (400 MHz, MeOD) δ: 9.55 (d, J = 0.7 Hz, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.8 Hz,\\n1H), 7.76 (s, 1H), 7.64 (dd, J = 0.6, 5.8 Hz, 1H),\\n7.52 (d, J = 7.7 Hz, 1H), 7.08–7.01 (m, 2H),\\n6.81 (s, 2H), 4.65–4.60 (m, 2H), 4.53–4.41 (m, 3H),\\n4.27–4.25 (m, 2H), 4.02–3.61 (m, 29H), 2.96–2.76\\n(m, 10H), 2.51 (s, 3H), 2.27–2.21 (m, 1H), 2.15–2.08\\n(m, 1H), 2.04 (s, 3H), 1.06 (s, 9H). 13C NMR (101 MHz,\\nMeOD) δ: 174.4, 173.1, 172.3, 163.0, 161.0, 158.0, 152.8, 151.8,\\n151.3, 151.2, 149.1, 143.5, 139.0, 133.5, 132.8, 130.0, 128.4, 122.8,\\n121.7, 119.2, 119.1, 113.7, 106.6, 71.9, 71.6, 71.5, 71.4, 71.1, 70.9,\\n69.4, 60.7, 59.2, 58.1, 58.0, 56.6, 52.8, 49.8, 39.4, 38.9, 37.4,\\n36.5, 27.0, 22.3, 16.0. HRMS m/z calcd for C56H76N8O12S 1084.53, found 1085.5658 [M + H+].
\\n \\n \\n 2-(2-(Hex-5-en-1-yloxy)ethoxy)ethan-1-ol\\n(<bold>33</bold>)\\n To a suspension of NaH (2.5 equiv) in DMF\\n(1 M) and THF (1 M),\\ndiethylene glycol (5 equiv) was added at 0 °C under nitrogen.\\nAfter 45 min 6-bromo-1-hexene (1 equiv) was added dropwise at 0 °C.\\nThen the ice bath was removed, and the reaction mixture was stirred\\novernight at rt under nitrogen. Distillate water was added, and the\\nreaction mixture was acidified with HCl 1 M up to pH 2. The product\\nwas extracted with CHCl3. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThe resulting oil was purified by flash column chromatography using\\na gradient from 50% to 100% of ethyl acetate in heptane to obtain\\nthe desired compound 33 as an oil. Yield: 552 mg, 49%. 1H NMR (400 MHz, CDCl3) δ: 5.85–5.74\\n(m, 1H), 5.03–4.96 (m, 1H), 4.96–4.91 (m, 1H), 3.75–3.56\\n(m, 8H), 3.46 (t, J = 6.7 Hz, 2H), 2.09–2.03\\n(m, 2H), 1.65–1.56 (m, 2H), 1.48–1.39 (m, 2H). 13C NMR (101 MHz, CDCl3) δ: 138.8, 114.7,\\n72.6, 71.5, 70.6, 70.3, 62.0, 33.6, 29.2, 25.5.
\\n \\n \\n 2-(2-(Hex-5-en-1-yloxy)ethoxy)acetic\\nAcid (<bold>34</bold>)\\n A mixture of 30 (1 equiv), BAIB (2.2\\nequiv), TEMPO (0.22 equiv)\\nin ACN/H2O (1:1) (0.5 M) was stirred overnight at rt. The\\nday after the solvent was evaporated, the crude was resuspended in\\nDCM and washed with H2O. The organic phases were combined,\\ndried over MgSO4, filtered, and evaporated to dryness.\\nThe resulting product was purified by flash column chromatography\\nusing a gradient from 0% to 20% of MeOH in DCM to obtain compound 34 as an oil. Yield: 472 mg, 81%. 1H NMR (400 MHz,\\nCDCl3) δ: 5.84–5.73 (m, 1H), 5.03–4.97\\n(m, 1H), 4.96–4.92 (m, 1H), 4.15 (s, 2H), 3.76–3.73\\n(m, 2H), 3.63–3.59 (m, 2H), 3.52 (t, J = 6.6\\nHz, 2H), 2.10–2.03 (m, 2H), 1.65–1.57 (m, 2H), 1.49–1.40\\n(m, 2H). 13C NMR (101 MHz, CDCl3) δ: 173.2,\\n138.7, 114.8, 71.7, 71.6, 69.7, 69.1, 33.6, 28.9, 25.4.
\\n \\n \\n (2<italic>R</italic>,4<italic>S</italic>)-1-((<italic>R</italic>)-2-(2-(2-(Hex-5-en-1-yloxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide\\n(<bold>35</bold>)\\n Following general method B, compound 35 was obtained from compounds 34 and 2a (synthesized accordingly to literature61). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid to obtain 35 as white powder. Yield: 14.7 mg, 30%. 1H NMR\\n(400 MHz, CDCl3) δ: 8.64 (s, 1H), 7.28–7.23\\n(m, 4H), 5.74–5.63 (m, 1H), 4.93–4.82 (m, 2H), 4.65\\n(t, J = 7.9 Hz, 1H), 4.49–4.21 (m, 4H), 4.03–3.85\\n(m, 3H), 3.59–3.47 (m, 5H), 3.40–3.34 (m, 2H), 2.47–2.39\\n(m, 4H), 2.06–1.91 (m, 3H), 1.54–1.45 (m, 2H), 1.37–1.29\\n(m, 2H), 0.87 (s, 9H). 13C NMR (101 MHz, CDCl3) δ: 171.6, 170.8, 150.7, 148.1, 138.7, 138.4, 132.0, 130.7,\\n129.6, 128.3, 114.8, 71.5, 71.4, 70.4, 70.3, 69.8, 58.6, 57.5, 56.7,\\n43.3, 36.0, 34.9, 33.6, 29.1, 26.5, 25.4, 16.0. MS m/z calcd for C32H46N4O6S 614.31, found 615.4 [M + H+].
\\n \\n \\n 2-(2-(Allyloxy)ethoxy)ethan-1-ol\\n(<bold>38</bold>)\\n To a mixture of NaOH (1 equiv) in dioxane\\n(0.3 M), diethylene glycol\\n(2 equiv) and allyl bromide (1 equiv) were added dropwise. The mixture\\nwas heated overnight at 55 °C. Then the reaction mixture was\\ndried over MgSO4, filtered, and evaporated in vacuum. The\\ncrude was purified by flash column chromatography using a gradient\\nfrom 50% to 100% of ethyl acetate in heptane to obtain compound 38 as an oil. Yield: 329 mg, 54%. 1H NMR (400 MHz,\\nCDCl3) δ: 5.90–5.80 (m, 1H), 5.21 (qd, J = 1.6, 17.2 Hz, 1H), 5.14–5.10 (m, 1H), 3.96 (td, J = 1.4, 5.7 Hz, 2H), 3.68–3.59 (m, 4H), 3.56–3.52\\n(m, 4H). 13C NMR (101 MHz, CDCl3) δ: 134.5,\\n117.3, 72.6, 72.2, 70.4, 69.4, 61.7.
\\n \\n \\n 3,6,9,12-Tetraoxapentadec-14-en-1-ol\\n(<bold>39</bold>)\\n Following the same procedure applied for\\ncompound 38, from tetraethylene glycol and allyl bromide\\ncompound 39 was obtained as oil. Yield: 3.29 g, 68%.\\nAnalytical data matched\\nthose previously reported.75
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-(Allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>40</bold>)\\n Following general method E, compound 40 was obtained from compounds 38 and 2d (synthesis detailed in the Supporting Information). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 21.7\\nmg, 42%. 1H NMR (500 MHz, MeOD) δ: 8.91 (s, 1H),\\n8.52 (t, J = 5.9 Hz, 1H), 7.55 (dd, J = 3.4, 9.4 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.10\\n(d, J = 1.6 Hz, 1H), 7.06 (dd, J = 1.6, 7.8 Hz, 1H), 5.98–5.90 (m, 1H), 5.30 (ddt, J = 1.7, 6.9, 8.6 Hz, 1H), 5.18 (ddt, J = 1.4, 4.5, 5.2 Hz, 1H), 4.79 (d, J = 9.6 Hz, 1H),\\n4.66 (t, J = 8.3 Hz, 1H), 4.54–4.43 (m, 3H),\\n4.31–4.26 (m, 2H), 4.07–4.04 (m, 2H), 3.97–3.94\\n(m, 2H), 3.90–3.82 (m, 2H), 3.80–3.76 (m, 2H), 3.69–3.66\\n(m, 2H), 2.53 (s, 3H), 2.29–2.24 (m, 1H), 2.17–2.10\\n(m, 1H), 1.42–1.30 (m, 4H), 1.07 (s, 10H). MS m/z calcd for C33H45FN4O7S 660.30, found 661.3 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((3,6,9,12-Tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>41</bold>)\\n Following general method E, compound 41 was obtained from compounds 39 and 2d (synthesis detailed in the Supporting Information). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 26.9\\nmg, 46%. 1H NMR (500 MHz, MeOD) δ: 8.91 (s, 1H),\\n8.51 (t, J = 6.1 Hz, 1H), 7.56 (dd, J = 3.2, 9.6 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.10\\n(d, J = 1.6 Hz, 1H), 7.06 (dd, J = 1.7, 7.7 Hz, 1H), 5.97–5.89 (m, 1H), 5.30 (ddt, J = 1.7, 6.9, 8.7 Hz, 1H), 5.19–5.16 (m, 1H), 4.79\\n(d, J = 9.1 Hz, 1H), 4.66 (t, J =\\n8.2 Hz, 1H), 4.56–4.43 (m, 3H), 4.30–4.26 (m, 2H), 4.04–4.02\\n(m, 2H), 3.97–3.94 (m, 2H), 3.90–3.82 (m, 2H), 3.80–3.77\\n(m, 2H), 3.73–3.70 (m, 2H), 3.69–3.64 (m, 7H), 3.62–3.59\\n(m, 2H), 2.54 (s, 3H), 2.30–2.25 (m, 1H), 2.16–2.11\\n(m, 1H), 1.42–1.31 (m, 4H), 1.07 (s, 9H). MS m/z calcd for C37H53FN4O9S 748.35, found 749.4 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-(Allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>42</bold>)\\n Following general method E, compound 42 was obtained from compounds 38 and 2c (synthesized as previously reported29). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 15.8\\nmg, 61%. 1H NMR (500 MHz, MeOD) δ: 8.91 (s, 1H),\\n8.50 (t, J = 5.9 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 7.06 (dd, J = 1.7, 8.0 Hz, 1H), 5.98–5.90 (m, 1H), 5.30 (ddt, J = 1.8, 6.9, 8.6 Hz, 1H), 5.20–5.16 (m, 1H), 4.71–4.64\\n(m, 2H), 4.54–4.44 (m, 4H), 4.31–4.27 (m, 2H), 4.06\\n(td, J = 1.4, 5.6 Hz, 2H), 3.97–3.95 (m, 2H),\\n3.87–3.78 (m, 5H), 3.69–3.66 (m, 3H), 2.53 (s, 4H),\\n2.28–2.23 (m, 1H), 2.16–2.10 (m, 1H), 1.71–1.56\\n(m, 4H), 1.06 (s, 9H). 13C NMR (101 MHz, MeOD) δ:\\n174.3, 171.6, 167.3, 158.2, 152.9, 149.2, 136.2, 133.6, 133.0, 130.2,\\n128.5, 122.9, 120.8, 117.4, 113.9, 73.2, 71.9, 71.1, 71.0, 70.7, 69.5,\\n60.9, 59.7, 59.6, 39.8, 39.0, 37.4, 26.9, 18.2, 18.1, 16.0, 15.0,\\n14.9. MS m/z calcd for C34H45N5O7S 667.30, found 668.4 [M\\n+ H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((3,6,9,12-Tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>43</bold>)\\n Following general method E, compound 43 was obtained from compounds 39 and 2c (synthesized as previously reported29). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 15.3\\nmg, 52%. 1H NMR (500 MHz, MeOD) δ: 8.91 (s, 1H),\\n8.49 (t, J = 6.1 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 1.6 Hz, 1H), 7.06 (dd, J = 1.6, 7.7 Hz, 1H), 5.97–5.89 (m, 1H), 5.30 (ddt, J = 1.6, 6.9, 8.6 Hz, 1H), 5.19–5.16 (m, 1H), 4.71–4.64\\n(m, 2H), 4.55–4.43 (m, 3H), 4.31–4.26 (m, 2H), 4.03\\n(td, J = 1.4, 5.6 Hz, 2H), 3.97–3.94 (m, 2H),\\n3.87–3.77 (m, 4H), 3.73–3.71 (m, 2H), 3.69–3.64\\n(m, 7H), 3.62–3.59 (m, 2H), 2.54 (s, 3H), 2.30–2.25\\n(m, 1H), 2.16–2.10 (m, 1H), 1.71–1.56 (m, 4H), 1.06\\n(s, 9H). MS m/z calcd for C38H53N5O9S 755.36, found 756.5\\n[M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-Acetamido-3,3-dimethylbutanoyl)-<italic>N</italic>-(2-(2-(2-(allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>44</bold>)\\n Following general method E, compound 44 was obtained from compounds 38 and 2b (synthesized accordingly to literature29). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 12.7\\nmg, 53%. 1H NMR (400 MHz, MeOD) δ: 8.91 (s, 1H),\\n7.52 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 7.04 (dd, J = 1.4, 7.8 Hz, 1H), 5.99–5.88 (m, 1H), 5.30 (dq, J = 1.7, 6.9 Hz, 1H), 5.18 (dq, J = 1.3,\\n4.5 Hz, 1H), 4.67–4.60 (m, 2H), 4.55–4.42 (m, 3H), 4.30–4.25\\n(m, 2H), 4.05 (dt, J = 1.4, 5.6 Hz, 2H), 3.97–3.90\\n(m, 3H), 3.85–3.77 (m, 3H), 3.68–3.65 (m, 2H), 2.53\\n(s, 3H), 2.28–2.20 (m, 1H), 2.16–2.07 (m, 1H), 2.03\\n(s, 3H), 1.06 (s, 9H). 13C NMR (101 MHz, MeOD) δ:\\n174.4, 173.1, 172.3, 158.0, 152.8, 149.1, 136.1, 133.5, 132.8, 130.0,\\n128.4, 122.8, 117.3, 114.0, 113.8, 73.1, 71.8, 70.9, 70.6, 69.4, 60.7,\\n59.1, 57.9, 39.4, 38.9, 36.5, 27.0, 22.3, 15.9. MS m/z calcd for C31H44N4O7S 616.29, found 617.4 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((3,6,9,12-Tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>45</bold>)\\n Following general method E, compound 45 was obtained from compounds 39 and 2b (synthesized accordingly to literature29). The crude was purified by HPLC using a gradient of 5% to 95% v/v\\nacetonitrile in 0.1% aqueous solution of formic acid. Yield: 13.8\\nmg, 53%. 1H NMR (400 MHz, MeOD) δ: 8.92 (s, 1H),\\n7.52 (d, J = 7.9 Hz, 1H), 7.08 (s, 1H), 7.04 (dd, J = 1.5, 7.8 Hz, 1H), 6.01–5.88 (m, 1H), 5.29 (dq, J = 1.7, 6.9 Hz, 1H), 5.17 (dq, J = 1.3,\\n4.5 Hz, 1H), 4.67–4.60 (m, 2H), 4.56–4.39 (m, 2H), 4.29–4.25\\n(m, 2H), 4.03 (dt, J = 1.4, 5.7 Hz, 2H), 3.97–3.90\\n(m, 3H), 3.85–3.76 (m, 3H), 3.73–3.63 (m, 9H), 3.62–3.58\\n(m, 2H), 2.53 (s, 3H), 2.28–2.21 (m, 1H), 2.16–2.08\\n(m, 1H), 2.04 (s, 3H), 1.06 (s, 9H). 13C NMR (101 MHz,\\nMeOD) δ: 174.4, 173.1, 172.2, 158.0, 152.8, 149.1, 136.1, 133.5,\\n132.8, 130.0, 128.4, 122.8, 117.2, 113.7, 73.1, 71.8, 71.6, 71.5,\\n71.1, 70.8, 70.6, 69.4, 60.7, 59.1, 58.0, 39.4, 38.9, 36.5, 27.0,\\n22.3, 15.9. MS m/z calcd for C35H52N4O9S 704.35, found 705.4\\n[M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>46</bold>)\\n Following general method C, from 69 (0.033 mmol) and 1c (0.033 mmol) compound 46 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid\\nas white powder. Yield: 5.3 mg, 16%. 1H NMR (400 MHz, MeOD)\\nδ: 9.61 (s, 1H), 8.95 (s, 1H), 8.71 (d, J =\\n6.2 Hz, 1H), 7.97 (s, 1H), 7.85 (d, J = 6.0 Hz, 1H),\\n7.53 (d, J = 8.2 Hz, 1H), 7.10–7.07 (m, 2H),\\n6.90 (s, 2H), 4.78 (s, 1H), 4.66 (t, J = 8.5 Hz,\\n1H), 4.54–4.44 (m, 3H), 4.32–4.29 (m, 2H), 4.00–3.81\\n(m, 14H), 3.76 (s, 3H), 3.55 (s, 4H), 3.45 (s, 4H), 3.30–3.27\\n(m, 2H), 2.53 (s, 3H), 2.32–2.26 (m, 1H), 2.18–2.10\\n(m, 1H), 1.44–1.30 (m, 4H), 1.08 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 174.2, 171.8, 164.4, 162.2, 161.2, 157.8,\\n153.0, 149.0, 146.4, 142.3, 140.0, 133.5, 132.9, 129.9, 128.2, 122.9,\\n122.3, 120.8, 118.2, 113.5, 106.8, 80.3, 78.0, 71.0, 70.9, 69.1, 67.2,\\n60.9, 58.8, 58.7, 58.2, 57.5, 56.9, 50.7, 50.1, 39.4, 39.0, 37.6,\\n37.3, 26.9, 15.8, 14.1, 14.0, 14.0, 13.9. HRMS m/z calcd for C52H65FN8O9S 996.46, found 997.4810 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-(4-(2,5-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>47</bold>)\\n Following general method C, from 69 (0.033 mmol) and 1d (synthesis detailed in\\nthe Supporting Information) (0.033 mmol)\\ncompound 47 was obtained after purification by HPLC using\\na gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution\\nof formic acid as white powder. Yield: 5.3 mg, 5%. 1H NMR\\n(400 MHz, MeOD) δ: 9.52 (s, 1H), 8.91 (s, 1H), 8.62 (d, J = 5.7 Hz, 1H), 7.65 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.23–7.19 (m, 2H), 7.10 (d, J = 1.2 Hz, 1H), 7.06 (dd, J = 1.6, 7.7 Hz, 1H),\\n6.98 (s, 1H), 4.78 (s, 1H), 4.66 (t, J = 8.5 Hz,\\n1H), 4.55–4.45 (m, 3H), 4.32–4.28 (m, 2H), 3.95–3.89\\n(m, 2H), 3.88–3.80 (m, 7H), 3.76–3.71 (m, 8H), 2.78–2.71\\n(m, 10H), 2.53 (s, 3H), 2.30–2.24 (m, 1H), 2.17–2.11\\n(m, 1H), 1.42–1.31 (m, 4H), 1.07 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 174.1, 171.7, 163.3, 158.0, 153.7, 152.8,\\n151.4, 150.6, 144.2, 139.2, 133.5, 133.0, 128.4, 124.1, 122.8, 121.5,\\n120.3, 115.8, 113.9, 80.3, 71.0, 70.8, 69.8, 69.3, 60.8, 58.7, 58.2,\\n56.7, 56.5, 54.3, 53.5, 39.4, 38.9, 37.4, 37.3, 26.9, 15.9, 14.1,\\n14.0, 13.9. HRMS m/z calcd for C52H65FN8O9S 996.46, found\\n997.4712 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((5-(4-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>51</bold>, VZ185)\\n Following general method C, from 70 (0.02 mmol) and 1c (0.02 mmol) compound 51 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid\\nas white powder. Yield: 4.8 mg, 24%. 1H NMR (400 MHz, MeOD)\\nδ: 9.56 (d, J = 0.8 Hz, 1H), 8.92 (s, 1H),\\n8.71 (d, J = 5.8 Hz, 1H), 7.78 (s, 1H), 7.65 (dd, J = 0.7, 5.8 Hz, 1H), 7.52 (d, J = 7.7\\nHz, 1H), 7.07–7.03 (m, 2H), 6.86 (s, 2H), 4.79 (s, 1H), 4.67\\n(t, J = 12.4 Hz, 1H), 4.55–4.47 (m, 3H), 4.18\\n(s, 2H), 4.14 (t, J = 6.0 Hz, 2H), 3.96–3.82\\n(m, 8H), 3.74 (s, 3H), 3.18–2.78 (m, 10H), 2.53 (s, 3H), 2.32–2.25\\n(m, 1H), 2.19–2.12 (m, 1H), 1.98–1.91 (m, 2H), 1.79–1.71\\n(m, 2H), 1.68–1.61 (m, 2H), 1.42–1.29 (m, 4H), 1.08\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 174.2, 171.8,\\n171.4, 163.1, 161.0, 158.0, 152.8, 151.7, 151.2, 149.1, 143.6, 138.8,\\n133.6, 132.8, 129.7, 128.0, 122.5, 121.7, 119.2, 113.1, 106.6, 80.3,\\n78.0, 71.0, 69.2, 60.8, 59.2, 58.7, 58.1, 56.4, 53.1, 49.8, 39.3,\\n38.9, 37.4, 37.3, 30.1, 26.9, 25.1, 15.9, 14.1, 14.0, 13.8. HRMS m/z calcd for C53H67FN8O8S 994.48, found 995.5024 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((5-(4-(2,5-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)piperazin-1-yl)pentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>52</bold>)\\n Following general method C, from 70 (0.02 mmol) and 1d (0.02 mmol) compound 52 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid\\nas white powder. Yield: 4 mg, 20%. 1H NMR (400 MHz, MeOD)\\nδ: 9.53 (s, 1H), 8.92 (s, 1H), 8.62 (d, J =\\n5.7 Hz, 1H), 7.65 (s, 1H), 7.53 (d, J = 7.4 Hz, 1H),\\n7.23–7.19 (m, 2H), 7.05 (s, 2H), 7.02 (s, 1H), 4.79 (s, 1H),\\n4.68 (t, J = 8.4 Hz, 1H), 4.55–4.47 (m, 3H),\\n4.15 (t, J = 5.9 Hz, 2H), 3.92–3.82 (m, 7H),\\n3.75 (s, 3H), 3.72 (s, 3H), 3.18–2.91 (m, 10H), 2.53 (s, 3H),\\n2.32–2.26 (m, 1H), 2.19–2.12 (m, 1H), 2.00–1.93\\n(m, 2H), 1.87–1.78 (m, 2H), 1.70–1.62 (m, 2H), 1.43–1.30\\n(m, 4H), 1.08 (s, 9H). 13C NMR (101 MHz, MeOD) δ:174.2,\\n171.8, 163.3, 157.9, 153.7, 152.8, 151.4, 150.5, 149.2, 144.1, 139.3,\\n133.6, 132.9, 129.6, 127.9, 122.6, 121.5, 120.1, 116.0, 115.6, 113.1,\\n80.4, 78.1, 71.0, 69.0, 60.9, 58.7, 58.4, 58.2, 56.8, 56.5, 56.2,\\n53.1, 51.8, 39.3, 38.9, 37.4, 37.3, 29.8, 26.9, 24.7, 15.9, 14.1,\\n14.0, 13.9. HRMS m/z calcd for C53H67FN8O8S 994.48, found\\n995.4771 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-((1-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)azetidin-3-yl)amino)-2-oxoethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>55</bold>)\\n Following general method B, from the 71 (0.02 mmol) and 1e (0.02 mmol) compound 55 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia\\nas white powder. Yield: 2.4 mg, 12%. 1H NMR (400 MHz, MeOD)\\nδ: 9.56 (d, J = 0.6 Hz, 1H), 8.93 (s, 1H),\\n8.71 (d, J = 6.0 Hz, 1H), 7.80 (s, 1H), 7.63 (dd, J = 0.6, 5.8 Hz, 1H), 7.55 (d, J = 8.1\\nHz, 1H), 7.12–7.08 (m, 2H), 6.89 (s, 2H), 4.80–4.64\\n(m, 3H), 4.60–4.31 (m, 11H), 4.23 (s, 2H), 4.06 (t, J = 4.0 Hz, 2H), 3.98 (s, 6H), 3.91–3.80 (m, 2H),\\n3.74 (s, 3H), 2.53 (s, 3H), 2.31–2.25 (m, 1H), 2.18–2.10\\n(m, 1H), 1.42–1.30 (m, 4H), 1.07 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 174.3, 171.8, 166.7, 160.7, 157.8, 152.9,\\n151.8, 151.3, 149.2, 143.3, 140.4, 139.2, 133.4, 133.0, 130.0, 128.2,\\n123.0, 121.7, 119.1, 118.6, 113.4, 106.8, 71.5, 71.5, 71.0, 69.2,\\n60.9, 60.9, 58.7, 58.2, 56.9, 49.5, 39.4, 38.9, 37.5, 37.3, 29.5,\\n26.9, 15.9, 13.9. HRMS m/z calcd\\nfor C51H61FN8O10S 996.42,\\nfound 997.4452 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2-((1-(2,5-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)azetidin-3-yl)amino)-2-oxoethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>56</bold>)\\n Following general method B, from 71 (0.02 mmol) and 1f (synthesis detailed in\\nthe Supporting Information) (0.02 mmol)\\ncompound 56 was obtained after purification by HPLC using\\na gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution\\nof ammonia as white powder. Yield: 3.3 mg, 17%. 1H NMR\\n(400 MHz, MeOD) δ: 9.39 (s, 1H), 8.80 (s, 1H), 8.49 (d, J = 5.7 Hz, 1H), 7.54 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.10 (s, 1H), 7.04 (d, J = 5.6 Hz,\\n1H), 6.99 (s, 1H), 6.98–6.94 (m, 2H), 4.66 (s, 1H), 4.60–4.51\\n(m, 2H), 4.45–4.32 (m, 5H), 4.27–4.15 (m, 6H), 4.10\\n(s, 2H), 3.93 (t, J = 4.1 Hz, 2H), 3.81 (s, 3H),\\n3.78–3.67 (m, 2H), 3.63 (s, 3H), 3.58 (s, 3H), 2.40 (s, 3H),\\n2.19–2.10 (m, 1H), 2.05–1.98 (m, 1H), 1.30–1.16\\n(m, 4H), 0.94 (s, 9H). 13C NMR (101 MHz, MeOD) δ:\\n174.2, 173.2, 171.8, 167.1, 166.6, 163.2, 157.8, 153.4, 153.0, 152.9,\\n151.4, 150.6, 149.2, 143.8, 139.5, 133.4, 133.0, 130.0, 128.2, 127.1,\\n123.0, 121.5, 120.9, 120.0, 116.1, 115.6, 113.4, 80.3, 78.0, 71.5,\\n71.5, 71.0, 69.2, 69.1, 60.9, 58.7, 58.2, 56.8, 56.6, 55.9, 49.5,\\n49.5, 40.7, 39.4, 38.9, 37.4, 37.3, 26.9, 15.9, 14.0. HRMS m/z calcd for C51H61FN8O10S 996.42, found 997.4435 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((5-((1-(2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)azetidin-3-yl)amino)-5-oxopentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>61</bold>)\\n Following general method B, from 72 (0.025 mmol) and 1e (0.025 mmol) compound 61 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid\\nas white powder. Yield: 4.5 mg, 18%. 1H NMR (400 MHz, MeOD)\\nδ: 9.45 (d, J = 0.5 Hz, 1H), 8.81 (s, 1H),\\n8.61 (d, J = 5.7 Hz, 1H), 7.68 (s, 1H), 7.54 (d, J = 5.5 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H),\\n6.96–6.91 (m, 2H), 6.74 (s, 2H), 4.69 (s, 1H), 4.58 (t, J = 8.5 Hz, 1H), 4.54–4.32 (m, 5H), 4.24 (s, 2H),\\n4.11–4.01 (m, 4H), 3.85 (s, 6H), 3.81–3.70 (m, 3H),\\n3.63 (s, 3H), 2.42 (s, 3H), 2.28 (t, J = 6.5 Hz,\\n2H), 2.21–2.15 (m, 1H), 2.08–2.01 (m, 1H), 1.85–1.78\\n(m, 4H), 1.32–1.20 (m, 4H), 0.96 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 176.1, 174.3, 171.7, 163.0, 160.7, 157.9,\\n152.8, 151.7, 151.3, 149.1, 148.6, 143.4, 139.4, 139.1, 133.6, 132.8,\\n129.7, 128.2, 128.0, 122.5, 121.7, 120.9, 119.2, 118.9, 113.0, 106.7,\\n80.3, 78.0, 71.0, 68.8, 61.1, 60.8, 58.7, 58.2, 56.7, 56.1, 40.9,\\n39.3, 38.9, 37.4, 37.3, 36.4, 29.8, 29.5, 26.9, 23.4, 15.9, 14.1,\\n14.0, 13.9. HRMS m/z calcd for C52H63FN8O9S 994.44, found\\n995.4634 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((5-((1-(2,5-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)azetidin-3-yl)amino)-5-oxopentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>62</bold>)\\n Following general method B, from 72 (0.025 mmol) and 1f (0.025 mmol) compound 62 was obtained after purification by HPLC using a gradient\\nof 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid\\nas white powder. Yield: 5.8 mg, 23%. 1H NMR (400 MHz, MeOD)\\nδ: 9.51 (d, J = 0.7 Hz, 1H), 8.91 (s, 1H),\\n8.62 (d, J = 6.0 Hz, 1H), 7.64 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.19 (dd, J = 0.8, 5.8\\nHz, 1H), 7.14 (s, 1H), 7.06–7.02 (m, 2H), 7.01 (s, 1H), 4.79\\n(s, 1H), 4.69 (t, J = 8.5 Hz, 1H), 4.58–4.43\\n(m, 4H), 4.13 (s, 2H), 4.01–3.81 (m, 10H), 3.74 (s, 3H), 3.71\\n(s, 3H), 3.47 (s, 1H), 2.52 (s, 3H), 2.36 (t, J =\\n6.5 Hz, 2H), 2.31–2.24 (m, 1H), 2.20–2.11 (m, 1H), 1.92\\n(t, J = 2.6 Hz, 4H), 1.46–1.29 (m, 4H), 1.08\\n(s, 9H). 13C NMR (101 MHz, MeOD) δ: 175.8, 174.3,\\n171.7, 171.6, 171.4, 163.2, 157.9, 153.2, 152.8, 152.8, 151.3, 150.5,\\n149.1, 144.1, 139.3, 133.6, 132.8, 129.7, 128.0, 124.7, 122.5, 121.5,\\n120.2, 116.1, 115.6, 115.0, 113.0, 80.3, 78.0, 71.1, 68.8, 61.8, 60.8,\\n58.7, 58.2, 57.2, 56.6, 56.5, 41.4, 39.3, 38.9, 37.4, 37.3, 36.4,\\n29.8, 26.9, 23.5, 15.9, 14.1, 14.0, 13.9. HRMS m/z calcd for C52H63FN8O9S 994.44, found 995.4664 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-(2-(2,2-Diethoxyethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>67</bold>)\\n Following general method E, compound 67 was obtained after reaction between 2-(2,2-diethoxyethoxy)ethan-1-ol\\n(synthesized accordingly to literature76) and 2d as white powder. Yield: 92 mg, 87%. 1H NMR (400 MHz, CDCl3) δ: 8.66 (s, 1H), 7.31 (d, J = 7.7 Hz, 1H), 6.95 (dd, J = 1.2, 7.7\\nHz, 1H), 6.88 (s, 1H), 4.66 (t, J = 7.6 Hz, 2H),\\n4.55–4.40 (m, 4H), 4.23–4.13 (m, 2H), 3.97–3.87\\n(m, 3H), 3.71–3.49 (m, 7H), 2.50 (s, 3H), 2.44–2.34\\n(m, 1H), 2.13–2.05 (m, 1H), 1.35–1.26 (m, 4H), 1.18\\n(t, J = 7.1 Hz, 6H), 0.93 (s, 9H). 13C\\nNMR (101 MHz, CDCl3) δ: 170.8, 170.3, 170.1, 156.9,\\n150.4, 148.6, 132.4, 131.8, 129.8, 126.9, 122.1, 112.9, 101.0, 79.5,\\n72.0, 70.3, 70.1, 68.0, 62.5, 62.3, 58.7, 57.5, 56.7, 39.1, 36.5,\\n35.6, 26.4, 16.2, 15.4, 13.9, 13.8, 13.7. MS m/z calcd for C34H49FN4O8S 692.33, found 693.3 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-<italic>N</italic>-(2-((5,5-Dimethoxypentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((<italic>S</italic>)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide\\n(<bold>68</bold>)\\n A mixture of 2d (0.075 mmol,\\n1 equiv), 5-bromo-1,1-dimethoxypentane 66 (synthesized\\naccordingly to literature77) (0.112 mmol,\\n1.8 equiv), and K2CO3 (0.225 mmol, 3 equiv)\\nin DMF (0.5 mL) was stirred at 70 °C overnight. The reaction\\nmixture was diluted with water and extracted with DCM, dried over\\nMgSO4, filtered and evaporated to dryness. The crude was\\npurified by flash column chromatography using a gradient from 0% to\\n20% of methanol in DCM to obtain the desired compound as white powder.\\nYield: 23 mg, 55%. 1H NMR (400 MHz, MeOD) δ: 8.91\\n(s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 1.5 Hz, 1H), 7.03 (s, 1H), 4.80 (d, J = 8.6 Hz, 1H), 4.68 (t, J = 8.3 Hz, 1H), 4.57–4.42\\n(m, 4H), 4.11 (t, J = 6.2 Hz, 2H), 3.81–3.62\\n(m, 2H), 3.36 (s, 6H), 2.53 (s, 3H), 2.31–2.25 (m, 1H), 2.21–2.14\\n(m, 1H), 1.95–1.88 (m, 2H), 1.76–1.68 (m, 2H), 1.67–1.58\\n(m, 2H), 1.38–1.29 (m, 4H), 1.08 (s, 9H). 13C NMR\\n(101 MHz, MeOD) δ: 174.2, 171.7, 171.4, 158.0, 152.8, 149.1,\\n133.6, 132.8, 129.6, 127.9, 122.4, 113.0, 106.2, 80.3, 71.1, 69.2,\\n60.8, 58.1, 55.8, 53.5, 43.8, 39.3, 38.9, 37.3, 33.4, 30.1, 26.9,\\n22.3, 15.9, 14.1, 14.0, 13.9, 13.2. MS m/z calcd for C33H47FN4O7S 662.31, found 663.5 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)-2-(2-(2-oxoethoxy)ethoxy)benzyl)pyrrolidine-2-carboxamide\\n(<bold>69</bold>)\\n Following general method F, compound 69 was obtained from 67 (0.066 mmol) and directly\\nused in the next step without any further purification. MS m/z calcd for C30H39FN4O7S 618.15, found 619.3 [M + H+].
\\n \\n \\n (2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-<italic>N</italic>-(4-(4-methylthiazol-5-yl)-2-((5-oxopentyl)oxy)benzyl)pyrrolidine-2-carboxamide\\n(<bold>70</bold>)\\n Following general method F, compound 70 was obtained from 68 (0.04 mmol) and directly\\nused in the next step without any further purification. MS m/z calcd for C31H41FN4O6S 616.27, found 617.3 [M + H+].
\\n \\n \\n 2-(2-(2-(((2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)acetic\\nAcid (<bold>71</bold>)\\n Following general method G, compound 71 was obtained from compound 69 (0.04 mmol)\\nand was used in the next step without any further purification. Quantitative\\nyield. MS m/z calcd for C30H39FN4O8S 634.25, found 635.3 [M\\n+ H+].
\\n \\n \\n 5-(2-(((2<italic>S</italic>,4<italic>R</italic>)-1-((<italic>S</italic>)-2-(1-Fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)pentanoic\\nAcid (<bold>72</bold>)\\n Following general method G, compound 72 was obtained from compound 70 (0.05 mmol)\\nand was used in the next step without any further purification. Quantitative\\nyield. MS m/z calcd for C31H41FN4O7S 632.27, found 633.3 [M\\n+ H+].
\\n \\n \\n Biology. Cell Culture\\n Human cell\\nlines HeLa and Hek293\\nwere purchased from ATCC and propagated in DMEM medium (Gibco), supplemented\\nwith 10% fetal bovine serum (FBS) (Gibco), l-glutamine (Gibco),\\n100 μg mL–1 of penicillin/streptomycin. RI-1\\ncells, purchased from DSMZ, were propagated in RPMI medium (Gibco),\\nsupplemented with 10% fetal bovine serum (FBS) (Gibco), l-glutamine, 100 μg mL–1 of penicillin/streptomycin.\\nCells were kept at 37 °C and 5% of CO2. EOL-1 and\\nA-204 cell lines were purchased from ATCC and DSMZ. EOL-1 cells were\\ngrown in RPMI supplemented with 10% fetal calf serum. A-204 cells\\nwere grown in McCoy’s medium supplemented with 10% fetal calf\\nserum. All cell lines were routinely tested for mycoplasma contamination\\nusing MycoAlert kit from Lonza.
\\n \\n \\n Testing Compounds in Cells\\n HeLa and Hek293 cells were\\nseeded at either 0.3 × 106 or 0.5 × 106 cells per well in 6-well plate in 2 mL of medium. At 80% confluency,\\ncells were treated with compounds at the desired concentration, with\\nfinal DMSO concentration of 0.1% v/v. Cells were incubated at 37 °C\\nand 5% of CO2 for the desired time before harvesting. For\\nprotein extraction from adherent cells, cells were washed twice with\\nDPBS (Gibco) and lysed with RIPA buffer (Sigma-Aldrich), supplemented\\nwith cOmplete Mini EDTA-free protease inhibitor cocktail (Roche).\\nInsoluble material was removed by centrifugation at 14 000\\nrpm for 15 min at 4 °C. Supernatant was collected and protein\\nconcentration was quantified by Bradford assay (Thermo Scientific\\nno. 23200, mean of two replicates). For RI-1 cells, (1–1.5)\\n× 106 cells/mL were seeded in 6-well plate in 2 mL\\nof medium. The day after, cells were treated with compounds at the\\ndesired concentration, with final DMSO concentration of 0.1% v/v.\\nBefore harvesting, cells were incubated at 37 °C and 5% CO2 for the desired time. Cells were washed twice with DPBS (Gibco)\\nand lysed with RIPA buffer (Sigma-Aldrich), supplemented with cOmplete\\nMini EDTA-free protease inhibitor cocktail (Roche). Lysates were sonicated\\n(10 s) and centrifuged at 14 000 rpm for 20 min at 4 °C.\\nSupernatant was collected, and protein concentration was quantified\\nby Bradford assay (mean of two replicates). For EOL-1 and A-204 cell\\nline, 20 000 cells were seeded in 300 μL per well in\\na 24-well plate and incubated at 37 °C overnight. Compounds were\\nadded from DMSO stock solution using a digital dispenser D300 (Tecan),\\nnormalizing for added DMSO, and cells were incubated at 37 °C\\nfor 18 h. Medium was removed, and cells were washed with PBS and lysed\\nin 80 μL of lysis buffer (1% Triton, 350 mM KCl, 10 mM Tris,\\npH 7,4, phosphatase-protease inhibitor cocktail (Thermo Scientific\\nno. 1861281), 10 mM DTT, Benzonase 0.5 μL/ml (Novagen no. 70746\\n10KU, 25 U/μL)) for 30 min on ice before insoluble debris was\\npelleted by centrifugation.
\\n \\n \\n Small Interfering RNA\\n HeLa cells were seeded at 0.3\\n× 106 cells per well in 6-well plates in 2 mL of medium,\\nwith a goal to achieve 70% of confluence on the day of transfection.\\nBRD7, BRD9, and CRBN targeting siRNA (L-020297-00-0005 ON-TARGETplus\\nhuman BRD7, L-014250-02-0005 ON-TARGETplus human BRD9, and L-021086-00-0005\\nON-TARGETplus human CRBN SMARTpool, 5 nmol, Dharmacon) were prepared\\nas a 20 μM solution in RNase-free 1× siRNA buffer (Dharmacon).\\nNontargeting siRNA (On-TARGETplus control pool, nontargeting pool\\nD-001810-10-05, 5 nmol, Dharmacon) was used as negative control. Medium\\nwas replaced on the day of transfection. siRNA solution (5 μL)\\nof BRD7, BRD9, or CRBN targeting siRNA, negative control, and vehicle\\ncontrol (1× siRNA buffer) were added to 250 μL of Opti-mem\\n(Gibco), prepared in duplicate. Lipofectamine RNAiMax (5 μL,\\nThermo Fisher Scientific) was added to 250 μL of Opti-mem in\\nanother tube, also in duplicate. The two solutions were combined,\\nincubated for 20 min at rt, and added to the wells. Plates were incubated\\nat 37 °C and 5% CO2 for 48 h before harvesting as\\ndescribed above.
\\n \\n \\n Mechanistic Evaluation\\n Cells were\\nseeded in 6-well\\nplates with 5 × 105 cells per well in 2 mL of complete\\nmedium to aim for 80% confluence on the day of treatment. At time\\nzero, ML4924 was added at final concentration of 3 μM with 0.1%\\nv/v of DMSO. DMSO was added to the remaining wells in order to match\\nconcentration of vehicle in all wells. After 3 h, MG132 (50 μM)\\nor VH298 (100 μM) was added to the designated wells at a final\\nconcentration of 0.1% v/v of DMSO. Again, DMSO was added to the remaining\\nwells to match vehicle concentration. At t = 3.5\\nh, VZ185 or cisVZ185 was added at 100 nM and 0.1%\\nv/v of DMSO, matching the DMSO concentration in the remaining wells\\n(final concentration 0.3% v/v). Plates were incubated for an additional\\n4 h at 37 °C and 5% CO2 before harvesting as described\\nbefore. The experiment was performed in duplicate.
\\n \\n \\n Immunoblotting\\n Proteins were resolved by SDS–PAGE\\non NuPage 4–12% Bis-Tris Midi Gel (Invitrogen) and transferred\\nto Amersham Protran 0.45 NC nitrocellulose membrane (GE Healthcare)\\nusing wet transfer. The membrane was blocked with 5% w/v milk in Tris-buffered\\nsaline (TBS) with 0.1% w/v Tween-20. The following primary antibodies\\nat the given concentration were used: anti β-actin (Cell Signaling\\nTechnology, 4970S, 13E5) 1:2000, anti-Brd9 (Bethyl A303-781A) 1:1000,\\nanti-Brd7 (Bethyl A302-304A) 1:1000, VHL rabbit Ab (Cell Signaling\\nTechnology, no. 68547S) 1:1000, anti-CRBN (Novus Bio, NBP 1-9-1810)\\n1:1000, anti-actin hFAB rhodamine antibody, Bio-Rad, no. 12004164.\\nFollowing incubation with horseradish peroxidase-conjugated secondary\\nantibody (Cell Signaling Technology) or IRDye secondary antibody (Licor),\\nbands were developed using Amersham ECL Prime Western blotting detection\\nkit on Amersham Hyperfilm ECL film (Amersham) or ChemiDoc imaging\\nsystem (Bio-Rad). ImageJ software was used for band quantification,\\nand the last was reported as relative amount as ratio of each protein\\nband relative to the lane’s loading control. Then the values\\nobtained were normalized to 0.1% DMSO vehicle control. Alternatively,\\nprotein levels were determined on a WES capillary electrophoresis\\ninstrument (Proteinsimple) using a BRD9 antibody (Bethyl A303-781A)\\nand a GAPDH antibody (Abcam no. ab9485) for normalization. DC50 and half-lives were determined by assuming a linear model\\nbetween the two data points across 50% protein level mark.
\\n \\n \\n Proliferation\\nAssays\\n A-204 cells (1 × 103) were seeded\\nper well of 384-well plates. After overnight incubation,\\ncompounds were added to the cells at logarithmic dose series using\\nthe HP digital dispenser D300 (Tecan), normalizing for added DMSO.\\n1 day and 8 days after seeding cellular ATP content was measured using\\nCellTiterGlo (Promega). Measurements after 8 days were divided by\\nthe measurement after 1 day (i.e., the T0 plate) to derive fold proliferation. Data were analyzed with GraphPad\\nPrism software to obtain EC50 values.
\\n \\n \\n MS Proteomics\\n \\n Sample\\nPreparation\\n RI-1 cells were seeded at 2 ×\\n106 cells/mL in a 10 cm plate 12 h before treatment. Cells\\nwere treated with 0.1% DMSO as vehicle control and with 100 nM VZ185\\nand 100 nM cisVZ185 as negative control. Cells were\\nincubated at 37 °C and 5% CO2 for 4 h before harvesting.\\nCells were washed twice with DPBS (Gibco) and lysed with 0.5 mL of\\n100 mM Tris, pH 8.0, 4% (w/v) SDS, supplemented with cOmplete Mini\\nEDTA-free protease inhibitor cocktail (Roche). Lysates were sonicated\\n(2 × 10 s) and centrifuged at 14 000 rpm for 20 min at\\n4 °C. The supernatant fraction of the cell extract was collected,\\nand protein concentration was quantified by BCA assay (Thermo Fisher\\nScientific). Further sample processing, digestion, desalting, TMT\\n10-plex isobaric labeling were performed as previously described.5 After labeling, the peptides from the 9 samples\\nwere pooled together in equal proportion. The pooled sample was fractionated\\ninto 20 discrete fractions using high pH reverse-phase chromatography\\non an XBridge peptide BEH column (130 Å, 3.5 μm, 2.1 mm\\n× 150 mm, Waters) on an Ultimate 3000 HPLC system (Thermo Scientific/Dionex).\\nA mixture of buffer A (10 mM ammonium formate in water, pH 9) and\\nB (10 mM ammonium formate in 90% CH3CN, pH 9) was used\\nover a linear gradient of 5% to 60% buffer B over 60 min at a flow\\nrate of 200 μL/min. The peptides eluted from the column were\\ncollected in 80 fractions before concatenation into 20 fractions based\\non the UV signal of each fraction. All the fractions were dried in\\na Genevac EZ-2 concentrator and resuspended in 1% formic acid for\\nMS analysis.
\\n \\n \\n nLC–MS/MS Analysis\\n The fractions\\nwere analyzed\\nsequentially on a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer\\n(Thermo Scientific) coupled to an UltiMate 3000 RSLCnano UHPLC system\\n(Thermo Scientific) and EasySpray column (75 μm × 50 cm,\\nPepMap RSLC C18 column, 2 μm, 100 Å, Thermo Scientific).\\nA mix of buffer A (0.1% formic acid in H2O) and B (0.08%\\nformic acid in 80% CH3CN) was used over a linear gradient\\nfrom 5% to 35% buffer B over 125 min using a flow rate of 300 nL/min.\\nColumn temperature was set at 50 °C. The Q Exactive HF hybrid\\nquadrupole-Orbitrap mass spectrometer was operated in data dependent\\nmode with a single MS survey scan from 335 to 1600 m/z followed by 15 sequential m/z dependent MS2 scans. The 15 most intense precursor ions\\nwere sequentially fragmented by higher energy collision dissociation\\n(HCD). The MS1 isolation window was set to 0.7 Da and the resolution\\nset at 120 000. MS2 resolution was set at 60 000. The\\nAGC targets for MS1 and MS2 were set at 3e6 ions and 1e5 ions, respectively.\\nThe normalized collision energy was set at 32%. The maximum ion injection\\ntimes for MS1 and MS2 were set at 50 and 200 ms, respectively.
\\n \\n \\n Peptide\\nand Protein Identification and Quantification\\n The raw MS\\ndata files for all 20 fractions were merged and searched\\nagainst the Uniprot-sprot-Human-Canonical database by Maxquant software\\n1.6.0.16 for protein identification and TMT reporter ion quantitation.\\nThe identifications were based on the following criteria: enzyme used\\ntrypsin/P; maximum number of missed cleavages equal to 2; precursor\\nmass tolerance equal to 10 ppm; fragment mass tolerance equal\\nto 20 ppm. Variable modifications: oxidation (M), acetyl (N-term),\\ndeamidation (NQ), Gln → pyro-Glu (Q N-term). Fixed modifications:\\ncarbamidomethyl (C). The data were filtered by applying a 1% false\\ndiscovery rate followed by exclusion of proteins with less than two\\nunique peptides. Quantified proteins were filtered if the absolute\\nfold-change difference between the three DMSO replicates was \\n≥1.5.
\\n \\n \\n \\n Protein Expression and Purification\\n Human proteins\\nVHL (UniProt accession number P40337), ElonginC (Q15369), and ElonginB\\n(Q15370) and the bromodomain (residues 134–239) for BRD9 (Q9H8M2)\\nwere used for all protein expression. His6-tagged constructs\\nwere transformed into Escherichia coli BL21(DE3),\\nand expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG). E. coli cells\\nwere lysed using a pressure cell homogenizer (Stansted Fluid Power);\\nlysates were clarified by centrifuge and loaded onto a HisTrap FF\\naffinity column (GE Healthcare). Following elution, His-tags were\\nremoved with TEV protease and samples were loaded onto a second Ni\\naffinity column to obtain tag-free protein. Following dialysis in\\na low-salt buffer, BRD9 bromodomain and VCB complex proteins were\\nfurther purified using cation exchange (Resource S; GE Healthcare)\\nor anion exchange (Resource Q; GE Healthcare) chromatography, respectively.\\nThis was followed by a final polish by size-exclusion chromatography\\nusing a Superdex-75 16/600 column (GE Healthcare) equilibrated with\\n20 mM HEPES, pH 7.5, 100 mM sodium chloride, and 1 mM\\nTCEP.
\\n \\n \\n Crystallization and Structure Solution of BRD9 Bromodomain Binary\\nComplex\\n The binary complex of Brd9-BD:5 was\\ngenerated by incubating 500 μM Brd9 bromodomain with 750 μM\\ncompound 5 (from a 100 mM stock in DMSO). Crystals were\\ngrown using the hanging-drop vapor diffusion method by mixing equal\\nvolumes of binary complex solution and a crystallization solution\\ncontaining 24% PEG 3350 and 0.2 M NH4F. Small needle crystals\\nappeared within 48 h but took approximately 2 weeks to reach a suitable\\nsize for harvesting. Crystals were flash-frozen in liquid nitrogen\\nusing 20% ethylene glycol in liquor solution as a cryoprotectant.\\nDiffraction data were collected at Diamond Light Source beamline I24\\nusing a Pilatus 6M-F detector at a wavelength of 0.98962 Å. Data\\nwere indexed and integrated using XDS,78 and scaling and merging were performed with AIMLESS79 in CCP4i.80 The structure was\\nsolved by molecular replacement using MOLREP81 and a search model derived from a BRD9 bromodomain structure (PDB\\nentry 5I40).\\nThe initial model underwent iterative rounds of model building and\\nrefinement with COOT82 and REFMAC5,83 respectively. Compound 5 geometry\\nrestraints for refinement were prepared with the PRODRG84 server. Model geometry and steric clashes were\\nvalidated using the MOLPROBITY server;85 Ramachandran plots indicate that 97.7% of backbone torsion angles\\nare in the favored region and there are no outliers. The structure\\nhas been deposited in the Protein Data Bank (PDB) with accession code 6HM0; data collection\\nand refinement statistics are presented in Supporting Information Table S1.
\\n \\n \\n AlphaLISA Proximity Assay\\n All assays were performed\\nat room temperature in 384-well plates with a final assay volume of\\n25 μL per well, as described previously.5 All reagents were prepared as 5× stocks diluted in\\n50 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% (w/v) bovine\\nserum albumin, and 0.02% (w/v) 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate\\n(CHAPS). Plates were sealed by transparent film and briefly centrifuged\\nat 100g between additions of reagents. Biotinylated\\nVCB (100 nM final) and His6-BRD9-bromodomain (100 nM\\nfinal) were incubated with a range of PROTAC concentrations (0.5–2000 nM;\\ntwo-in-one serial dilution) for 1 h. Streptavidin-coated donor\\nbeads and nickel chelate acceptor beads (PerkinElmer) were added to\\na final concentration of 10 μg mL–1, and plates were incubated for another hour. Plates were read on\\na PHERAstar FS (BMG Labtech) using an optic module with an excitation\\nwavelength of 680 nm and emission wavelength of 615 nm.\\nIntensity values were plotted against PROTAC concentration on a log10 scale using GraphPad Prism, version 7. To obtain biotinylated-VCB,\\nprotein was mixed with EZ-link NHS-PEG4-biotin (Thermo\\nScientific) in a 1:1 molar ratio and incubated at room temperature\\nfor 1 h. The reaction was quenched using 1 M Tris-HCl, pH 7.5,\\nand unreacted NHS-biotin was removed with a PD-10 MiniTrap desalting\\ncolumn (GE Healthcare) equilibrated with 20 mM HEPES, pH 7.5,\\n150 mM NaCl, and 1 mM DTT.
\\n \\n \\n Fluorescence Polarization\\n All measurements were taken\\nusing a PHERAstar FS (BMG LABTECH) with fluorescence excitation and\\nemission wavelengths (λ) of 485 and 520 nm, respectively. FP\\ncompetitive binding assays were run in triplicate in 384-well plates\\n(Corning 3575) using a total well volume of 15 μL (ref (63)). Each well solution contained\\n15 nM VCB protein, 10 nM FAM-labeled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, Kd = 3 nM as measured by a direct FP titration),\\nand decreasing concentrations of PROTAC (14-point serial 2-fold dilutions\\nstarting from 50 μM PROTAC) or PROTAC:bromodomain (14-point\\nserial 2-fold dilutions starting from 50 μM PROTAC:100 μM\\nbromodomain into wells containing an additional 1 μM bromodomain)\\nin 100 mM Bis-Tris propane, 100 mM NaCl, 1 mM TCEP, pH 7, with a final\\nDMSO concentration of 1%. Control wells containing VCB and peptide\\nwith no compound (zero displacement), or peptide in the absence of\\nprotein (maximum displacement), were also included. Control values\\nwere used to obtain the percentage of displacement which was graphed\\nagainst log[PROTAC]. Average IC50 values and the standard\\nerror of the mean (SEM) were determined for each titration using Prism\\n7. Dissociation constants Kd were back-calculated\\nfrom the measured IC50 values using a displacement binding\\nmodel, as described previously.68
\\n \\n \\n Isothermal\\nTitration Calorimetry\\n ITC experiments were\\nperformed in an ITC200 microcalorimeter (GE Healthcare) as described\\npreviously.5 Titrations were carried out\\nat 25 °C while stirring at 750 rpm and were performed as reverse\\nmode (protein in the syringe and the ligand in the cell). Compounds\\nwere diluted in ITC buffer (20 mM Bis-Tris propane, 100 mM NaCl, 1\\nmM tris(2-carboxyethyl)phosphine (TCEP), pH 7.5) from 10 mM\\nDMSO stock solutions to a final concentration of 20 μM (0.2%\\nDMSO). Each run consisted of 19 injections of 2 μL of protein\\nsolution (ITC buffer with 0.2% DMSO) at a rate of 0.5 μL/s at\\n120 s time intervals. An initial injection of 0.4 μL was made\\nand discarded during data analysis. BRD9 bromodomain (200 μM,\\nin the syringe) was first titrated into the PROTAC (20 μM, in\\nthe cell); at the end of the titration, the excess of solution was\\nremoved from the cell and the syringe was washed and dried. VCB complex\\n(168 μM, in the same buffer) was loaded in the syringe and titrated\\ninto the complex PROTAC-bromodomain. The concentration of the complex\\nin the cell (C) after the first titration (16.8 μM),\\nwas calculated as follows:![]()
where C0 is the\\ninitial concentration of the PROTAC in the cell (20 μM), Vcell is the volume of the sample cell (200.12\\nμL), and Vinj is the volume of titrant\\ninjected during the first titration (38.4 μL). Titrations for\\nthe binary complex PROTAC-VCB were performed by first adding buffer\\n(38.4 μL) to the solution of PROTAC (20 μM, in the cell)\\nby a single ITC injection. After removal of the excess solution from\\nthe cell, VCB complex (168 μM, in the same buffer) was loaded\\ninto the (washed and dried) syringe and titrated into the diluted\\nPROTAC solution. The data were fitted to a single binding-site model\\nusing the MicroCal PEAQ-ITC analysis software provided by the manufacturer\\nto obtain the stoichiometry n, the dissociation constant Kd, and the enthalpy of binding ΔH.
\\n \\n \\n Live Cell Kinetic Analysis of BRD7 and BRD9\\nDegradation\\n HEK293 cells stably expressing LgBiT protein\\ncultured in DMEM (Gibco)\\nsupplemented with 10% fetal bovine serum (Seradigm) and maintained\\nat 37 °C and 5% CO2 were genome-edited using CRISPR/Cas9\\nto generate endogenous HiBiT-BRD7 or HiBiT-BRD9 fusions. Clonal populations\\nwere isolated, and cells were plated in 96-well tissue culture plates\\nat a density of 2 × 104 cells per well in 100 μL\\nof growth medium. Following overnight incubation at 37 °C and\\n5% CO2 medium was replaced with CO2-independent\\nmedium (Gibco) containing 20 μM Endurazine, an extended time-released\\nsubstrate (Promega), and plates were incubated at 37 °C, 5% CO2, for 2.5 h before addition of a 3-fold serial dilution of\\n1 μM final concentration VZ185 compound. Plates retaining the\\nplate lids were then read every 5 min for a period of 24 h on a GloMax\\nDiscover (Promega) set to 37 °C. Degradation traces for each\\nconcentration were plotted in GraphPad Prism, and the degradation\\nportion of each curve was fitted to a one-component exponential decay\\nmodel to obtain degradation parameters, rate, and Dmax, as previously described.69
\\n \\n \\n NanoBRET Ubiquitination of BRD7 and BRD9\\n Clonal populations\\nof edited HEK293 cells expressing HiBiT-BRD7 or HiBiT-BRD9 were plated\\nin tissue 96-well tissue culture plates at a density of 2 × 104 cells per well in 100 μL of growth medium and incubated\\novernight at 37 °C, 5% CO2. Following treatment with\\n1 μM VZ185 for the indicated time frames, medium was replaced\\nwith Opti-MEM (Gibco) containing 200 μg/mL digitonin, 1:200\\ndilution of primary anti-Ub antibody (Enzo Life Sciences, BML-PW8810),\\n1:500 dilution of secondary anti-mouse Alexa 594 antibody (Cell Signaling\\nTechnologies, 8890), and 20 μM NanoGlo (Promega) substrate.\\nAdditional control wells received no antibodies (control for background\\nNanoBRET) or no primary antibody (control for specificity). Plates\\nwere placed on an orbital shaker for 10 min, and NanoBRET measurements\\nwere collected on a CLARIOstar (BMG Labtech). Background subtracted\\nNanoBRET ratios were generated by calculating the ratios of acceptor\\nsignal to donor signal in both the presence and absence of the Alexa594\\nantibody and computing the difference. Background subtracted BRET\\nratios were expressed in milliBRET units by multiplying by 1000.
\\n \\n \\n \\n