4. Materials and Methods
Section I
4.1 Chemistry: synthesis and characterization of BRD7/9-targeting PROTACs (Dundee project)
Synthesis. Chemicals, commercially available, were purchased from Apollo Scientific, Sigma-Aldrich, Fluorochem or Manchester Organics and used without any further purification. All reactions were carried out using anhydrous solvents. Analytical thin-layer chromatography (TLC) was performed on precoated TLC plates (layer 0.20 mm silica gel 60 with fluorescent indicator (UV 254: Merck). The TLC plates were air-dried and revealed under UV lamp (254/365 nm). Flash-column chromatography was performed using prepacked silica gel cartridges (230-400 mesh, 40-63 mm; SiliCycle) using a Teledyne ISCO Combiflash Companion or Combiflash Retrieve using the solvent mixtures stated for each synthesis as mobile phase. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed with either an Agilent HPLC 1100 series connected to a Bruker Daltonics MicroTOF or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole spectrometer or a Waters 2795 connected to a Waters ZQ Micromass spectrometer, all instruments were connected to a diode array detector. All the final compounds used in all the experiments were evaluated after preparative LC-MS separations with a Waters X-bridge C18 column (50 mm × 2.1 mm × 3.5 mm particle size); flow rate, 0.5 mL/min with a mobile phase of water/MeCN + 0.1% NH4OH or water/MeCN + 0.1% CHOOH; 95/5 water/MeCN was initially held for 0.5 min followed by a linear gradient from 95/5 to 5/95 water/MeCN over 3.5 min which was then held for 2 min. The purity of all the compounds was evaluated using the abovementioned analytical LC-MS system and yield a purity >95%. 1H-NMR and 13C-NMR
MHz) or on a Bruker DPX-400 spectrometer (1H at 400.1 MHz, 13C at 101 MHz). Chemical shifts (δ) are expressed in ppm reported using residual solvent as the internal reference in all cases. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), multiplet (m), or a combination thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz.
General method A: A mixture of aldehyde (1 eq), amine (HCl salt, 1.1 eq) and TEA (1.1 eq) in DMF (0.2 M) was stirred at rt. After 15 min, NaBH(OAc)3 (1.5 eq) was added and the reaction was stirred overnight at rt under nitrogen. The solvent was evaporated under reduced pressure to give the corresponding crude, which was purified to yield the desired compound.
General method B: To a mixture of aryl bromide (1 eq) in dioxane (0.2 M), Pd(dppf)Cl2 (0.1 eq), bis(pinacolato)diboron (1.2 eq) and KOAc (3 eq) were added. The mixture was heated under microwave conditions at 140 °C for 40 min. Then aryl iodide 4 (1 eq) and a degassed solution of K2CO3 2M (2 eq) were added. The reaction mixture was heated in microwave at 120 °C for 30 min. The resulting mixture was filtered through celite and washed several times with DCM. The organic phase was washed with H2O and brine, dried over MgSO4, filtered and evaporated in vacuum. The crude was purified by flash column chromatography using a gradient from 0% to 20% of MeOH in DCM to obtain the desired compound. Then the intermediate was dissolved in a 1:1 mixture of HCl 4N in dioxane/DCM and stirred at rt for 3 h. The solvents were evaporated to dryness to obtain the desired compound as hydrochloride salt.
General method C: To a solution of acid (1 eq) in DMF (0.2 M), HATU (1 eq), HOAt (1 eq) and amine (1 eq) were added. After stirring at rt for 5 min, DIPEA (5 eq) was added. The reaction mixture was stirred at rt for 2 h. Then the solvent was evaporated under reduced pressure to give the corresponding crude, which was purified to yield the final compound.
General method D: To a stirred solution of oxalyl chloride (1.5 eq) in DCM (0.3 M), DMSO (2 eq) was added dropwise at -78 ˚C. After 10 min at -78 ˚C, the starting alcohol (1 eq) in DCM (0.3 M) was added. The reaction was stirred at -78 ˚C for 1h, TEA (10 eq) was added dropwise and temperature was increased to rt. After 1 h, the reaction mixture was diluted with DCM (10 mL), washed with a saturated solution of ammonium chloride (2 x 5 mL). The organic phases were combined, washed with brine, dried over MgSO4, filtered and evaporated to dryness. Then the crude material was dissolved in DMF (0.05 M) and BrdL1 (1 eq) and TEA (1.1 eq) were added. After 15 min, NaBH(OAc)3 was added and the reaction mixture was stirred overnight at rt under nitrogen. The solvent was evaporated under reduced pressure and the corresponding crude was purified by preparative HPLC to yield the desired compound.
General method E: To a solution of the starting alcohol (1 eq) and trimethylamine (1.5 eq) in DCM (0.2 M), methansulfonyl chloride (1.2 eq) was added at 0 °C. The reaction mixture was stirred at 0 °C for 3h. The reaction was quenched with a saturated solution of ammonium chloride (5 mL) and extracted with DCM (3 x 15 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. The crude (1.5 eq) was suspended in DMF (0.2M), and the proper VHL2-4 derivative (1 eq) and K2CO3 (3 eq) were added. The mixture was heated overnight at 70 °C. The reaction mixture was diluted with water and extracted with DCM. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. The crude was purified by flash column chromatography using a gradient from 0% to 20% of MeOH in DCM to yield the desired compound.
General method F: To NaHDMS 1M in THF (2.5 eq), glycol (5 eq) was added at 0 °C under nitrogen in seal vial. The reaction mixture is stirred at 0 °C for 45 min. Then bromide (1 eq) and DMF (0.5 M) were added and the mixture was heated in microwave at 130 °C for 2h. The solvent was removed in vacuum. A 5% solution of NaHSO4 was added (3 mL) and the reaction mixture was extracted with DCM (3 x 15 mL). The organic phases were combined, dried over
MgSO4, filtered and evaporated to dryness. The crude was purified by flash column chromatography using a gradient from 0% to 5-10% of MeOH in DCM to yield the desired compound.
General method G: A 1M aqueous solution of HCl (1 mL) was added to the VHL-acetal linker (0.06 mmol) in THF (1 mL). The reaction mixture was heated at 50 °C for 2 h. The solvent was evaporated under reduced pressure and the product was extracted with DCM/MeOH (9:1) (3 x 10 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. The crude was used directly without any further purification.
General method H: Aldehyde (1 eq) was dissolved in a t-BuOH: water (4:1) mixture (0.3 M) and 2-methyl-2-butene 2M in THF (4 eq), sodium phosphate dibasic (1 eq) and sodium chlorite (2 eq) were sequentially added. The reaction mixture was stirred at rt for 4 h. 1M HCl aqueous solution was added and the mixture was extracted with DCM (3 x 10 mL). The pooled organic phases were dried over MgSO4, filtered and evaporated to dryness. The crude was used directly without any further purification.
2,7-naphthyridin-1(2H)-one (2).
To a DMF solution (48 mL) of 4-methyl-3-cyanopyridine (4 g, 34 mmol, 1 eq), DMF acetal (4.02 g, 34 mmol, 1 eq) was added. The reaction mixture was heated under reflux at 160 ºC overnight. Then, the solvent was evaporated and the solid was dissolved in glacial acetic acid (24 mL) and sulfuric acid (24 mL). The reaction mixture was heated at 130 ºC for 2 h. The mixture was neutralized at 0 ºC with an aqueous solution of NH4OH and the product was extracted with CHCl3/MeOH (9:1). The solvent was evaporated and the product was used directly without any further purification. Yield: 3.69 g, 74%. ¹H-NMR (400 MHz, CDCl3) d:
N HN
9.57 (s, 1H), 8.73 (d, J = 5.5 Hz, 1H), 7.37 (d, J = 5.5 Hz, 1H), 7.29 (d, J = 7.1 Hz, 1H), 6.48 (d, J = 7.2 Hz, 1H). Analytical data matched those previously reported.171 MS m/z calcd for C8H6N2O 146.05.
4-iodo-2,7-naphthyridin-1(2H)-one (3).
A mixture of 2 (3.69 g, 25 mmol, 1 eq) and iodine (6.34 g, 25 mmol, 1 eq) in a 0.4 N aqueous solution of NaOH (0.15 M final conc.) was heated at 80 °C for 5 h. Citric acid was added up to neutral pH (dark precipitate), then a 10% aqueous solution of Na2SO3 was added to quench I2. The precipitate was filtered and washed with water. The product was used directly without any further purification. Yield: 3.6 g, 53%. ¹H-NMR (400 MHz, DMSO) d: 10.08 (s, 1H), 9.68 (d, J = 5.6 Hz, 1H), 8.70 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H). Analytical data matched those previously reported.172 MS m/z calcd for C
8H5IN2O 271.94, found 273 [M + H+].
4-iodo-2-methyl-2,7-naphthyridin-1(2H)-one (4).
A DMF (20 mL) solution of 3 (1.2 g, 4.4 mmol, 1 eq) and NaH (325 mg, 8.8 mmol, 2 eq) was stirred 30 min at 0 °C and then CH3I (2.5 g, 18 mmol, 1.6 eq) was added. The reaction mixture was stirred at 0 °C for 5 h. Water was added, and the formed precipitate was filtered and dried in vacuum. The product was used directly without any further purification. Yield: 1.2 g, 95%.
N HN O I N N O I
= 5.5 Hz, 1H), 3.52 (s,3H); 13C-NMR (101 MHz, DMSO) d: 160.3, 152.0, 150.3, 144.8, 143.0, 122.4, 120.7, 67.9, 36.2. MS m/z calcd for C9H7IN2O 285.96, found 287.1 [M + H+].
tert-butyl 4-(4-bromo-2,6-dimethoxybenzyl)piperazine-1-carboxylate (6).
Following general method A, compound 6 was obtained from the commercially available 4-bromo-2,6-dimethoxybenzaldehyde (5, 300 mg, 1.22 mmol) and boc-piperazine (251 mg, 1.34 mmol). The reaction was quenched with saturated solution of NaHCO3 (2 mL), extracted with DCM (3 x 15 mL), washed with water and brine. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 495 mg, 97%. ¹H-NMR (500 MHz, CDCl3) d: 6.69 (s, 2H), 3.78 (s, 6H), 3.62 (s, 2H), 3.38 (t, J = 5.0 Hz, 4H), 2.41 (t, J = 4.4 Hz, 4H), 1.43 (s, 9H); 13C-NMR (125 MHz, CDCl3) d: 159.9, 154.7, 122.8, 107.7, 79.8, 56.1, 51.8, 48.5, 31.0, 28.5. MS m/z calcd for C18H27BrN2O4 414.12, found 415.2 [M + H+].
4-(3,5-dimethoxy-4-(piperazin-1-ylmethyl)phenyl)-2-methyl-2,7-naphthyridin-1(2H)-one (BrdL1, 7). Br N O O N Boc
Following general method B, compound BrdL1 was obtained from compound 4 (110 mg, 0.38 mmol) and 6 (160 mg, 0.38 mmol) as brown powder. Yield: 50 mg, 55%. ¹H-NMR (400 MHz, CD3OD) d: 9.67 (s, 1H), 8.76 (d, J = 6.9 Hz, 1H), 8.27 (s, 1H), 8.21 (d, J = 6.9 Hz, 1H), 6.95 (s, 2H), 4.62 (s, 2H), 4.03 (s, 6H), 3.81 (s, 3H), 3.73 (s, 8H); 13C-NMR (101 MHz, CDCl
3) d: 161.5, 161.4, 148.6, 146.3, 145.5, 141.9, 139.7, 122.9, 117.9, 107.1, 105.8, 57.2, 50.8, 49.7, 41.9, 37.9. MS m/z calcd for C22H26N4O3 394.2, found 395.3 [M + H+].
2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol (9).
To a THF (40 mL) solution of LAH (40 mmol, 6 eq), a solution of 8 (1.4 g, 6.71 mmol, 1 eq) in THF was added at 0 °C. The reaction mixture was heated at 55 °C overnight, cooled at 0 ˚C and quenched with water (2.58 mL), 15% NaOH aqueous solution (1.3 mL) and water (4.8 mL). The precipitated aluminium hydroxide was filtered off under vacuum and washed with a mixture of CHCl3/H2O 8:2. The crude product was purified by flash chromatography using a mixture of 0-20% MeOH/NH3 in DCM. Yield: 41%. Analytical data matched those previously reported.72 MS m/z calcd for C
11H12N2OS 220.07. N N O N O O NH2+Cl -S N OH H2N
tert-butyl (2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (10).
(4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (630 mg, 2.73 mmol) and 9 (600 mg, 2.73 mmol) were reacted according to general method C to afford compound 10 that was purified by flash column chromatography using a gradient from 0% to 20% of methanol in DCM. Yield: 200 mg, 78%. Analytical data matched those previously reported.72 MS m/z calcd for C21H27N3O5S 433.52, found 434 [M + H+].
(2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-ium chloride (11).
To a DCM (4 mL) solution of 10 (915 mg, 2.11 mmol), a 4 M HCl solution in dioxane (4 mL) in was added and the mixture was stirred at rt for 3 h. Then, the solvent was evaporated and the product was dried in vacuum. Yield: 773 mg, quant. Analytical data matched those previously reported.72 MS m/z calcd for C
16H20ClN3O3S 369.09. (S)-1-((2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-aminium chloride (12). N O H N S N Boc HO OH N H2 + Cl -O H N S N HO OH
Intermediate 10 (765 mg, 2.07 mmol) and Boc-L-tert-leucine (478 mg, 2.07 mmol) were reacted according to general method C and the Boc derivative of 12 was obtained after flash column chromatography using a gradient from 0% to 20% of methanol in DCM. Then, the crude was dissolved in DCM (4 mL) and a 4 M HCl solution in dioxane (4 mL) was added. The reaction mixture was stirred at rt for 1 h and the solvent was evaporated. The product was dried in vacuum. Yield: 973.5 mg, quant. Analytical data matched those previously reported.72 MS m/z calcd for C22H31ClN4O4S 482.18.
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VHL2, 13).
To a mixture of 12 (100 mg, 0.2 mmol, 1 eq) and DIPEA (57 mg, 0.44 mmol, 2.2 eq) in DMF (500 µL), 1-acetylimidazole (33 mg, 0.3 mmol, 1.5 eq) was added. The reaction mixture was stirred at rt for 3 h. Then, the solvent was evaporated and the product was purified by preparative HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain compound 13. Yield: 50 mg, 51%. Analytical data matched those previously reported.71 MS m/z calcd for C H NOS 488.21.
N O H N S N O NH3+Cl -HO OH N O H N S N O NH HO OH O
(2S,4R)-1-((R)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide ( VHL3, 14).
Following general method C, compound VHL3 was obtained from compound 12 (200 mg, 0.4 mmol, 1 eq) and 1-cianocyclopropane-1-carboxylic acid (46 mg, 0.4 mmol, 1 eq). The crude was purified by flash column chromatography using a gradient from 0% to 20% of methanol in DCM, affording VHL3. Yield: 116 mg, 50%. Analytical data matched those previously reported.71 C
27H33N5O5S 539.22, found 540.3 [M + H+].
(2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VHL4, 15).
Following general method C, compound VHL4 was obtained from compound 12 (200 mg, 0.4 mmol, 1 eq) and 1-fluorocyclopropane-1-carboxylic acid (43 mg, 0.4 mmol, 1 eq). The crude
N O H N S N O HO OH O NC NH N O H N S N O HO OH O F NH
was purified by flash column chromatography using a gradient from 0% to 20% of methanol in DCM, affording VHL4. Yield: 123 mg, 56%. ¹H NMR (500 MHz, CDCl3) d: 8.66 (s, 1H), 8.00 (t, J = 6.5 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.98 (d, J = 1.5 Hz, 1H), 6.97 - 6.93 (m, 1H), 6.88 (dd, J = 1.9, 7.8 Hz, 1H), 4.75 (t, J = 7.8 Hz, 1H), 4.55 - 4.49 (m, 2H), 4.42 (d, J = 8.5 Hz, 1H), 4.14 (dd, J = 5.5, 14.7 Hz, 1H), 4.01 (d, J = 11.7 Hz, 1H), 3.57 (dd, J = 3.7, 11.4 Hz, 1H), 2.57 - 2.46 (m, 4H), 2.10 - 2.04 (m, 1H), 1.35 - 1.25 (m, 4H), 0.85 (s, 10H); 13C-NMR (CDCl3, 125MHz) δ: 172.6, 171.5, 170.5, 155.6, 150.3, 148.5, 133.6, 131.5, 123.8, 120.9, 118.6, 70.1, 57. 8, 56.6, 40.0, 35.0, 26.1, 20.6 16.23, 13.8. MS m/z calcd for C26H33FN4O5S 532.22, found 533.3 [M + H+]. 3-chloro-7-nitro-1H-indole (17).
To a THF (1.54 mL) solution of 7-nitroindole (250 mg, 1.54 mmol, 1 eq), 0.1N aqueous solution of HCl (20 µL, 2 eq) and N-chlorosuccinimide (210 mg, 1.57 mmol, 1.02 eq) were added. The reaction mixture was stirred at rt for 5 h. Then, water (3 mL) was added to the reaction mixture and the crystalline precipitate was collected by filtration. Next, the precipitate was washed with H2O, MeOH/ H2O and finally dried in vacuum to give compound 17 as a yellow crystalline powder. Yield: 285 mg, 94%. ¹H-NMR (400 MHz, DMSO) d: 12.13 (s, 1NH), 8.20 (dd, J = 0.6, 8.1 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 2.7 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H); 13C-NMR (101 MHz, DMSO) d: 133.0, 128.7, 127.1, 125.9, 125.8, 119.9, 119.6, 105.5. MS m/z calcd for C8H5ClN2O2 196.00. H N NO2 Cl
3-chloro-1H-indol-7-amine (18).
To a 2-propanol solution (2.2 mL) of 17 (150 mg, 0.76 mmol, 1 eq), Fe powder (128 mg, 2.29 mmol, 6 eq) and an aqueous solution (460 µL) of NH4Cl were added. The reaction mixture was stirred in a sealed vial overnight at 80 °C. The reaction mixture was filtered off through celite and washed with ethyl acetate (3 x 10 mL). The organic phase was washed with H2O and brine, dried over MgSO4 and filtered. The crude was used directly for the next step without any further purification. Yield: quant. ¹H-NMR (400 MHz, CD3OD) d: 7.21 (s, 1H), 6.98 - 6.92 (m, 2H), 6.59 (dd, J = 1.7, 6.7 Hz, 1H); 13C-NMR (101 MHz, CD
3OD) d: 133.8, 127.4, 127.1, 121.8, 121.7, 109.0, 108.7, 106.6. MS m/z calcd for C8H7ClN2 166.03, found 167.1 [M + H+].
N-(3-chloro-1H-indol-7-yl)-4-formylbenzenesulfonamide (20).
To ethyl acetate (5 mL) solution of compound 18 (105 mg, 0.63 mmol, 1 eq), 4-formylbenzenesulfonyl chloride 19 (142 mg, 0.69 mmol, 1.1 eq) and pyridine (101 µL, 1.26 mmol, 2 eq) were added. The reaction mixture was stirred at rt for 3 h, diluted with ethyl acetate (5 mL) and washed successively with a 1N HCl(aq), H2O, saturated aqueous NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and evaporated in vacuum. The crude was purified by flash column chromatography using a gradient from 10% to 60% of ethyl acetate
H N NH2 Cl H N NH S Cl O O H O
in heptane to obtain compound 20. Yield: 104.5 mg, 62%. ¹H-NMR (400 MHz, DMSO) d: 11.11 (s, 1NH), 10.22 (d, J = 2.2 Hz, 1NH), 10.03 (s, 1COH), 8.02 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 2.6 Hz, 1H), 7.28 (dd, J = 0.6, 8.0 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.77 - 6.74 (m, 1H); 13C-NMR (101 MHz, DMSO) d: 192.6, 144.2, 138.9, 130.2, 129.7, 127.8, 126.4, 123.3, 123.1, 121.6, 120.2, 116.8, 116.7, 115.4, 103.8. MS m/z calcd for C15H11ClN2O3S 334.02, found 335.1 [M + H+].
4-(aminomethyl)-N-(3-chloro-1H-indol-7-yl)benzenesulfonamide (Indisulam derivative, 21).
Compound 20 (110 mg, 0.33 mmol, 1 eq) was dissolved in a saturated solution of NH4OAc in EtOH (6.6 mL), prepared as follow: 25ml of EtOH were heated to boil then NH4OAc was added until saturation, then NH3 30% (2.64 mL) was added. After 15 min, NaBCNH3 (0.09 mmol, 3 eq) was added and the reaction mixture was heated at 100 °C for 15 minutes. The solvent was removed in vacuum and the crude was dissolved in DCM/MeOH, washed successively with saturated aqueous NaHCO3 and 15% aqueous NaOH up to pH 8. The solvent was evaporated in vacuum to give the corresponding crude, which was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain compound 21. Yield: 42 mg, 35%. ¹H-NMR (400 MHz, CD3OD) d: 8.58 (d, J = 2.8 Hz, 1NH), 7.79 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.36 (dd, J = 0.7, 8.0 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), H N NH S Cl O O H2N
6.92 (t, J = 7.8 Hz, 1H), 6.75 - 6.70 (m, 1H), 4.14 (s, 2H); 13C-NMR (101 MHz, CD3OD) d: 141.0, 140.9, 131.8, 130.2, 129.1, 128.3, 123.3, 122.8, 120.9, 119.1, 117.0, 106.3, 43.9. MS
m/z calcd for C15H14ClN3O2S 335.05, found 336.1 [M + H+].
2-(2-(benzyloxy)ethoxy)ethan-1-ol (24).
To a DMF (11 mL) mixture of NaH (660 mg, 16.5 mmol, 2.5 eq) , diethylene glycol (3.5 g, 33 mmol, 5 eq) was added at 0 ˚C under nitrogen. After 45 min benzyl bromide (1.1 g, 6.6 mmol, 1 eq) was added dropwise at 0 ˚C. Then the ice bath was removed, and the reaction mixture was stirred overnight at rt under nitrogen. The reaction was quenched with a saturated solution of ammonium chloride (3 mL) and extracted with ethyl acetate (3 x 10 mL) and DCM (3 x 15 mL). The organic phases were combined, washed with brine, dried over MgSO4, filtered and evaporated to dryness. The resulting oil was purified by flash column chromatography using a gradient from 60% to 100% of ethyl acetate in heptane to yield the compound 24 as an oil. Yield: 1.04g, 80%. ¹H-NMR (500 MHz, CDCl3) d: 7.35 - 7.26 (m, 5H), 4.57 (s, 2H), 3.74 - 3.67 (m, 4H), 3.65 - 3.60 (m, 4H); 13C-NMR (125 MHz, CDCl
3) d: 138.2, 128.6, 128.0, 73.5, 72.8, 70.6, 69.6, 61.9. MS m/z calcd for C11H16O3 196.11, found 197.2 [M + H+].
1-phenyl-2,5,8,11-tetraoxatridecan-13-ol (25).
Under the same conditions used to synthetized 24, compound 25 was obtained from the reaction between tetraethylene glycol and benzyl bromide. Yield: 1.7 g, 91%. ¹H-NMR (500
O OH
O
O O O
MHz, CDCl3) d: 7.35 - 7.26 (m, 5H), 4.56 (s, 2H), 3.72 - 3.58 (m, 16H); 13C-NMR (125 MHz, CDCl3) d: 138.6, 128.7, 128.1, 73.6, 72.9, 70.7, 69.8, 62.1. MS m/z calcd for C15H24O5 284.16.
tert-butyl 2-(2-(2-(benzyloxy)ethoxy)ethoxy)acetate (26).
To a stirred DCM (5 mL) solution of 24 (500 mg, 2.55 mmol, 1eq), 37% aqueous solution of NaOH (6 mL) was added, followed by tert-butylbromo acetate (2 g, 10.19 mmol, 4 eq) and TBAB (838 mg, 2.6 mmol, 1.02 eq). The reaction mixture was stirred overnight at rt and then extracted with EtOAc (3 x 10 mL). The organic phases were combined, washed with brine, dried over MgSO4, filtered and evaporated to dryness. The resulting oil was purified by flash column chromatography using a gradient from 20% to 60% of ethyl acetate in heptane to yield 26 as an oil. Yield: 670 mg, 85%. ¹H-NMR (500 MHz, CDCl3) d: 7.35 - 7.26 (m, 5H), 4.56 (s, 2H), 4.02 (s, 2H), 3.73 - 3.67 (m, 6H), 3.64 - 3.62 (m, 2H), 1.46 (s, 9H). Analytical data matched those previously reported.173 MS m/z calcd for C
17H26O5 310.18.
tert-butyl 1-phenyl-2,5,8,11,14-pentaoxahexadecan-16-oate (27).
Following method used to synthetized 26, compound 27 was obtained by reacting compound 25 (850 mg, 2.98 mmol) with tert-butylbromo acetate (2.34 g, 10.19 mmol, 4 eq) and TBAB (983 mg, 2.6 mmol, 1.02 eq). Yield: 770 mg, 65%. ¹H-NMR (500 MHz, CDCl3) d: 7.35 - 7.26 (m, 5H), 4.56 (s, 2H), 4.00 (s, 2H), 3.71 - 3.61 (m, 16H), 1.46 (s, 9H); 13C-NMR (125 MHz, CDCl3) d: 170.0, 138.6, 128.7, 128.1, 81.8, 73.6, 71.0, 70.9, 69.8, 69.4, 28.4. MS m/z calcd for C H O O O O O O O O O O O O O
tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy) acetate (28).
Compound 26 (670 mg, 2.15 mmol) was dissolved in ethanol (43 mL) and hydrogenation (catalyst: 10% Pt/C) was carried out using H-Cube at 65 ˚C, at 8 atm and at 1 mL/min. The solvent was evaporated under vacuum and the crude material was directly used in the next step without any further purification. Yield: 470 mg, 93%. ¹H-NMR (400 MHz, CDCl3) d: 4.01 (s, 2H), 3.75 - 3.60 (m, 8H), 1.47 (s, 9H); 13C-NMR (101 MHz, CDCl
3) d: 169.6, 81.6, 72.7, 70.8, 70.3, 68.9, 61.5, 28.1. MS m/z calcd for C10H20O5 220.13.
tert-butyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate (29).
An ethanol (38 mL) solution of compound 27 (770 mg, 1.9 mmol) was hydrogenated (catalyst: 10% Pt/C) using H-Cube at 65 ˚C, at 8 atm and at 1 mL/min. The solvent was evaporated under vacuum and the crude material was directly used in the next step without any further purification. Yield: 561 mg, 92%. ¹H-NMR (400 MHz, CDCl3) d: 4.01 (s, 2H), 3.73 - 3.58 (m, 16H), 1.46 (s, 9H); 13C-NMR (101 MHz, CDCl 3) d: 169.9, 81.7, 72.7, 70.9, 70.8, 70.5, 69.2, 61.9, 28.3. MS m/z calcd for C14H28O7 308.18. tert-butyl 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)acetate (31). O O O O HO O O O O HO O O
Following the general method D, compound 31 was obtained from 28 (70 mg, 0.3 mmol) and BrdL1 (20 mg, 0.05 mmol). The final compound was purified by HPLC (gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia) and isolated as a white powder. Yield: 8.8 mg, 32%. ¹H-NMR (500 MHz, CDCl3) d: 9.69 (d, J = 0.8 Hz, 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, 2H), 3.81 (s, 6H), 3.70 - 3.60 (m, 11H), 2.65 - 2.56 (m, 10H), 1.46 (s, 9H); 13C-NMR (125 MHz, CDCl3) d: 170.0, 161.8, 160.0, 152.0, 151.3, 142.1, 136.1, 135.5, 120.8, 118.4, 117.8, 113.9, 105.7, 81.9, 71.0, 70.7, 69.3, 58.1, 56.2, 53.7, 52.6, 48.9, 37.4, 28.4. MS m/z calcd for C32H44N4O7 596.32, found 597.34 [M + H+].
tert-butyl 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-tetraoxatetradecanoate (32).
Following the general method D, compound 32 was obtained by reacting 29 (97 mg, 0.3 mmol) and BrdL1 (22 mg, 0.05 mmol). The desired compound was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia and isolated as white powder. Yield: 10.7 mg, 34%. ¹H-NMR (500 MHz, CDCl3) d: 9.69 (d, J = 0.8 Hz, 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), 3.81 (s, N N O O N N O O O O O N N O O N N O O O O O O O
6H), 3.73 - 3.57 (m, 19H), 2.67 - 2.55 (m, 10H), 1.46 (s, 9H); 13C-NMR (125 MHz, CDCl3) d: 170.0, 161.8, 160.0, 152.0, 151.3, 142.1, 136.2, 120.8, 118.4, 117.8, 105.7, 81.9, 71.1, 70.9, 70.7, 69.4, 69.2, 58.0, 56.3, 53.8, 52.4, 48.9, 37.5, 28.5. MS m/z calcd for C36H52N4O9 684.37, found 685.39 [M + H+]. tert-butyl 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-pentaoxaheptadecanoate (33).
Following general method D, the reaction between compound 30 and BrdL1 (20 mg, 0.05 mmol) led to the formation of compound 33 that was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia as white powder. Yield: 24.6 mg, 73%. ¹H-NMR (500 MHz, CDCl3) d: 9.68 (d, J = 0.9 Hz, 1H), 8.69 (d, J = 5.6 Hz, 1H), 7.42 (dd, J = 0.8, 5.6 Hz, 1H), 7.26 (s, 1H), 6.52 (s, 2H), 3.99 (s, 2H), 3.80 (s, 6H), 3.72 - 3.57 (m, 23H), 2.67 - 2.53 (m, 10H), 1.45 (s, 9H); 13C-NMR (125 MHz, CDCl
3) d: 170.0, 161.8, 160.0, 152.0, 151.2, 142.1, 136.1, 135.6, 120.8, 118.4, 117.8, 113.7, 105.7, 81.8, 71.0, 70.9, 70.6, 69.3, 69.2, 58.0, 56.2, 53.9, 52.5, 48.9, 37.4, 28.4. MS m/z calcd for C38H56N4O10728.40, found 729.42 [M + H+]. 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 acid (34). N N O O N N O O O O O O O O
A mixture of compound 31 (8.8 mg, 0.015 mmol), TFA (0.5 mL) and DCM (0.5 mL) was stirred at rt for 3 h. Then the solvent was evaporated and the crude was kept under high vacuum overnight. The isolated product was used in the next step without any further purification. Quantitative yield. MS m/z calcd for C28H36N4O7 540.26, found 541.4 [M + H+].
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 acid (35).
A mixture of compound 32 (11 mg, 0.016 mmol), TFA (0.5ml) and DCM (0.5ml) was stirred at rt for 3 h. Then the solvent was evaporated and the crude was dried under high vacuum overnight. The obtained product was used directly without any further purification. Quantitative yield. MS m/z calcd for C34H48N4O10 672.34, found 673.36 [M + H+].
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 acid (36). N N O O N N O O O OH O N N O O N N O O O O O OH O N N O N N O O O O O O OH O
A mixture of compound 33 (25 mg, 0.034 mmol), TFA (0.5 mL) and DCM (0.5 mL) was stirred at rt for 3 h. Then the solvent was evaporated; the crude was dried under high vacuum overnight and used directly without any further purification. Quantitative yield. MS m/z calcd for C34H48N4O10 672.34, found 673.36 [M + H+].
(2S,4R)-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-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VZ48).
Following the general method C, compound VZ48 was obtained from 34 (0.015 mmol) and VHL1 (7 mg, 0.015 mmol; synthetized accordingly to literature48). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain VZ48 as white powder. Yield: 6.3 mg, 44%. ¹H-NMR (400 MHz, CD3OD) d: 9.44 (s, 1H), 8.80 (s, 1H), 8.59 (d, J = 5.5 Hz, 1H), 7.66 (s, 1H), 7.54 (d, J = 5.9 Hz, 1H), 7.37 (q, J = 7.8 Hz, 4H), 6.73 (s, 2H), 4.66 (s, 1H), 4.52 - 4.26 (m, 4H), 4.05 - 3.95 (m, 4H), 3.86 - 3.72 (m, 8H), 3.69 - 3.59 (m, 9H), 2.96 - 2.73 (m, 10H), 2.40 (s, 3H), 2.21 - 2.14 (m, 1H), 2.06 - 1.97 (m, 1H), 0.99 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.3, 172.0, 171.7, 163.0, 161.0, 152.8, 151.7, 151.2, 149.1, 143.5, 140.3, 139.0, 133.3, 131.6, 130.5, 130.4, 129.5, 129.0, 121.7, 119.2, 119.0, 106.6, 72.2, 71.3, 71.1, 71.0, 69.1, 60.9, 58.2, 58.1, 57.9, 56.6, 52.8, 52.4, 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 H O NH N S OH O O O O N N O O N N O
(2S,4R)-1-((S)-2-(tert-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-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VZ52).
Following the general method C, compound VZ52 was obtained from 35 (0.016 mmol) and VHL1 (7.6 mg, 0.016 mmol, synthetized accordingly to literature48). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain VZ52 as white powder. Yield: 4.9 mg, 30%. ¹H-NMR (400 MHz, CD3OD) d: 9.44 (s, 1H), 8.81 (s, 1H), 8.60 (d, J = 5.6 Hz, 1H), 7.67 (s, 1H), 7.54 (d, J = 5.6 Hz, 1H), 7.37 (q, J = 9.0 Hz, 4H), 6.73 (s, 2H), 4.63 (s, 1H), 4.53 - 4.26 (m, 4H), 4.02 - 3.94 (m, 4H), 3.84 - 3.72 (m, 8H), 3.66 - 3.50 (m, 17H), 2.89 - 2.67 (m, 10H), 2.41 (s, 3H), 2.20 - 2.14 (m, 1H), 2.06 - 1.97 (m, 1H), 0.98 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.4, 172.0, 171.7, 163.0, 161.0, 152.9, 151.7, 151.2, 149.1, 143.5, 140.3, 139.0, 138.6, 133.4, 131.5, 130.5, 130.4, 129.5, 129.0, 121.7, 119.2, 119.1, 106.6, 72.3, 71.7, 71.6, 71.5, 71.4, 71.1, 71.0, 68.9, 60.9, 58.2, 58.0, 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+]. (2S,4R)-1-((S)-2-(tert-butyl)-20-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7- naphthyridin-4-yl)benzyl)piperazin-1-yl)-4-oxo-6,9,12,15,18-pentaoxa-3-azaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VZ49). N O NH N S OH O O O O N H O O N N O O N N O
Following general method C, compound VZ49 was obtained by the reaction between 36 (0.034 mmol) and VHL1 (16 mg, 0.034 mmol, synthetized accordingly to literature48). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain VZ49 as white powder. Yield: 12.8 mg, 35%. ¹H-NMR (400 MHz, CD3OD) d: 9.43 (s, 1H), 8.81 (s, 1H), 8.59 (d, J = 5.6 Hz, 1H), 7.66 (s, 1H), 7.54 (d, J = 5.6 Hz, 1H), 7.37 (q, J = 9.3 Hz, 4H), 6.72 (s, 2H), 4.63 (s, 1H), 4.53 - 4.27 (m, 4H), 4.03 - 3.94 (m, 4H), 3.84 - 3.71 (m, 8H), 3.66 - 3.52 (m, 21H), 2.89 - 2.68 (m, 10H), 2.41 (s, 3H), 2.20 - 2.13 (m, 1H), 2.09 - 1.97 (m, 1H), 0.98 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.4, 172.0, 171.7, 170.1, 163.0, 161.0, 152.8, 151.7, 151.2, 149.0, 143.5, 140.3, 139.0, 138.5, 133.4, 131.5, 130.5, 130.4, 129.5, 129.0, 121.7, 119.2, 119.1, 106.6, 72.3, 71.7, 71.6, 71.5, 71.4, 71.1, 71.0, 69.0, 68.9, 60.8, 58.1, 56.6, 52.8, 43.7, 39.0, 37.4, 37.1, 27.0, 15.9. HRMS m/z calcd for C56H76N8O12S 1084.53, found 1085.5578 [M + H+]. 2-(2-(hex-5-en-1-yloxy)ethoxy)ethan-1-ol (38).
To a suspension of NaH (600 mg, 15 mmol, 2.5 eq) in DMF (6 mL) and THF (6 mL), diethylene glycol (3 g, 31 mmol, 5 eq) was added at 0 ˚C under nitrogen. After 45 min 6-bromo-1-hexene (1 g, 6 mmol, 1 eq) was added dropwise at 0 ˚C. Then the ice bath was removed, and the reaction mixture was stirred overnight at rt under nitrogen. Distillate water (4 mL) was added and the reaction mixture was acidified with HCl 1M up to pH 2. The product was extracted with CHCl3 (3 x 20 mL). The organic phases were combined, dried over MgSO4, filtered and
N O NH N S OH O O O N N O O N N O O N H O O O O O HO
evaporated to dryness. The resulting oil was purified by flash column chromatography using a gradient from 50% to 100% of ethyl acetate in heptane to obtain the desired compound 38 as an oil. Yield: 552 mg, 49%. ¹H-NMR (400 MHz, CDCl3) d: 5.85 - 5.74 (m, 1H), 5.03 - 4.96 (m, 1H), 4.96 - 4.91 (m, 1H), 3.75 - 3.56 (m, 8H), 3.46 (t, J = 6.7 Hz, 2H), 2.09 - 2.03 (m, 2H), 1.65 - 1.56 (m, 2H), 1.48 - 1.39 (m, 2H); 13C-NMR (101 MHz, CDCl3) d: 138.8, 114.7, 72.6, 71.5, 70.6, 70.3, 62.0, 33.6, 29.2, 25.5. MS m/z calcd for C10H20O3 188.14.
2-(2-(hex-5-en-1-yloxy)ethoxy)acetic acid (39).
A mixture of 38 (542 mg, 2.88 mmol, 1 eq), BAIB (2 g, 6.33 mmol, 2.2 eq), TEMPO (99 mg, 0.63 mmol, 0.22 eq) in ACN/H2O (1:1) (5.6 mL) was stirred overnight at rt. The solvent was then evaporated, the crude product was resuspended in DCM (15 mL) and washed with H2O (5 mL). The organic phase was dried over MgSO4, filtered and evaporated to dryness. The resulting product was purified by flash column chromatography using a gradient from 0% to 20% of MeOH in DCM to obtain compound 39 as an oil. Yield: 472 mg, 81%. ¹H-NMR (400 MHz, CDCl3) d: 5.84 - 5.73 (m, 1H), 5.03 - 4.97 (m, 1H), 4.96 - 4.92 (m, 1H), 4.15 (s, 2H), 3.76 - 3.73 (m, 2H), 3.63 - 3.59 (m, 2H), 3.52 (t, J = 6.6 Hz, 2H), 2.10 - 2.03 (m, 2H), 1.65 - 1.57 (m, 2H), 1.49 - 1.40 (m, 2H); 13C-NMR (101 MHz, CDCl
3) d: 173.2, 138.7, 114.8, 71.7, 71.6, 69.7, 69.1, 33.6, 28.9, 25.4. MS m/z calcd for C10H18O4 202.12.
(2R,4S)-1-((R)-2-(2-(2-(hex-5-en-1-yloxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (40).
HO O O
Following the general method C, compound 40 was obtained from compound 39 (16 mg, 0.08 mmol) and VHL1 (40 mg, 0.08 mmol, synthetized accordingly to literature48). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain 40 as white powder. Yield: 14.7 mg, 30%. ¹H-NMR (400 MHz, CDCl3) d: 8.64 (s, 1H), 7.28 - 7.23 (m, 4H), 5.74 - 5.63 (m, 1H), 4.93 - 4.82 (m, 2H), 4.65 (t, J = 7.9 Hz, 1H), 4.49 - 4.21 (m, 4H), 4.03 - 3.85 (m, 3H), 3.59 - 3.47 (m, 5H), 3.40 - 3.34 (m, 2H), 2.47 - 2.39 (m, 4H), 2.06 - 1.91 (m, 3H), 1.54 - 1.45 (m, 2H), 1.37 - 1.29 (m, 2H), 0.87 (s, 9H); 13C-NMR (101 MHz, CDCl 3) d: 171.6, 170.8, 150.7, 148.1, 138.7, 138.4, 132.0, 130.7, 129.6, 128.3, 114.8, 71.5, 71.4, 70.4, 70.3, 69.8, 58.6, 57.5, 56.7, 43.3, 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+].
(2R,4S)-1-((R)-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-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VZ55).
A mixture of 40 (14.7 mg, 0.024 mmol, 1 eq), osmium tetroxide 4% in H2O (3 µL, 0.0005 mmol, 0.02 eq), sodium periodate (20 mg, 0.096 mmol, 4 eq), pyridine (4 µL, 0.048 mmol, 2
N H N O HN N S OH O O O O N H N O HN N S OH O O N N O O N N O O O
eq) in dioxane/ H2O (3:1) (0.25 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 5 mL). The pooled organic phases were dried over MgSO4, filtered and evaporated to dryness. The crude was dissolved in DCE (1 mL) and a mixture of BrdL1 (7.8 mg, 0.018 mmol, 1 eq) and TEA (3 µL, 0.02 mmol, 1.1 eq) in DCE (0.02M) was added. After 15 min, NaBH(OAc)3 (5.7 mg, 0.027 mmol, 1.5 eq) was added and the reaction mixture was stirred at rt for 4 h under nitrogen. The solvent was evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia. Compound VZ55 was obtained as a white powder. Yield: 6.9 mg, 54%. ¹H-NMR (400 MHz, CDCl3) d: 9.68 (s, 1H), 8.69 (d, J = 5.6 Hz, 1H), 8.66 (s, 1H), 7.41 (dd, J = 0.6, 5.6 Hz, 1H), 7.37 - 7.32 (m, 4H), 7.27 (s, 1H), 6.53 (s, 2H), 4.72 (t, J = 8.0 Hz, 1H), 4.59 - 4.49 (m, 3H), 4.35 - 4.29 (m, 1H), 4.07 - 3.95 (m, 3H), 3.85 - 3.80 (m, 8H), 3.67 - 3.55 (m, 8H), 3.43 (t, J = 6.4 Hz, 2H), 2.74 - 2.37 (m, 14H), 2.15 - 2.09 (m, 1H), 1.60 - 1.47 (m, 4H), 1.36 - 1.27 (m, 2H), 0.94 (s, 9H); 13C-NMR (101 MHz, CDCl 3) d: 171.5, 170.9, 170.5, 168.6, 161.6, 159.8, 151.8, 151.1, 150.4, 148.6, 141.9, 138.3, 136.0, 131.7, 131.1, 129.6, 128.3, 120.6, 118.1, 117.6, 105.5, 71.4, 71.3, 70.6, 70.1, 58.6, 58.2, 57.2, 56.9, 56.1, 52.5, 51.7, 48.6, 43.4, 37.3, 36.1, 35.2, 29.4, 26.6, 26.0, 24.2, 16.2. HRMS m/z calcd for C53H70N8O9S994.50, found 995.5178 [M + H+].
2-(2-(allyloxy)ethoxy)ethan-1-ol (43).
To a mixture of NaOH (165 mg, 4.13 mmol, 1 eq) in dioxane (13 mL), diethylene glycol (877 mg, 8.26 mmol, 2 eq) and allyl bromide (500 mg, 4.13 mmol, 1 eq) were added dropwise. The mixture was heated overnight at 55 °C. Then the reaction mixture was dried over MgSO4,
HO
using a gradient from 50% to 100% of ethyl acetate in heptane to obtain compound 43 as an oil. Yield: 329 mg, 54%. ¹H-NMR (400 MHz, CDCl3) d: 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 (m, 4H); 13C-NMR (101 MHz, CDCl 3) d: 134.5, 117.3, 72.6, 72.2, 70.4, 69.4, 61.7. MS m/z calcd for C7H14O3146.09. 3,6,9,12-tetraoxapentadec-14-en-1-ol (44).
Following the same procedure applied for compound 43, compound 44 was obtained by reacting tetraethylene glycol (2 eq) and allyl bromide (1 eq). Yield: 3.29 g, 68%. Analytical data matched those previously reported.174
(2S,4R)-N-(2-(2-(2-(allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1- fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (45).
Following general method E, compound 45 was obtained from compound 43 (1 eq) and VHL4 (1 eq). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 21.7 mg, 42%. ¹H NMR (500 MHz, CD3OD) d: 8.91 (s, 1H), 8.52 (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 O O O O HO O O H N O N S N HO O HN O O F
(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), 4.66 (t, J = 8.3 Hz, 1H), 4.54 - 4.43 (m, 3H), 4.31 - 4.26 (m, 2H), 4.07 - 4.04 (m, 2H), 3.97 - 3.94 (m, 2H), 3.90 - 3.82 (m, 2H), 3.80 - 3.76 (m, 2H), 3.69 - 3.66 (m, 2H), 2.53 (s, 3H), 2.29 - 2.24 (m, 1H), 2.17 - 2.10 (m, 1H), 1.42 - 1.30 (m, 4H), 1.07 (s, 10H). MS m/z calcd for C33H45FN4O7S 660.30. (2S,4R)-N-(2-((3,6,9,12-tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5- yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (46).
Following general method E, compound 46 was obtained from the reaction of compound 44 (1 eq) and VHL4 (1 eq). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 26.9 mg, 46%. ¹H NMR (500 MHz, CD3OD) d: 8.91 (s, 1H), 8.51 (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 (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 (d, J = 9.1 Hz, 1H), 4.66 (t, J = 8.2 Hz, 1H), 4.56 - 4.43 (m, 3H), 4.30 - 4.26 (m, 2H), 4.04 - 4.02 (m, 2H), 3.97 - 3.94 (m, 2H), O O H N O N S N HO O HN O O F O O
(m, 2H), 2.54 (s, 3H), 2.30 - 2.25 (m, 1H), 2.16 - 2.11 (m, 1H), 1.42 - 1.31 (m, 4H), 1.07 (s, 9H). MS m/z calcd for C37H53FN4O9S 748.35.
(2S,4R)-N-(2-(2-(2-(allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1- cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (47).
In keeping with general method E, the reaction between compound 43 (1 eq) and VHL3 (1 eq) led to the formation of derivative 47. The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 15.8 mg, 61%. ¹H NMR (500 MHz, CD3OD) d: 8.91 (s, 1H), 8.50 (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 (m, 2H), 4.54 - 4.44 (m, 4H), 4.31 - 4.27 (m, 2H), 4.06 (td, J = 1.4, 5.6 Hz, 2H), 3.97 - 3.95 (m, 2H), 3.87 - 3.78 (m, 5H), 3.69 - 3.66 (m, 3H), 2.53 (s, 4H), 2.28 - 2.23 (m, 1H), 2.16 - 2.10 (m, 1H), 1.71 - 1.56 (m, 4H), 1.06 (s, 9H). 13 C-NMR (101 MHz, CD3OD) d: 174.3, 171.6, 167.3, 158.2, 152.9, 149.2, 136.2, 133.6, 133.0, 130.2, 128.5, 122.9, 120.8, 117.4, 113.9, 73.2, 71.9, 71.1, 71.0, 70.7, 69.5, 60.9, 59.7, 59.6, 39.8, 39.0, 37.4, 26.9, 18.2, 18.1, 16.0, 15.0, 14.9. MS m/z calcd for C34H45N5O7S 667.30, found 668.4 [M + H+]. O O H N O N S N HO O HN O O CN
(2S,4R)-N-(2-((3,6,9,12-tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5- yl)benzyl)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (48).
Following general method E, compound 48 was obtained from compound 44 (1 eq) and VHL3 (1 eq). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 15.3 mg, 52%. ¹H NMR (500 MHz, CD3OD) d: 8.91 (s, 1H), 8.49 (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 (m, 2H), 4.55 - 4.43 (m, 3H), 4.31 - 4.26 (m, 2H), 4.03 (td, J = 1.4, 5.6 Hz, 2H), 3.97 - 3.94 (m, 2H), 3.87 - 3.77 (m, 4H), 3.73 - 3.71 (m, 2H), 3.69 - 3.64 (m, 7H), 3.62 - 3.59 (m, 2H), 2.54 (s, 3H), 2.30 - 2.25 (m, 1H), 2.16 - 2.10 (m, 1H), 1.71 - 1.56 (m, 4H), 1.06 (s, 9H). MS m/z calcd for C38H53N5O9S 755.36, found 756.5 [M + H+].
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-N-(2-(2-(2-(allyloxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (49). O O H N O N S N HO O HN O O CN O O
Following general method E, compound 49 was obtained from compound 43 (13 mg, 0.06 mmol) and VHL2 (18.8 mg, 0.038 mmol). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 12.7 mg, 53%. ¹H-NMR (400 MHz, CD3OD) d: 8.91 (s, 1H), 7.52 (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, 4.5 Hz, 1H), 4.67 - 4.60 (m, 2H), 4.55 - 4.42 (m, 3H), 4.30 - 4.25 (m, 2H), 4.05 (dt, J = 1.4, 5.6 Hz, 2H), 3.97 - 3.90 (m, 3H), 3.85 - 3.77 (m, 3H), 3.68 - 3.65 (m, 2H), 2.53 (s, 3H), 2.28 - 2.20 (m, 1H), 2.16 - 2.07 (m, 1H), 2.03 (s, 3H), 1.06 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.4, 173.1, 172.3, 158.0, 152.8, 149.1, 136.1, 133.5, 132.8, 130.0, 128.4, 122.8, 117.3, 114.0, 113.8, 73.1, 71.8, 70.9, 70.6, 69.4, 60.7, 59.1, 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 +]. (2S,4R)-N-(2-((3,6,9,12-tetraoxapentadec-14-en-1-yl)oxy)-4-(4-methylthiazol-5- yl)benzyl)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (50). O O H N O N S N HO O HN O O
Following general method E, compound 50 was obtained from compound 44 (18 mg, 0.06 mmol) and VHL2 (20 mg, 0.04 mmol). The crude was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid. Yield: 13.8 mg, 53%. ¹H-NMR (400 MHz, CD3OD) d: 8.92 (s, 1H), 7.52 (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, 4.5 Hz, 1H), 4.67 - 4.60 (m, 2H), 4.56 - 4.39 (m, 2H), 4.29 - 4.25 (m, 2H), 4.03 (dt, J = 1.4, 5.7 Hz, 2H), 3.97 - 3.90 (m, 3H), 3.85 - 3.76 (m, 3H), 3.73 - 3.63 (m, 9H), 3.62 - 3.58 (m, 2H), 2.53 (s, 3H), 2.28 - 2.21 (m, 1H), 2.16 - 2.08 (m, 1H), 2.04 (s, 3H), 1.06 (s, 9H); 13C-NMR (101 MHz, CD3OD) d: 174.4, 173.1, 172.2, 158.0, 152.8, 149.1, 136.1, 133.5, 132.8, 130.0, 128.4, 122.8, 117.2, 113.7, 73.1, 71.8, 71.6, 71.5, 71.1, 70.8, 70.6, 69.4, 60.7, 59.1, 58.0, 39.4, 38.9, 36.5, 27.0, 22.3, 15.9. MS m/z calcd for C35H52N4O9S704.35, found 705.4 [M + H+].
(2S,4R)-N-(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-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (VZ95). O O H N O N S N HO O O O O O HN
A mixture of 45 (21.7 mg, 0.03 mmol, 1 eq), osmium tetroxide 4% in H2O (42 µL, 0.0065 mmol, 0.2 eq), sodium periodate (26 mg, 0.12 mmol, 4 eq), pyridine (5 µL, 0.06 mmol, 2 eq) in dioxane/ H2O (3:1) (0.3 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 10 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. Then, following general method A, compound VZ95 was synthesized from the aldehyde derivative (0.015 mmol) and BrdL1 (6.5 mg, 0.015 mmol). The solvent was removed in vacuum to give the corresponding crude, which was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to yield compound VZ95 as white powder. Yield: 7 mg, 45%. ¹H-NMR (400 MHz, CD3OD) d: 9.54 (s, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.8 Hz, 1H), 8.58 (s, 1NH), 7.75 (s, 1H), 7.63 (dd, J = 0.6, 5.7 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.10 - 7.04 (m, 2H), 6.79 (s, 2H), 4.78 (s, 1H), 4.66 (t, J = 8.4 Hz, 1H), 4.55 - 4.42 (m, 3H), 4.32 - 4.23 (m, 2H), 3.97 - 3.77 (m, 14H), 3.73 - 3.66 (m, 7H), 2.85 - 2.68 (m, 10H), 2.52 (s, 3H), 2.30 - 2.24 (m, 1H), 2.16 - 2.09 (m, 1H), 1.46 - 1.27 (m, 4H), 1.07 (s, 9H); 13C-NMR (101 MHz, CD3OD) d: 174.2, 171.7, 171.5, 171.3, 170.2, 163.0, 161.0, 158.0, 152.8, 151.7, 151.2, 149.1, 143.6, 138.9, 138.1, 133.5, 132.9, 130.0, 128.4, 122.9, 121.7, 119.2, 113.8, 106.5, 79.2 (d, 1J CF = 233 Hz), 71.8, 71.4, 71.0, 70.8, 69.4, 69.3, 60.8, 58.7, 58.3, 58.2, 56.5, 53.3, 53.0, 39.5, 39.0, 37.4, 37.3, 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 O O O O O N N O NH S N O N O N H O F OH
(2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-N-(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 (VZ96).
A mixture of 47 (15.8 mg, 0.023 mmol, 1 eq), osmium tetroxide 4% in H2O (30 µL, 0.005 mmol, 0.2 eq), sodium periodate (20 mg, 0.1 mmol, 4 eq), pyridine (4 µL, 0.05 mmol, 2 eq) in dioxane/ H2O (3:1) (0.25 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 10 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. The obtained aldehyde derivative (0.024 mmol) was condensed with BrdL1 (10 mg, 0.024 mmol) according to general method A to afford derivative VZ96 that was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid as white powder. Yield: 11 mg, 44%. ¹H-NMR (400 MHz, CD3OD) d: 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), 7.10 - 7.03 (m, 2H), 6.79 (s, 2H), 4.71 - 4.63 (m, 2H), 4.56 - 4.42 (m, 3H), 4.33 - 4.23 (m, 2H), 3.98 - 3.66 (m, 21H), 2.86 - 2.69 (m, 10H), 2.52 (s, 3H), 2.30 - 2.23 (m, 1H), 2.16 - 2.08 (m, 1H), 1.70 - 1.55 (m, 4H), 1.06 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.1, 171.5, 170.1, 167.1, 163.0, 161.0, 158.1, 152.8, 151.7, 151.2, 149.1, 143.5, 138.9, 138.1, 133.4, 132.9, 130.1, 128.4, 122.8, 121.7, N N O O O O O N N O NH S N O N O N H O NC OH
53.2, 53.0, 39.5, 38.9, 37.7, 37.4, 26.8, 18.1, 18.0, 16.0, 14.8. HRMS m/z calcd for C55H69N9O10S 1047.49, found 1048.4493 [M + H+]. (2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-N-(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 (VZ97).
A mixture of 48 (15.3 mg, 0.02 mmol, 1 eq), osmium tetroxide 4% in H2O (25.7 µL, 0.04 mmol, 0.2 eq), sodium periodate (20 mg, 0.1 mmol, 4 eq), pyridine (4 µL, 0.05 mmol, 2 eq) in dioxane/ H2O (3:1) (0.25 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 10 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. Then, following general method A, from the obtained aldehyde derivative (0.02 mmol) was condensed with BrdL1 (8.6 mg, 0.02 mmol) to afford compound VZ97 that was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia. Yield: 9 mg, 39%. ¹H-NMR (400 MHz, CD3OD) d: 9.55 (d, J = 0.7 Hz, 1H), 8.89 (s, 1H), 8.70 (d, J = 5.9 Hz, 1H), 7.74 (s, 1H), 7.64 (dd, J = 0.7, 5.8 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.09 - 7.02 (m, 2H), 6.76 (s, 2H), 4.69 - 4.63 (m, 2H), 4.55 - 4.42 (m, 3H), 4.34 - 4.23 (m, 2H), 3.95 (t, J = 4.5 Hz, 2H), 3.89 (s, 6H), 3.84 - 3.75 (m, 6H), 3.73 - 3.70 (m, 5H), 3.69 - 3.58 (m, 9H), 2.68 - 2.56 (m, 10H), 2.52 (s, 3H), 2.32 N N O O O O O N N O NH S N O N O N H O NC OH O O
- 2.22 (m, 1H), 2.17 - 2.09 (m, 1H), 1.71 - 1.55 (m, 4H), 1.06 (s, 9H); 13C-NMR (101 MHz, CD3OD) d: 174.1, 171.5, 167.2, 163.1, 161.0, 158.1, 152.8, 151.7, 151.2, 149.2, 143.7, 138.8, 137.2, 133.5, 133.0, 130.2, 128.4, 122.8, 121.7, 120.7, 119.5, 119.3, 113.9, 113.7, 106.5, 71.8, 71.6, 71.6, 71.4, 71.1, 70.9, 69.7, 69.4, 60.8, 59.5, 58.7, 58.1, 56.3, 54.2, 53.4, 39.6, 38.9, 37.4, 37.3, 26.8, 18.1, 18.0, 16.0, 14.9. HRMS m/z calcd for C59H77N9O12S 1035.54, found 1036.5169 [M + H+].
(2S,4R)-N-(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-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (VZ98).
A mixture of 46 (27 mg, 0.04 mmol, 1 eq), osmium tetroxide 4% in H2O (45 µL, 0.007 mmol, 0.2 eq), sodium periodate (31 mg, 0.144 mmol, 4 eq), pyridine (6 µL, 0.07 mmol, 2 eq) in dioxane/ H2O (3:1) (0.4 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 0.5 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. Then, following general method A, the obtained aldehyde derivative (0.018 mmol) was reacted with BrdL1 (7.74 mg, 0.018 mmol) to afford compound VZ98. The crude compound was purified by HPLC using a
N N O O O O O N N O NH S N O N O N H O F OH O O
white powder. Yield: 9.5 mg, 46%. ¹H-NMR (400 MHz, CD3OD) d: 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 (m, 2H), 6.77 (s, 2H), 4.78 (s, 1H), 4.69 - 4.63 (m, 1H), 4.55 - 4.42 (m, 3H), 4.31 - 4.23 (m, 2H), 3.97 - 3.57 (m, 29H), 2.71 - 2.60 (m, 10H), 2.52 (s, 3H), 2.30 - 2.24 (m, 1H), 2.17 - 2.08 (m, 1H), 1.43 - 1.29 (m, 4H), 1.07 (s, 9H); 13C-NMR (101 MHz, CD3OD) d: 174.2, 171.6, 171.3, 163.0, 161.0, 158.0, 152.8, 151.7, 151.2, 149.1, 143.6, 138.8, 137.3, 133.5, 132.9, 130.0, 128.4, 122.8, 121.7, 119.4, 119.3, 113.8, 113.2, 106.5, 79.1 (d, 1J CF = 232 Hz), 73.7, 71.8, 71.6, 71.5, 71.4, 71.0, 70.8, 69.6, 69.4, 62.2, 60.8, 58.7, 58.6, 58.2, 56.3, 54.0, 53.3, 39.5, 38.9, 37.4, 37.3, 26.9, 16.0, 14.1, 14.0, 13.9. HRMS m/z calcd for C58H77FN8O12S 1128.54, found 1129.5445 [M + H+].
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-N-(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 (VZ117).
A mixture of 49 (12.7 mg, 0.02 mmol, 1 eq), osmium tetroxide 4% in H2O (25 µL, 0.004 mmol, 0.2 eq), sodium periodate (17 mg, 0.08 mmol, 4 eq), pyridine (3 µL, 0.04 mmol, 2 eq) in dioxane/ H2O (3:1) (0.2 mL) was stirred at rt for 48 h. Water (0.5 mL) was then added to the reaction and the product was extracted with DCM (3 x 10 mL). The pooled organic phases
N N O O O O O N N O NH S N O N O N H O OH
were dried over MgSO4, filtered and evaporated to dryness. The so obtained aldehyde derivative (12 mg, 0.02 mmol, 1 eq) was reacted, following general method A, with BrdL1 (8.6 mg, 0.02 mmol) and compound VZ117 was obtained after purification by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid as white powder. Yield: 3.3 mg, 17%. ¹H-NMR (400 MHz, CD3OD) d: 9.55 (d, J = 0.7 Hz, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.9 Hz, 1H), 7.76 (s, 1H), 7.64 (dd, J = 0.7, 5.9 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 1.7, 7.7 Hz, 1H), 6.79 (s, 2H), 4.65 - 4.60 (m, 2H), 4.54 - 4.42 (m, 3H), 4.28 - 4.25 (m, 2H), 3.96 - 3.65 (m, 21H), 2.86 - 2.67 (m, 10H), 2.52 (s, 3H), 2.27 - 2.21 (m, 1H), 2.15 - 2.08 (m, 1H), 2.04 (s, 3H), 1.05 (s, 9H); 13C-NMR (101 MHz, CD3OD) d: 174.4, 173.1, 172.3, 163.1, 161.0, 158.0, 152.8, 151.7, 151.2, 149.1, 143.6, 138.9, 133.5, 132.9, 130.0, 128.4, 122.8, 121.7, 119.2, 113.7, 106.5, 71.8, 71.4, 71.1, 70.8, 69.4, 69.2, 60.7, 59.2, 58.2, 58.0, 56.5, 53.2, 53.0, 49.8, 39.4, 38.9, 37.4, 36.5, 27.0, 22.3, 16.0. HRMS m/z calcd for C52H68N8O10S 996.48, found 997.5008 [M + H+].
14-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4- yl)benzyl)piperazin-1-yl)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)-3,6,9,12-tetraoxatetradecanamide (VZ118).
A mixture of 50 (14 mg, 0.02 mmol, 1 eq), osmium tetroxide 4% in H2O (25 µL, 0.004 mmol, 0.2 eq), sodium periodate (17 mg, 0.08 mmol, 4 eq), pyridine (3 µL, 0.04 mmol, 2 eq) in
N N O O O O O N N O NH S N O N O N H O OH O O
dioxane/ H2O (3:1) (0.2 mL) was stirred at rt for 48 h. Then water (0.5 mL) was added to the reaction and the product was extracted with DCM (3 x 10 mL). The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. Then, following general method A, the obtained aldehyde derivative (0.02 mmol) was condensed with BrdL1 (8.6 mg, 0.02 mmol) to afford compound VZ118. The crude compound was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid and isolated as white powder. Yield: 3.7 mg, 17%. ¹H-NMR (400 MHz, CD3OD) d: 9.55 (d, J = 0.7 Hz, 1H), 8.90 (s, 1H), 8.70 (d, J = 5.8 Hz, 1H), 7.76 (s, 1H), 7.64 (dd, J = 0.6, 5.8 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.08 - 7.01 (m, 2H), 6.81 (s, 2H), 4.65 - 4.60 (m, 2H), 4.53 - 4.41 (m, 3H), 4.27 - 4.25 (m, 2H), 4.02 - 3.61 (m, 29H), 2.96 - 2.76 (m, 10H), 2.51 (s, 3H), 2.27 - 2.21 (m, 1H), 2.15 - 2.08 (m, 1H), 2.04 (s, 3H), 1.06 (s, 9H); 13C-NMR (101 MHz, CD 3OD) d: 174.4, 173.1, 172.3, 163.0, 161.0, 158.0, 152.8, 151.8, 151.3, 151.2, 149.1, 143.5, 139.0, 133.5, 132.8, 130.0, 128.4, 122.8, 121.7, 119.2, 119.1, 113.7, 106.6, 71.9, 71.6, 71.5, 71.4, 71.1, 70.9, 69.4, 60.7, 59.2, 58.1, 58.0, 56.6, 52.8, 49.8, 39.4, 38.9, 37.4, 36.5, 27.0, 22.3, 16.0. HRMS m/z calcd for C56H76N8O12S 1084.53, found 1085.5658 [M + H+]. 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)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide (VZ109). N N O O O O O N N O N H H N N O O HN O O
Following the general method C, compound VZ109 was obtained from compound 34 (0.03 mmol) and POMA (9.25 mg, 0.02 mmol, synthetized accordingly to literature175). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain compound VZ109 as yellow powder. Yield: 5.6 mg, 33%. ¹H-NMR (400 MHz, CD3OD) d: 9.54 (d, J = 0.8 Hz, 1H), 8.70 (d, J = 6.0 Hz, 1H), 8.11 (s, 1NH), 7.84 (s, 1H), 7.73 (dd, J = 0.6, 6.0 Hz, 1H), 7.55 - 7.49 (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 (s, 6H), 3.77 - 3.67 (m, 9H), 3.51 - 3.46 (m, 4H), 3.39 (s, 4H), 3.20 (s, 4H), 3.01 (t, J = 4.8 Hz, 2H), 2.94 - 2.67 (m, 4H); 13C-NMR (101 MHz, CD 3OD) d: 174.6, 173.4, 171.6, 170.6, 169.1, 162.7, 161.1, 150.9, 149.8, 147.9, 144.0, 140.1, 137.2, 133.9, 121.8, 119.6, 118.5, 117.9, 112.2, 111.4, 106.7, 71.7, 71.3, 71.0, 67.7, 57.5, 56.8, 51.8, 51.1, 50.2, 50.0, 42.6, 39.4, 37.5, 32.2, 23.8. HRMS m/z calcd for C43H50N8O10 838.36, found 839.3796 [M + H+]. 14-(4-(2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4- yl)benzyl)piperazin-1-yl)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)-3,6,9,12-tetraoxatetradecanamide (VZ110).
Following the general method C, compound VZ110 was obtained from compound 35 (0.03 mmol) and POMA (9.25 mg, 0.02 mmol, synthetized accordingly to literature175). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain VZ110 as yellow powder. Yield: 10 mg, 54%. ¹H-NMR (400
N N O O O O O O O N N O N H H N N O O HN O O
MHz, CD3OD) d: 9.53 (s, 1H), 8.70 (d, J = 5.7 Hz, 1H), 8.15 (s, 1NH), 7.77 (s, 1H), 7.64 (d, J = 5.7 Hz, 1H), 7.59 - 7.53 (m, 1H), 7.17 - 7.13 (m, 1H), 7.08 - 7.04 (m, 1H), 6.86 (s, 2H), 5.07 (dd, J = 5.5, 12.5 Hz, 1H), 4.29 (s, 2H), 4.01 (s, 2H), 3.96 (s, 6H), 3.72 - 3.63 (m, 17H), 3.54 (s, 4H), 3.25 (s, 4H), 3.02 (s, 4H), 2.94 - 2.67 (m, 6H); 13C-NMR (101 MHz, CD 3OD) d: 174.6, 173.5, 171.5, 170.6, 169.2, 163.0, 161.0, 151.7, 151.2, 148.0, 143.4, 139.8, 139.2, 137.3, 133.9, 121.7, 119.1, 118.7, 118.1, 112.1, 111.4, 108.1, 106.7, 72.0, 71.4, 71.1, 68.5, 57.6, 56.8, 52.4, 51.6, 50.2, 49.9, 42.5, 39.4, 37.5, 32.2, 23.8. HRMS m/z calcd for C47H58N8O12 926.42, found 927.4396 [M + H+].
N-(4-(N-(3-chloro-1H-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 (VZ89).
Following the general method C, compound VZ89 was obtained from compound 34 (0.04 mmol) and Indisulam derivative (21, 12 mg, 0.04 mmol). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid and compound VZ89 was obtained as a white powder. Yield: 10.8 mg, 34%.¹H-NMR (400 MHz, CD3OD) d: 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, 3H), 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, 1H), 4.49 (s, 2H), 4.28 (s, 2H), 4.11 (s, 2H), 3.94 (s, 6H), 3.76 - 3.66 (m, 7H), 3.63 (t, J=5.3 Hz, 2H), 3.22 (s, 4H), 2.88 (s, 4H), 2.73 (t, J = 5.1 Hz, 2H); 13C-NMR (101 MHz, CD3OD) d: 173.0, 166.4, 163.0, 161.0, 151.7, 151.2, 145.5, 143.3, N N O O N H O S H N O O HN Cl O O N N O
140.0, 139.4, 139.1, 132.0, 128.8, 128.7, 128.2, 123.4, 122.7, 121.6, 120.9, 119.4, 119.1, 118.8, 117.0, 107.5, 106.7, 106.2, 71.9, 71.4, 71.3, 68.7, 57.6, 56.8, 52.5, 51.3, 49.9, 43.0, 37.5. HRMS m/z calcd for C43H48ClN7O8S857.30, found 858.3111 [M + H+].
N-(4-(N-(3-chloro-1H-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 (VZ90).
Following the general method C, compound VZ90 was obtained from compound 35 (0.03 mmol) and Indisulam derivative (21, 10 mg, 0.03 mmol). The crude product was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to obtain compound VZ90 as white powder. Yield: 11.1 mg, 37%. ¹H-NMR (400 MHz, CD3OD) d: 9.54 (d, J = 0.6 Hz, 1H), 8.69 (d, J = 5.7 Hz, 1H), 8.30 (s, 1NH), 7.75 (s, 1H), 7.68 - 7.62 (m, 3H), 7.41 (d, J = 8.2 Hz, 2H), 7.34 (dd, J = 0.8, 8.1 Hz, 1H), 7.27 (s, 1H), 6.92 (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, 2H), 3.94 (s, 6H), 3.73 - 3.67 (m, 7H), 3.65 - 3.59 (m, 4H), 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, CD 3OD) d: 173.1, 166.7, 163.1, 161.0, 151.7, 151.2, 145.5, 143.4, 139.7, 139.2, 139.0, 131.9, 128.9, 128.6, 128.2, 123.4, 122.7, 121.6, 121.0, 119.3, 119.2, 118.9, 117.0, 108.0, 106.7, 106.2, 72.0, 71.3, 71.3, 71.2, 68.3, 57.5, 56.8, 52.3, 51.5, 49.9, 42.9, 37.6. HRMS m/z calcd for C47H56ClN7O10S945.35, found 946.3676 [M + H+]. N N O O N H O S H N O O HN Cl O O O O N N O
tert-butyl 4-(4-bromo-2,5-dimethoxybenzyl)piperazine-1-carboxylate (52).
Following general method A, compound 52 was obtained from the commercially available 4-bromo-2,5-dimethoxybenzaldehyde 51 (120 mg, 0.5 mmol) and boc-piperazine (100 mg, 0.54 mmol). The reaction was quenched with saturated solution of NaHCO3 (156 mg, 0.74 mmol) extracted with DCM (3 x 10 mL), washed with water and brine. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 123 mg, 72%. ¹H-NMR (400 MHz, CDCl3) d: 7.01 (s, 1H), 6.99 (s, 1H), 3.82 (s, 3H), 3.73 (s, 3H), 3.48 (s, 2H), 3.41 (t, J = 4.7 Hz, 4H), 2.39 (t, J = 4.3 Hz, 4H), 1.42 (s, 9H); 13C-NMR (101 MHz, CDCl3) d: 154.9, 152.3, 150.1, 126.5, 116.2, 114.5, 109.9, 79.6, 57.0, 56.4, 55.7, 52.9, 31.0, 28.5. MS m/z calcd for C18H27BrN2O4 414.12, found 415.2 [M + H+]. 4-(2,5-dimethoxy-4-(piperazin-1-ylmethyl)phenyl)-2-methyl-2,7-naphthyridin-1(2H)-one (BrdL2, 53). Br N N O Boc O N NH2+Cl -O N O N O
Following general method B, compound BrdL2 was obtained from compound 4 (83 mg, 0.29 mmol) and 52 (123.6 mg, 0.29 mmol) as brown powder. Yield: 72.3 mg, 55%. ¹H-NMR (400 MHz, CD3OD) d: 9.65 (s, 1H), 8.71 (d, J = 6.2 Hz, 1H), 8.18 (s, 1H), 7.81 (d, J = 6.7 Hz, 1H), 7.54 (s, 1H), 7.24 (s, 1H), 3.99 (s, 3H), 3.88 - 3.64 (m, 16H). 13C-NMR (101 MHz, CDCl
3) d: 163.2, 156.4, 153.6, 152.7, 151.3, 150.5, 144.1, 127.6, 124.1, 121.5, 120.3, 116.3, 115.7, 115.5, 56.9, 56.6, 56.4, 53.8, 37.4, 30.7. MS m/z calcd for C22H26N4O3 394.2, found 395.3 [M + H+].
tert-butyl (1-(4-bromo-2,6-dimethoxybenzyl)azetidin-3-yl)carbamate (55).
Following general method A, compound 55 was obtained from the commercially available 4-bromo-2,6-dimethoxybenzaldehyde 5 (129 mg, 0.52 mmol) and 3-(Boc-amino)azetidine 54 (100 mg, 0.58 mmol). The reaction was quenched with saturated solution of NaHCO3 (5 mL), extracted with DCM (3 x 10 mL), washed with water and brine. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 120 mg, 78%. ¹H-NMR (400 MHz, CDCl3) d: 6.72 (s, 2H), 4.33 (s, 1H), 3.87 - 3.80 (m, 10H), 3.57 (s, 2H), 1.40 (s, 9H); 13C-NMR (101 MHz, CDCl3) d: 159.6, 155.3, 124.1, 107.9, 103.3, 79.8, 59.7, 56.2, 46.0, 41.1, 28.5. MS m/z calcd for C17H25BrN2O4 400.1, found 401.1 [M + H+].
tert-butyl (1-(4-bromo-2,5-dimethoxybenzyl)azetidin-3-yl)carbamate (56). Br O N O N H Boc
Following general method A, compound 56 was obtained from the commercially available 4-bromo-2,5-dimethoxybenzaldehyde 51 (129 mg, 0.52 mmol) and 3-(Boc-amino)azetidine 54 (100 mg, 0.58 mmol). The reaction was quenched with saturated solution of NaHCO3 (5 mL), extracted with DCM (3 x 10 mL), washed with water and brine. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 150 mg, 72%. ¹H-NMR (400 MHz, CDCl3) d: 7.02 (s, 1H), 6.88 (s, 1H), 4.38 - 4.31 (m, 1H), 3.86 (s, 3H), 3.77 (s, 3H), 3.73 - 3.66 (m, 4H), 2.91 (s, 2H), 1.43 (s, 9H); 13C-NMR (101 MHz, CDCl 3) d: 155.1, 151.7, 150.2, 115.9, 113.8, 109.9, 62.5, 57.1, 57.0, 56.2, 41.9, 31.1, 28.5. MS m/z calcd for C17H25BrN2O4 400.1, found 401.1 [M + H+]. 4-(4-((3-aminoazetidin-1-yl)methyl)-3,5-dimethoxyphenyl)-2-methyl-2,7-naphthyridin-1(2H)-one (BrdL3, 57).
Following general method B, compound BrdL3 was obtained from compound 4 (125 mg, 0.44 mmol) and 55 (176 mg, 0.44 mmol) as brown powder. Yield: 77 mg, 50%. ¹H-NMR (400 MHz,
Br N O N H Boc O O N O N N NH3+Cl -O
CD3OD) d: 9.67 (s, 1H), 8.76 (dd, J = 0.6, 6.7 Hz, 1H), 8.27 (s, 1H), 8.20 (d, J = 5.5 Hz, 1H), 6.94 (s, 2H), 4.70 - 4.36 (m, 7H), 4.03 (s, 6H), 3.80 (s, 3H); 13C-NMR (101 MHz, CD
3OD) d: 161.3, 160.9, 149.0, 146.2, 145.8, 140.8, 139.2, 123.0, 122.9, 117.7, 106.9, 106.6, 58.9, 58.1, 57.0, 40.1, 37.9. MS m/z calcd for C21H24N4O3 380.18, found 381.3 [M + H+].
4-(4-((3-aminoazetidin-1-yl)methyl)-2,5-dimethoxyphenyl)-2-methyl-2,7-naphthyridin-1(2H)-one (BrdL4, 58).
Following general method B, compound BrdL4 was obtained from compound 4 (125 mg, 0.44 mmol) and 56 (176 mg, 0.44 mmol) as brown powder. Yield: 55.5 mg, 60%. ¹H-NMR (400 MHz, CD3OD) d: 9.51 (s, 1H), 8.56 (d, J = 6.7 Hz, 1H), 7.99 (s, 1H), 7.56 (d, J = 6.4 Hz, 1H), 7.31 (s, 1H), 7.06 (s, 1H), 4.60 - 4.29 (m, 7H), 3.86 (s, 3H), 3.68 (s, 3H), 3.64 (s, 3H); 13C-NMR (101 MHz, CD3OD) d: 160.2, 152.2, 151.4, 147.2, 145.0, 144.4, 140.1, 124.7, 122.1, 118.8, 115.4, 115.1, 114.6, 113.2, 55.6, 55.4, 49.0, 46.5, 42.6, 36.4. MS m/z calcd for C21H24N4O3 380.18, found 381.3 [M + H+]. 1-(4-bromo-2,6-dimethoxyphenyl)-N,N-dimethylmethanamine (60). O N O N O N NH3+Cl -Br N O O
A mixture of dimethylamine hydrochloride (150 mg, 1.84 mmol, 1.1 eq), NaOAc (151 mg, 1.84 mmol, 1.5 eq) and AcOH (70 µL, 1.22 mmol, 1 eq) in DMF (5 mL) was stirred at rt. After 10 min, aldehyde 5 (300 mg, 1.22 mmol, 1 eq) was added and stirring is prolonged for 30 min. Then, NaBH(OAc)3 (530 mg, 2.5 mmol, 1.5 eq) was added and the reaction was stirred overnight at rt under nitrogen. The reaction was quenched with saturated solution of NaHCO3 (3 mL), extracted with DCM (3 x 15 mL), washed with water and brine. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 368 mg, 100%. Analytical data matched those previously reported.107 MS m/z calcd forC
11H16BrNO2 273.04.
1-(4-bromo-2,5-dimethoxyphenyl)-N,N-dimethylmethanamine (61).
A mixture of dimethylamine hydrochloride (150 mg, 1.84 mmol, 1.1 eq), NaOAc (151 mg, 1.84 mmol, 1.5 eq) and AcOH (70 µL, 1.22 mmol, 1 eq) in DMF (5 mL) was stirred at rt. After 10 min, aldehyde 51 (300 mg, 1.22 mmol, 1 eq) was added and stirring was prolonged for 30 min. NaBH(OAc)3 (530 mg, 2.5 mmol, 1.5 eq) was then added and the reaction was stirred overnight at rt under nitrogen. The reaction was quenched with saturated solution of NaHCO3 (3 mL), extracted with DCM (3 x 10 mL), washed with water and brine. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness to give the desired compound without any further purification as sticky oil. Yield: 392 mg, 100%. ¹H-NMR (400 MHz, CDCl3) d: 7.18 (s, 1H), 7.09 (s, 1H), 3.87 (s, 5H), 3.79 (s, 3H), 2.49 (s, 6H); 13C-NMR
Br
N O
