Compound 4

Synthetic procedure: See article for the definitive version of this procedure and for full experimental details.

A 1 L round bottom flask was charged with (R)-3-bromo-6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one 15 (15.6 g, 31.9 mmol), SPhos (786 mg, 1.92 mmol), Pd(OAc)₂ (215 mg, 0.958 mmol), (4-(((tert-butoxycarbonyl)amino)methyl)phenyl)boronic acid (12.0 g, 47.9 mmol) and K₃PO₄ (13.6 g, 63.8 mmol) and was degassed by evacuating and refilling the flask with N₂ three times using a Schlenk manifold. Under a N₂ atmosphere, n-butanol (128 mL) was added before the flask was evacuated and refilled with N₂ three times using a Schlenk manifold. The reaction mixture was heated at 100 °C for 2 h before being cooled to RT and concentrated in vacuo. The residue was partitioned between water (350 mL) and EtOAc (250 mL). The phases were separated and the aqueous phase was extracted using EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄, concentrated in vacuo and the residue was purified by flash chromatography (Biotage KP-Sil 340 g cartridge, 0-100% EtOAc in cyclohexane then 0-20% MeOH in EtOAc), the impure fractions were re-purified using a GraceResolv silica 80 g cartridge, 0-100% EtOAc in cyclohexane then 0-20% MeOH in EtOAc) to give (R)-tert-butyl 4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)benzylcarbamate (15.4 g, 78%) as a colourless foam. LCMS: R_T = 1.37 min, m/z = 615 [M+H]⁺. A solution of (R)-tert-butyl 4-(6-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-2-methyl-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-3-yl)benzylcarbamate (8.73 g, 14.2 mmol) in DCM (14 mL) and TFA (14 mL) was stirred for 90 min before the reaction was purified using 4 x 10 g SCX-2 cartridges in parallel (1:10 MeOH in DCM then 1:3 7 M in NH₃ in MeOH in DCM). The basic phases were combined and concentrated to give the crude product (4.89 g). The DCM/MeOH phases were concentrated and the residue was purified using 4 x 10 g SCX-2 cartridges in parallel (1:10 MeOH in DCM then 1:3 7 M in NH₃ in MeOH in DCM) and the basic phases were combined and concentrated to give the crude product (2.62 g). The combined crude products (7.51 g) were purified by flash chromatography (GraceResolve silica 120 g cartridge, 0-30% dilute NH₃ in MeOH in DCM) to give the title compound (6.9 g, 94%) as a colourless solid after freeze drying. LCMS: R_T = 0.71 min (purity >99% at 254 nm), m/z = 515 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 8.03 – 7.92 (m, 1H), 7.69 – 7.43 (m, 4H), 7.30 – 7.21 (m, 4H), 7.19 – 7.12 (m, 1H), 4.87 (s, 1H), 4.10 (s, 3H), 4.07 – 3.86 (m, 3H), 3.81 (s, 2H), 3.70 – 3.61 (m, 1H), 3.28 – 3.12 (m, 2H), 2.93 – 2.83 (m, 1H), 2.66 – 2.52 (m, 2H), 2.31 (br. s, 2H), 1.57 – 1.23 (m, 4H), 1.21 (d, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 169.07 + 169.05 (conformers), 156.24 + 156.19 (conformers), 147.27, 146.68, 146.56, 145.04, 136.00, 134.59 + 134.41 (conformers), 129.20, 128.20 + 128.16 (conformers), 127.37, 126.86, 125.95 + 125.89 (conformers), 125.31, 69.35 + 69.30 (conformers), 52.94, 45.29, 41.07 + 40.96 (conformers), 40.25 + 40.23 (conformers), 39.51, 36.92, 36.20 + 35.99 (conformers), 35.02 + 34.91 (conformers), 34.27 + 34.14 (conformers), 22.06 + 21.88 (conformers). HRMS (TOF MS ES+): m/z [M + H]⁺ Calcd for C₂₉H₃₅N₆O₃ 515.2771, found 515.2773.

PubChemID:

348266823

MDL Molfile:

41589_2018_BFnchembio2528_MOESM10_ESM.mol

ChemDraw:

41589_2018_BFnchembio2528_MOESM11_ESM.cdx