At 0 °C and under nitrogen protection, 2,6-dichlorobenzoic acid (0.1 g, 0.52 mmol) was dissolved in dichloromethane (5 mL), N,N-dimethylformamide (3.83 mg, 52.35 μmol) was added as a catalyst, followed by the slow addition of oxalyl dichloride (0.20 g, 1.57 mmol). The reaction mixture was added dropwise at 0 °C and stirred in the range of 0 to 20 °C for 2 hours. The progress of the reaction was monitored by thin layer chromatography (TLC, unfolding reagent ratio of petroleum ether:ethyl acetate=5:1, Rf=0.77) and after confirming the complete consumption of the feedstock, the solvent was removed under reduced pressure to afford 2,6-dichlorobenzoyl chloride (0.11 g, 0.37 mmol, 70% yield), and the product was ready to be used in the next step of the reaction without further purification. 28g-int (65 mg, 0.17 mmol, in hydrochloride form) and N,N-diisopropylethylamine (DIEA, 87.3 mg, 0.68 mmol) were dissolved in dichloromethane (10 mL), and dichloromethane (5 mL) was slowly added dropwise at 0 °C and under nitrogen protection to a 2,6-dichlorobenzoyl chloride (88.43 mg, 0.42 mmol) solution. The reaction mixture was stirred at 0-20 °C for 16 h. Liquid chromatography-mass spectrometry (LCMS) analysis showed that 28g-int was completely consumed and 28h-int was produced.After removing the solvent under reduced pressure, the crude product was purified by preparative high-performance liquid chromatography (HPLC) to give pure 28h-int.LCMS (electrospray ionization in positive ion mode, ESI+) data: m/ z 521.3 ([M+H]+), retention time (RT): 1.1 min. HPLC purification conditions: a Gilson 281 semi-preparative HPLC system was used; mobile phase A: an aqueous solution containing 0.075% trifluoroacetic acid (TFA, v/v), mobile phase B: acetonitrile (ACN) with a gradient elution program (phase B ratio: 20% at 0 min, 40% at 12 min ); column: YMC-Actus Triart C18 (150 mm×30 mm, 5 μm); flow rate: 20 mL/min; detection wavelength: 220 nm and 254 nm.
