10% |
In N,N-dimethyl-formamide; at 18 - 25℃; |
To a solution of <strong>[737-31-5]sodium 3,5-diacetamido-2,4,6-triiodobenzoate</strong> (99 mg, 0.155 mmol) in DMF (2 mL) was added intermediate A (170 mg, 0.155 mmol) at RT in one portion. The reaction was stirred at RT overnight. After the conversion of intermediate A was ~70% as indicated by TLC, the reaction mixture was poured into icy-water (12 mL). After stirring for 30 min, the suspension was filtrated. The filter cake was washed with water (2 mL×4). The solid was dissolved in ethyl acetate, dried over anhydrous Na2SO4, filtrated and concentrated.200 mg of crude Formula 2 product was obtained. Crude Formula 2 product was dissolved in ethyl acetate (1 mL) at RT. The EtOAc solution of the crude 2 was slowly added into a solution of hexane (2 mL) dropwise and stirred at RT for 30 min. The solid formed was collected by vacuum filtration. The filter cake was washed with hexane to provide 130 mg of Formula 2 product. TLC analysis showed that the product contained ~5% of intermediate A. Formula 2 product was further purified by recrystallization in ethyl acetate/hexane (0.75 mL/0.75 mL) to produce 95 mg of pure Formula 2 product as off-white solid. TLC showed a single spot. HPLC analysis showed 90% purity (Note 1). The 95 mg of Formula 2 product was further purified multiple times by silica gel column (70 mg, HPLC: 93%), by recrystallization in MTBE/ethyl acetate = 3 mL/1 mL at 40 C (35 mg, HPLC: 88%), and by preparative TLC (25 mg. HPLC: 99%, Note 2).25 mg of Formula 2 product was combined with another batch of Formula 2 product (24 mg.3125-037) to provide 48 mg of Formula 2 product as yellow solid. The yield was 10%. The analytical data is depicted in Figure 3 (mass spectrum of the compound represented by Formula 2), Figure 4 (HPLC chromatogram of the compound represented by Formula 2), and Figure 5 (1H-NMR spectrum of the compound represented by Formula 2). LCMS (ESI+): m/z 1146 (M+Na); HPLC: 99.2%; H-NMR (300 MHz, CDCl3) ^ ^8.00 (brs, 1H), 7.75 (brs, 1H), 6.20-6.45 (m, 2H), 6.20-6.05 (m, 1H), 5.90- 6.00 (m, 1H), 5.55-5.45 (m, 1H), 5.45-5.35 (m, 1H), 5.30-5.20 (m, 1H), 5.20-5.05 (m, 1H), 4.80-4.60 (m, 2H), 4.50-4.35 (m, 2H), 4.25-4.15 (m, 1H), 4.00-3.70 (m, 2H), 3.70-3.60 (m, 1H), 3.60-3.50 (m, 1H), 3.50-3.20 (m, 7H), 3.20-3.05 (m, 4H), 2.90- 2.70 (m, 2H), 2.65-2.50 (m, 1H), 2.40-2.25 (m, 8H), 2.15-2.05 (m, 1H), 2.00-1.90 (m, 3H), 1.90-1.65 (m, 14H), 1.65-1.55 (m, 6H), 1.55-1.40 (m, 6H), 1.40-1.20 (m, 7H), 1.20-0.80 (m, 18H). Batch Summary of Step 2 NOTES 1. The HPLC purity may not be true. It was found that the compound of Formula 2 was not stable under some HPLC conditions. The HPLC purity is greatly dependent upon the HPLC mobile phases. With MeOH/H2O/0.05% of TFA buffer, the HPLC purity is 90%. With MeOH/H2O mobile phase, the HPLC purity is 91.7%. With acetonitrile/H2O mobile phase, the HPLC purity is 99%. The same stability issues in HPLC mobile phases were also observed for rapamycin itself. 2. HPLC condition for the compound of Formula 2 |