Description
E-64d is an irreversible, membrane-permeable inhibitor of lysosomal and cytosolic cysteine proteases and has diverse biological activities. It is a synthetic analog of E-64 and prodrug form of E-64c that inhibits calpain and the cysteine proteases cathepsins F, -K, -B, -H, and -L. E-64d (20-200 μM) induces cell cycle arrest at the G
2/M phase in A431 human epidermoid carcinoma cells. It inhibits protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells with an IC
50 value of 0.5 μM. E-46d also inhibits entry of vesicular stomatitis virus (VSV) particles pseudotyped with severe acute respiratory syndrome coronavirus (SARS-CoV) or SARS-CoV-2 spike glycoprotein into Vero cells, an effect that is reduced by expression of the serine protease TMPRSS2.
Synthesis
General procedure for the synthesis of ethyl (2S,3S)-3-(((S)-1-(isopentylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)ethylene oxide-2-carboxylate from rel-(2R,3R)-3-(ethoxycarbonyl)oxirane-2-carboxylate and N1-isopentyl-L-leucinamide under nitrogen protection: under nitrogen protection (2S,3S)-3-((S)-3-(isopentylamino)-1-(isopentylamino)oxirane-2-carboxylic acid (25g, 0.16mol) and (S)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)-1-(isopentylamino)carbamoyl)ethylene oxide-2-carboxylic acid was prepared. -(ethoxycarbonyl)oxirane-2-carboxylic acid (25 g, 0.16 mol) and (S)-1-(isoamylamino)-2-amino-4-methyl-1-oxopentane hydrochloride (38 g, 0.16 mol) were dissolved in dichloromethane (750 mL) and cooled to 0 °C (ice water bath). Subsequently 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (65 g, 0.17 mol) and diisopropylethylamine (DIPEA, 56 mL, 0.31 mol) were added. The reaction mixture was stirred at 0 °C for 1 h. After removing the ice bath, the reaction mixture was continued to be stirred for 2 h at room temperature. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane (750 mL), washed sequentially with saturated sodium bicarbonate solution (2 times) and saturated sodium chloride solution (1 time), the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a yellow viscous oily crude product. The crude product was purified by a silica gel column (60 mm × 300 mm), eluting with a gradient of 10% to 50% ethyl acetate in hexane solution. The combined eluents (2.5 L) were collected and concentrated under reduced pressure to give E64d (53 g, 100% yield) as a white solid. Recrystallization of E64d (36 g) in methyl tert-butyl ether in 1% ethanol (MTBE, 535 mL) afforded white short needle-like crystals of E64d (25 g, 69% yield) with a melting point of 123-124 °C.1H NMR (500 MHz, CDCl3) δ 6.68 (d, J=8.4 Hz, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), 6.08 (br s, 1H), and 6.08 (br s, 1H). 4.36-4.40 (m, 1H), 4.21-4.28 (m, 2H), 3.67 (d, J=1.9 Hz, 1H), 3.46 (d, J=1.9 Hz, 1H), 3.19-3.30 (m, 2H), 1.49-1.65 (m, 4H), 1.38 (q, J=7.4 Hz, 2H), 1.30 (t, J= 7.0 Hz, 3H), 0.89-0.93 (m, 12H); ESI MS m/z 343.2 (M+H)+; elemental analysis (C17H30N2O5) is in accordance with the theoretical values; HPLC purity is in accordance with the requirements.
References
[1] ELEANOR B. MCGOWAN Thomas C D Edward Becker. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d[J]. Biochemical and biophysical research communications, 1989, 158 2: Pages 432-435. DOI:
10.1016/s0006-291x(89)80065-8[2] D WILCOX R W M. Inhibition of cysteine proteinases in lysosomes and whole cells.[J]. Biochemical Journal, 1992, 285 ( Pt 2): 495-502. DOI:
10.1042/bj2850495[3] NOBORU MIZUSHIMA B L Tamotsu Yoshimori. Methods in mammalian autophagy research.[J]. Cell, 2010, 140 3: 313-326. DOI:
10.1016/j.cell.2010.01.028
