EST Chemical Properties

Melting point:

126.2°C

Boiling point:

477.88°C (rough estimate)

Density 

1.0657 (rough estimate)

refractive index 

1.5800 (estimate)

storage temp. 

-20°C

solubility 

Soluble in DMSO, DMF or ethanol

pka

13.32±0.40(Predicted)

form 

White solid

color 

Fine needles from EtOH

biological source

synthetic (organic)

Stability:

Stable for 1 year from date of purchase as supplied. Solutions in DMSO or ethanol may be stored at -20°C for up to 1 month.

Safety Information

Hazard Codes 

Xi

Risk Statements 

36/37/38

Safety Statements 

26-36-24/25

RIDADR 

3077

WGK Germany 

2

RTECS 

RR0404300

HS Code 

29242990

Toxicity

cyt-ham:lng 200 mg/L IYKEDH 17,815,86

MSDS

• Language:English Provider:SigmaAldrich

EST Usage And Synthesis

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 G₂/M phase in A431 human epidermoid carcinoma cells. It inhibits protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells with an IC₅₀ 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.

Uses

E-64d has been used as a lysosomal inhibitor in human acute promyelocytic leukemia NB4 cells and Huh-7?cells. It has also been used as a protease inhibitor in free calcium physiological tyrode solution for perfusion into the isolated heart samples.

Uses

E-64d is an inhibitor of cathepsins B and L as well as a potential inhibitor of calpain. E-64d has been shown to inhibit lysosomal proteases. E-64d has been used in combination with Prepstatin A to interfere with autolysosomal digestion. E-64d displays neurovascular and neuronal protective effects after focal cerebral ischemia in rats.

Definition

ChEBI: An L-leucine derivative that is the amide obtained by formal condensation of the carboxy group of (2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid with the amino group of N-(3-methylb tyl)-L-leucinamide.

Biochem/physiol Actions

E-64d is an epoxysuccinyl peptide and an inhibitor of cysteine protease cathepsin B, calpains 1 and 2. E-64d by its cathepsin B protease inhibition functionality, may serve as a potential drug for treating traumatic brain injury (TBI) and Alzheimer′s disease (AD). It inhibits gametocyte surface antigen resulting in a decreased oocyst production in Plasmodium falciparum.

Safety Profile

Moderately toxic by intraperitoneal route. An experimental teratogen. Experimental reproductive effects. Mutation data reported. Whenheated to decomposition it emits toxic fumes of NOx.

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.

storage

Store at -20°C

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

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