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Khatua, Kaustav ; Alugubelli, Yugendar R. ; Yang, Kai S. , et al. bioRxiv,2023.04.11.536467. DOI: 10.1101/2023.04.11.536467
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Abstract: Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallog. analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.
Purchased from AmBeed: 5669-19-2 ; 1149-26-4 ; 7304-32-7 ; 16966-09-9 ; 3590-48-5 ; 38377-38-7 ; 848133-35-7 ; 103260-65-7 ; 27746-99-2 ; 51021-87-5 ; 127-17-3 ; 540750-29-6 ; 40056-18-6 ; 5704-66-5
CAS No. : | 1149-26-4 | MDL No. : | MFCD00008922 |
Formula : | C13H17NO4 | Boiling Point : | No data available |
Linear Structure Formula : | - | InChI Key : | CANZBRDGRHNSGZ-NSHDSACASA-N |
M.W : | 251.28 | Pubchem ID : | 726987 |
Synonyms : |
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Chemical Name : | Z-L-Val-OH |
Signal Word: | Warning | Class: | |
Precautionary Statements: | P261-P280-P305+P351+P338 | UN#: | |
Hazard Statements: | H302+H312+H332-H315-H319-H335 | Packing Group: | |
GHS Pictogram: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
98% | [0197] To a stuffed solution of compound I (10 g, 39.84 mmol, 1 eq) in DMF (100 mL) was added DIPEA (19.7 mL, 119.5 mmol, 3 eq) and HATU (18.17 g, 47.8 mmol, 1.2 eq) at 0C and the resulting mixture was stuffed for 15 mm. To the mixture was added ammonium chloride (10.7 g, 199.2 mmol, 5 eq) and the resulting mixture was allowed to stir at 23C for another 16 h. The mixture was poured into ice cold water (500 mL), the organic components were extracted with EtOAc (3 x 500 mL) and the combined organic layers were washed with aqueous ammonium chloride solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The crude product was recrystallized from ethanol to obtain the title compound (9.8 g, 98%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) oe 7.35 (m, 6H), 7.12 (d, 1H, J = 9 Hz), 7.01 (s, 1H), 5.03 (s, 2H), 3.80 (t, 1H, J = 8 Hz), 1.95 (m, 1H), 0.85 (m, 6H). LCMS: mlz = 251.2 [M+Hj , RT = 2.81 minutes, (Program P1, Column Y). | |
93% | General procedure: To a colorless solution of 150mg (0.50mmol) of Cbz-L-Phe-OH 3a in 10mL of THF were added 67muL (0.70mmol, 1.4equiv) of ClCO2Et and 209muL (1.5mmol, 3.0equiv) of Et3N at 0C. After stirring for 30min at 0C, 0.75 ml of a 1.0M aqueous solution of NH4Cl (0.75mmol, 1.5equiv) were added at 0C to the colorless suspension. The mixture was stirred for 30min at 0C and 5mL of H2O was added to the resulted mixture. The colorless clear solution was extracted with 30mL of EtOAc and the aqueous layer was extracted with 20mL of EtOAc. The organic layers were combined, washed with 5mL of brine, and dried over anhydrous MgSO4. The crude product was chromatographed on silica gel with EtOAc to afford 129mg (86% yield) of Cbz-L-Phe-NH2 4a. 4.3.7 Cbz-l-Val-NH2 4d 117?mg (93%); >99% ee; coloress solid; mp: 172-175?C; [alpha]D25?=?+17.8 (c 0.99, DMSO); 1H NMR (400?MHz, DMSO-d6): delta 0.83 (d, J?=?6.8?Hz, 3H, CH3), 0.86 (d, J?=?6.8?Hz, 3H, CH3), 1.94 (ddd, J?=?6.7, 6.8, 6.8?Hz, 1H, CH(CH3)2), 3.80 (dd, J?=?6.7, 8.8?Hz, 1H, CHCO), 5.03 (s, 2H, OCH2C6H5), 7.03 (br, 1H, CONHA), 7.16 (d, J?=?8.8?Hz, 1H, NHCH), 7.29-7.39 (m, 6H, CONHB, C6H5); 13C NMR (100?MHz, DMSO-d6): delta 18.0, 19.3, 30.1, 60.0, 65.3, 127.6, 127.7, 128.3, 137.1, 156.1, 173.2; IR (KBr, vmax/cm-1): 3381 (CONH), 3319 (CONH), 3203 (CONH), 1654 (CON); HRMS (ESI-TOF): Calcd for C13H18N2O3Na (M+Na)+: 273.1210, found: 273.1193; The enantiomeric ratio was determined by HPLC (Chiralcel AD: hexane/2-propanol?=?90/10): Tr 11.5?min. | |
63.8% | Step 1. To a solution of 27-1 (8 g, 31.8 mmol) and Et3N (6.43 g, 63.6 mmol) in dry THF (100 mL) at -78C was added dropwise a solution of ethyl chloro formate (3.45 g, 31.8 mmol) in THF (10 mL). After the reaction was stirred at -78C for 1 h, NH3.H20 (5 mL) was added. The reaction was warmed to r.t. over 2 h. White solid was collected by filtration and dried to give 27-2 (5.1 g, 63.8%). |
53.15% | With pyridine; di-tert-butyl dicarbonate; ammonium bicarbonate; In 1,4-dioxane; at 0 - 20℃; | To a solution of (S)-2-(benzyloxycarbonylamino)-3-methylbutanoic acid (10.0 g, 39.84 mmol) in 1,4-dioxane (50 mL) was added di-tert-butyl dicarbonate (11.95 g, 51.79mmol) in portions at 0C. Then pyridine (2 mL, 25.76 mmol) was added drop-wise at 0C, followed by ammonium bicarbonate (3.94 g, 50.43 mmol) in portions. The reaction was stirred at room temperature overnight. The mixture was poured into ice water and filtered. The filter cake was washed with water and dried under vacuum. The filter cake was then re-crystallized from dioxane/water (80 mL, 9: 1, v/v) to give (S)-benzyl (1-amino-3-methyl-1-oxobutan-2-yl)carbamate(5.3 g, 53.15% yield) as a white solid. LC-MS (ESI) found: 251 [M+Hf?, ee>99%, (CHIRALPAK AS-H, 15% ethanol/ hexane) |
General procedure: The appropriate N-protected amino/peptide acid (1.0 mmol) was dissolved in THF, to which NMM (1.5 mmol) and ECF (1.5 mmol) were added at -15 C, followed by the addition of NH4HCO3 (1.5 mmol) to obtain the corresponding amide. The reaction mixture was stirred until the completion of the reaction and the progress of the reaction was checked by TLC. After the removal of THF, the product was extracted into ethyl acetate (15 mL) and the organic layer was washed with dilute HCl solution (10 mL) or citric acid solution (10 mL) in the case of the Boc-protected compounds, then with Na2CO3 solution (15 mL x 2), water (15 mL) and brine (15 mL). The organic extract was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The amide thus formed was then treated with P2S5 (0.5 mmol) to form the thioamide under ultrasonic conditions for 2-3 hrs. The resulting thioamide in dry THF was reacted with chloroacetaldehyde (1.0 mmol) under reflux conditions for 2 hrs. N-Protected thiazoles were obtained in good yield after simple acid-base work up and purified through column chromatography using EtOAc:hexane (3:7) as an eluent. |
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