Paxlovid

Description: Paxlovid is an antiviral medication developed by Pfizer which acts as an orally active 3C-like protease inhibitor. Paxlovid had been further developed prior to the COVID-19 pandemic for other diseases including SARS.

• Catalog: OFC2628280408
• CAS: 2628280-40-8
• Category: Fluorinated APIs
• Synonyms: Nirmatrelvir
• Purity: 98%

• IUPAC Name: (1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
• InChI: InChI=1S/C23H32F3N5O4/c1-21(2,3)16(30-20(35)23(24,25)26)19(34)31-10-13-14(22(13,4)5)15(31)18(33)29-12(9-27)8-11-6-7-28-17(11)32/h11-16H,6-8,10H2,1-5H3,(H,28,32)(H,29,33)(H,30,35)/t11-,12-,13-,14-,15-,16+/m0/s1
• InChI Key: LIENCHBZNNMNKG-OJFNHCPVSA-N
• Isomeric SMILES: CC1([C@@H]2[C@H]1[C@H](N(C2)C(=O)[C@H](C(C)(C)C)NC(=O)C(F)(F)F)C(=O)N[C@@H](C[C@@H]3CCNC3=O)C#N)C

• Molecular Formula: C23H32F3N5O4
• Molecular Weight: 499.53
• Appearance: White to off-white solid powder

• XLogP3-AA: 2.2
• Hydrogen Bond Donor Count: 3
• Hydrogen Bond Acceptor Count: 8
• Rotatable Bond Count: 7
• Exact Mass: 499.24063901 g/mol
• Monoisotopic Mass: 499.24063901 g/mol
• Topological Polar Surface Area: 131Ų
• Heavy Atom Count: 35
• Formal Charge: 0
• Complexity: 964

Case Study

An Effective Synthesis Method for Niramatrelvir (PF-07321332)

Shekhar C, et al. Tetrahedron Chem, 2022, 4, 100033.

Nirmatrelvir, a tripeptide protein mimetic, is a crucial compound in antiviral therapy. This case study outlines the efficient synthesis process for Nirmatrelvir, starting with the production of the critical bicyclic amino acid.
Synthesis of Bicyclic Amino Acid
The synthesis began with Boc trans-4-hydroxy L-proline benzylester (compound 8) through a four-step process:
Mesylation: The hydroxy group of compound 8 was mesylated using methanesulfonyl chloride, triethylamine, and DMAP, resulting in compound 9 in quantitative yield.
Phenylselenylation: Compound 9 was treated with diphenyldiselenide and sodium borohydride to yield phenylselenyl derivative 10.
Oxidation and Elimination: Oxidation of selenide 10 with hydrogen peroxide, followed by elimination with pyridine, produced alkene 11.
Cyclopropanation: Alkene 11 underwent cobalt(II)-catalyzed dimethylcyclopropanation using 2,2-dichloropropane, zinc metal, zinc bromide, and a Co(II)-complex to form the target bicyclic dimethylcyclopropyl amino acid fragment (compound 4).
To facilitate peptide coupling, compound 4 was converted into two derivatives:
Free amine benzyl ester (compound 12) using 4 N hydrochloric acid.
Boc-protected free acid (compound 13) using Pd/C-H2.
Peptide Coupling and Dipeptide Formation
The amine hydrochloride of bicyclic fragment 12 was coupled with N-trifluoroacetyl L-tert-leucine (compound 14) using HATU, NMM, and DMAP, yielding dipeptide 15.
Synthesis of Cyano Lactam Residue
L-glutamic acid underwent the following transformations to form the cyano lactam residue:
Protection and Esterification: Silylation with TMSCl, esterification with methanol, and Fmoc protection in the presence of sodium carbonate produced protected glutamic acid (compound 16).
Mono-alkylation: Compound 16 was mono-alkylated with bromoacetonitrile using LiHMDS in THF, forming cyano compound 17.
Cyclization: The cyano diester 17 was cyclized to lactam 18 using CoCl2.6H2O and sodium borohydride.
Reduction and Oxidation: The ester in lactam 18 was reduced to alcohol 6 using sodium borohydride in THF (2:1). Oxidation with Dess-Martin periodinane (DMP) then produced aldehyde 19.
Nitrile Formation: A one-pot conversion of aldehyde 19 to nitrile 20 was achieved using iodine and ammonia solution. Finally, Fmoc-deprotection of compound 20 with diethylamine yielded cyano amine fragment 7.
Final Coupling and Formation of Nirmatrelvir
Dipeptide 15 was debenzylated using Pd/C, H2 in methanol to produce dimer acid 5. The amine 7 was then coupled with dimer acid 5 using HATU, NMM, and DMAP, resulting in the target molecule, Nirmatrelvir (compound 1).

Efficacy of Nirmatrelvir Against SARS-CoV-2 Variants

Greasley SE, et al. JBC, 2022, 298, 101972.

Nirmatrelvir (PF-07321332) is a protease inhibitor targeting the main protease (M^pro) of SARS-CoV-2, which is a reversible, covalent inhibitor that binds to the active site of M^pro. The compound has been designed to inhibit the proteolytic activity of M^pro, thereby halting the replication of the virus. This case study highlights the robustness of Nirmatrelvir as an antiviral agent against SARS-CoV-2 variants.
Methods
Protein Expression and Purification: Full-length wild-type M^pro of the original Washington variant (USA-WA1/2020) and various mutant forms of M^pro (K90R, G15S, P132H) were expressed and purified. The near homogeneity of these proteins was confirmed by size exclusion chromatography and protein blotting analysis.
Enzyme kinetics: The catalytic activity of wild-type and mutant M^pro proteins was determined using a fluorescence resonance energy transfer-based cleavage assay.
Inhibition assay: The inhibitory potency of Nirmatrelvir against wild-type and mutant M^pro proteins was evaluated.
Structural Analysis: The crystal structures of Nirmatrelvir bound to wild-type and mutant M^pro were analyzed at high resolution.
Results
Catalytic Activity: The catalytic efficiencies of the mutant M^pro proteins K90R, G15S, and P132H were comparable to the wild-type M^pro. This indicates that these mutations do not significantly affect the enzymatic activity of M^pro.
Inhibition by Nirmatrelvir:Nirmatrelvir showed potent inhibition of both wildtype (mean Ki of 0.93 nM) and mutant M^pro enzymes. The Ki values for K90R (1.05 nM), G15S (4.07 nM), and P132H (0.64 nM) variants were similar to the wildtype, except for G15S, which showed a statistically significant shift in potency (p < 0.0005).
Structural Insights: Crystal structures revealed that the binding mode of Nirmatrelvir is consistent across wildtype and mutant M^pro proteins. The mutations K90R, G15S, and P132H are distal to the Nirmatrelvir-binding pocket and do not cause significant structural changes around the binding site. HDX-MS analysis further confirmed that these mutations do not lead to notable alterations in the solution-phase structural dynamics of M^pro.