Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

Tale of Twisted Molecules. Atropselective Photoreactions: Taming Light Induced Asymmetric Transformations through Non-biaryl Atropisomers.

Photochemical transformations are a powerful tool in organic synthesis to access structurally complex and diverse synthetic building blocks. However, this great potential remains untapped in the mainstream synthetic community due to the challenges associated with stereocontrol originating from excited state(s). The finite lifetime of an excited state and nearly barrierless subsequent processes present significant challenges in manipulating the stereochemical outcome of a photochemical reaction. Several methodologies were developed to address this bottleneck including photoreactions in confined media and preorganization through noncovalent interactions resulting in stereoenhancement. Yet, stereocontrol in photochemical reactions that happen in solution in the absence of organized assemblies remained largely unaddressed. In an effort to develop a general and reliable methodology, our lab has been exploring non-biaryl atropisomers as an avenue to perform asymmetric phototransformations. Atropisomers are chiral molecules that arise due to the restricted rotation around a single bond (chiral axis) whose energy barrier to rotation is determined by nonbonding interactions (most often by steric hindrance) with appropriate substituents. Thus, atropisomeric substrates are chirally preorganized during the photochemical transformation and translate their chiral information to the expected photoproducts. This strategy, where "axial to point chirality transfer" occurs during the photochemical reaction, is a hybrid of the successful Curran's prochiral auxiliary approach involving atropisomers in thermal reactions and the Havinga's NEER principle (nonequilibrating excited-state rotamers) for photochemical transformations. We have investigated this strategy in order to probe various aspects such as regio-, enantio-, diastereo-, and chemoselectivity in several synthetically useful phototransformations including 6π-photocyclization, 4π-ring closure, Norrish-Yang photoreactions, Paternò-Büchi reaction, and [2 + 2]- and [5 + 2]-photocycloaddition. The investigations detailed in this Account clearly signify the scope of our strategy in accessing chirally enriched products during phototransformations. Simple design modifications such as tailoring the steric handle in atropisomers to hold reactive units resulted in permanently locked/traceless axial chirality in addition to incorporating multiple stereocenters in already complex scaffolds obtained from phototransformation. Further improvements allowed us to employ low energy visible light rather than high energy UV light without compromising the stereoenrichment in the photoproducts. Continued investigations on atropisomeric scaffolds have unraveled new design features, with outcomes that are unique and unprecedented for excited state reactivity. For example, we have established that reactive spin states (singlet or triplet excited state) profoundly influence the stereochemical outcome of an atropselective phototransformation. In general, the photochemistry and photophysics of atropisomeric substrates differ significantly from their achiral counterparts irrespective of having the same chromophore initiating the excited state reactivity. The ability of axially chiral chromophores to impart stereoenrichment in the intramolecular photoreactions appears to be promising. A challenging endeavor for the "axial to point chirality transfer" strategy is to enhance stereoenrichment or alter chemical reactivity in intermolecular photoreactions. Insights gained from our investigations will serve as a platform to venture into more complicated yet fruitful research in terms of broad synthetic utility.

Enantiospecific photochemical 6π-ring closure of α-substituted atropisomeric acrylanilides--role of alkali metal ions.

Direct irradiation of atropisomeric α-substituted acrylanilides in the presence of alkali metal ions gave high ee values in the 3,4-dihydro-2-quinolin-2-one photoproduct, while in the absence of alkali metal ions, racemic photoproduct was observed. The heavy atom effect leading to enhanced triplet yields alters the reactive pathway leading to the observed enantioselectivity in the photoproduct.

Enantiospecific 6π-photocyclization of atropisomeric α-substituted acrylanilides in the solid-state: role of crystalline confinement on enantiospecificity.

Direct irradiation of optically-pure axially-chiral α-substituted acrylanilides in the crystalline state leads to 3,4-dihydro-2-quinolin-2-one photoproduct with an enantiomeric ratio of 85 : 15 while a racemic mixture of photoproduct is observed in solution.

Reactive spin state dependent enantiospecific photocyclization of axially chiral α-substituted acrylanilides.

The enantiomeric ratio (e.r.) in the 3,4-dihydroquinolin-2-one photoproduct during 6π-photocyclization of α-substituted axially chiral ortho-tert-butyl-acrylanilides depends on the nature of the reactive spin state (singlet or triplet), where the singlet-spin state reactivity gives a racemic mixture and the triplet reactivity gives an e.r. value >95 : 5.

Enantiospecific photochemical Norrish/Yang type II reaction of nonbiaryl atropchiral alpha-oxoamides in solution--axial to point chirality transfer.

Alpha-oxoamides 1 with o-tert-butyl substitution on the N-phenyl moiety were found to be stable axially chiral atropisomers. These axially chiral alpha-oxoamides undergo enantiospecific photochemical gamma-Hydrogen abstraction in CHCl(3) to yield beta-lactams with high enantioselectivity (e.r. approximately 90:10) in solution. The extent of enantioselectivity was found to be dependent on the reaction temperature.

6pi-Photocyclization of O-tert-butylacrylanilides. N-substitution dictates the regiochemistry of cyclization.

O-tert-Butylacrylanilides with N-H substitution undergo 6pi-photocyclization at the unsubstituted ortho carbon, whereas the corresponding N-methyl derivatives cyclize at the ortho carbon bearing the tert-butyl with the eventual loss of 2-methylpropene.

Light-induced transfer of molecular chirality in solution: enantiospecific photocyclization of molecularly chiral acrylanilides.

Molecularly chiral o-tert-butylacrylanilides undergo enantiospecific 6pi-photocyclization to yield 3,4-dihydroquinolin-2-ones with very high enantioselectivity (>90%) in solution. The photocyclization results in the removal of the ortho tert-butyl substituent, presumably via a zwitterionic intermediate. Beta-substitution in the alkene is found to be critical for the transfer of molecular chirality (axial chirality) in the reactant to point chirality in the photoproduct(s).