Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (Mpro) is a promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 Mpro inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of Mpro. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 Mpro. Among the four stereoisomers, (R,R)-18 exhibited a markedly higher inhibitory activity against Mpro than the other isomers. Reaction kinetics and computational docking studies suggest that the R configuration of the CFA warhead is crucial for the rapid covalent inhibition of Mpro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.


Introduction
The outbreak of the novel coronavirus disease 2019 (COVID- 19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1,2 poses a severe threat to global public health and economy. Although efficacious vaccines are now being administered worldwide, novel SARS-CoV-2 variants of interest and concern continue to emerge. 3 Thus, the development of antiviral agents against SARS-CoV-2 is urgently needed to tackle and end the current pandemic. 4,5 SARS-CoV-2 has a singlestranded genomic RNA that encodes polyproteins pp1a and pp1ab. Two cysteine proteases, the main protease (M pro , also known as 3C-like protease, 3CL pro ) and the papain-like protease (PL pro ), are excised from these polyproteins and further digest the polyproteins into nonstructural proteins, including crucial components of the viral replication-translation machinery. Although both proteases are essential for viral replication, the predominant role of M pro in polyprotein processing and the lack of a human homolog has led to extensive studies to establish it as an attractive drug target against SARS-CoV-2. 6,7 M pro recognizes glutamine and a hydrophobic amino acid residue at the S1 and S2 pockets in the active site, respectively, and cleaves amide bonds primarily within the Leu-GlnY(Ser, Ala, Gly) sequence (Y: cleavage site). 8 This substrate specicity is conserved across other coronaviruses, including SARS-CoV-1 and middle east respiratory syndrome coronavirus (MERS-CoV). 9,10 Therefore, current efforts to develop SARS-CoV-2 M pro inhibitors are extensively guided by the previous molecular designs, especially those targeting SARS-CoV-1 M pro . 5,6 Most of the reported SARS-CoV-2 M pro inhibitors are peptidomimetics based on the Leu-Gln sequence, combined with an electrophilic warhead, such as a-ketoamide, 11 aldehyde, [12][13][14][15] ketone, [16][17][18][19] vinyl sulfone, 8 and nitrile, 20,21 for the covalent capture of the sulydryl group of the catalytic Cys145. Nirmatrelvir (PF-07321332), developed by Pzer, is one of the most advanced compounds in this category and has been recently approved by regulatory ageincies as a combination with ritonavir (Fig. 1A). 21 Other than peptidomimetics, several Ugi multicomponent reaction (MCR)generated dipeptidic compounds have been reported as inhibitors against SARS-CoV-1 and SARS-CoV-2 M pro (Fig. 1B). [22][23][24] Covalent inhibition of proteins has been a powerful strategy for achieving potent and sustained pharmacological efficacy. 25 To date, a number of acrylamide-based covalent inhibitors targeting a noncatalytic cysteine of proteins have been clinically approved or under clinical trials for cancer treatment. 26,27 In development of covalent inhibitors, high target selectivity should be ensured to minimize the risk of toxicity due to off-target labeling. 28 This point is also crucial for designing covalent inhibitors targeting SARS-CoV-2 M pro , as medications for infectious diseases generally require a broader safety margin than cancer treatment. We have recently introduced a-chloro-uoroacetamide (CFA) as a weakly reactive, cysteine-directed warhead for covalent inhibition of proteins including tyrosine kinases. [29][30][31] We found that the CFA derivatives exhibited higher selectivity in covalent modication toward the targeted tyrosine kinases than the structurally related acrylamide-based inhibitors. We also demonstrated that the CFA-thiol adduct is hydrolyzed under neutral aqueous conditions to reversibly generate an unmodied cysteine, which could contribute to high target selectivity by eliminating the solvent-exposed offtarget labeling. These desirable features led us to envision CFA as a valuable tool for designing selective covalent SARS-CoV-2 M pro inhibitors.

Results and discussion
Wang et al. recently reported Jun8-76-3A as a reversible inhibitor of SARS-CoV-2 M pro . 23 The molecular architecture of Jun8-76-3A is based on ML188-(R) that was previously developed as an inhibitor of SARS-CoV-1 M pro . 22 The crystal X-ray structure analysis revealed that the 3-pyridyl group of Jun8-76-3A resides in the S1 glutamine binding pocket of SARS-CoV-2 M pro by forming a hydrogen bond with His163. The 4-biphenyl group occupies the S2 pocket that prefers hydrophobic amino acid residues like leucine and phenylalanine. The furan-2carboxamide moiety exerts a bifurcated interaction with the backbone NH of Gly143 in the S1 0 pocket, wherein the catalytic Cys145 resides. Based on the same Ugi MCR-generated dipeptide scaffold, London et al. reported an acrylamide-based covalent SARS-CoV-2 M pro inhibitor 1. 24 We also employed this dipeptide scaffold as the starting point for the development of a CFA-based covalent inhibitor against M pro . Ugi MCR using racemic chlorouoroacetic acid, along with amine, aldehyde, and isocyanide, proceeded smoothly to yield CFA compound 3 as a mixture of diastereomers in a single step. The two diastereomers, 3A (less polar isomer) and 3B (more polar isomer), were separable by silica gel chromatography ( Fig. 2A). The inhibitory activity of these compounds was assessed by an enzymatic assay using the recombinant SARS-CoV-2 M pro and the uorogenic substrate Ac-Abu-Tle-Leu-Gln-(4-methylcoumaryl-7-amide). 8 This substrate allowed robust uorescent reading compared to the FRET-based substrate possessing an Edans/Dabcyl donor-acceptor pair. 32 We found that 3A and 3B displayed different inhibitory activities against M pro (Fig. 2B); 3B exhibited moderate inhibitory activity (IC 50 ¼ 1.05 mM), whereas 3A was virtually inactive (IC 50 > 20 mM). In the case of ML188 and Jun8-76-3A, it has been reported that the R conguration of the pyridyl a-carbon is crucial for M pro inhibition. 22,23 Considering that both 3A and 3B contain the R conguration at this stereocenter, our results suggest that the chirality at the CFA stereocenter also substantially impacts their inhibitory activities. Non-reactive analogs of 3 such as acetamide 4 and  diuoroacetamide 5 were found to be inactive (IC 50 > 20 mM), indicating that the covalent attachment at CFA contributes to the M pro inhibitory activity of 3B in a stereospecic manner. In the same assay, we also found that acrylamide 1 showed a weaker activity (IC 50 ¼ 2.62 mM) than 3B.
This observation encouraged us to subsequently conduct an SAR study to improve the potency of CFA dipeptides (Fig. 3A). We rst evaluated the effect of the para-substituent of the benzene ring (R 1 ) on inhibitory activity. As this moiety occupies the S2 pocket that primarily accommodates a leucine residue, we screened compounds 6-9 with a hydrophobic para-substituent. In the assay, less polar diastereomer A was inactive (IC 50 > 20 mM), whereas more polar diastereomer B showed moderate activity in all the cases. Among them, 9B possessing a penta-uorosulfanyl (SF 5 ) group, a thermally and chemically stable bioisostere for a tert-butyl group, 33 exhibited a slightly higher M pro inhibitory activity (IC 50 ¼ 0.90 mM) than 3B. Next, we investigated the effect of the heteroaromatic ring (R 2 ) on the activity. The replacement of the pyridine ring of 3B with a diazine increased the potency of 11-12, whereas the 3-pyridazinyl derivative 10 was almost inactive (IC 50 > 20 mM as a diastereomixture). 5-Pyrimidine 12 was the only compound with separable diastereomers, and 12B exhibited the highest activity in the series (IC 50 ¼ 0.25 mM). The effect of the substituent R 3 was also investigated. The introduction of a tert-butyl group, as observed in the structure of ML188, reduced the potency (14B, IC 50 ¼ 2.70 mM). Replacement of the 3-uorophenyl group of 3 to a 3-pyridyl group slightly increased the activity (16, IC 50 ¼ 0.73 mM as a diastereomixture). The introduction of a 7-isoquinoline ring resulted in a slight loss of activity (17B, IC 50 ¼ 1.43 mM). Combining the SAR results, we synthesized compound 18 and 19 as structurally optimized CFA inhibitors (Fig. 3B). The diastereomers were separable in both cases and the active isomers 18B and 19B exhibited potent M pro inhibitory activities (IC 50 ¼ 0.21 and 0.14 mM, respectively). Although 19B was marginally more active than 18B, we chose 18 for further investigations because the chromatograhic separation of the diastereomixture of 18 was much easier than that of 19. Fortunately, we were able to determine the relative conguration of 18B by X-ray crystallography (Fig. S1 †). The analyzed structure revealed that 18B has a syn-conguration, where the 5-pyrimidinyl group and the CFA uorine atom are oriented toward the same direction in the drawn structure (Fig. 3C). Consequently, 18A was determined to be an anti-isomer.
To elucidate the relationship between the CFA chirality and the inhibitory activity, we next attempted to obtain the four stereoisomers of 18 in enantiomerically pure form using optically active chlorouoroacetic acid. 34 Wakselman previously reported the preparation of optically active chlorouoroacetyl chloride based on the chromatographic separation of the diastereomers of CFA 20 derived from (S)-(À)-1-phenethylamine. 35 However, the isolation of diastereomers of 20 proved to be laborious in our attempts due to poor separation by silica gel chromatography. Thus, we established a new method employing (R)-(À)-2-phenylglycinol as a chiral resolving agent (Scheme 1). We found that the diastereomers of CFA 21, readily accessible from commercially available (R)-(À)-2-phenylglycinol and ethyl chlorouoroacetate, were easily separated by silica gel chromatography and afforded pure 21A and 21B on a multigram scale. X-ray crystallography determined the absolute conguration at the CFA chiral center to be R in 21B (Fig. S2 †). Isolated 21A and 21B were subjected to hydrolysis in reuxing 3 M H 2 SO 4 /1,4-dioxane to afford (S)-and (R)-chlorouoroacetic acid, respectively. Finally, Ugi MCR using enantiopure chlorouoroacetic acids and subsequent chromatographic separation of the diastereomers yielded a set of four stereoisomers of 18.
Having the four stereoisomers of 18 in hand, we evaluated their inhibitory activities against SARS-CoV-2 M pro (Fig. 4). We found that (R,R)-18 (the former R indicates the absolute conguration at the Ugi MCR-generated stereocenter, and the latter at CFA) exclusively exhibited strong activity (IC 50 ¼ 0.056 mM) among the four stereoisomers ( Fig. 4B and D). Notably, this activity is much higher than that of (R,S)-18 possessing an (S)-CFA warhead (IC 50 ¼ 10.62 mM), indicating that the chirality at CFA greatly inuences the inhibitory activity. The IC 50 values of the other isomers (S,R)-18 and (S,S)-18 were 2.24 and 14.04 mM, respectively. To further investigate these results, we performed a kinetic analysis of M pro inhibition by (R,R)-18 and its diastereomers (S,R)-and (R,S)-18. 36 All three isomers inhibited the hydrolysis of the uorogenic substrate by M pro in a timedependent manner, indicating an irreversible mode of action ( Fig. 4C and S3-S5 †). The kinetic parameters for irreversible inactivation of M pro are summarized in Fig. 4D. As expected, the most potent isomer (R,R)-18 inactivated M pro with much higher efficiency (k inact /K I ¼ 4167 M À1 s À1 ) than the diastereomers (S,R)-and (R,S)-18 (k inact /K I ¼ 66.0 and 20.0 M À1 s À1 , respectively). (R,R)-18 exhibited both a higher k inact value and a lower K I value than those of (R,S)-18, suggesting that the CFA chirality largely affected the reaction rate of covalent modication as well as the binding affinity with M pro . In comparison between (R,R)-18 and (S,R)-18, the binding constant K I was signicantly different (1.34 and 64.7 mM, respectively), whereas the inactivation rates were similar (k inact ¼ 0.0056 and 0.0043 s À1 , respectively). This marked difference in K I value was consistent with previous studies of ML188 and Jun8-76-3A, where the R conguration at the a-pyridine chiral center was crucial for the inhibitory activities. 22,23 We also determined the kinetic parameters of acrylamide 1 (Fig. S6 †). Despite higher thiol reactivity of acrylamide than CFA, 29 the k inact value of 1 was signicantly smaller than (R)-CFA. This result implies the mismatched disposition of the acrylamide warhead in the 1-M pro binding complex for the Michael addition of the thiol group of Cys145.
To gain further insight into the effect of CFA chirality, we performed docking simulations of the stereoisomers of 18 against M pro . Based on the X-ray cocrystal structure of (R)-1 with SARS-CoV-2 M pro (PDB code 7NW2), 24 we examined the possible binding poses and affinities of each of the four isomers, (R,R)-, (R,S)-, (S,R)-, and (S,S)-18 in the same pocket of M pro . The predicted binding affinities for the stereoisomers fell into the range of 2 kcal mol À1 difference, but there found no clear correlation with the experimental result. To nd out the cause of the higher reactivity of (R,R)-18, we assessed the occurrences of "reactive conformations" in the predicted poses for the four isomers, where we dened the pose as reactive for the S N 2 reaction (backside attack of Cys145 to the CFA carbon) if the C/S distance <4.5Å and Cl-C/S angle >150 . We found a large number of reactive conformations (46 out of 183 poses) exclusively in the low binding energy region for (R,R)-18 among the four stereoisomers (Fig. S7 †). The most stable pose of (R,R)-18 was S N 2-reactive, where the Cl-C/S angle is nearly 180 (Fig. 4E). In the complex, the CFA uorine and the carbonyl oxygen were close proximity to the backbone NH of Gly143, which is thought to constitute the oxyanion hole in the S1 0 pocket. 37 This result suggests the potential contribution of NH/F hydrogen bond to facilitate the reaction. The bound structure of (R,R)-18 was well aligned to the X-ray crystal structure of a dichloroacetamide inhibitor Jun9-62-2R covalently bound to M pro , which was recently reported by Wang et al. (Fig. S8 †). 38 On the other hand, the most stable pose of (R,S)-18 was S N 2-non-reactive due to the unpreferable orientation of the C-Cl bond for the nucleophilic attack by Cys145. The rotation of the CFA unit of (R,S)-18 could give an S N 2-reactive pose, but the resulting rotamer lacks any stabilizing interaction with the M pro active site. Taken together, we infer that (R,R)-18 is more likely to become S N 2-reactive as the NH/F hydrogen bond forces the C-Cl bond to a favorable orientation in the binding complex. The NH/F hydrogen bond in the (R,R)-18-M pro complex could also contribute to increasing the electrophilic reactivity of the CFA warhead.
The inhibitory activity of (R,R)-18 against other cysteine proteases was evaluated (Fig. 5A). (R,R)-18 potently inhibited SARS-CoV-1 M pro with an IC 50 value of 0.094 mM. This result was not surprising because the structures of M pro are highly conserved between SARS-CoV-1 and SARS-CoV-2 (96% identity of the amino acid sequence). (R,R)-18 did not inhibit SARS-CoV-2 papain-like protease (PL pro ) (<1% inhibition at 20 mM). Human cathepsin B and L were not inhibited by 20 mM of (R,R)-18, whereas the noncovalent inhibitor Jun8-76-3A weakly inhibited cathepsin B (55% inhibition at 20 mM). To assess the covalent binding properties of the CFA inhibitor, we designed a clickable activity-based probe (R,R)-22 (Fig. 5B). Introduction of the ethynyl group was well tolerated, and (R,R)-22 exhibited potent inhibitory activity against SARS-CoV-2 M pro (IC 50 ¼ 0.11 mM) comparable to that of (R,R)-18 (IC 50 ¼ 0.056 mM). In the gelbased in vitro assay, the recombinant SARS-CoV-2 M pro was treated with (R,R)-22 (37 C, 1 h) and conjugated to rhodamineazide by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The in-gel uorescence analysis showed that M pro could be detected at concentrations as low as 10 nM by 2.5 mM of (R,R)-22, and 50 nM of M pro was labeled by (R,R)-22 in a concentration-dependent manner (0.1-10 mM). Incubation of (R,R)-22 with A431 cell lysate spiked with M pro (50 nM) showed the main band at $34 kDa. This band disappeared aer preincubation with the covalent inhibitors PF-00835231 (ref. 16) and (R,R)-18 (5 mM, 30 min), but not with the reversible inhibitor Jun8-76-3A ( Fig. 5C and S9 †). These results demonstrate the ability of the CFA-type inhibitor to covalently modify M pro in the complex human proteome. We also synthesized acrylamide probe 23 as an alkynylated analog of 1 and evaluated its proteome-wide activity under live cell conditions ( Fig. 5D and S10 †). Treatment of A431 cells with (R,R)-22 or 23 (1-10 mM, 37 C, 1 h) and the subsequent in-gel uorescence analysis revealed that acrylamide 23 exhibited a higher level of off-target labeling compared to 22, suggesting a low indiscriminate reactivity of the dipeptidic CFA inhibitor in live cells.

Conclusions
In summary, we have developed a novel irreversible inhibitor of SARS-CoV-2 M pro using CFA as a reactive warhead. The convenient synthesis by Ugi MCR facilitated the rapid SAR study to identify the dipeptidic CFA compound 18 and 19 as potent M pro inhibitors. Careful analysis of the diastereoselective inhibition of 18 and establishment of the synthetic protocol for enantiopure CFA derivatives revealed that (R,R)-18 exclusively shows strong inhibitory activity among the stereoisomers. The detailed reaction kinetics and computational studies suggest that the uorine atom of the CFA warhead could participate in the reaction by forming an NH/F hydrogen bond in the M pro active site that induces the stereoselective activation of (R,R)-18 for covalent modication. This reaction activation represents another possible utility of the CFA warhead, achievable by the unique characteristics of uorine atoms, such as the solid electron-withdrawing ability. 39,40 We believe that the prominent role of the CFA chirality in cysteine conjugation would provide an additional opportunity for the improvement of potency and selectivity of covalent inhibitors. Further application of the CFA chirality to the selective inhibition of other disease-associated proteins is ongoing and will be reported in due course.

Data availability
Additional gures as described in the text, experimental procedures, synthetic procedures, and spectral data of the target compounds are available in the ESI. † Crystallographic data for 21B and 18B have been deposited in the Cambridge Crystallographic Data Centre (CCDC) under accession numbers CCDC 2124399 and 2124400, respectively.

Conflicts of interest
There are no conicts to declare.