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Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity

The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.


■ INTRODUCTION
The ongoing COVID-19 pandemic is a timely reminder that direct-acting antivirals are urgently needed. Despite the expeditious development of mRNA vaccines, SARS-CoV-2 is likely to remain a significant public health concern in the foreseeable future for several reasons. First, variant viruses with escape mutations continue to emerge, which compromise the efficacy of vaccines. 1 Second, a portion of the population opt out of vaccination based on their religious beliefs, concerns of long-term side effects, or other reasons. As such, it is unpredictable when or whether herd immunity can be achieved. Third, the durability of COVID vaccines is currently unknown. Therefore, antivirals are important complements of vaccines to combat both current COVID-19 pandemic and future coronavirus outbreaks.
In combating the COVID-19 pandemic, researchers from different disciplines work relentlessly to discover countermeasures. Drug repurposing led to the identification of remdesivir as the first FDA-approved SARS-CoV-2 antiviral. EIDD-2801, another viral polymerase inhibitor discovered through a similar approach, is in human clinical II/III trials. 2 Among the drug targets exploited, viral polymerases including the main protease (M pro ) and the papain-like protease (PL pro ) are the most extensively studied. 3 The M pro is a cysteine protease and digests the viral polyprotein at more than 11 sites during the viral replication. M pro functions as a dimer and has a unique preference for glutamine at the substrate P1 position. M pro is a validated high-profile antiviral drug target, and M pro inhibitors have demonstrated potent antiviral activity in cell cultures and animal models ( Figure 1). 4−8 Two Pfizer M pro inhibitors PF-07304814 and PF-07321332 are advanced to phase I clinical trial. 9,10 Additional promising leads are listed in Table 1, which are in different stages of translational development. The success of fast-track development of SARS-CoV-2 M pro inhibitors is a result of accumulated expertise and knowledge in targeting SARS-CoV M pro and similar picornavirus 3C-like (3CL) proteases over the years. 11 Despite the tremendous progress in developing M pro inhibitors, the selectivity profiling has thus far been largely neglected. It is essential to address the target selectivity issue early on to avoid catastrophic failures in the later clinical studies. Cysteine protease inhibitor has yet received FDA approval, and the lack of target specificity might be the culprit.
The majority of current reported SARS-CoV-2 M pro inhibitors are peptidomimetic covalent inhibitors with a reactive warhead such as ketone, aldehyde, or ketoamide. 11 Some of the promising examples include the Pfizer compounds PF-07304814 (the parent compound PF-00835231), 10 11a, 12 GC-376, 7,13 the deuterated GC-376 (D2-GC-376), 5 6e, 6j, 14 MI-09, MI-30, 4 and MPI8 15 (Figure 1). Although the high reactivity of these reactive warheads, especially the aldehyde, confers potent activities in the enzymatic assay and antiviral assay, it inevitably leads to off-target side effects through reaction with some host proteins. 16−19 For example, we and others have shown that GC-376 is a potent inhibitor of cathepsin L (Table 1). 17,20 A recent study revealed that MP18, an analogue of GC-376 with an aldehyde warhead, inhibits cathepsins B, L, and K with IC 50 values of 1.2, 230, and 180 nM, respectively. 15 The off-target effect is also a potential concern for some of the most advanced M pro inhibitors including the clinical candidate PF-07304814, 21 compounds 6j and 6e which showed in vivo antiviral efficacy against MERS-CoV-2 infection in mice, and compound 11a with potent in vitro antiviral activity (Table 1). 22 All of these compounds are potent inhibitors of cathepsin L. The high reactivity of the aldehyde warhead might confer the lack of target specificity, and the design of covalent inhibitors with a high target specificity remains a daunting task.
We report herein the rational design of covalent M pro inhibitors with novel cysteine reactive warheads and high target specificity. Specifically, guided by the X-ray crystal structure of SARS-CoV-2 M pro with 23R (Jun8-76-3A) (PDB code 7KX5), which was one of the most potent noncovalent M pro inhibitors developed from our earlier study, 23 we systematically explored a number of novel electrophiles in the replacement of the P1′ furyl substitution in 23R. The aim is to identify C145 reactive electrophiles with both potent M pro inhibition and high target selectivity. This effort led to the discovery of several novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. One of the most potent lead compounds Jun9-62-2R (dichloroacetamide) inhibited SARS-CoV-2 M pro with an IC 50 of 0.43 μM and viral replication with an EC 50 of 2.05 μM in Caco2-hACE2 cells. Significantly, unlike GC-376, Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) are highly selective toward M pro and do not inhibit the host calpain I, cathepsins B, K, L, caspase-3, and trypsin. X-ray crystal structure of SARS-CoV-2 M pro with Jun9-62-2R (dichloroacetamide) and Jun9-57-3R (chloroacetamide) revealed that the C145 forms a covalent adduct with the reactive warheads. Overall, the discovery of these di-and trihaloacetamides as novel cysteine reactive warheads sheds light on the feasibility of developing SARS-CoV-2 M pro inhibitors with high target specificity over tested calpain and cathepsins and a high cellular selectivity index. These novel compounds represent  ■ RESULTS AND DISCUSSION Synthesis of Covalent M pro Inhibitors. The covalent M pro inhibitors were synthesized by the one-pot Ugi fourcomponent reaction (Ugi-4CR) as shown for Jun9-62-2 ( Figure 2) with yields from 33% to 88%. For compounds with potent enzymatic inhibition, the diastereomers were subsequently separated by chiral HPLC. The absolute stereochemistry of Jun9-57-3R and Jun9-62-2R was determined by X-ray crystallography, and the stereochemistry for the diastereomers of Jun9-90-4, Jun9-89-2, Jun9-89-4, and Jun9-88-6 was tentatively assigned based on their relevant retention times in chiral HPLC.
Rational Design of Covalent M pro Inhibitors. 23R was designed based on the superimposed X-ray crystal structure of GC-376 with ML188 and UAWJ254. 23,24 The X-ray crystal structure showed that the furyl substitution at the P1′ position of 23R is in close proximity with the catalytic cysteine 145 (3.4 Å between C145 sulfur and the C-2 carbon of furyl, PDB code 7KX5) ( Figure 3A), suggesting that replacement of furyl with a reactive warhead might lead to covalent inhibitors ( Figure 3B). 23R is an ideal lead candidate for the design of covalent M pro inhibitors for several reasons: (1) the P1, P2, and P3 substitutions have already been optimized; (2) the designed compounds can be expeditiously synthesized by the one-pot Ugi-4CR; (3) a diversity of cysteine reactive warheads are commercially available and can be promptly introduced at the P1′ position to react with the C145.
Although a number of thiol-reactive warheads have been exploited in the development of covalent protease and kinase inhibitors, 25−27 we decided to focus on pharmacologically compliant reactive warheads from the FDA-approved drugs. The majority of FDA-approved thiol-reactive drugs are kinase inhibitors including ibrutinib, osimertinib, zanubrutinib, acalabrutinib, dacomitinib, neratinib, and afatinib ( Figure  3C). 25 As such, acrylamide and 2-butynamide were chosen as reactive warheads in our initial design of covalent SARS-CoV-2 M pro inhibitors ( Figure 3B). Chloroacetamide was also chosen as it was previously explored by Pfizer for the development of SARS-CoV and SARS-CoV-2 M pro inhibitors (Pfizer compound 12) ( Figure 3C). 21 Chloroacetamide is frequently used as a reactive warhead for designing chemical probes for target pull down. 28 Finally, we included azidomethylene as it was previously shown to be a relatively unreactive cysteine warhed. 29,30 The fluoroacetamide was included as a control.
For the haloacetamide series of compounds, the reference compound Jun9-54-1 with the classical chloroacetamide reactive warhead had potent inhibition against SARS-CoV-2 M pro with an IC 50 of 0.17 μM. However, it was cytotoxic in both Vero E6 cells and Calu-3 cells with CC 50 values less than 3.5 μM. To increase the cellular selectivity index, we reasoned that substituted chloroacetamides or haloacetamides might have reduced cellular cytotoxicity while maintaining potent M pro inhibition. It was found that Jun9-77-1 with the 2chloropropanamide warhead was not active (IC 50 > 20 μM). Encouragingly, compound Jun9-62-2R with the dichloroacetamide warhead had potent inhibition against M pro with an IC 50 of 0.43 μM while being noncytotoxic to Vero E6 cells (CC 50 > 100 μM). In comparison, the corresponding diastereomer Jun9-62-2S was not active (IC 50 > 20 μM), which is consistent with the predicted binding mode ( Figure  3A). Given these promising results, we further designed two     Journal of the American Chemical Society pubs.acs.org/JACS Article As the tribromoacetamide is sterically hindered, the mechanism of action of Jun9-88-6R might involve the nucleophilic attack of the carbonyl by the C145 thiol to give a thiohemiketal intermediate, followed by a 1,2-shift of the sulfur to displace one bromide ( Figure S2). To provide additional lines of evidence to support the proposed mode of action of covalent binding, we performed three additional experiments. First, to demonstrate the reversibility of the binding of Jun9-62-2R to M pro , we incubated 10 μM SARS-CoV-2 M pro with 10 μM Jun9-62-2R for 2 h and monitored the enzymatic activity of M pro following 100-fold dilution of the mixture. It was found that no enzymatic activity was recovered ( Figure 5C). In contrast, the mixture with our previously developed noncovalent inhibitor 23R showed nearly complete recovery of enzymatic activity after dilution ( Figure 5C). These results suggest that the binding of Jun9-62-2R is irreversible while the binding of 23R is reversible. Second, we repeated the FRET assay of Jun9-62-2R with different preincubation times and found that longer preincubation time gave lower IC 50 values ( Figure 5D). These data are consistent with the mode of action of covalent inhibitors. 31 In contrast, preincubation of M pro with the noncovalent inhibitor 23R did not lead to significant changes of the IC 50 value ( Figure 5D). Third, we used native mass spectrometry to detect the covalent adducts of M pro with Jun9-62-2R, Jun9-89-2R, Jun9-88-6R, and Jun9-89-4R. The expected mass shifts of 482 Da and 526 Da were observed for Jun9-62-2R and Jun9-89-2R, respectively ( Figure 5E,F). Interesting, the expected dibromoacetamide conjugate was not observed for Jun9-88-6R, suggesting this conjugate might not be stable. Instead, the mass shift corresponding to the monobromo thiol adduct was observed ( Figure 5G). For Jun9-89-4R, the mass shifts for both the chlorobromo and chloro thiol adducts were observed ( Figure 5H).
To further profile the cellular M pro inhibition, we tested these four compounds in our recently developed FlipGFP assay. 18,32 Briefly, the GFP is split into two parts, the β1−9 template and the β10−11 strands. The β10 and β11 strands were engineered with K5-E5 linker such that they are restrained in the parallel form. When the linker is cleaved by M pro , β10 and β11 adopt antiparallel conformation, which allows association with the β1−9 template, leading to the recovery of the GFP signal. In the FlipGFP assay, the GFP signal is proportional to the M pro enzymatic activity. It was found that all four compounds led to dose-dependent inhibition of the GFP signal with EC 50 values of 0.96 μM  Figure 6C).
Profiling the Target Selectivity against Host Proteases. Lack of target specificity is one of the major reasons that many cysteine protease inhibitors failed in the clinical trials. To profile the target specificity of these SARS-CoV-2 M pro inhibitors with a novel reactive warhead, we selected Jun9-62-2R and Jun9-88-6R as representative examples and included the canonical GC-376 with an aldehyde reactive warhead for comparison. The results showed that GC-376 had potent inhibition of the host proteases including calpain I,  Table S1). Jun9-57-3R and Jun9-62-2R have nearly identical chemical features to their noncovalent progenitor 23R (Jun8-76-3A) (PDB code 7KX5). As such, the binding poses are very similar. The pyridyl ring binds to the S1 pocket of M pro , where it forms a hydrogen bond with His163. This hydrogen bond is critical for coordinating the Gln side chain of its substrate, a residue it is uniquely selective for. Consequently, a hydrogen bond acceptor at this position confers tremendous potency to M pro inhibitors. The phenylpyrrole (Jun9-57-3R) or biphenyl (Jun9-62-2R) moieties insert into the hydrophobic S2 pocket where they form nonpolar contacts and stack with the catalytic base, His41. An amide group linking the pyridyl ring to an αmethylbenzene group accepts a hydrogen bond from the main chain of Glu166. This α-methylbenzene group flips down toward the core of the substrate channel, where it forms additional π-stacking interactions with the biphenyl or phenylpyrrole moieties. The key distinction between Jun9-62-2R, Jun9-57-3R, and analogues Jun8-76-3A and ML188 is the presence of an electrophilic chloroacetamide warhead, which forms a covalent adduct with the catalytic cysteine Cys145 ( Figure 8C,D). The short distance of this covalent bond (1.8 Å) allows the inhibitor to press further into the

■ CONCLUSION
The majority of the reported M pro inhibitors contain the aldehyde reactive warhead, which is known to have nonspecific reactivity toward host proteins. 16−19 It should be noted that both of the Pfizer M pro inhibitors that are currently in clinical trials do not contain the aldehyde warhead. 9,10 As such, we are interested in developing SARS-CoV-2 M pro inhibitors with high target specificity. A highly specific M pro inhibitor is also needed for target validation as it separates the effect of M pro inhibition from host protease inhibition such as cathepsin L. It is known that host cathepsin L is important in SARS-CoV-2 replication in Vero E6 cells, which are TMPRSS2-negative, but not in Calu-3 cells, which are TMPRSS2-positive. 36 In this study, we report the discovery of dichloroacetamide, dibromoacetamide, 2-bromo-2,2-dichloroacetamide, 2-chloro-2,2-dibromoacetamide, and tribromoacetamide as novel cysteine reactive warheads. To the best of our knowledge, these warheads have not been explored in cysteine protease inhibitors. The most promising lead compounds Jun9-62-2R with the dichloroacetamide warhead and Jun9-88-6R with the tribromoacetamide inhibited SARS-CoV-2 M pro with IC 50 values of 0.43 μM and 0.08 μM, respectively. These two compounds also showed potent inhibition against SARS-CoV-2 in both Vero E6 and Caco2-hACE2 cells with EC 50 values in the single-digit micromolar to submicromolar range. Significantly, both Jun9-62-2R and Jun9-88-6R had high target specificity toward M pro and did not inhibit the host proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, caspase-3, and trypsin. In comparison, GC-376 was not selective and inhibited calpain I, cathepsin B, cathepsin K, and cathepsin L with comparable potency as M pro . Regarding the translational potential of the di-and trihaloacetamidecontaining M pro inhibitors, the widely used antibiotic chloramphenicol contains the dichloroacetamide, suggesting Jun9-62-2R might be tolerated in vivo. Follow-up studies will optimize the in vitro and in vivo pharmacokinetic properties and in vivo antiviral efficacy of these novel compounds in SARS-CoV-2 infection animal models. Other potential strategies include developing selective M pro inhibitors including allosteric inhibitors 37,38 or targeting the more reactive Cys44 at the S2 binding pocket. 39,40 Overall, these novel compounds represent valuable chemical probes for target validation and promising drug candidates for translational development as SARS-CoV-2 antivirals.
■ ASSOCIATED CONTENT
Additional figures and tables describing the experimental materials, methods, X-ray data set, synthesis, and characterization of the M pro inhibitors (PDF)

Notes
The authors declare the following competing financial interest(s): Jun Wang is an inventor of a filed patent claiming the use of Jun9-62-2R and related compounds as potential COVID-19 antiviral drugs.