Exploration of P1 and P4 Modifications of Nirmatrelvir: Design, Synthesis, Biological Evaluation, and X-ray Structural Studies of SARS-CoV-2 Mpro Inhibitors

Abstract

We report the Synthesis, biological evaluation, and X-ray structural studies of a series of SARS-CoV-2 Mpro inhibitors based upon the X-ray structure of nirmatrelvir, an FDA approved drug that targets the main protease of SARS-CoV-2. The studies involved examination of various P4 moieties, P1 five- and six-membered lactam rings to improve potency. In particular, the six-membered P1 lactam ring analogs exhibited high SARS-CoV-2 Mpro inhibitory activity. Several compounds effectively blocked SARSCoV-2 replication in VeroE6 cells. One of these compounds maintained good antiviral activity against variants of concern including Delta and Omicron variants. A high-resolution X-ray crystal structure of an inhibitor bound to SARS-CoV-2 Mpro was determined to gain insight into the ligand-binding properties in the Mpro active site.

Graphical Abstract

1. Introduction

The global COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has lasted for over three years, resulting in worldwide crisis encompassing both health and economics.^1,2 With nearly 7 million global deaths and 769 million cases reported to date, the outbreak is far from over. SARS-CoV-2 continues to cause a large number of severe illnesses, hospitalizations, and deaths worldwide.^3,4,5 The development of COVID-19 vaccines and the large-scale vaccination efforts resulted in much needed relief from the early devastating situation.^6,7 However, achieving herd immunity using vaccines has remained quite uncertain.^8,9 Also, the emergence of several deadly variants caused many concerns regarding the possible return of the pandemic.^10,11 Therefore, the continued development of effective antivirals is critically important to prevent COVID-related ailments, hospitalization and to prepare for potential drug-resistant SARS-CoV-2.^12,13 Recently, the first orally active SARS-CoV-2 main protease (Mpro) inhibitor drug nirmatrelvir (1, Figure 1) was approved in combination with ritonavir (2), a CYP3A4 inhibitor to boost pharmacokinetic properties.^14,15 Molnupiravir (3), a ribonucleoside derivative that blocks RNA-dependent RNA polymerase (RdRp) activity has also been approved recently.^16,17 Both drugs have been shown to reduce the risk of hospitalization and deaths for COVID-related illnesses.^18,19

Figure 1.

Structure of nirmatrelvir 1, showing P1-P4 moieties and structures of compounds 2-4.

SARS-CoV-2 is an enveloped single-stranded RNA virus whose genome sequencing revealed nearly 82% genome similarities to previous SARS-CoV-1.^20,21 Also, SARS-CoV-2 shows 90% resemblance to SARS-CoV-1 in essential enzymes, including Mpro or chymotrypsin-like protease (3CLpro).^22,23 Both Mpro and PLpro are cysteine protease, and they process the CoV replicase polyproteins PP1a and PP1b into non-structural proteins essential for viral replication and transcription of the genome. The homodimeric enzyme main protease is highly conserved. It contains a Cys-His catalytic dyad in its active site where the nucleophilic thiolate Cys145 has been exploited in inhibitor design by forming a covalent bond with a range of electrophilic functionalities.^24,25 The past and present research efforts focus on the development of covalent inhibitors with electrophilic warhead groups such as: α-ketoamides, aldehydes, esters, α-hydroxymethylketones and benzothiazoles.^26,27 Nirmatrelvir is the first orally active main protease inhibitor drug which is a peptidomimetic derivative with a nitrile functionality as the warhead.^14,15 The other key features of nirmatrelvir include, a trifluoro acetamide group, two peptide bonds, and a gem-dimethyl cyclopropyl proline derivative which is inherent to Hepatitis C virus protease inhibitor, boceprevir. Incidentally, nirmatrelvir has been designed and developed based upon X-ray structural studies of boceprevir-bound main protease and other structural leads.^28,29 Nirmatrelvir was reported to inhibit SARS-CoV-2 Mpro with an inhibitory Ki value of 3.1 nM and antiviral EC₅₀ of 74.5 nM in VeroE6 cell lines when co-treated with the P-glycoprotein inhibitor CP-100356 to prevent drug efflux.^14,15 Nirmatrelvir is clinically used as a combination therapy (named Paxlovid) with ritonavir, a well-known pharmacokinetic booster which inhibits CYP3A4 enzyme to slow down metabolic degradation.^30,31 Paxlovid has become the drug of choice for treatment of SARS-CoV-2 infection and COVID-19, but it is far from ideal as it presents significant risks of drug -drug interactions and viral rebound-effect.^32,33 Therefore, it is very important to develop better antiviral drugs with improved drug properties. As part of our continuing studies for the development of potent and effective coronavirus protease inhibitor, we have investigated the effect of various P4-substituents of nirmatrelvir in combination with 5-membered and 6-membered P1 lactam ring to improve Mpro inhibitory and antiviral activity of the corresponding derivatives. Herein, we report the results of studies which show that several derivatives with a larger 6-membered P1 lactam ring exhibited very potent SARS-CoV-2 Mpro inhibitory activity as well as potent low nanomolar antiviral activity. To obtain insight into the ligand-binding site interactions, we have determined high resolution X-ray crystal structure of the inhibitor-bound to the main protease of SARS-CoV-2.

2. Results and discussion

We and others have determined high resolution X-ray structure crystal of SARS-CoV-2 Mpro and nirmatrelvir complex.^14,34 The key interactions between nirmatrelvir and the Mpro active site are highlighted in Figure 2. The warhead P1’ nitrile functionality forms a covalent bond with the active site Cys145, resulting in the formation of a thioimidate adduct in the S1 and S1’ subsites. The P1 5-membered lactam NH forms a hydrogen bond with Glu166 side chain carboxylic acid. The lactam carbonyl oxygen also forms a hydrogen bond with the His163 located in the S1 subsite. The P2 bicycloproline and P3 tert-leucine form a dipeptide bond in nirmatrelvir. The bicycloproline nests in the hydrophobic pocket surrounding His41, Met49, Tyr54, Met165, Asp187, Arg188, and Gln189, the P3 tert-butyl group is mostly solvent exposed. The P3 amide carbonyl oxygen forms a hydrogen bond with backbone NH of Glu166 while the P4 trifluoro acetamide carbonyl oxygen forms a hydrogen bond with Glu166 amide in S4 subsite. Thus, the formation of a covalent bond with Cys145 and extensive ligand binding site interactions are responsible for nirmatrelvir inhibitory activity. ^14,34 Based upon this structure analysis, we speculated that a six-membered P1-lactam could fill in the hydrophobic pocket in the S1 subsite more effectively while maintaining the key hydrogen bonding interactions with His163 and Glu166. We have further speculated that other non-polar P4 groups may fill in the S4 subsite effectively and improve potency. For these studies, we synthesized and evaluated both 5 and 6 membered lactams in combination with various P4 groups and compared in vitro activity against nirmatrelvir.

Figure 2.

X-ray crystal structure of Nirmatrelvir (orange) bound to SARS-CoV-2 Main protease (grey) (PDB 7RFW).

Our synthetic strategy for the preparation of various P4 and P1 modified derivatives of nirmatrelvir is shown in Scheme 1. We planned to couple carboxylic acid derived from methyl ester 6a-f with amino nitrile derivative derived from lactam derivatives 7 and 8. The synthesis of lactam derivative 7 was achieved using the recent protocol developed by us.³⁵ The synthesis of 6-membered lactam derivative 8 was carried out using commercially available N-Boc-L-(+)-glutamic acid dimethyl ester 9 as shown in Scheme 2. Treatment of diester 9 with 2.2 equivalents of LiHMDS at −78°C provided the corresponding enolate which was alkylated with bromopropionitrile to furnish alkylated product 10 in 40% yield as a single diastereomer.^36,37 The high degree of diastereoselectivity and stereochemical outcome was previously reported by Hanessian and co-workers.³⁸ Selective reduction of nitrile group with a sodium borohydride-cobaltous chloride complex provided the corresponding amine which cyclized to 6-membered lactam derivative 11 in 68% yield. Methyl ester 11 was then converted to nitrile derivative 8 in a two-step sequence. Reaction of 11 with ammonium hydroxide in MeOH at 23 °C for 12 h provided the corresponding carboxamide derivative. The resulting carboxamide was treated with tosyl chloride and pyridine at 23°C for 18 h to furnish the nitrile derivative 8 in 58% yield over 2-steps.

Scheme 1.

Synthetic strategy for inhibitors 5a-i

Scheme 2.

Asymmetric synthesis of nitrile derivative 8.

The synthesis of P4 carboxamide derivatives in combination with the P1 5- and 6-membered lactam rings are achieved with minimum racemization of the tert-leucine side chain utilizing the synthetic route developed by us recently for the synthesis of nirmatrelvir.³⁵ As shown in Scheme 3, dipeptide methyl ester 6a was prepared in multigram quantity by coupling commercially available optically active bicyclic proline methyl ester 12 with Boc-tert-leucine 13a as described previously.¹⁵ Treatment of Boc-derivative 6a with 10% ZnCl₂ and acetic anhydride in CH₂Cl₂ at 23 °C for 12 h provided the acetamide derivative 14a in 81% yield with minimum racemization (< 5%). Similarly, reaction of 6a with ZnCl₂ and benzoic anhydride, pivalic anhydride, trifluoroacetic anhydride, and trichloroacetic anhydride furnished the respective amide derivatives 14b-e in good to excellent yields (74–96%). Saponification of these methyl esters with LiOH in THF/H₂O at 23 °C for 1 h yielded (61–95%) the corresponding acids 15a-e. The amine coupling partners were prepared from the corresponding aminonitrile derivatives 7 and 8 via Boc-deprotection using trifluoroacetic acid (TFA). The resulting amines were coupled with acids 15a-e using HATU and NMM in DMF at 23°C for 12 h furnished inhibitors 5a-f in moderate to good yields (21–64%).

Scheme 3.

Synthesis of inhibitors 5a-f.

Synthesis of various P4-carbamate derivatives and bis-trifluoroacetamide derivatives is shown in Scheme 4. Coupling of bicycloproline derivative 12 with P4-substituted tert-leucine derivatives 13a-f provided the carbamate derivatives 6a-f in good yields. Saponification of 6a with aqueous LiOH at 23 °C for 1 h followed by coupling of the resulting acid with amine derived from nitrile 7 furnished Boc-derivative 5g in 62% yield. We attempted to convert Boc-derivative 5g to nirmatrelvir using our previously described procedure with ZnCl₂ and trifluoroacetic anhydride.³⁵ Interestingly, reaction of 5g with 10% ZnCl₂ in the presence of trifluoracetic anhydride at 23 °C for 12 h did not provide nirmatrelvir 1, instead bis-amide derivative 5h was obtained in 85% yield. Carbamate derivatives 6b-f were converted to various P1 5-membered lactam analogs 5i-m using a two-steps sequence involving saponification of esters 6b-f followed by coupling of the resulting acids with amine derivative from 7 after removal of Boc-group with trifluoroacetic acid. Carbamate derivatives 5g-m were obtained in good yields. Similarly, 3-pyridyl and 4-pyridyl carbamates 6d and 6e were converted to compounds 5n and 5o, respectively by saponification of 6d and 6e followed by coupling of the resulting acids with amine derived from 8 after treatment with TFA as described above. Both carbamates 5n and 5o were obtained in 34% yield.

Scheme 4.

Synthesis of inhibitors 5g-o.

We have prepared a series of P4-N-amide derivatives to assess stereoelectronic effect and hydrogen bonding capabilities of the amide NH group in the S4 subsite. The structure and biological activity of these inhibitors are shown in Table 1. We utilized our previously reported assay protocols for SARS-CoV-2 Mpro inhibitory activity and antiviral activity in veroE6 cells.^{39, 40} As shown in Table 1, in-house prepared nirmatrelvir 1, exhibited a SARS-CoV-2 Mpro inhibitory activity of 0.26 nM. It showed an antiviral EC₅₀ value of 2.0 μM. Remdesivir displayed antiviral EC₅₀ of 3.2 μM in the same assay. In comparison nirmatrelvir was reported to have an average Ki value of 3.1 nM and antiviral activity of 74.5 nM in veroE6 cells.¹⁴ We examined the effect N-acetamide group in compound 5a in place of the N-trifluoroacetamide group in 1. Compound 5a exhibited comparable Mpro inhibitory activity to 1 (entry 2). The incorporation of N-benzamide group, 5b, also resulted in substantial loss of both Mpro inhibitory and antiviral activity (entry 3). Replacement of the trifluoroacetamide with the bulky pivalamide group in compound 5c caused nearly 10-fold loss of Mpro inhibitory activity. However, compound 5c exhibited comparable antiviral activity, EC₅₀ 2.3 μM vs 2.0 μM for 1 (entries 1 and 4). We next examined the effect of increasing the size of the halogen from N-trifluoro to N-trichloro group in compound 5d. This replacement resulted in significant improvement of both Mpro inhibitory and antiviral activity. Compound 5d displayed very similar in vitro activity as nirmatrelvir 1 (entries 1 and 5). We then investigated the effect of a 6-membered lactam at P1 position as it evident from the X-ray crystal structure of SARS-CoV-2 Mpro bond nirmatrelvir that a larger ring can be accommodated in the S1-subsite.

Table 1.

SARS-CoV-2 3CLpro inhibition by nirmatrelvir derivatives 5a-h.

Entry	Inhibitor	Ki (nM)a	EC50 (μM)b
1		0.26	2.0
2.		0.44	4.0
3		36.9	17
4		22.1	2.3
5		0.2	1.6
6		0.04	0.26
7		1.1	0.27
8.		32,300	100

^aK_i values were determined from fitting inhibition data to the Morrison equation as described.³⁹

^bEC₅₀ values are means of at least four experiments.

Indeed, compound 5e containing a P4 6-membered lactam led to more than 6-fold improvement of Mpro inhibitory activity as well as 7-fold improvement in antiviral activity over nirmatrelvir 1 (entry 6). Incorporation of P4 trichloromethyl group in place of trifluoromethyl provided inhibitor 5f which displayed very potent Mpro inhibitory activity (Ki 1.1 nM) and over 7-fold improvement in antiviral activity over nirmatrelvir 1 (entry 7). We have also evaluated P2 trifluoroacetamide derivative 5h. However, this compound showed no appreciable Mpro inhibitory or antiviral activity. This result suggests the important role of the P2 NH group for binding of nirmatrelvir in the Mpro active site.

We have also investigated the feasibility of various P4 carbamate and urea derivatives that can impact steric and electronic effects. The X-ray crystal structure of nirmatrelvir-bound to SARS-CoV-2 Mpro suggests that the P4 carbamate NH group can effectively form a hydrogen bond with the Glu166 backbone carbonyl group. Also, a suitable alkyl or aryl group could fill the S4 subsite. The biological evaluation results are shown in Table 2. The P4 tert-butyloxycarbonyl group containing inhibitor 5g showed a significant loss in Mpro inhibitory and antiviral activity as compared to nirmatrelvir 1 (entry 1). Incorporation of a benzyloxy carbonyl group in compound 5i resulted in significant improvement of Mpro inhibitory activity compared to the Boc-group containing derivative 5g (entry 2). Compound 5i also exhibited very potent antiviral activity with EC₅₀ value of 290 nM. The effect of pyridyl methyl group over the benzyl group was then evaluated in compounds 5j-l. Interestingly, the 3-pyridyl methyl carbamate 5k displayed significantly enhanced activity over the 4-pyridylmethyl carbamate 5j and 2-pyridylmethyl carabamate 5l (entries 3–5). Carbamate 5k also exhibited very potent antiviral activity with an EC₅₀ value of 35 nM, which is over an 8-fold improvement of antiviral activity compared to benzyl carbamate derivative 5i. The current structure-activity relationship (SAR) indicates that the benzyloxy group fills the S4 subsite more effectively than the tert-butyloxy group in 5g. Also, the position of nitrogen atom in the pyridyl ring is very important. However, the actual contribution of the 3-pyridyl group in compound 5k in the Mpro active site is not clear. Further expansion of SAR and X-ray structural studies are the subject of current investigation. Incorporation of tert-butyl urea at P4 resulted in compound 5m which exhibited potent Mpro inhibitory activity. However, this compound exhibited 3-fold loss of antiviral activity compared to the corresponding Boc-derivative 5g (entries 1 and 6). We have examined the effect of P4 3-and 4-pyridyl carbamate in combination with 6-membered lactam. Compounds 5n with 3-pyridylmethyl carbamate showed comparable Mpro inhibitiory activity over compound 5k with 5-membered lactam. However, compound 5n displayed significant loss of antiviral activity over 5k (entries 4 and 7). Carbamate derivative 5o containing 4-pyridylmethyl carbamate showed 4-fold improvement of Mpro inhibitory activity but exhibited comparable antiviral activity (entries 3 and 8). Overall, benzyl and 3-pyridylmethyl carbamates 5i, 5k, and amide derivatives 5e, 5f exhibited very potent antiviral activity comparable to nirmatrelvir. We have also examined and compared anti-SARS-CoV-2 activity of compound 5f against variants of concern and the result of this investigation is shown in Table 3.⁴¹ Interestingly, compound 5f maintained excellent antiviral activity against Alpha and Delta SARS-CoV-2 variants. However, compound 5f lost 3–5-fold antiviral activity against beta, gamma and omicron variants. Remdesivir and nirmatrelvir displayed comparable antiviral activity.

Table 2.

SARS-CoV-2 3CLpro inhibition by nirmatrelvir derivatives 5g-o.

Entry	Inhibitor	Ki (nM)a	EC50 (μM)b
1		7.7	30.7
2		0.025	0.29
3.		32.2	29.7
4.		0.09	0.035
5.		0.49	40.1
6.		3.3	100
7.		0.094	3.4
8.		7.8	27.1

^aK_i values were determined from fitting inhibition data to the Morrison equation as described.³⁹

^bEC₅₀ values are means of at least four experiments.

Table 3.

Anti-SARS-CoV-2 activity of compound 5f against VOCs (variants of concern)

EC50(μM)
Compound	SARS-CoV-205−2N (Wuhan)	SARS-CoV-2QK2 (Alpha)	SARS-CoV-2TY8 (Beta)	SARS-CoV-2TY7 (Gamma)	SARS-CoV-2K1734 (Delta)	SARS-CoV-2929 (Omicron)
Compound 5f (GRL-018–22)	0.7 ± 0.1	0.8 ± 0.2	2.40 ± 0.09	2.0 ± 0.1	0.6 ± 0.2	4.0 ± 1.2
Remdesivir	3.8 ± 1.9	3.5 ± 0.1	2.8 ± 0.1	2.6 ± 0.1	2.9 ± 0.1	4.2 ± 0.5
Nirmatrelvir	3.0 ± 0.1	2.5 ± 0.1	3.7 ± 0.2	4.0 ± 0.7	2.9 ± 0.2	3.1 ± 0.2

EC₅₀ values were determined by WST-8 assays, using VeroE6 cells. Data was collected on day-3 post infection (day-7, Omicron).

We also evaluated compound 5e in our immunocytochemistry assay to assess antiviral activity and comapre with the activity of nirmatrelvir at the cellular level. We utilized primary antibody IgG fraction isolated from a COVID-19 convalescent patient in this assay. These patients possess high titers of neutralizing antibodies as well as SARS-CoV-2-binding IgG antibodies.^40,42 As can be seen in Figure 3, robust cellular cytoskeleton filamentors actin (F-actin) were observed as mesh-like structures (in red) and a lot of nuclei (in blue) were formed which suggest that cells were healthy and replicating. As shown, the exposure of VeroE₆ cells to SARS-CoV-2^WK−521 resulted in the destruction of the F-actin structure and viral infection of the cells (stained green, top right panel). However, when VeroE6 cells are exposed to SARS-CoV-2^WK−521 in the presence of 100 μM and 10 μM concentration of compound 5e and nirmatrelvir, there were hardly any infected cells. While some infected cells were observed in the presence of 1 μM compound 5e, nirmatrelvir exposed culture at 1 μM showed complete viral breakthrough (infected cells, stained green). Compound 5e exerted potent Mpro inhibitory activity (Ki 40 pM) and antiviral activity (EC₅₀ 0.26 μM). Also, it significantly reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity.

Figure 3.

Inhibitor 5e and nirmatrelvir potently blocked the infectivity and cytopathic effect of SARS-CoV-2^WK−521 in VeroE6^TMPRSS2 cells.

To better understand the molecular interactions of the 6-membered lactam of inhibitor 5e with the SARS-CoV-2 Mpro active site, we determined the X-ray structure of Mpro in complex with 5e to 1.9 Å resolution.⁴³ Our X-ray crystallography data clearly shows strong and continuous electron density of compound 5e inside the Mpro active site as shown in Figure 4. The overall structure is very similar to that of the nirmatrelvir SARS-CoV-2 Mpro complex.¹⁴ The key interactions between inhibitor 5e and Mpro active site residues are highlighted in Figure 5. Compound 5e occupies the substrate binding pockets from S1 to S4. It forms seven direct hydrogen bonds with various Mpro residues. In particular, compound 5e forms two hydrogen bonds with the backbone carbonyl and amide NH of Glu166 involving the P4 amide NH and P3 amide carbonyl group respectively. The backbone carbonyl of His164 also forms a hydrogen bond with the P2 amide NH of inhibitor 5e. The six membered lactam NH forms two hydrogen bonds with Glu166 side chain carboxylate as well as the backbone carbonyl of Phe140. The lactam carbonyl group accepts a hydrogen bond from the His163 side chain imidazole ε2-nitrogen assuming it is protonated. The active site Cys145 sulfur atom performs a nucleophilic attack on the nitrile functionality of 5e forming a covalent bond, and the resulting imidate forms a hydrogen bonds with the backbone NH of Cys145. Apart from hydrogen bonding interactions, compound 5e fills the hydrophobic pockets particularly the S1-S3 subsites of Mpro active site and is involved in van der Waals interactions.

Figure 4.

X-ray crystal structure of SARS-CoV-2 3CLpro covalently bound to compound 5e through Cys145 (PDB:8UND). (Left Panel) Polder electron density omit map (mF_obs − DF_model ) surrounding 5e contoured at (+) 4.0 σ and represented as a solid surface colored in Khaki. The enzyme 3CLpro is depicted as a gray surface.

Figure 5.

X-ray structure of inhibitor 5e-bound to SARS-CoV-2 Mpro. The inhibitor carbon atoms are shown in orange and the Mpro carbon atoms are shown in grey. Hydrogen bond interactions between 5e and Mpro are indicated by dotted lines (PDB ID:8UND).

The main difference between nirmatrelvir and inhibitor 5e is the presence of a six-membered lactam in place of the 5-membered lactam in 1. We therefore compared both hydrogen bonding distances and van der Waals interactions of the 5-membered lactam of nirmatrelvir and the six-membered lactam of inhibitor 5e in the Mpro active site. This structural analysis is shown in Figure 6. Interestingly, the P1 lactam carbonyl and NH groups of inhibitors 5e form very strong hydrogen bonds with the His163 side chain, Glu166 carboxylic acid chain, and Phe140 backbone NH, (2.7 Å, 2.7 Å and 3.2 Å respectively, Figure 5A) compared to 5-membered lactam in 1 (2.8 Å. 3.2 Å and 3.2 Å respectively, Figure 5B). The lactam rings in both compounds 1 and 5e engage in van der Waals interaction with Asn142, Phe140 and Gly141. The extra methylene group in the lactam ring of 5e makes stronger van der Waals interaction with these residues in the S1 subsite. Both enhanced hydrogen bonding and van der Waals interactions of six membered lactam in compound 5e contributed to the high Mpro affinity and antiviral activity compared to inhibitor 1.

Figure 6.

Comparison of the P1 six-membered lactam moiety of inhibitor 5e (5A-top, orange carbon atoms) with the P1 5-membered lactam moiety of nirmatrelvir (5B-bottom, cyan carbon chain) inside the S1 subsite. Both ligands form extensive van der Waals interactions in the S1 subsite. Also, both inhibitors form strong hydrogen bonds in a similar fashion (black dotted lines).

3. Conclusions

In summary, we have modified P1 and P4 ligands of nirmatrelvir, an FDA approved drug for COVID-19 treatment. Our investigation has been aimed at improving inhibitor activity by modification of inhbitor interactions within the S1 subsite and filling in the hydrophobic pocket in the S4 subsite. In particular, we examined various alkyl, aryl, halogenated acetamide, carbamate and urea derivatives as the P4 ligands in combination with 5-membered and 6-membered lactams as the P1 ligands. The P4 amide derivatives are significantly more potent than carbamate and urea derivatives examined in this study. Several inhibitors with a larger 6-membered lactam ring with halogenated P4 amides showed significant enhancement of inhibitory and antiviral activity over nirmatrelvir. Compound 5e with a P1 6-membered lactam and P4 trifluoro acetamide groups exhibited 3-fold improvement in antiviral activity in VeroE6 cells. This compound maintained very good activity against variants concerns such as Delta and Omicron variants, maintaining similar antiviral activity as remdesivir. To obtain insight into the ligand-binding site interactions for the 6-membered lactam in the S1 subsite, we determined the high resolution X-ray crystal structure of inhibitor 5e-bound to SARS-CoV-2 Mpro. The structural analysis showed that the 6-membered lactam ring functionality forms stronger hydrogen bonding compared to the 5-membered lactam in nirmatrelvir in the S1 subsite. The 6-membered lactam ring also fills the hydrophobic pocket more effectively than nirmatrelvir’s 5-membered lactam ring. Our investigation revealed the potency enhancing effect of the P1 6-membered lactam in this series of compounds. The current result will further aid the optimization of nirmatrelvir derivatives and related compounds for Mpro inhibition. Design and evaluation of novel Mpro inhibitors are in progress in our laboratories.

4. Experimental Section

All chemicals and solvents were purchased from commercial suppliers and were used as received unless otherwise stated. All reactions were carried out under an argon atmosphere in either flame or oven-dried (120 °C) glassware. TLC analysis was conducted using glass-backed thin-layer silica gel chromatography plates (60 Å, 250 μm thickness, F254 indicator). Column chromatography was performed using silica gel, 230– 400 mesh, 60 Å pore diameter. Isolated yields and yields based on the recovered starting material (brsm) were determined following purification. Proton Nuclear Magnetic Resonance NMR (¹H NMR) spectra and carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on Bruker AV-III-400HD and Bruker AVIII-800 spectrometers. Chemical shifts for protons are reported in parts per million and are references to the NMR solvent peak (CDCl₃: δ 7.26). Chemical shifts for carbons are reported in parts per million and are referenced to the carbon resonances of the NMR solvent (CDCl₃: δ77.16). Data are reported as (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sep = septet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublets of doublets, td = triplet of doublets, dq = doublet of quartets, qd = quartet of doublets, dt = doublet of triplets, brs = broad singlet). All coupling constants are measured in hertz (Hz). Optical rotations were measured on a Rudolph’s AUTOPOL-III automatic digital polarimeter with a sodium lamp and are reported as follows: [α]λ T °C (c = g/100 mL, solvent). High-resolution mass spectrometry (HRMS) spectra were recorded under positive electron spray ionization (ESI+) at Agilent 6550 Q-TOF LC/MS instrument at the Purdue University Analytical Mass Spectrometry Facility.

Dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-(2-cyanoethyl)pentanedioate (10):

To a solution of N-Boc-L(+)-gluatamic dimethyl ester 9 (3.0 g, 10.9 mmol) in anhydrous THF (30 mL) was added the solution of lithium hexamethyldisilazide (1M solution in THF, 24 mL, 23.56 mmol) dropwise under argon atmosphere at −78 °C. After 1 h of stirring at −78 °C, 3-bromopropionitrile (2.19 g, 1.36 mmol) solution in anhydrous THF (10 mL) was added dropwise to the reaction mixture while maintaining the temperature under −78 °C. The reaction mixture was stirred at −78 °C for an additional 2 h and quenched with cold methanol (30 mL). The crude mixture was stirred for 30 minutes at −78 °C followed by addition of cold solution of 1:6 mixture of acetic acid and THF (3 mL AcOH/ 18 mL THF). After a further 30 min of stirring at −78 °C, the cooling bath was removed, and the reaction mixture was allowed to warm up to 23 °C. The crude mixture was diluted with EtOAc, washed with brine. The organic phase was dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (25% EtOAc/hexanes) to give the ester 10 (1.42 g, 40%) as clear oil. ¹H NMR (400 MHz, CDCl₃) δ 5.03 (d, J = 8.7 Hz, 1H), 4.44 – 4.27 (m, 1H), 3.73 (s, 3H), 3.70 (s, 3H), 2.67 – 2.55 (m, 1H), 2.38 (td, J = 7.2, 4.1 Hz, 2H), 2.07 – 1.90 (m, 4H), 1.43 (s, 9H); ESI-MS (m/z): [M + H]⁺ : 329.1.

Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopiperidin-3-yl)propanoate (11):

To a vigorously stirring solution of ester 10 (1.30 g, 3.96 mmol) in anhydrous MeOH (26 mL) was added CoCl₂·6H₂O (565 mg, 2.38 mmol). The resulting pink solution was cooled to 0 °C and NaBH₄ (903 mg, 23.76 mmol) was added portion-wise. After 15 minutes, the ice bath was removed, and the reaction mixture was warmed to 23 °C. After 20 h, the solvents were evaporated in vacuo and the crude mixture was poured into a cold solution of 1M citric acid (50 mL). The residual mixture was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with saturated sodium bicarbonate solution and brine. The organic phase was dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (50–100% EtOAc/hexanes) to give the lactam 11 (800 mg, 68%) as fluffy white solid. ¹H NMR (400 MHz, CDCl₃) δ 6.19 (s, 1H), 5.62 (d, J = 8.5 Hz, 1H), 4.46 – 4.19 (m, 1H), 3.71 (s, 3H), 3.29 (dp, J = 6.9, 2.2 Hz, 2H), 2.38 – 2.22 (m, 2H), 2.13 (dd, J = 11.2, 5.5 Hz, 1H), 1.87 (s, 1H), 1.84 – 1.69 (m, 2H), 1.60 – 1.50 (m, 1H), 1.42 (s, 9H); ESI-MS (m/z): [M + H]⁺ : 301.1.

Tert-butyl ((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)carbamate (8):

Boc-protected amino ester 11 (300 mg, 1.0 mmol) was dissolved in a 1: 1 mixture of MeOH (5.0 mL) and NH₄OH (28–30% NH₃, 5.0 mL) at 23 °C. The reaction mixture was stirred for 12 h. Upon completion, the solvents were evaporated in vacuo and the crude primary amide was used for the subsequent reaction without further purification. To a solution of the above primary amide (285 mg, 1.0 mmol) in 2.5 mL CH₂Cl₂ at 0 °C were added p-toluene sulfonyl chloride (381 mg, 2.0 mmol) and pyridine (402 μL, 5.0 mmol). The resulting solution was warmed to 23 °C and stirred for 18 h. The reaction was quenched by saturated sodium bicarbonate solution and extracted with CH₂Cl₂ (3 × 5 mL). The combined organic layers were washed with 1N HCl, saturated sodium bicarbonate solution and brine. The organic phase was dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (0–2% MeOH/CH₂Cl₂) to give the nitrile 8 (150 mg, 58% over two steps) as fluffy white solid. ¹H NMR (400 MHz, CDCl₃) δ 6.61 (s, 1H), 6.21 (d, J = 7.6 Hz, 1H), 4.68 (d, J = 8.6 Hz, 1H), 3.29 (ddt, J = 9.0, 4.8, 2.1 Hz, 2H), 2.46 – 2.26 (m, 2H), 2.10 – 1.96 (m, 1H), 1.87 (dq, J = 13.6, 4.5 Hz, 2H), 1.80 – 1.64 (m, 1H), 1.55 (ddd, J = 13.8, 8.1, 3.0 Hz, 1H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 173.9, 154.8, 119.1, 80.7, 42.1, 40.9, 37.7, 34.9, 28.2, 27.1, 21.5; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₁₃H₂₂N₃O₃ 268.16609, found: 268.16614.

Methyl(1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (6a):

To a stirred solution of Boc-L-tert-leucine 13a (1.35 g, 5.85 mmol) and methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride 12 (1.0 g, 4.88 mmol) in 40 mL DMF at 0 °C were added EDC·HCl (1.12 g, 5.85 mmol), HOBt (896 mg, 5.85 mmol), and N-methylmorpholine (1.30 mL, 12.20 mmol). After 10 minutes, the ice bath was removed, and the reaction mixture was stirred at 23 °C for 5 h. The reaction then diluted with diethyl ether and washed with cold H₂O. The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (5–10% EtOAc/hexanes) to give the dipeptide methyl ester 6a (1.37 g, 74%) as white sticky solid. [α]_D²⁰ −88.0 (c 1.73, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆) δ 8.29 (s, 1H), 6.69 (d, J = 8.0 Hz, 1H), 4.19 (s, 1H), 4.04 (d, J = 12.0 Hz, 1H), 3.92 (d, J = 12.0 Hz, 1H), 3.79 – 3.63 (m, 1H), 3.29 (s, 3H), 1.52 – 1.48 (m, 1H), 1.40 – 1.38 (m, 1H), 1.33 (s, 9H), 0.99 (s, 3H), 0.92 (s, 9H), 0.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.9, 170.5, 156.3, 78.6, 59.1, 58.9, 52,3, 47.4, 34.4, 29.9, 28.4, 27.4, 26.6, 26.1, 19.3, 12.5; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₀H₃₅N₂O₅ 383.2546, found: 383.2548.

Methyl(1R,2S,5S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (6b):

To a stirred solution of Cbz-L-tert-leucine 13b (100 mg, 0.377 mmol) and methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride 12 (78 mg, 0.377 mmol) in 3.0 mL of DMF at 0 °C were added HATU (172 mg, 0.452 mmol) and DIPEA (0.076 mL, 0.452 mmol). After 10 minutes, the ice bath was removed, and the reaction mixture was stirred at 23 °C for 12 h. The reaction then diluted with diethyl ether and washed with cold H₂O. The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (1–2% MeOH/CH₂Cl₂) to give the carbamate derivative 6b (112 mg, 71%) as white fluffy solid. ¹H NMR (400 MHz, CDCl₃) δ 7.34 – 7.29 (m, 5H), 5.37 (d, J = 10.0 Hz, 1H), 5.06 (d, J = 5.4 Hz, 2H), 4.45 (s, 1H), 4.26 (d, J = 10.0 Hz, 1H), 3.90 (t, J = 2.8 Hz, 2H), 3.74 (s, 3H), 1.44 (s, 1H), 1.02 (s, 12H), 0.94 (s, 1H), 0.86 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 170.4, 156.3, 136.3, 128.4, 127.8, 66.8, 59.2, 59.1, 52.2, 47.6, 35.2, 30.2, 27.3, 26.2, 19.3, 12.4; LRMS-ESI (m/z) [M+H]⁺ 417.5.

Methyl (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(((pyridin-2-ylmethoxy)carbonyl)amino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (6c):

Following the procedure described for the preparation of carbamate derivative 6b using (S)-3,3-dimethyl-2-(((pyridin-2-ylmethoxy)carbonyl)amino)butanoic acid 13c (181 mg, 0.68 mmol) and proline derivative 12 (140 mg, 0.68 mmol)afforded the acid 6c (200 mg, 71%) as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.57 – 8.56 (m, 1H), 7.67 (td, J = 7.7, 1.8 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.20 (dd, J = 7.5, 5.1 Hz, 1H), 5.48 (d, J = 10.1 Hz, 1H), 5.18 (d, J = 4.1 Hz, 2H), 4.46 (s, 1H), 4.27 (d, J = 10.0 Hz, 1H), 3.90 – 3.89 (m, 2H), 3.75 (s, 3H), 1.45 (s, 1H), 1.04 (s, 9H), 1.03 (s, 3H), 0.96 (s, 1H), 0.87 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 170.3, 156.3, 156.0, 149.2, 136.6, 122.6, 121.2, 67.3, 59.2, 52.2, 47.6, 35.3, 30.2, 27.3, 26.2, 19.4, 12.4; LRMS-ESI (m/z) [M+H]⁺ 418.2.

Methyl (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(((pyridin-3-ylmethoxy)carbonyl)amino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (6d):

Following the procedure described for the preparation of carbamate derivative 6b using (S)-3,3-dimethyl-3-(((pyridin-2-ylmethoxy)carbonyl)amino)butanoic acid 13d (194 mg, 0.729 mmol) and proline derivative 12 (150 mg, 0.729 mmol) afforded the acid 6d (220 mg, 78%) as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.59 (s, 2H), 7.66 (dd, J = 7.8, 1.9 Hz, 1H), 7.29 – 7.27 (m, 1H), 5.39 (d, J = 9.9 Hz, 1H), 5.08 (d, J = 4.8 Hz, 2H), 4.45 (s, 1H), 4.24 (d, J = 10.0 Hz, 1H), 3.89 (d, J = 2.8 Hz, 2H), 3.74 (s, 3H), 1.45 (s, 1H), 1.03 (s, 3H), 1.03 (s, 9H), 0.94 (s, 1H), 0.85 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.7, 170.3, 156.0, 149.4, 135.6, 123.3, 64.2, 59.2, 52.2, 47.6, 35.2, 30.2, 27.3, 26.2, 19.3, 12.3; LRMS-ESI (m/z) [M+H]⁺ 418.2.

Methyl(1R,2S,5S)-3-((S)-3,3-dimethyl-2-(((pyridin-4-ylmethoxy)carbonyl)amino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (6e):

Following the procedure described for the preparation of carbamate derivative 6b using (S)-3,3-dimethyl-4-(((pyridin-2-ylmethoxy)carbonyl)amino)butanoic acid 13e (65 mg, 0.243 mmol) and proline derivative 12 (50 mg, 0.243 mmol) afforded the acid 6e (72 mg, 71%) as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.57 – 8.55 (m, 2H), 7.20 – 7.18 (m, 2H), 5.48 (d, J = 10.0 Hz, 1H), 5.13 – 5.01 (m, 2H), 4.46 (s, 1H), 4.24 (d, J = 10.0 Hz, 1H), 3.88 – 3.87 (m, 2H), 3.75 (s, 3H), 1.44 (d, J = 1.7 Hz, 1H), 1.04 (s, 9H), 1.02 (s, 3H), 0.95 (s, 1H), 0.83 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.7, 170.2, 155.8, 149.9, 145.5, 121.4, 64.8, 59.3, 59.2, 52.2, 47.7, 35.3, 30.2, 27.3, 26.2, 26.1, 19.3, 12.3; LRMS-ESI (m/z) [M+H]⁺ 418.2.

Methyl(1R,2S,5S)-3-((S)-2-acetamido-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo [3.1.0]hexane-2-carboxylate (14a):

To a stirred solution of methyl (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate 6a (100 mg, 0.261 mmol) in 3 mL CH₂Cl₂ at 23 °C were added ZnCl₂ (13 mg, 0.10 mmol) and acetic anhydride (0.1 mL, 1.04 mmol). After 12 h, the reaction mixture was quenched with H₂O and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (30% EtOAc/hexanes) to give acetamide 14a (68 mg, 81%) white sticky solid. ¹H NMR (400 MHz, CDCl₃) δ 6.01 (d, J = 9.7 Hz, 1H), 4.57 (d, J = 9.6 Hz, 1H), 4.45 (s, 1H), 3.92 – 3.90 (m, 2H), 3.75 (s, 3H), 1.98 (s, 3H), 1.46 (dd, J = 4.0, 1.4 Hz, 1H), 1.04 (s, 3H), 1.03 (s, 9H), 0.94 (s, 1H), 0.89 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 170.3, 169.6, 59.2, 56.9, 52.2, 47.7, 35.3, 30.2, 27.3, 26.2, 26.1, 23.1, 19.4, 12.4; LRMS-ESI (m/z) [M+H]⁺ 325.2.

Methyl(1R,2S,5S)-3-((S)-2-benzamido-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo [3.1.0]hexane-2-carboxylate (14b):

Following the procedure described for the preparation of compound 14a with a slight modification of conducting the reaction with benzoic anhydride (118 mg, 0.52 mmol) and ZnCl₂ (18 mg, 0.131 mmol) at 40 °C afforded the benzamide 14b (93 mg, 92%) as a white sticky solid. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (dt, J = 7.1, 1.4 Hz, 2H), 7.47 – 7.43 (m, 1H), 7.38 (dd, J = 8.2, 6.6 Hz, 2H), 6.70 (d, J = 9.5 Hz, 1H), 4.78 (d, J = 9.6 Hz, 1H), 4.43 (s, 1H), 4.01 – 3.90 (m, 2H), 3.73 (s, 3H), 1.45 (d, J = 4.7 Hz, 1H), 1.08 (s, 9H), 1.00 (s, 3H), 0.99 (s, 1H), 0.84 (s, 3H); ¹³C NMR (100 MHz, CDCl3) δ 171.7, 170.2, 166.9, 134.0, 131.5, 128.5, 128.4, 126.9, 59.3, 57.2, 52.2, 47.7, 35.8, 30.2, 27.3, 26.5, 26.4, 26.1, 19.3, 12.3; LS-MS (ESI, m/z): [M + Na]⁺ : 409.4.

Methyl(1R,2S,5S)-3-((S)-3,3-dimethyl-2-pivalamidobutanoyl)-6,6-dimethyl-3-azabicyclo [3.1.0]hexane-2-carboxylate (14c):

Following the procedure described for the preparation of compound 14a with a slight modification of conducting the reaction with pivalic anhydride (97 mg, 0.52 mmol) and ZnCl₂ (18 mg, 0.131 mmol) at 40 °C afforded the tert-butyl amide 14c (71 mg, 74%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 6.13 (d, J = 9.6 Hz, 1H), 4.53 (d, J = 9.6 Hz, 1H), 4.40 (d, J = 6.1 Hz, 1H), 3.92 – 3.82 (m, 2H), 3.70 (d, J = 1.7 Hz, 3H), 1.39 (s, 1H), 1.13 (s, 1H), 1.11 (s, 9H), 0.98 (s, 3H), 0.97 (s, 9H), 0.88 (s, 1H), 0.81 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.8, 171.8, 170.4, 60.7, 59.1, 56.5, 52.1, 47.5, 38.7, 36.4, 35.3, 32.6, 30.1, 27.3, 26.3, 26.2, 26.1, 19.2, 12.2; LS-MS (ESI, m/z): [M + Na]⁺ : 389.3.

Methyl (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (14d):

Following the procedure described for the preparation of compound 14a with trifluoroacetic anhydride (0.76 mL, 5.46 mmol) and ZnCl₂ (25 mg, 0.18 mmol) at 23 °C afforded the trifluoroacetamide 14d (623 mg, 91%) as a white sticky solid. [α]_D²⁰ −93.75 (c 0.16, CHCl₃). ¹H NMR (400 MHz, CDCl₃) 7.09 (d, J = 9.5 Hz, 1H), 4.52 (d, J = 9.5 Hz, 1H), 4.41 (s, 1H), 3.89 (dd, J = 10.2, 4.6 Hz, 1H), 3.76 (d, J = 10.3 Hz, 1H), 3.70 (s, 3H), 1.43 (s, 1H), 1.01 (s, 9H), 1.00 (s, 3H), 0.96 – 0.93 (m, 1H), 0.83 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.4, 168.3, 156.8 (q, J = 37.6 Hz), 115.7 (q, J = 287.7 Hz), 59.4, 57.6, 52.2, 47.7, 35.9, 30.1, 27.2, 26.2, 26.1, 25.9, 19.3, 12.2; HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₁₇H₂₆F₃N₂O₄ 379.1839, found: 379.1840.

Methyl (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trichloroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (14e):

Following the procedure described for the preparation of compound 14a with a slight modification of conducting the reaction with trichloroacetic anhydride (153 mg, 0.50 mmol) and ZnCl₂ (18 mg, 0.131 mmol) at 40 °C afforded the trichloroacetamide 14e (102 mg, 96%) as a white sticky solid. ¹H NMR (400 MHz, CDCl₃) δ 7.26 – 7.24 (m, 1H), 4.47 (d, J = 6.9 Hz, 2H), 3.92 (dd, J = 10.2, 4.4 Hz, 1H), 3.82 (d, J = 10.3 Hz, 1H), 3.75 (d, J = 4.9 Hz, 3H), 1.47 (s, 1H), 1.07 (s, 9H), 1.03 (s, 3H), 0.98 (s, 1H), 0.87 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.5, 168.6, 161.6, 92.4, 60.8, 59.4, 59.1, 52.3, 47.7, 47.0, 36.4, 32.7, 30.1, 27.2, 26.3, 26.1, 26.0, 25.6, 19.4, 12.3; LSMS (ESI, m/z): [M + Na]⁺ : 449.2.

(1R,2S,5S)-3-((S)-2-Acetamido-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxylic acid (15a):

To a vigorously stirring solution of amide derivative 15a (40 mg, 0.12 mmol) in a 1:3 mixture of THF/H₂O (0.15 M) at 0 °C was added LiOH·H₂O (26 mg, 0.62 mmol). After 1 h, the solvent evaporated under reduced pressure. The reaction mixture was diluted with ethyl acetate and neutralized with 1 N HCl. The solution was extracted with EtOAc (3 × 10 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the corresponding acid 15a (30 mg, 78%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 6.67 (d, J = 9.5 Hz, 1H), 4.60 (d, J = 9.6 Hz, 1H), 4.42 (d, J = 3.4 Hz, 1H), 3.99 – 3.87 (m, 2H), 1.99 (s, 3H), 1.61 (dd, J = 13.7, 7.5 Hz, 1H), 1.05 (s, 3H), 1.01 (s, 9H), 0.94 (s, 1H), 0.89 (s, 3H).

(1R,2S,5S)-3-((S)-2-Benzamido-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxylic acid (15b):

Following the ester hydrolysis procedure described for the preparation of carboxylic acid 15a using ester 14b (75 mg, 0.19 mmol) afforded the acid 15b (44 mg, 61%) as a white fluffy solid. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (d, J = 7.6 Hz, 2H), 7.47 (d, J = 7.3 Hz, 1H), 7.40 (t, J = 7.6 Hz, 2H), 7.07 (d, J = 9.7 Hz, 1H), 4.84 (d, J = 9.5 Hz, 1H), 4.44 (s, 1H), 4.05 (d, J = 10.5 Hz, 1H), 3.93 (dd, J = 10.4, 5.1 Hz, 1H), 1.50 (d, J = 7.2 Hz, 1H), 1.43 (t, J = 6.2 Hz, 1H), 1.07 (s, 9H), 1.01 (s, 3H), 0.86 (s, 3H).

(1R,2S,5S)-3-((S)-3,3-Dimethyl-2-pivalamidobutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxylic acid (15c):

Following the procedure described for the preparation of carboxylic acid 15a using ester 14c (65 mg, 0.18 mmol) afforded the acid 15c (60 mg, 95%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 6.27 (d, J = 9.6 Hz, 1H), 4.61 (d, J = 9.7 Hz, 1H), 4.44 (s, 1H), 4.02 (d, J = 10.5 Hz, 1H), 3.86 (dd, J = 10.5, 5.4 Hz, 1H), 1.47 (dd, J = 7.5, 5.2 Hz, 1H), 1.29 (ddd, J = 13.4, 6.9, 1.6 Hz, 1H), 1.17 (d, J = 3.2 Hz, 9H), 1.04 (d, J = 2.4 Hz, 4H), 1.00 (s, 9H), 0.84 (s, 3H).

(1R,2S,5S)-3-((S)-3,3-Dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (15d):

Following the ester hydrolysis procedure described for the preparation of carboxylic acid 15a using ester 14d (600 mg, 1.59 mmol) afforded the crude compound 15d (481 mg, 83%) as a white solid. The crude residue was crystallized using a 1:4 mixture of hexanes and THF. [α]_D²⁰ −73.14 (c 0.458, MeOH). ¹H NMR (400 MHz, CDCl₃) δ 9.38 (bs, 1H), 7.57 (d, J = 9.5 Hz, 1H), 4.62 (d, J = 9.6 Hz, 1H), 4.45 (s, 1H), 3.93 (dd, J = 10.4, 5.1 Hz, 1H), 3.87 (d, J = 10.4 Hz, 1H), 1.58 (d, J = 7.6 Hz, 1H), 1.54–1.25 (m, 1H), 1.06 (s, 3H), 1.04 (s, 9H), 0.88 (s, 3H).

(1R,2S,5S)-3-((S)-3,3-Dimethyl-2-(2,2,2-trichloroacetamido)-butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (15e):

Following the procedure described for the preparation of carboxylic acid 15a using ester 14e (50 mg, 0.12 mmol) afforded the acid 15e (45 mg, 94%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.64 (s, 1H), 7.39 (d, J = 9.2 Hz, 1H), 4.52 – 4.48 (m, 2H), 3.91 – 3.89 (m, 2H), 1.60 (d, J = 7.5 Hz, 1H), 1.54 – 1.49 (m, 1H), 1.05 (d, J = 3.6 Hz, 3H), 0.88 (s, 9H), 0.87 (s, 3H).

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (1):

To a solution of tert-butyl ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamate 7 (318 mg, 1.26 mmol) in CH₂Cl₂ (8.3 mL) at 0 °C was added TFA (5.0 mL) and the solution was stirred for 2 h at 23 °C. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (192 mg, 1.26 mmol) was coupled to the acid 15d (457 mg, 1.26 mmol) using the coupling agent HATU (573 mg, 1.51 mmol) in the presence of N-methylmorphine (0.352 mL, 2.5 mmol) in DMF (6.2 mL) at 0 °C. After 10 minutes, the ice bath was removed, and the reaction mixture was stirred at 23 °C for 12 h. The solvent was then evaporated in vacuo, and the residue was dissolved in CH₂Cl₂. The organic layer was washed with water and brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (1–2% MeOH/CH₂Cl₂) to give nirmatrelvir 1 (460 mg, 73%) as white fluffy solid. [α]_D²⁰ −74.36 (c 1.13, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (d, J = 8.4 Hz, 1H), 9.00 (d, J = 8.5 Hz, 1H), 7.64 (s, 1H), 4.95 (ddd, J = 10.9, 8.5, 5.1 Hz, 1H), 4.39 (d, J = 8.6 Hz, 1H), 4.14 (s, 1H), 3.89 (dd, J = 10.4, 5.5 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 3.12 (t, J = 9.0 Hz, 1H), 3.02 (td, J = 9.3, 7.0 Hz, 1H), 2.41 – 2.34 (m, 1H), 2.11 (dddd, J = 20.8, 12.2, 8.7, 3.8 Hz, 2H), 1.70 (ddt, J = 17.1, 9.0, 4.3 Hz, 2H), 1.54 (dd, J = 7.6, 5.3 Hz, 1H), 1.31 (s, 1H), 1.01 (s, 3H), 0.96 (s, 9H), 0.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.8, 171.0, 167.8, 157.3 (q, J = 36.7, 36.2 Hz), 119.9, 117.7 – 114.5 (m), 60.4, 58.5, 48.0, 38.1, 37.1, 34.9, 34.5, 30.6, 27.7, 27.2, 26.1, 19.2, 12.7; ¹⁹F NMR (376 MHz, DMSO-d₆) δ −74.5; HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₂₃H₃₃F₃N₅O₄ 500.2479, found: 500.2480.

(1R,2S,5S)-3-((S)-2-Acetamido-3,3-dimethylbutanoyl)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5a):

Compound 5a was prepared in two steps in 21% yield by boc-deprotection of lactam residue 7 (25 mg, 0.097 mmol) followed by coupling to the acid 15a (30 mg, 0.097 mmol) utilizing the same procedure as described for compound 1.¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 4.97 – 4.91 (m, 1H), 4.28 (d, J = 8.0 Hz, 1H), 4.09 (s, 1H), 3.86 – 3.76 (m, 2H), 3.12 (t, J = 8.0 Hz, 1H), 3.05 – 2.98 (m, 1H), 2.45 – 2.36 (m, 1H), 2.17 – 2.07 (m, 1H), 1.81 (s, 3H), 1.73 – 1.64 (m, 2H), 1.52 (q, J = 4.0 Hz, 1H), 1.27 – 1.21 (m, 2H), 1.00 (s, 3H), 0.91 (s, 9H), 0.84 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 178.0, 171.4, 169.93, 169.86, 120.2, 60.3, 57.4, 47.9, 38.2, 37.2, 34.7, 34.6, 30.9, 28.9, 28.0, 27.3, 26.9, 26.3, 22.5, 19.3, 13.1; HRMS (ESI): m/z calcd for C₂₃H₃₅N₅O₄ [M+H]+ 446.2767 found 446.2763.

(1R,2S,5S)-3-((S)-2-Benzamido-3,3-dimethylbutanoyl)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5b):

Compound 5b was prepared in two steps in 53% yield by boc-deprotection of lactam residue 7 (30 mg, 0.12 mmol) followed by coupling to the acid 15b (44 mg, 0.12 mmol) utilizing the same procedure as described for compound 1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 (d, J = 8.5 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.81 – 7.76 (m, 2H), 7.64 (s, 1H), 7.49 (t, J = 7.3 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 5.01 – 4.93 (m, 1H), 4.55 (d, J = 8.7 Hz, 1H), 4.14 (s, 1H), 3.91 (d, J = 3.2 Hz, 2H), 3.13 (t, J = 9.1 Hz, 1H), 3.05 (dd, J = 9.4, 7.0 Hz, 1H), 2.45 – 2.35 (m, 1H), 2.12 (ddt, J = 21.5, 11.2, 5.4 Hz, 2H), 1.69 (tdd, J = 12.6, 10.5, 10.1, 4.6 Hz, 2H), 1.55 (dt, J = 6.7, 3.0 Hz, 1H), 1.28 (d, J = 7.6 Hz, 1H), 1.00 (s, 12H), 0.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.9, 171.3, 169.5, 167.5, 134.4, 131.6, 128.4, 128.1, 120.0, 60.3, 58.1, 47.9, 38.1, 37.1, 34.9, 34.5, 30.7, 27.8, 27.2, 26.9, 26.2, 19.2, 12.9; HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ calcd for C₂₈H₃₇N₅O₄Na 530.2737, found: 530.2739.

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-pivalamidobutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5c):

Compound 5c was prepared in two steps in 48% yield by boc-deprotection of lactam residue 7 (32 mg, 0.13 mmol) followed by coupling to the acid 15c (45 mg, 0.13 mmol) utilizing the same procedure as described for compound 1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J = 8.5 Hz, 1H), 7.65 (s, 1H), 6.85 (d, J = 9.3 Hz, 1H), 4.94 (ddd, J = 10.8, 8.4, 5.2 Hz, 1H), 4.42 (d, J = 9.3 Hz, 1H), 4.10 (s, 1H), 3.85 (dd, J = 10.3, 5.4 Hz, 1H), 3.71 (d, J = 10.4 Hz, 1H), 3.12 (t, J = 9.2 Hz, 1H), 3.03 (td, J = 9.3, 7.0 Hz, 1H), 2.39 (dd, J = 9.6, 3.9 Hz, 1H), 2.17 – 2.02 (m, 2H), 1.69 (ddt, J = 12.3, 9.8, 6.1 Hz, 2H), 1.51 (dd, J = 7.6, 5.2 Hz, 1H), 1.27 (d, J = 7.6 Hz, 1H), 1.06 (s, 9H), 0.99 (s, 3H), 0.90 (s, 9H), 0.79 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.9, 177.8, 171.2, 169.6, 120.0, 60.1, 56.5, 47.7, 38.5, 38.1, 37.1, 34.9, 34.4, 30.6, 27.6, 27.2, 26.7, 26.1, 19.1, 12.7; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₆H₄₂N₅O₄ 488.32366, found: 488.32375.

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trichloroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5d):

Compound 5d was prepared in two steps in 52% yield by boc-deprotection of lactam residue 7 (25 mg, 0.10 mmol) followed by coupling to the acid 15e (41 mg, 0.10 mmol) utilizing the same procedure as described for compound 1. ¹H NMR (400 MHz, DMSO-d₆) δ 9.00 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 4.95 (dd, J = 5.4, 2.2 Hz, 1H), 4.37 (d, J = 8.8 Hz, 1H), 4.16 (s, 1H), 3.91 (dd, J = 10.4, 5.5 Hz, 1H), 3.66 (d, J = 10.5 Hz, 1H), 3.16 – 3.08 (m, 1H), 3.03 (td, J = 9.3, 6.9 Hz, 1H), 2.37 (dd, J = 9.3, 4.6 Hz, 1H), 2.16 – 2.02 (m, 2H), 1.70 (ddt, J = 12.9, 10.0, 6.8 Hz, 2H), 1.56 (dd, J = 7.6, 5.3 Hz, 1H), 1.32 (s, 1H), 1.22 (d, J = 6.5 Hz, 1H), 1.01 (s, 3H), 0.97 (s, 9H), 0.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.8, 171.0, 167.8, 161.9, 119.9, 92.8, 60.5, 59.3, 48.0, 38.2, 37.1, 35.7, 34.4, 30.6, 27.6, 27.2, 26.4, 26.1, 19.2, 12.8; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₃H₃₃Cl₃N₅O₄ 548.1598, found: 548.1604.

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5e):

Compound 5e was prepared in two steps in 64% yield by boc-deprotection of lactam residue 7 (94 mg, 0.35 mmol) followed by coupling to the acid 15d (127 mg, 0.35 mmol) utilizing the same procedure as described for compound 1. ¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (d, J = 8.4 Hz, 1H), 8.97 (d, J = 8.2 Hz, 1H), 4.99 (ddd, J = 10.4, 8.2, 5.7 Hz, 1H), 4.39 (d, J = 8.5 Hz, 1H), 4.15 (s, 1H), 3.88 (dd, J = 10.4, 5.5 Hz, 1H), 3.66 (d, J = 10.5 Hz, 1H), 3.06 (dt, J = 8.2, 3.6 Hz, 2H), 2.35 – 2.17 (m, 2H), 1.88 – 1.78 (m, 1H), 1.71 (tdd, J = 18.2, 8.7, 5.2 Hz, 2H), 1.54 (dd, J = 7.7, 5.3 Hz, 2H), 1.40 – 1.31 (m, 1H), 1.27 (d, J = 7.6 Hz, 1H), 1.00 (s, 3H), 0.97 (s, 9H), 0.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.2, 171.0, 167.7, 157.4, 120.1, 117.6, 60.3, 58.5, 47.9, 41.5, 38.2, 37.0, 34.9, 34.7, 30.7, 27.6, 26.6, 26.1, 21.6, 19.2, 12.7; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₄H₃₅F₃N₅O₄ 514.26409, found: 514.26396.

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trichloroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5f):

Compound 5f was prepared in two steps in 37% yield by boc-deprotection of lactam residue 8 (18 mg, 0.07 mmol) followed by coupling to the acid 15e (28 mg, 0.07 mmol) utilizing the same procedure as described for compound 1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 2.8 Hz, 1H), 4.99 (ddd, J = 10.4, 8.2, 6.0 Hz, 1H), 4.38 (d, J = 8.8 Hz, 1H), 4.17 (s, 1H), 3.91 (dd, J = 10.5, 5.5 Hz, 1H), 3.65 (d, J = 10.4 Hz, 1H), 3.11 – 3.02 (m, 2H), 2.25 (dddd, J = 33.2, 14.5, 10.7, 5.7 Hz, 2H), 1.88 – 1.79 (m, 1H), 1.71 (dddd, J = 17.8, 13.3, 8.7, 5.3 Hz, 2H), 1.55 (dd, J = 7.6, 5.2 Hz, 2H), 1.37 (dd, J = 13.9, 11.1 Hz, 2H), 1.29 (d, J = 7.6 Hz, 1H), 1.01 (s, 3H), 0.98 (s, 9H), 0.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.3, 170.9, 167.8, 161.9, 120.1, 92.8, 60.4, 59.3, 47.9, 41.5, 38.3, 37.0, 35.8, 34.7, 30.7, 27.5, 26.5, 26.1, 21.6, 19.2, 12.8; HRMS (ESI/Q-TOF) m/z: [M + H]⁺ calcd for C₂₄H₃₅Cl₃N₅O₄ 562.1749, found: 562.1764.

Tert-butyl ((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5g):

To a solution of lactam 7 (37 mg, 0.15 mmol) in CH₂Cl₂ (1.0 mL) at 0 °C was added TFA (0.6 mL) and the solution was stirred for 1 h at 23 °C. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (22 mg, 0.15 mmol) was coupled to the acid 6a (54 mg, 0.15 mmol) using EDC·HCl (34 mg, 0.18 mmol), HOBt (27 mg, 0.18 mmol) and N-methylmorphine (24 μL, 0.22 mmol) in DMF (1.8 mL) at 0 °C. After 10 minutes, the ice bath was removed, and the reaction mixture was stirred at 23 °C for 12 h. The solvent was then evaporated in vacuo, and the residue was dissolved in CH₂Cl₂. The organic layer was washed with water and brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (1–2% MeOH/ CH₂Cl₂) to give the amide 5g (46 mg, 62% over 2-steps) as white fluffy solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (d, J = 8.6 Hz, 1H), 7.63 (s, 1H), 6.64 (d, J = 9.1 Hz, 1H), 4.93 (ddd, J = 10.9, 8.5, 5.1 Hz, 1H), 4.13 (s, 1H), 3.98 (d, J = 9.2 Hz, 1H), 3.89 – 3.77 (m, 2H), 3.14 – 2.96 (m, 2H), 2.38 (dt, J = 10.2, 4.9 Hz, 1H), 2.18 – 2.01 (m, 2H), 1.76 – 1.60 (m, 2H), 1.51 (dd, J = 7.7, 5.1 Hz, 1H), 1.32 (s, 9H), 1.25 (d, J = 7.6 Hz, 1H), 1.00 (s, 3H), 0.89 (s, 9H), 0.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.8, 171.3, 170.3, 156.3, 120.0, 78.5, 60.1, 59.1, 47.8, 38.1, 37.0, 34.4, 34.3, 30.6, 28.4, 27.8, 27.2, 26.7, 26.2, 19.2, 12.8; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₆H₄₂N₅O₅ 504.31857, found: 504.31869.

(1R,2S,5S)-N-((S)-1-Cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-N-(2,2,2-trifluoroacetyl)-3-azabicyclo[3.1.0] hexane-2-carboxamide (5h):

Following the procedure described for the preparation of compound 14d with trifluoloroacetic anhydride (14 mg, 0.1 mmol) and ZnCl₂ (5 mol%) afforded the compound 5h (10 mg, 85%) as a white sticky solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.69 (s, 1H), 9.41 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 4.87 (s, 1H), 4.43 (d, J = 8.4 Hz, 1H), 3.92 (dd, J = 10.6, 5.2 Hz, 1H), 3.82 (d, J = 10.6 Hz, 1H), 3.13 – 3.05 (m, 2H), 2.74 – 2.68 (m, 1H), 2.42 (dt, J = 9.3, 4.6 Hz, 1H), 2.25 (dd, J = 14.6, 10.3 Hz, 1H), 2.11 – 1.99 (m, 1H), 1.75 – 1.58 (m, 2H), 1.45 (d, J = 7.5 Hz, 1H), 1.00 (s, 4H), 0.94 (s, 9H), 0.86 (s, 3H); ¹³C NMR (200 MHz, DMSO-d₆) δ 178.0, 168.2, 160.3, 157.4, 157.4, 156.5, 156.3, 136.0, 131.0, 127.8, 116.9, 116.5, 115.1, 114.2, 79.5, 79.3, 79.1, 59.0, 54.7, 47.8, 35.0, 31.2, 29.4, 27.5, 26.6, 26.5, 26.4, 26.1, 22.5, 19.4, 12.4; HRMS (ESI/LTQ) m/z: [M + H]⁺ calcd for C₂₅H₃₂F₆N₅O₅ 596.2307, found: 596.2321.

Benzyl ((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5i):

To a vigorously stirring solution of amide derivative 6b (65 mg, 0.16 mmol) in a 1:3 mixture of THF/H₂O (3.0 mL) at 0 °C was added LiOH·H₂O (20 mg, 0.5 mmol). After 1 h, the solvent evaporated under reduced pressure. The reaction mixture was diluted with ethyl acetate and neutralized with 1 N HCl. The solution was extracted with EtOAc (3 × 5 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude acid (58 mg, 92%) as a white solid.

To a solution of tert-butyl((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamate 7 (20 mg, 0.08 mmol) in CH₂Cl₂ (1.0 mL) at 0 °C was added TFA (0.2 mL) and the solution was stirred for 2 h at 23 °C. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (10 mg, 0.064 mmol) was coupled to the above crude acid (26 mg, 0.064 mmol) using the coupling agent HATU (38 mg, 0.1 mmol) and DIPEA (0.13 ml, 0.65 mmol) in DMF (2.0 mL) at 0 °C. After 10 minutes, the ice bath was removed, and the reaction mixture was stirred at 23 °C for 12 h. The solvent was then evaporated in vacuo, and the residue was dissolved in CH₂Cl₂. The organic layer was washed with water and brine, dried over Na₂SO₄, concentrated under reduced pressure and the residue was purified by column chromatography (1–2% MeOH/ CH₂Cl₂) to give compound 5i (14 mg, 40%) as white fluffy solid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.0 Hz, 1H), 7.28 – 7.38 (m, 6H), 5.78 (s, 1H), 5.43 (d, J = 8.5 Hz, 2H), 5.05 (d, J = 3.22 Hz, 2H), 4.88–4.92 (m, 1H), 4.26 (s, 2H), 3.90 (s, 1H), 3.29–3.36 (m, 2H), 2.51–2.57 (m, 1H), 2.30–2.37 (m, 2H), 1.86–2.01 (m, 1H), 1.54–1.83 (m, 1H), 1.35–1.37 (t, J = 2.5 Hz, 1H), 1.05 (s, 3H), 0.96 (s, 9H), 0.85 (s, 3H); ¹³C NMR (200 MHz, CDCl₃) δ 171.5, 171.3, 170.2, 167.6, 156.3, 136.4, 128.52, 128.49, 128.0, 118.3, 67.0, 60.6, 59.3, 48.3, 40.4, 37.6, 35.3, 29.7, 27.9, 26.4, 26.2, 19.3, 14.1, 13.4, 12.7; HRMS (ESI/LTQ) m/z. [M+H]⁺ calcd for C₂₉H₃₉N₅O₅: 538.3029; found 538.3020.

Pyridin-2-ylmethyl((2S)-1-((1R,2S,5S)-2-((1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl) carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5j):

(S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanenitrile (12 mg, 0.079 mmol) was coupled with the corresponding acid (32 mg, 0.079 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5j (15 mg, 35%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 (d, J = 8.5 Hz, 1H), 8.50 – 8.49 (m, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.64 (s, 1H), 7.47 (d, J = 8.9 Hz, 1H), 7.32 – 7.27 (m, 2H), 5.03 (q, J = 13.8 Hz, 2H), 4.94 (ddd, J = 10.9, 8.5, 5.2 Hz, 1H), 4.13 (s, 1H), 4.06 (d, J = 8.9 Hz, 1H), 3.84 (dd, J = 10.2, 5.4 Hz, 1H), 3.76 (d, J = 10.3 Hz, 1H), 3.11 (t, J = 9.1 Hz, 1H), 3.02 (td, J = 9.3, 7.0 Hz, 1H), 2.39 (dt, J = 10.1, 4.9 Hz, 1H), 2.17 – 2.03 (m, 2H), 1.73 – 1.64 (m, 1H), 1.51 (dd, J = 7.6, 5.2 Hz, 1H), 1.26 (d, J = 7.5 Hz, 2H), 0.99 (s, 3H), 0.93 (s, 9H), 0.79 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 178.0, 171.4, 169.9, 157.2, 156.8, 149.5, 137.3, 123.2, 121.4, 120.1, 66.7, 60.4, 59.8, 48.0, 38.3, 37.2, 34.8, 34.6, 30.8, 27.9, 27.4, 26.8, 26.3, 19.3, 13.0; HRMS (ESI): m/z calcd for C₂₈H₃₈N₆O₅ [M+H]+ 539.2981 found 539.2975.

Pyridin-3-ylmethyl((2S)-1-((1R,2S,5S)-2-((1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl) carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5k):

(S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanenitrile (13 mg, 0.081 mmol) was coupled with the corresponding acid (33 mg, 0.081 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5k (14 mg, 32%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (d, J = 8.5 Hz, 1H), 8.51 (d, J = 15.1 Hz, 2H), 7.71 (d, J = 8.1 Hz, 1H), 7.63 (s, 1H), 7.41 – 7.34 (m, 2H), 5.09 – 5.00 (m, 2H), 4.95 (d, J = 13.5 Hz, 1H), 4.10 (s, 1H), 4.04 (d, J = 8.7 Hz, 1H), 3.82 (d, J = 6.0 Hz, 1H), 3.76 (d, J = 10.4 Hz, 1H), 3.11 (t, J = 9.1 Hz, 1H), 3.02 (t, J = 8.4 Hz, 1H), 2.38 (d, J = 9.9 Hz, 1H), 2.09 (ddd, J = 24.7, 13.1, 5.4 Hz, 2H), 1.67 (d, J = 10.5 Hz, 1H), 1.51 (t, J = 6.5 Hz, 1H), 1.24 (dd, J = 11.0, 7.5 Hz, 2H), 1.00 (s, 3H), 0.91 (s, 9H), 0.80 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 178.0, 171.4, 169.9, 156.8, 149.5, 136.0, 131.1, 123.9, 120.1, 63.7, 60.3, 59.8, 48.0, 38.2, 37.2, 34.7, 34.6, 30.8, 28.0, 27.4, 26.8, 26.3, 19.3, 13.0; HRMS (ESI): m/z calcd for C₂₈H₃₈N₆O₅ [M+H]+ 539.2981 found 539.2976.

Pyridin-4-ylmethyl((2S)-1-((1R,2S,5S)-2-((1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl) carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5l):

(S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanenitrile (12 mg, 0.074 mmol) was coupled with the corresponding acid (30 mg, 0.074 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5l (14 mg, 35%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 (d, J = 8.5 Hz, 1H), 8.52 (s, 2H), 7.64 (s, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.33 – 7.22 (m, 2H), 5.12 – 5.00 (m, 2H), 4.94 (ddd, J = 10.9, 8.5, 5.2 Hz, 1H), 4.12 (s, 1H), 4.03 (dd, J = 18.6, 8.0 Hz, 1H), 3.88 – 3.69 (m, 2H), 3.14 – 3.01 (m, 2H), 2.14 – 2.04 (m, 2H), 1.70 – 1.66 (m, 1H), 1.51 (dd, J = 7.7, 5.3 Hz, 1H), 1.27 – 1.23 (m, 2H), 0.98 (s, 3H), 0.93 (s, 9H), 0.77 (s, 3H); ¹³C NMR (125 MHz, DMSO-d6) δ 178.0, 171.4, 169.9, 156.7, 150.0, 146.9, 121.9, 120.1, 64.2, 60.4, 59.8, 48.0, 38.3, 37.2, 34.8, 34.6, 30.8, 27.9, 27.4, 26.8, 26.3, 19.3, 12.9; LRMS-ESI (m/z) [M+H]⁺ 539.4.

(1R,2S,5S)-3-((S)-2-(3-(Tert-butyl)ureido)-3,3-dimethylbutanoyl)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (5m):

(S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanenitrile (13 mg, 0.087 mmol) was coupled with the corresponding acid (32 mg, 0.087 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5m (14 mg, 32%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H), 5.17 (s, 1H), 5.06 (q, J = 5.96 Hz, 1H), 4.97 (s, 1H), 4.33 (d, J = 9.54 Hz, 1H), 4.28 (s, 1H), 4.12 (d, J = 10.14 Hz, 1H), 3.89–3.92 (m, 1H), 3.27–3.33 (m, 1H), 2.50–2.54 (m, 1H), 2.30–2.40 (m, 2H), 1.93–1.97 (m, 1H), 1.80–1.84 (m, 1H), 1.55 (t, J = 2.54 Hz, 1H), 1.43–1.45 (m, 1H), 1.32–1.34 (t, J = 2.12 Hz, 1H), 1.24 (s, 9H), 1.03 (s, 3H), 0.96 (s, 9H), 0.92 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 179.1, 173.1, 171.2, 157.5, 119.4, 60.5, 58.0, 50.2, 48.4, 40.4, 38.9, 37.6, 34.7, 34.5, 30.2, 29.4, 28.0, 26.7, 26.3, 19.3, 12.6; HRMS (ESI): m/z calcd for C₂₆H₄₂N₆O₄ [M+H]+ 503.3345; found 503.3337.

Pyridin-3-ylmethyl((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl) carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5n):

(S)-2-Amino-3-((S)-2-oxopiperidin-3-yl)propanenitrile (12 mg, 0.073 mmol) was coupled with the corresponding acid (27 mg, 0.073 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5n (15 mg, 34%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J = 8.0 Hz, 1H), 8.54 (s, 1H), 8.50 (d, J = 4.0 Hz, 1H), 7.74 – 7.71 (m, 1H), 7.47 (s, 1H), 7.39 – 7.35 (m, 2H), 5.09 (d, J = 12.0 Hz, 1H), 5.00 – 4.94 (m, 2H), 4.12 (s, 1H), 4.04 (d, J = 8.0 Hz, 1H), 3.85 – 3.75 (m, 2H), 3.05 (t, J = 4.0 Hz, 2H), 2.34 – 2.21 (m, 2H), 1.84 – 80 (m, 1H), 1.75 – 1.64 (m, 1H), 1.51 (t, J = 4.0 Hz, 2H), 1.38 – 1.34 (m, 1H), 1.23 (d, J = 8.0 Hz, 2H), 0.99 (s, 3H), 0.92 (s, 9H), 0.79 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 172.4, 171.4, 169.8, 156.8, 149.4, 136.1, 133.2, 123.9, 120.3, 63.7, 60.2, 59.8, 47.9, 41.6, 38.3, 37.1, 34.8, 34.7, 30.9, 27.8, 26.8, 26.3, 26.2, 21.8, 19.3, 12.9; HRMS (ESI): m/z calcd for C₂₉H₄₀N₆O₅ [M+H]+ 553.3138 found 553.3132.

Pyridin-4-ylmethyl((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl) carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5o):

(S)-2-Amino-3-((S)-2-oxopiperidin-3-yl)propanenitrile (13 mg, 0.074 mmol) was coupled with the corresponding acid (30 mg, 0.074 mmol) utilizing the same procedure as described for compound 5i to afford the compound 5o (14 mg, 34%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J = 8.2 Hz, 1H), 8.51 (s, 2H), 7.60 – 7.40 (m, 2H), 7.27 (d, J = 5.4 Hz, 2H), 5.14 – 4.91 (m, 3H), 4.13 (s, 1H), 4.06 (d, J = 8.9 Hz, 1H), 3.88 – 3.72 (m, 2H), 3.12 – 3.01 (m, 2H), 2.35 – 2.18 (m, 2H), 1.83 (dd, J = 12.8, 4.0 Hz, 1H), 1.78 – 1.63 (m, 2H), 1.55 – 1.44 (m, 2H), 1.23 (d, J = 7.1 Hz, 2H), 0.98 (s, 3H), 0.94 (s, 9H), 0.76 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 172.4, 171.3, 169.8, 156.7, 150.0, 146.8, 121.9, 120.3, 64.2, 60.2, 59.8, 47.9, 41.6, 38.3, 37.1, 34.84, 34.79, 30.9, 27.8, 26.9, 26.3, 26.2, 21.8, 19.3, 12.9; HRMS (ESI): m/z calcd for C₂₉H₄₀N₆O₅ [M+H]+ 553.3138 found 553.3131.

Expression and Purification of SARS-CoV-2 main protease

The gene encoding the SARS-CoV-2 main protease (Mpro), also known as 3C like protease (3CLpro) was obtained from the polyprotein sequence (Accession #: MN908947). The expression construct was designed to contain an N-terminal hexa-histidine tag. This was followed by polyprotein residues 3259–3569 to reflect the sequence of the 3CL^pro enzyme in addition to the nsp4/5 autocleavage site. The 3CLpro nucleotide sequence was codon optimized for expression in E. Coli, synthesized and subcloned into a pET11a vector by BioBasic. This expression construct produced a protein with its authentic N- and C-termini.

This plasmid containing the SARS-CoV-2 3CLpro construct was transformed into E. coli BL21 (DE3) cells by electroporation. A single colony of the transformed cells was used to inoculate 150 mL of Super LB media (3 g monobasic potassium phosphate, 6 g dibasic sodium phosphate, 20 g tryptone, 5 g yeast extract, 5 g NaCl per 1 L of water, 100 μg/mL Carbenicillin, pH adjusted to 7.20 using 10 M NaOH). This preculture was incubated overnight for 12 hours at 37 °C with shaking. The expression culture consisted of 500 mL Super LB media supplemented with 100 μg/mL Carbenicillin, 12.5 mL 8% lactose, 5 mL 60% glycerol, 2.5 mL 10% glucose, and 12.5 mL of preculture that was incubated at 25 °C for 24 hours. The cells were harvested via centrifugation at 16,800 x g for 40 minutes to yield 7.2 g L⁻¹ of cells. The cell pellet was resuspended in 5 mL Lysis Buffer (25 mM HEPES, 0.05 mM EDTA, 5 mM β-ME, 1 mg/mL lysozyme) per 1 g of pelleted cells using a manual homogenizer. Homogenized cell suspension was sonicated for a total of 10 minutes at an amplitude of 60% for periods of 10 s punctuated by 20 s delays using a Branson digital sonifier. The lysate produced was clarified by centrifugation for 16,080 x g for 45 minutes at 4 °C.

Clarified lysate was injected onto a 60 mL Q-Sepharose Fast Flow (GE Healthcare) strong anion-exchange column equilibrated in Buffer A (25 mM HEPES pH 7.50, 0.05 mM EDTA, 5 mM β-ME). Protein was recovered from the flow-through and an elution phase to ~45 % Buffer B (50 mM HEPES pH 7.5, 1 M Ammonium sulfate, 0.05 mM EDTA, 5 mM β-ME) over ~5 column volumes. Protein fractions were pooled based upon those with the highest specific activity as well as by visual inspection of purity using SDS-PAGE gels. Solid Ammonium sulfate was gradually added to the protein pool to a final concentration of 1 M while stirring on ice. Precipitation was removed via centrifugation at 16,000 x g for 45 minutes at 4 °C. Supernatant was checked for activity prior to injection onto a 60 mL Phenyl Sepharose 6 Fast Flow Hi Sub (GE Healthcare) hydrophobic interaction column that had been equilibrated in Buffer B. Protein was eluted using a linear gradient to 100% Buffer A over ~12 column volumes. Protein eluted at ~0.6 column volumes after reaching 100% Buffer B. Fractions containing CoVID-19 3CL^pro were assessed for purity and pooled based on the criteria above. The pH of the pool was adjusted to pH 5.5 using a 1 M MES solution while stirring on ice. This protein was syringe-filtered with a 0.45 μm SFCA membrane prior to injection onto an 8 mL Mono S (GE Healthcare) equilibrated in Buffer C (20 mM MES monohydrate pH 5.50, 0.05 mM EDTA, 5 mM β-ME) for cation-exchange chromatography. Protein was eluted using a gradient to ~60% Buffer D (50 mM MES monohydrate pH 5.5, 1 M NaCl, 0.05 mM EDTA, 5 mM β-ME) over ~46 column volumes. Fractions were pooled using the same criteria. This pool was concentrated using a 10,000 MWCO spin concentrator (Millipore) before being injected onto a 300 mL HiLoad 26/60 Superdex 200 prep grade column (GE Healthcare) for size-exclusion and desalting into Buffer E (25 mM HEPES pH 7.5, 10% Glycerol, 2.5 mM DTT). Protein was pooled based on the above criteria, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. The protein concentration was determined by measuring absorbance at 280 nm and calculating the concentration using a molar extinction coefficient of 32,890 M⁻¹ cm⁻¹ determined from ProtParam (Expasy).

SARS-CoV-2 3CLpro inhibition assays

Inhibition of SARS-CoV-2 3CLpro by designed compounds was assessed similar to our previously published methods using a continuous fluorescence assay and the FRET-based substrate UIVT3 (HiLyte Fluor488^™–ESATLQSGLRKAK-QXL520™-NH₂) which was synthesized by Anaspec, Fremont, CA.^40,44 Briefly, the assay buffer consisted of 50 mM HEPES pH 7.5, 0.1 mg/mL BSA, 0.01% Triton X-100, 2 mM DTT, 1% DMSO and a final enzyme concentration of 50 nM to 200 nM depending on inhibitory potency. Kinetic assays were performed in Costar 3694 EIA/RIA 96-well half-area, flat bottom, black polystyrene plates (Corning, Corning, NY) at 25 °C. The increase in fluorescence intensity was measured at an emission wavelength of 528 nm (20 nm bandwidth) using an excitation wavelength of 485 (bandwidth 20 nm) using a CLARIOstar Plate Reader (BMG Labtech, Cary, NC). The initial rates of the reactions were determined from the slopes of the Relative Fluorescence Units (RFU) versus time (RFU min⁻¹). Inhibition data were collected and analyzed as described previously.⁴⁰

X-ray Structure determination of inhibitor 5e (GRL-190–21) bound to SARS-CoV-2 3CLpro.

Co-crystals of SARS-CoV-2 3CLpro with 5e (GRL-190–21) were grown at 4 °C using the sitting-drop or hanging-drop vapor diffusion method as described previously.^40,44 Briefly, 1 μL of ~110 μM SARS-CoV-2 3CLpro in 25 mM HEPES pH 7.5, 2.5 mM DTT containing ~150 μM 5e (GRL-190–21) was mixed with 2 μL of reservoir solution which contained a constant concentration of 3 mM DTT, 50 mM MES pH 6.0, 1% MPD, and varying concentrations of PEG-10,000 and KCl. Crystals that formed were removed using nylon loops and transferred into 3 μL of a cryo-solution containing the crystallization solution and inhibitor that was supplemented with 30% MPD. Crystals were soaked in the resulting solution for about 30 minutes and then flash-frozen in liquid nitrogen for X-ray data collection.

X-ray diffraction data were collected on crystals using Life Sciences Collaborative Access Team (LS-CAT) beamline 21-ID-F at the Advanced Photon Source, Argonne National Laboratory. X-ray data were indexed, integrated and scaled using the HKL2000 software package.⁴⁵ The software package PHENIX was used for structure determination via molecular replacement and iterative rounds of structural refinement.⁴⁶ Inhibitor coordinates and restraints were generated using eLBOW (PHENIX).⁴⁷ Manual modeling building was performed using Coot.⁴⁸ Automated structural refinement was performed using the Refine module avilalbe in PHENIX. The X-ray data collection and refinement statistics for the SARS-CoV-2:3Clpro GRL-190–21 inhibitor complex are summarized in Table 4.⁴³ The resulting coordinates and associated reflection files are deposited in the PDB under accession number 8UND.

Highlights.

• Design and synthesis of potent SARS-CoV-2 Mpro inhibitors are reported.

• Inhibitors with a six-membered P1 lactam exhibited improved antiviral activity.

• Inhibitors maintained potent antiviral activity against variants of concern.

• An inhibitor exhibited improved antiviral profile in immunocytochemistry assays.

• The X-ray crystal structure of SARS-CoV-2 Mpro inhibitor complex was determined.

Abreviations used

CYP

Cytochrome p450

MPro

Main Protease

PDB

Protein data base

SAR

Structure-Activity Relationship

SARS

CoV-2 Severe Acute Respiratory Syndrome Coronavirus-2

RdRp

RNA-dependent RNA polymerase

VOCs

Variants of concern

Data Availability

X-ray crystallographic data for compound 5e has been deposited at the PDB (8UND). Spectral data, assay, and X-ray data supporting this article are available for public use.