Novel Alkynylamide-Based Nonpeptidic Allosteric Inhibitors for SARS-CoV-2 3-Chymotrypsin-like Protease

Abstract

Although the coronavirus disease 2019 (COVID-19) crisis has passed, there remains a necessity for continuous efforts toward developing more targeted drugs and preparing for potential future virus attacks. Currently, most of the drugs received authorization for the treatment of COVID-19 have exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness. It is still significant to develop novel, structurally diverse small-molecule antiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease (3CL^pro). Herein, we report a class of alkynylamide-based nonpeptidic 3CL^pro inhibitors that can be prepared conveniently by our previously developed one-pot synthetic method. The structure–activity relationships of alkynylamides as SARS-CoV-2 3CL^pro inhibitors have been carefully investigated and discussed in this study. The two stereoisomers of the resulting molecules exhibit stereoselective interaction with 3CL^pro, and the optimized compound (S,R)-4y inhibits 3CL^pro with high potency (IC₅₀ = 0.43 μM), low cytotoxicity, and acceptable cell permeability. Compound (S,R)-4y presents as a noncovalent inhibitor of 3CL^pro against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. The Lineweaver–Burk plots, binding energy, surface plasmon resonance, and molecular docking studies suggest that (S,R)-4y specifically binds to an allosteric pocket of the SARS-CoV-2 3CL^pro. These findings provide a novel class of nonpeptidic alkynylamide-based allosteric inhibitors with high selectivity against SARS-CoV-2 3CL^pro featured by a simplified one-pot synthesis at room temperature in air.

1

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to global public health and economies.^1,2 By the end of June. 2024, COVID-19 had caused more than 771 million confirmed cases and 6.96 million deaths worldwide.³ Although the current virus crisis has passed, the development of antiviral agents against SARS-CoV-2 is still needed to prevent future outbreaks and the new emerging variants. Most of the drugs have received emergency use authorization for the treatment of COVID-19 but exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness.^4,5 It is still meaningful to develop novel structurally diverse small-molecule antiviral drugs targeting 3CL^pro with a simple preparation procedure.

SARS-CoV-2 has a single-stranded genomic RNA that encodes the polyproteins pp1a and pp1ab.^6,7 These polyproteins are processed by two viral proteases, the 3-chymotrypsin-like protease (3CL^pro or main protease, M^pro) and papain-like protease (PL^pro), to generate a series of functional nonstructural proteins, which are essential for virus replication and transcription. The viral main protease (3CL^pro), an indispensable tool for an intact coronaviral replication cycle, can convert the viral polyproteins into functional proteins.^8,9 As an essential component for the formation of the coronavirus replication complex, this protease is an attractive target for the discovery of anticoronavirus small-molecule therapeutics and prophylactics.¹⁰

Recently reported 3CL^pro inhibitors primarily consist of polypeptide or peptide-like drugs that covalently bind to the Cys145 residue of 3CL^pro through electrophilic covalent warheads.¹¹⁻¹⁵ This class of compounds includes GC-376,¹⁶ AG7088,¹⁷ PF-00835231,¹⁸ Nirmatrelvir (PF-07321332),¹⁹ Leritrelvir,²⁰ and Simnotrelvir,²¹ which have been developed as clinical candidates or approved for the treatment of COVID-19 (Figure 1A). Despite their high affinity and selectivity, these peptide compounds often face challenges in further development due to poor membrane permeability, oral bioavailability, and complex synthetic procedures.²² Therefore, there is a pressing need for novel nonpeptidic small molecule inhibitors targeting 3CL^pro to combat COVID-19 and potential emerging coronaviruses. Several notable examples, including S-216722,²³ masitinib,²⁴ GC-14,²⁵ walrycin B,²⁶ JZD-07,²⁷ and WU-04²⁸ (Figure 1B), have been developed with remarkable inhibitory activity and bioavailability. However, the development of nonpeptide-like inhibitors of 3CL^pro remains limited, and a few examples completed Phase III clinical trials²⁸ or were launched to market²³ thus far. Consequently, this study has attempted to develop nonpeptidomimetic 3CL^pro inhibitors with good antiviral activity and drug-like properties as well as operationally simple preparation.

Figure 1.

Chemical structures of Representative SARS-CoV-2 3CL protease inhibitors.

The alkynylamide warheads have been extensively documented as a cysteine-directed warhead for covalent protein targeting.²⁹ The binding pocket of SARS-CoV-2 3CL^pro contains five subpockets with different hydrophobic properties, including S1′, S1, S2, S3, and S4, which forms an open and three-dimensional cavity.^21,30 Thus, inhibitors with a three-dimensional structure, which are named “spherical molecules”,³¹ might advantageously provide a matched structure. Quaternary carbon centers provide a ready access to three-dimensional molecules.³¹ To design a novel and stereochemically diverse molecular library containing alkynylamide for screening against SARS-CoV-2 3CL^pro, we attempted to assemble alkynylamide covalent warheads onto a quaternary carbon center (Figure 2A) through our previously developed one-pot synthetic methods.³²⁻³⁵ These synthetic methods can assemble vicinal two quaternary carbon centers connected by a rotatable σ-bond with several hydrophobic substituents (Figure 2B). With the covalent binding of the alkylamide warhead to the residue Cys145 in the S1′ pocket, the quaternary carbon center with tunable substituents may provide a good spatial match to other subpockets. Additionally, the structural diversity can be achieved by varying the synthetic building blocks for a structure–activity relationship (SAR) investigation. The surface of the S3/S4 pocket is more hydrophobic,²¹ and it has been reported that the SARS-CoV-2 3CL^pro inhibitor with aromatic groups, such as indoles and phenyl substitutes, can interact with amino acid residues on the surface of S3 and conserve water molecules on the surface of the S4 site.³⁰ Therefore, we chose aryl imines as the third building block to construct the desired molecules.

Figure 2.

(A) Design of nonpeptidic inhibitors for SARS-CoV-2 3CL^pro. (B) Hit compound searching for anti-SARS-CoV-2 3CL^pro based on one-pot synthesis of the stereochemically diverse covalent molecular library.

2. Results and Discussion

2.1. Chemical Synthesis

To screen molecules targeting SARS-CoV-2 3CL^pro, a 58-member library containing alkynylamide warheads was efficiently synthesized using our previously developed one-pot method.³²⁻³⁵ Briefly, the method involving aryl diazo compounds 1, amides containing covalent warhead 2, and imines 3 was employed to generate amino acid derivatives containing alkynylamide warheads as the desired products. The in situ-generated ylides from aryl diazo compounds 1 and nucleophiles amides 2 could be trapped by electrophile imines 3 to construct the desired products 4 containing the quaternary carbon center. The metal-associated carbene also readily undergoes an addition reaction to the electrophilic warheads³⁶ to give the main byproducts. Gratifyingly, it was found that the Rh-associated carbene intermediate had good compatibility with the electrophilic warheads to deliver the desired molecular scaffolds containing quaternary carbon centers and alkynylamide warheads. By varying the synthetic building blocks, we quickly assembled different covalent warheads and building blocks in the target molecules, such as alkynylamide, acrylamide, and chloroacetamide groups. The various warheads and synthetic building blocks provide a structural diversity of the resulting library for the hit hunting.

Briefly, the designed compounds were conveniently prepared by a slightly modified method we reported previously.³⁷ The substrates, diazo compounds 1a–d, were prepared from commercially available arylacetic acid,³⁸ and 1e was prepared from 2-bromo-1-phenylethan-1-one.³⁹ Imines 3a–w were readily prepared from commercially available amines and aldehydes.⁴⁰ Subsequently, an efficient one-pot synthesis of alkynyl amide-substituted α,β-diamino acid derivatives from diazoles 1, amines 2, and imines 3 was conducted using the rhodium-catalyzed reaction to obtain target compounds syn-4 and anti-4 as stereoisomers in one pot (Figure 3). The desired products were efficiently synthesized at room temperature in air and finished within 0.5–2 h, and the diastereomeric isomers were readily separated by chromatographic column. It is worthy to note that all these starting points are also commercially available.

Figure 3.

One-pot synthesis of covalent molecular for hit compound search.

2.2. Screening and SARs Analysis

To identify potent inhibitors against SARS-CoV-2 3CL^pro, we first screened our library using our previously established fluorescence resonance energy transfer (FRET)-based assay^41,42 at concentrations of 5 and 50 μM. Gratifyingly, it was found that compounds containing the alkynylamide moiety generally show good inhibitory activity over those containing other covalent warheads (see Figure S1). According to the structure of the compounds containing alkynylamide moiety, we divide it into four regions named R¹, R², R³, and R⁴ (top insert of Table 1). The SARs of the tested alkynylamide derivatives are summarized in Table 1. Both the two diastereomers (syn- and anti-) of 4a, 4b, and 4c showed high inhibitory activities against SARS-CoV-2 3CL^pro when R¹ was the phenyl group (78% vs 74%, 89% vs 86%, 84% vs 86%). Introducing chlorine into the R¹ ring (4d) further enhanced the activity. The equal inhibitory activity of the syn- and anti-isomers of 4a–d indicates that the molecules using aryl diazos as synthetic building blocks are not stereochemically selective to SARS-CoV-2 3CL^pro (Table 1). We then speculated that a potential hydrogen bond donor or acceptor may improve the interaction of one isomer with 3CL^pro and result in a better inhibitory activity than that of the other isomer, providing a stereochemical effect for SARS-CoV-2 3CL^pro.

Table 1. Inhibitory Activities of Alkynylamide Derivatives against SARS-CoV-2 3CL^pro at 5 μM.

Subsequently, based on 4d, we used α-pyridyl diazoacetate as a synthetic building block to replace aryl diazo to anchor a pyridyl ring in region R¹ (Table 2). It resulted in a significant improvement capable of enhancing the differentiation of the bioactivity of the two isomers (syn-4e, 9% vs anti-4e, 65%), and the anti-isomer gave much better inhibitory activity than the syn-isomer. Following this point, we investigated the SAR of the R² region, keeping R¹ as the pyridyl group to improve the potency of anti-isomers. Halogen substitutions on phenyl ring in imine building blocks exhibited favorable inhibitory activity and selectivity for anti-isomers (4g–j) except for the electron-donating methyl group (4f). Other heterocyclic rings such as thiophene, benzofuran, and naphthalene were introduced replacing of the halogen-substituted phenyl ring to enhance the activity and selectivity of anti-isomers. It was proved unsuccessful as these anti-isomers displayed reduced inhibitory activity at 5 μM upon replacement or a diminished difference of the inhibitory bioactivity of the two isomers (4k–s). To explore the SAR in region R³, we selected 4e as the basis for the subsequent structural transformation. The introduction of halogen at the para-position of the benzene ring in region R² and R³ did not result in a significant enhancement inhibitory activity of the anti-isomers of 4t–w. Next, we investigated the incorporation of chloride atoms at the meta-position of the benzene ring in region R³. The substitution with a 3-dichloride substituted phenyl group efficiently improved the inhibitory potency of anti-isomer from 65% to 87% at 5 μM (4x). Finally, introducing a 3,5-dichloride substituted phenyl group led to a significantly enhanced inhibitory activity for anti-4y and demonstrated much better potency than the syn-isomer (syn-4y, 8% vs anti-4y, 98%), showing a good stereochemical effect for SARS-CoV-2 3CL^pro.

Table 2. Inhibitory Activities of Alkynylamide Derivatives against SARS-CoV-2 3CL^pro at 5 μM^a.

^aNo syn-isomer was obtained due to the low yield with high diastereoselctivity (anti: syn > 5:1).

The aforementioned SAR study demonstrates that the alkynylamide warhead and the 3,5-dichloride substituted phenyl group at the R³ position were optimal choices for maintaining the potent inhibition of SARS-CoV-2 3CL^pro. With the set of R¹ and R³ fragments, we returned to further investigate SARs of R² by introducing various chloride substitutions (4z–ab) on the benzene ring based on compounds anti- and syn-4y. However, introduction of chloride atoms in R² did not give an improved activity. The thiophene ring in R² still gave a poor inhibitory activity (anti- and syn-4ac) even R³ is set as 3,5-dichloride substituted phenyl group. Thus, for the desired hit, R¹, R², and R³ are then optimized as pyridyl group, phenyl group, and 3,5-dichloride-substituted phenyl group, respectively, to give a good inhibitory activity for the anti-isomer and a good stereochemical effect (anti- and syn-4y).

Exploration of SARs involving other various covalent warheads, such as acrylamide (anti- and syn-4ad), chloroacetamide (anti- and syn-4ae), benzoamide (anti- and syn-4af), and cyclopropionamide (anti- and syn-4ag), did not exhibit enhanced inhibitory activity (Table 3). These findings underscore the significance of the alkynylamide warhead in these molecules. Replacement of methyl ester with ethyl ester at the R⁴ position resulted in loss of activity (anti- and syn-4ah). Consequently, anti-4y was selected as the hit molecule for subsequent investigations.

Table 3. Inhibitory Activities of Alkynylamide Derivatives against SARS-CoV-2 3CL^pro at 5 μM.

Based on the excellent inhibitory activity of anti-4y, we attempted to isolate its enantiomers using chiral high-performance liquid chromatography (HPLC) to yield the corresponding enantiomers (Figure 4) for further bioactive identification. The optimized separation conditions using a CHIRALFLASH IC column (Daicel) enabled us to obtain multihundred milligrams of enantiopure (R,S)-4y and (S,R)-4y, and the absolute configurations of these isomers were determined by comparing the experimental circular dichroism spectra with the theoretical calculations (See Figure S2). Then, the activity of the two isolated enantiomers was tested and 10 μM VK3 was selected as the positive control, whose inhibition rate was 31.65%. As shown in Figure 4, the results showed that (S,R)-4y had better activity against SARS-CoV-2 3CL^pro than (R,S)-4y (IC₅₀ = 0.43 μM vs 0.90 μM).

Figure 4.

Inhibitory activity and cytotoxicity of three inhibitors, anti-4y, (R,S)-4y, and (S,R)-4y.

2.3. Toxicity Evaluation of the Test Compounds to Vero-E6 Cells

The cytotoxicity of anti-4y, (R,S)-4y, and (S,R)-4y in Vero-E6 cells was then evaluated. As indicated in Figure 4, anti-4y was more toxic with a CC₅₀ value of 19.88 μM. Both (R,S)-4y and (S,R)-4y have a much less cytotoxicity to Vero-E6 cells with a CC₅₀ of 36.24 and 35.69 μM, respectively. Taken together, these results suggest that (S,R)-4y possesses a favorable safety profile as a potential inhibitor against SARS-CoV-2 3CL^pro.

2.4. Binding Studies by the Surface Plasmon Resonance Assay

To determine whether anti-4y, (R,S)-4y, and (S,R)-4y directly bind to SARS-CoV-2 3CL^pro, we performed binding studies using the SPR-based assay. As shown in Figure 5, anti-4y exhibited a dose-dependent binding affinity to immobilized SARS-CoV-2 3CL^pro with an equilibrium constant (K_D) of 14.10 μM (Figure 5A). The binding rate constant (k_a) and dissociation rate constant (k_d) were determined as 1.98 × 10³ M^–1 s^–1 and 2.79 × 10^–2 s^–1, respectively, suggesting slow binding and dissociation kinetics. Compared to anti-4y, the enantiomeric isomer (R,S)-4y displayed a relatively quick binding and dissociation to SARS-CoV-2 3CL^pro, with a K_D value of 186.00 μM and k_a and k_d values of 3.32 × 10³ M^–1 s^–1 and 6.18 × 10^–1 s^–1, respectively (Figure 5B). In contrast, (S,R)-4y demonstrated a relatively fast binding and slow dissociation with SARS-CoV-2 3CL^pro, as evidenced by the values of k_a, k_d, and K_D of 4.61 × 10³ M^–1 s^–1, 7.33 × 10^–2 s^–1, and 15.90 μM, respectively (Figure 5C). Those results indicate that (S,R)-4y can rapidly bind to protease.

Figure 5.

SPR binding curves between SARS-CoV-2 3CL^pro and different concentrations of three inhibitors. (A) anti-4y, (B) (R,S)-4y, and (C) (S,R)-4y.

2.5. Binding Studies by the Inhibition Ratio Curves and Mass Spectrometry

Given the alkynylamide covalent warhead in three selected inhibitors, including anti-4y, (R,S)-4y, and (S,R)-4y, we tried to determine whether these inhibitors could form a covalent bond with SARS-CoV-2 3CL^pro. We first performed the TDI assay according to the literature-described method.⁴³ The inhibition ratios of SARS-CoV-2 3CL^pro were measured when anti-4y, (R,S)-4y, and (S,R)-4y with different concentrations were incubated with the protease at 3 and 30 min, respectively. The results show that the inhibition of anti-4y, (R,S)-4y, and (S,R)-4y on SARS-CoV-2 3CL^pro was not in a time-dependent manner (Figure 6). The IC₅₀ values exhibited minor fluctuations, changing from 0.68 to 0.76 μM, 0.92 to 0.96 μM, and 0.41 to 0.48 μM in 30 min, respectively, suggesting that they are not covalent inhibitors of SARS-CoV-2 3CL^pro. Empirically, the ratio of IC₅₀ value (3 min/30 min) is more than 3-fold, suggesting a covalent interaction of the inhibitor with the protein.⁴¹ Then, the interaction patterns between anti-4y and (S,R)-4y with proteins were further studied by mass spectrometry (UHPLC-MS). The molecular weight of small molecule binding to SARS-CoV-2 3CL^pro protein was not detected in the experimental group (35,274 Da), and only the molecular weight signals of the protein (34,773 Da) were observed, further confirming that both inhibitors anti-4y and (S,R)-4y are noncovalent bound to SARS-CoV-2 3CL^pro protein (See Figure S2).

Figure 6.

Inhibition ratio curves of SARS-CoV-2 3CL^pro under the action of the inhibitors for 3 and 30 min. (A) anti-4y. (B) (R,S)-4y. (C) (S,R)-4y. Inhibitors anti-4y, (R,S)-4y, and (S,R)-4y exhibited allosteric inhibition against SARS-CoV-2 3CL^pro.

To uncover the inhibition mode of anti-4y, (R,S)-4y, and (S,R)-4y against SARS-CoV-2 3CL^pro, the enzyme kinetic experiment was performed to determine their mechanism and type of inhibition. As presented in Figure 7A–C, Lineweaver–Burk plots⁴⁴ (1/V versus 1/[S]) for the inhibitors of SARS-CoV-2 3CL^pro were obtained with several concentrations of anti-4y, (R,S)-4y, and (S,R)-4y. The result shows that both anti-4y and (R,S)-4y dose-dependently inhibit SARS-CoV-2 3CL^pro via a mixed-inhibition manner with K_i values of 0.31 and 1.35 μM, respectively.⁴⁵ However, for (S,R)-4y, it inhibits SARS-CoV-2 3CL^pro by uncompetitive inhibition with a Ki value of 0.28 μM.⁴⁴ These results suggest that both anti-4y and (R,S)-4y may bind to both the enzymic active site and allosteric sites of SARS-CoV-2 3CL^pro, whereas (S,R)-4y may specifically target allosteric sites of SARS-CoV-2 3CL^pro.

Figure 7.

Inhibition of SARS-CoV-2 3CL^pro by a noncompetitive mechanism. (A) Lineweaver–Burk plots were used to determine the kinetic mechanism for inhibition of the SARS-CoV-2 3CL^pro by anti-4y. (B) Lineweaver–Burk plots were used to determine the kinetic mechanism for inhibition of the SARS-CoV-2 3CL^pro by (R,S)-4y. (C) Lineweaver–Burk plots were used to determine the kinetic mechanism for inhibition of the SARS-CoV-2 3CL^pro by (S,R)-4y. (D) The competition assay curve between calpeptin and (S,R)-4y was determined by SPR.

To further investigate this possibility, we performed SPR experiments using a known covalent inhibitor, Calpeptin.⁴⁶ As shown in Figure 7D, the results indicated that even with the addition of the covalent inhibitor, the best active (S,R)-4y was still able to bind to the enzyme. This suggests that (S,R)-4y likely binds to a different site from that using the known covalent inhibitor, which is consistent with the observation that (S,R)-4y interacts with an allosteric site. Therefore, both the kinetic data and the SPR results support the conclusion that (S,R)-4y, along with anti-4y and (R,S)-4y, may act as uncompetitive inhibitors that bind to allosteric sites on SARS-CoV-2 3CL^pro.

2.6. Molecular Docking Simulations of (R,S)-4y and (S,R)-4y with SARS-CoV-2 3CL^pro

To further investigate the interaction model of these inhibitors with 3CL^pro and search potential allosteric binding sites, we used molecular docking (AutoDock4)⁴⁷ to predict the binding modes of inhibitors (R,S)-4y and (S,R)-4y at the active site (7U29) and two representative allosteric sites (PDB ID: 7AGA and 7AXM) of SARS-CoV-2 3CL^pro.⁴⁵ The binding energies computed by MMGBSA analysis and docking results of compounds (R,S)-4y and (S,R)-4y with 3CL^pro protein are depicted in Table 4 and Figure 8.

Table 4. Binding Energies Computed by MMGBSA Analysis of Inhibitors (R,S)-4y and (S,R)-4y.

binding site	compound	ΔG (kcal/mol)
7U29	(R, S)-4y	–45.11
	(S, R)-4y	–31.09
7AGA (1)	(R, S)-4y	–17.48
	(S, R)-4y	–23.08
7AXM (2)	(R, S)-4y	–32.82
	(S, R)-4y	–33.76

Figure 8.

SARS-CoV-2 3CL^pro inhibitor binding modes for (S,R)-4y and (R,S)-4y. (A) Active site (7U29). (B) Binding mode of (S,R)-4y and (R,S)-4y at the active site. (C) Interactions of (R,S)-4y with the surrounding residues at the active site. (D) Interactions of (S,R)-4y with the surrounding residues at the active site. (E) Allosteric site 1 (7AGA). (F) Binding mode of (S,R)-4y and (R,S)-4y at the allosteric site 1. (G) Interactions of (R,S)-4y with the surrounding residues at the allosteric site 1. (H) Interactions of (S,R)-4y with the surrounding residues at the allosteric site 1. (I) Allosteric site 2 (7AXM). (J) Binding mode of (S,R)-4y and (R,S)-4y at the allosteric site 2. (K) Interactions of (R,S)-4y with the surrounding residues at the allosteric site 2. (L) Interactions of (S,R)-4y with the surrounding residues at the allosteric site 2.

As shown in Figure 8A, at active site (7U29), it can be observed that both compounds (S,R)-4y and (R,S)-4y exhibit distinct spatial configurations and orientations within the same binding pocket, resulting in specific interactions with surrounding amino acid residues (Figure 8B). Compound (S,R)-4y forms a hydrogen forms a hydrogen bond with the residue Gly143 using the carbonyl group of alkynylamide moiety (Figure 8D). The compound (R,S)-4y forms a hydrogen bond with the residue Glu166 using the carbonyl group of the ester group moiety, it was also found the dichlorophenyl group forms a halogen bond with residue Gln192 (Figure 8C), enhancing the molecular binding force through this unique mode of interaction.

As shown in Figure 8E, at allosteric site 1 (7AGA), compound (S,R)-4y binds deeper in the pocket than compound (R,S)-4y (Figure 8F). Compound (R,S)-4y forms a hydrogen bond with residue VAL303 using the imide group, and the chloride atom on the phenyl group forms a halogen bond with residue TYR154 (Figure 8G). In contrast to (R,S)-4y, at the same site, compound (S,R)-4y forms hydrogen bonds with residue THR111 using the pyridine group and ASN151 using amine group (Figure 8H). It was also found the 6-chloropyridyl group forms a halogen bond with residue PHE294. By comparing the binding energies of the two compounds listed in Table 4, compound (S,R)-4y shows a better binding ability with 3CL^pro at allosteric site 1 than (R,S)-4y possibly due to its more interacting forces, which is consistent with the observation that (S,R)-4y has a better inhibitory activity than (R,S)-4y (Figure 4B). The interaction of the pyridyl group with residue THR111 in compound (S,R)-4y further illustrates that introducing pyridine groups in the desired product molecules facilitates the enhancement of their bioactivity.

Comparatively, at allosteric site 2 (7AXM, Figure 8I), the binding positions of compounds (R,S)-4y and (S,R)-4y in the pocket are essentially identical (Figure 8J). Compound (R,S)-4y forms a hydrogen bond with residue SER1 using the alkynylamide group, while the chloride atom on the phenyl group forms a halogen bond with residue ARG4 (Figure 8K). While compound (S,R)-4y forms hydrogen bonds with residue ASN214 using the imide group and ILE213 using alkynylamide group, respectively. Additionally, it was also found the chloride atom on the phenyl group forms a halogen bond with residue ARG4 (Figure 8L). By comparing the binding energies of compounds at both two binding sites listed in Table 4, it was found that allosteric site 2 appears to be potentially optimal for binding. At this binding site, both compounds utilize the alkynylamide group instead of imine groups to form hydrogen bonds with 3CL^pro protein. These results suggest that the relatively rigid and electrophilic alkynylamide group may serve as a hydrogen bond donor to develop an efficient allosteric inhibitor. The superior inhibitory activity of (S,R)-4y may be attributed to its specific interaction model.

2.7. Target Selectivity of Active Compounds

To test the target selectivity of the three selected inhibitors, we carried out an enzyme inhibition assay against the host proteases. One of the main reasons for the failure of many cysteine protease inhibitors in clinical trials is the lack of target specificity.⁴⁸ Among them, the selectivity of cathepsins is particularly important because they are present in the respiratory system, which is infected by virus.⁴⁹ Though inhibiting TMPRSS2, a host serine protease, could be a promising strategy for combating SARS-CoV-2 infection because of its critical role in facilitating viral entry into host cells by cleaving the spike protein, compounds target a host protease like TMPRSS2 carries potential risks. Inhibiting its enzymatic activity might disrupt normal physiological processes in the respiratory and gastrointestinal tracts, leading to adverse side effects. Additionally, other proteases, such as TMPRSS4, might compensate for the loss of TMPRSS2 function, potentially reducing the effectiveness of the inhibition strategy.^50,51 Therefore, four host proteases (CTSB, CTSL, CTRC, and TMPRSS2) were chosen to conduct the target selectivity assay of the active compounds. Excitingly, both (R,S)-4y and (S,R)-4y did not inhibit any of the four host proteases (CTSB, CTSL, CTRC, and TMPRSS2) at concentration up to 50 μM (Figure 9B,C). Only anti-4y showed some inhibition of CTRC at 50 μM (47.85%, Figure 9A). It may be due to the drug–drug interaction of the two isomers, but the mechanism is not clear. (S,R)-4y has a stronger inhibitory effect on SARS-CoV-2 3CL^pro, and it also has good target selectivity toward these host proteases. Thus, (S,R)-4y can serve as a promising hit for further development against SARS-CoV-2.

Figure 9.

Inhibitory activity of compounds against CTSB, CTSL, CTRC, and TMPRSS2. (A) anti-4y. (B) (R,S)-4y. (C) (S,R)-4y. GC376 is a positive control for CTSB and CTSL. Delanzomib is a positive control of CTRC, and Camostat mesylate is a positive control of TMPRSS2. ***P < 0.001.

2.8. Cellular Permeability of (S,R)-4y

To identify whether the compound can permeate the cell membrane, we conducted LC–MS analysis on the lysate obtained after cell membrane removal from samples incubated with compound (S,R)-4y and Vero-E6 cells for different times. As shown in Figure 10 (and also see Figure S4), in the positive control, the residence time of compound (S,R)-4y under ES⁺ was 3.37 min, and the molecular weight was 504 (Figure 10A). Compared with the control group (Figure 10B), after being incubated with Vero-E6 cells for 12 (Figure 10C) and 24 h (Figure 10D), a distinct absorption peak at 3.38 min was observed along with a consistent molecular weight of 504, which was consistent with the positive control group. These findings indicate that compound (S,R)-4y can effectively traverse the cell membrane.

Figure 10.

LC–MS analysis of the cellular permeability of compound (S,R)-4y. (A) Molecular weight of compound (S,R)-4y in LC–MS analysis. (B) Molecular weight of blank in LC–MS analysis. (C) Molecular weight of compound (S,R)-4y after incubation with Vero-E6 cells for 12 h in LC–MS analysis. (D) Molecular weight of compound (S,R)-4y after incubation with Vero-E6 cells for 24 h in LC–MS analysis.

To further investigate the permeability of compound (S,R)-4y, we evaluated its cellular permeability using the parallel artificial membrane permeability assay (PAMPA).⁵² The market drugs methothrexate (0.003 × 10^–6 cm/s) and propranolol (5.05 × 10^–6 cm/s) were employed as controls, respectively. In comparison to these controls, compound (S,R)-4y exhibited a PAMPA permeability of 0.2 × 10^–6 cm/s, indicating acceptable cell permeability (Table 5).

Table 5. Permeability Rate of Test Compounds in the PAMPA Assay (pH 7.4).

compound	PAMPA (10–6 cm·s–1)
(S,R)-4y	0.20
methothrexate	0.003
propranolol	5.05

3. Conclusions

In summary, we have successfully designed and developed a new class of nonpeptidic inhibitors of SARS-CoV-2 3CL^pro featuring an alkynylamide moiety. Based on the acetylene amide warhead, referring to the active site of the 3CL^pro (S1–S4), through a simple, our previously developed one-pot synthesis of structurally diverse molecules, followed by comprehensive SARs studies, we identified readily accessible (S,R)-4y as a potent inhibitor of 3CL^pro. Utilizing techniques such as TDI, mass spectrometry analysis, molecular docking, SPR studies and competitive experiments, we determined that the (S,R)-4y binds to SARS-CoV-2 3CL^pro in a noncovalent allosteric manner. The Lineweaver–Burk plots, binding energy, SPR, and molecular docking simulations shed light on the potential binding mode of (S,R)-4y within the substrate-binding pocket and the dimer interface. The inhibitor (S,R)-4y demonstrated strong activity against SARS-CoV-2 3CL^pro activity with low cytotoxicity and acceptable cell permeability. Thus, given its simple synthesis procedures, (S,R)-4y and its derivatives are promising for further development as antiviral and therapeutic candidates for COVID-19, offering advantages over peptide-like drugs in terms of reduced synthetic complexity and improved metabolic stability.

4. Materials and Methods

4.1. Purification of Compounds and Separation of Enantiomers

To a 10 mL oven-dried vial containing a magnetic stirring bar, imine 3 (0.2 mmol, 1 equiv), amine 2 (0.24 mmol, 1.2 equiv), 4 Å Ms (100 mg), rac-PPA (0.2 equiv), and Rh₂(OAc)₄ (0.004 mmol, 2.0 mol %) in DCM (1 mL), a solution of diazo compound 1b (0.24 mmol, 1.2 equiv) in DCM (1 mL) was added via a syringe pump over 1 h at room temperature. After addition, the reaction mixture was stirred for an additional 2.0 h under these conditions until consumption of the material (monitored by TLC). Then, the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EA, 5:1) to give products syn-4 and anti-4. The representative compound anti-4 was separated by chiral revolution, and the optical enantiomers (R,S)-4y and (S,R)-4y were obtained using the CHIRALFLASH IC column (Daicel) optimized separation conditions. HPLC (IC, hexane/isopropanol = 85/15, flow rate = 1.0 mL/min, λ = 254 nm) t_R = 10.930 min (R,S)-4y, 7.738 min (S,R)-4y. The ¹H, ¹³C NMR, and HRMS characterization data for compounds 4a-ah are provided in the Supporting Information.

4.1.1. Methyl (2S,3S)-2,3-Diphenyl-3-(phenylamino)-2-propiolamidopropanoate (syn-4a)

White solid, 39% yield, R_f = 0.3 (PE/EA = 5/1), syn-4a: ¹H NMR (400 MHz, chloroform-d): δ 7.40 (d, J = 7.30 Hz, 2H), 7.33 (m, J = 4.68 Hz, 9H), 7.05 (t, J = 7.65 Hz, 2H), 6.67–6.56 (m, 2H), 6.44 (d, J = 7.93 Hz, 2H), 5.66 (d, J = 4.92 Hz, 1H), 3.70 (s, 3H), 2.87 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 170.7, 152.3, 146.7, 138.5, 134.9, 129.0, 128.8, 128.7, 128.4, 128.3, 127.8, 127.0, 116.8, 112.6, 77.0, 74.8, 72.0, 62.0, 53.9. HRMS(ESI) calcd for C₂₅H₂₃N₂O₃ (M + H)⁺ 399.1703; found, 399.1700.

4.1.2. Methyl (2S,3R)-2,3-Diphenyl-3-(phenylamino)-2-propiolamidopropanoate (anti-4a)

White solid, 39% yield, R_f = 0.3 (PE/EA = 5/1), anti-4a: ¹H NMR (400 MHz, chloroform-d): δ 7.27 (m, 9H), 7.18–7.12 (m, 3H), 7.06 (d, J = 7.60 Hz, 2H), 6.65 (t, J = 7.35 Hz, 1H), 6.51 (d, J = 7.90 Hz, 2H), 5.75 (s, 1H), 3.85 (s, 3H), 2.73 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 170.6, 150.9, 145.9, 137.5, 135.1, 129.2, 128.5, 128.4, 128.3, 128.2, 128.0, 128.0, 118.1, 113.7, 77.2, 73.6, 69.9, 59.5, 53.6. HRMS(ESI) calcd for C₂₅H₂₃N₂O₃ (M + H)⁺ 399.1703; found, 399.1700.

4.1.3. Methyl (2S,3S)-3-(4-Chlorophenyl)-2-phenyl-3-(phenylamino)-2-propiolamidopropanoate (syn-4b)

White solid, 36% yield, R_f = 0.3 (PE/EA = 5/1), syn-4b: ¹H NMR (400 MHz, chloroform-d): δ 7.39–7.28 (m, 10H), 7.07 (t, J = 7.74 Hz, 2H), 6.63 (dd, J = 10.71, 5.89 Hz, 2H), 6.42 (d, J = 7.99 Hz, 2H), 5.66 (d, J = 5.53 Hz, 1H), 3.73 (s, 3H), 2.93 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 170.5, 152.2, 146.3, 137.2, 134.5, 134.1, 129.1, 129.0, 128.9, 128.8, 128.5, 126.8, 117.0, 112.6, 76.8, 75.0, 71.8, 61.4, 54.0. HRMS(ESI) calcd for C₂₅H₂₂ClN₂O₃[M + H]⁺: 433.1313; found, 433.1315.

4.1.4. Methyl (2S,3R)-3-(4-Chlorophenyl)-2-phenyl-3-(phenylamino)-2-propiolamidopropanoate (anti-4b)

White solid, 36% yield, R_f = 0.3 (PE/EA = 5/1), anti-4b: ¹H NMR (400 MHz, chloroform-d): δ 7.41–7.27 (m, 7H), 7.12 (d, J = 7.55 Hz, 5H), 6.71 (t, J = 7.40 Hz, 1H), 6.50 (d, J = 7.99 Hz, 2H), 5.78 (d, J = 7.19 Hz, 1H), 5.06 (d, J = 7.19 Hz, 1H), 3.90 (s, 3H), 2.80 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 170.4, 150.9, 145.6, 136.2, 135.0, 134.0, 129.3, 129.2, 128.6, 128.6, 128.1, 127.9, 118.2, 113.6, 77.2, 73.6, 69.7, 58.8, 53.7. HRMS(ESI) calcd for C₂₅H₂₂ClN₂O₃[M + H]⁺: 433.1313; found, 433.1315.

4.1.5. Methyl (2S,3S)-3-(Benzo[b]thiophen-2-yl)-2-phenyl-3-(phenylamino)-2-propiolamidopropanoate (syn-4c)

White solid, 34% yield, R_f = 0.3 (PE/EA = 5/1), syn-4c: ¹H NMR (400 MHz, chloroform-d): δ 8.41 (t, J = 2.26 Hz, 1H), 7.77 (d, J = 7.42 Hz, 1H), 7.70 (d, J = 7.38 Hz, 1H), 7.63 (dt, J = 8.57, 2.27 Hz, 1H), 7.46 (s, 1H), 7.32 (m, 3H), 7.20 (s, 1H), 7.10 (t, J = 8.07 Hz, 2H), 6.68 (t, J = 7.33 Hz, 1H), 6.56 (t, J = 8.99 Hz, 3H), 5.99 (d, J = 6.70 Hz, 1H), 3.89 (d, J = 1.84 Hz, 3H), 2.96 (d, J = 1.75 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 152.5, 151.7, 148.4, 145.7, 142.8, 139.7, 139.4, 137.5, 129.4, 129.3, 124.6, 124.5, 124.3, 123.7, 123.0, 122.4, 118.1, 112.7, 76.4, 76.0, 70.4, 59.0, 54.8. HRMS(ESI) calcd for C₂₇H₂₃N₂O₃S (M + H)⁺ 455.1424; found, 455.1423.

4.1.6. Methyl (2S,3S)-3-(Benzo[b]thiophen-2-yl)-2-phenyl-3-(phenylamino)-2-propiolamidopropanoate (anti-4c)

White solid, 34% yield, R_f = 0.3 (PE/EA = 5/1), anti-4c: ¹H NMR (400 MHz, chloroform-d): δ 8.28 (s, 1H), 7.77–7.68 (m, 3H), 7.40 (s, 1H), 7.33 (d, J = 6.82 Hz, 3H), 7.26 (s, 1H), 7.15 (s, 3H), 6.76 (t, J = 7.47 Hz, 1H), 6.64 (d, J = 7.96 Hz, 2H), 6.23 (d, J = 9.43 Hz, 1H), 4.77 (d, J = 9.43 Hz, 1H), 3.97 (s, 3H), 2.86 (d, J = 1.96 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.5, 150.9, 149.1, 144.8, 141.6, 139.6, 139.4, 139.2, 130.2, 129.5, 124.8, 124.6, 124.1, 123.8, 123.4, 122.3, 119.4, 113.8, 77.2, 74.4, 67.9, 55.4, 54.4. HRMS(ESI) calcd for C₂₇H₂₃N₂O₃S (M + H)⁺ 455.1424; found, 455.1423.

4.1.7. Methyl (2S,3S)-2,3-bis(4-Chlorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (syn-4d)

White solid, 38% yield, R_f = 0.3 (PE/EA = 5/1), syn-4d: ¹H NMR (400 MHz, chloroform-d): δ 7.40–7.28 (m, 9H), 7.27–7.23 (m, 2H), 7.01 (d, J = 6.31 Hz, 1H), 6.58 (d, J = 1.86 Hz, 1H), 6.30 (d, J = 1.78 Hz, 2H), 5.60 (d, J = 6.32 Hz, 1H), 3.76 (s, 3H), 2.95 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 170.2, 151.0, 147.3, 136.3, 135.5, 134.7, 133.9, 129.3, 128.8, 128.8, 128.5, 127.6, 117.9, 111.6, 76.9, 73.9, 69.4, 58.2, 54.0. HRMS(ESI) calcd for C₂₅H₂₁Cl₂N₂O₃ (M + H)⁺ 467.0924; found, 467.0929.

4.1.8. Methyl (2S,3R)-2,3-bis(4-Chlorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (anti-4d)

White solid, 38% yield, R_f = 0.3 (PE/EA = 5/1), anti-4d: ¹H NMR (400 MHz, chloroform-d): δ 7.31 (m, 6H), 7.22–7.18 (m, 2H), 7.15 (dd, J = 6.72, 2.98 Hz, 2H), 6.99 (s, 1H), 6.64 (t, J = 1.80 Hz, 1H), 6.37 (d, J = 1.75 Hz, 2H), 5.74 (d, J = 7.59 Hz, 1H), 5.46 (d, J = 7.64 Hz, 1H), 3.89 (s, 3H), 2.79 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.9, 152.6, 148.0, 136.8, 135.3, 134.7, 132.9, 129.2, 129.0, 128.8, 128.3, 127.5, 116.9, 110.8, 76.5, 75.7, 71.5, 61.6, 54.3. HRMS(ESI) calcd for C₂₅H₂₁Cl₂N₂O₃ (M + H)⁺ 467.0924; found, 467.0929.

4.1.9. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-phenyl-3-(phenylamino)-2-propiolamidopropanoate (syn-4e)

White solid, 32% yield, R_f = 0.3 (PE/EA = 3/1), syn-4e: ¹H NMR (400 MHz, chloroform-d): δ 8.41 (d, J = 2.79 Hz, 1H), 7.65 (dd, J = 8.37, 2.88 Hz, 1H), 7.38–7.26 (m, 7H), 7.06 (t, J = 7.65 Hz, 2H), 6.63 (t, J = 7.38 Hz, 1H), 6.45 (t, J = 7.47 Hz, 3H), 5.62 (d, J = 6.94 Hz, 1H), 3.80 (s, 3H), 2.96 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.7, 152.4, 151.4, 148.6, 145.9, 137.9, 137.3, 130.0, 129.2, 128.9, 128.7, 127.6, 124.2, 117.5, 112.7, 76.4, 75.8, 70.2, 62.0, 54.4. HRMS(ESI) calcd for C₂₄H₂₁ClN₃O₃ (M + H)⁺ 434.1266; found, 434.1271.

4.1.10. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-phenyl-3-(phenylamino)-2-propiolamidopropanoate (anti-4e)

White solid, 24% yield, R_f = 0.3 (PE/EA = 3/1), anti-4e: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.63 Hz, 1H), 7.59 (dd, J = 8.59, 2.50 Hz, 1H), 7.31 (d, J = 4.71 Hz, 5H), 7.09 (d, J = 7.01 Hz, 4H), 6.71 (t, J = 7.41 Hz, 1H), 6.55 (d, J = 7.93 Hz, 2H), 5.73 (d, J = 9.38 Hz, 1H), 4.63 (d, J = 9.78 Hz, 1H), 3.91 (s, 3H), 2.83 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.4, 150.8, 149.3, 145.0, 139.9, 136.0, 129.9, 129.4, 128.9, 128.7, 127.7, 123.1, 119.0, 114.0, 76.6, 74.3, 67.9, 58.8, 54.0. HRMS(ESI) calcd for C₂₄H₂₁ClN₃O₃ (M + H)⁺ 434.1266; found, 434.1271.

4.1.11. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-(phenylamino)-2-propiolamido-3-(p-tolyl)propanoate (syn-4f)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), syn-4f: ¹H NMR (400 MHz, chloroform-d): δ 8.41 (d, J = 2.68 Hz, 1H), 7.65 (dd, J = 8.50, 2.73 Hz, 1H), 7.33–7.27 (m, 2H), 7.14 (s, 4H), 7.09–7.01 (m, 2H), 6.61 (t, J = 7.32 Hz, 1H), 6.48–6.37 (m, 3H), 5.57 (d, J = 6.92 Hz, 1H), 3.78 (s, 3H), 2.95 (s, 1H), 2.32 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 169.8, 152.4, 151.4, 148.6, 146.0, 138.4, 137.9, 134.1, 130.1, 129.6, 129.1, 127.4, 124.1, 117.4, 112.7, 76.4, 75.7, 70.2, 61.8, 54.4, 21.1. HRMS(ESI) calcd for C₂₅H₂₃ClN₃O₃ (M + H)⁺ 448.1422; found, 448.1428.

4.1.12. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-phenyl-3-(phenylamino)-2-propiolamidopropanoate (anti-4f)

White solid, 28% yield, R_f = 0.3 (PE/EA = 3/1), anti-4e: ¹H NMR (400 MHz, chloroform-d): δ 8.22–8.15 (m, 1H), 7.59 (dd, J = 8.52, 2.63 Hz, 1H), 7.29 (s, 1H), 7.23 (d, J = 8.56 Hz, 1H), 7.12–7.07 (m, 4H), 6.95 (d, J = 7.89 Hz, 2H), 6.71 (t, J = 7.32 Hz, 1H), 6.55 (d, J = 8.00 Hz, 2H), 5.66 (d, J = 9.83 Hz, 1H), 4.54 (d, J = 9.98 Hz, 1H), 3.90 (s, 3H), 2.83 (s, 1H), 2.31 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.3, 150.8, 149.4, 145.0, 140.0, 138.7, 132.7, 129.5, 129.4, 127.5, 123.0, 119.0, 114.1, 76.6, 74.3, 67.9, 58.9, 53.8, 21.1. HRMS(ESI) calcd for C₂₅H₂₃ClN₃O₃ (M + H)⁺ 448.1422; found, 448.1428.

4.1.13. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-(4-fluorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (syn-4g)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), syn-4f: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.68 Hz, 1H), 7.64 (dd, J = 8.47, 2.73 Hz, 1H), 7.30 (d, J = 7.82 Hz, 2H), 7.25 (dd, J = 5.55, 3.08 Hz, 2H), 7.09–7.02 (m, 4H), 6.64 (t, J = 7.31 Hz, 1H), 6.46 (d, J = 6.68 Hz, 1H), 6.42 (d, J = 7.93 Hz, 2H), 5.62 (d, J = 6.76 Hz, 1H), 3.80 (s, 3H), 2.98 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 163.9, 161.5, 152.4, 151.5, 148.6, 145.7, 137.7, 133.1, 133.1, 129.8, 129.2, 129.1, 124.2, 117.7, 116.1, 115.8, 112.7, 76.3, 75.9, 70.2, 61.3, 54.5. ¹⁹F NMR (376 MHz, CDCl₃): δ −112.9, −112.9, −113.0, −113.0. HRMS(ESI) calcd for C₂₄H₂₀ClFN₃O₃ (M + H)⁺ 452.1172; found, 452.1177.

4.1.14. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-(4-fluorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (anti-4g)

White solid, 29% yield, R_f = 0.3 (PE/EA = 3/1), anti-4f: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.69 Hz, 1H), 7.63 (dd, J = 8.52, 2.73 Hz, 1H), 7.26 (s, 1H), 7.24 (d, J = 2.60 Hz, 1H), 7.15–7.09 (m, 4H), 7.01 (t, J = 8.54 Hz, 2H), 6.73 (t, J = 7.35 Hz, 1H), 6.52 (d, J = 8.00 Hz, 2H), 5.78 (d, J = 9.32 Hz, 1H), 4.66 (d, J = 9.36 Hz, 1H), 3.92 (s, 3H), 2.84 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 163.9, 161.5, 152.4, 151.5, 148.6, 145.7, 137.7, 133.1, 133.1, 129.8, 129.2, 129.1, 124.2, 117.7, 116.1, 115.8, 112.7, 76.3, 75.9, 70.2, 61.3, 54.5. ¹⁹F NMR (376 MHz, CDCl₃): δ −112.48, −112.50, −112.50, −112.52, −112.53, −112.54, −112.55. HRMS(ESI) calcd for C₂₄H₂₀ClFN₃O₃ (M + H)⁺ 452.1172; found, 452.1177.

4.1.15. Methyl (2S,3S)-3-(4-Chlorophenyl)-2-(6-chloropyridin-3-yl)-3-(phenylamino)-2-propiolamidopropanoate (syn-4h)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), syn-4h: ¹H NMR (400 MHz, chloroform-d): δ 8.41 (d, J = 2.70 Hz, 1H), 7.63 (dd, J = 8.51, 2.80 Hz, 1H), 7.35 (q, J = 8.18, 7.11 Hz, 4H), 7.27–7.21 (m, 2H), 6.99 (d, J = 8.38 Hz, 2H), 6.59 (d, J = 6.94 Hz, 1H), 6.35 (d, J = 8.41 Hz, 2H), 5.59 (d, J = 6.95 Hz, 1H), 3.80 (s, 3H), 2.97 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.5, 152.5, 151.5, 148.6, 144.5, 137.7, 136.8, 129.8, 129.0, 129.0, 128.8, 127.5, 124.2, 122.0, 113.7, 76.3, 76.0, 70.2, 62.0, 54.5. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.16. Methyl (2S,3R)-3-(4-Chlorophenyl)-2-(6-chloropyridin-3-yl)-3-(phenylamino)-2-propiolamidopropanoate (anti-4h)

White solid, 32% yield, R_f = 0.3 (PE/EA = 3/1), anti-4h: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.68 Hz, 1H), 7.61 (dd, J = 8.50, 2.71 Hz, 1H), 7.35–7.31 (m, 3H), 7.26 (d, J = 2.26 Hz, 1H), 7.20 (s, 1H), 7.13–7.09 (m, 2H), 7.04 (d, J = 8.82 Hz, 2H), 6.46 (d, J = 8.83 Hz, 2H), 5.73 (d, J = 9.38 Hz, 1H), 4.82 (d, J = 9.43 Hz, 1H), 3.91 (s, 3H), 2.83 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.4, 150.8, 149.2, 143.6, 139.7, 135.9, 130.3, 129.3, 129.0, 128.8, 127.6, 123.5, 123.2, 115.0, 76.6, 74.3, 67.9, 58.4, 54.1. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.17. Methyl (2S,3S)-3-(4-Bromophenyl)-2-(6-chloropyridin-3-yl)-3-(phenylamino)-2-propiolamidopropanoate (syn-4i)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), syn-4i: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 3.07 Hz, 1H), 7.63 (d, J = 8.23 Hz, 1H), 7.33 (dd, J = 15.24, 8.00 Hz, 5H), 7.23 (d, J = 7.05 Hz, 2H), 7.16–7.10 (m, 2H), 6.61 (d, J = 6.77 Hz, 1H), 6.38–6.27 (m, 2H), 5.59 (d, J = 6.92 Hz, 1H), 3.80 (d, J = 2.16 Hz, 3H), 2.97 (d, J = 2.22 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.5, 152.5, 151.5, 148.6, 144.9, 137.7, 136.8, 131.9, 129.8, 129.0, 128.8, 127.4, 124.2, 114.3, 109.1, 76.3, 76.0, 70.2, 62.0, 54.5. HRMS(ESI) calcd for C₂₄H₂₀BrClN₃O₃ (M + H)⁺ 512.0371; found, 512.0378.

4.1.18. Methyl (2S,3R)-3-(4-Bromophenyl)-2-(6-chloropyridin-3-yl)-3-(phenylamino)-2-propiolamidopropanoate (anti-4i)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), anti-4i: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (s, 1H), 7.63 (d, J = 8.46 Hz, 1H), 7.33 (d, J = 3.93 Hz, 4H), 7.26 (d, J = 1.94 Hz, 1H), 7.18 (d, J = 7.48 Hz, 3H), 7.13–7.08 (m, 2H), 6.41 (d, J = 8.20 Hz, 2H), 5.75 (d, J = 9.25 Hz, 1H), 4.85 (d, J = 9.29 Hz, 1H), 3.91 (d, J = 2.05 Hz, 3H), 2.83 (d, J = 2.07 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.4, 150.8, 149.2, 144.1, 139.6, 135.9, 132.1, 130.3, 129.0, 128.8, 127.6, 123.2, 115.4, 110.6, 76.6, 74.2, 67.9, 58.1, 54.1. HRMS(ESI) calcd for C₂₄H₂₀BrClN₃O₃ (M + H)⁺ 512.0371; found, 512.0378.

4.1.19. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-(3,4-dichlorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (syn-4j)

White solid, 38% yield, R_f = 0.3 (PE/EA = 3/1), syn-4j: ¹H NMR (400 MHz, chloroform-d): δ 8.39 (d, J = 2.73 Hz, 1H), 7.61 (dd, J = 8.45, 2.75 Hz, 1H), 7.44 (d, J = 8.28 Hz, 1H), 7.36 (d, J = 2.11 Hz, 1H), 7.33–7.28 (m, 2H), 7.14 (dd, J = 8.33, 2.11 Hz, 1H), 7.09 (t, J = 7.68 Hz, 2H), 6.67 (t, J = 7.32 Hz, 1H), 6.48 (d, J = 6.63 Hz, 1H), 6.41 (d, J = 7.97 Hz, 2H), 5.60 (d, J = 6.62 Hz, 1H), 3.81 (s, 3H), 3.01 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 152.5, 151.7, 148.4, 145.4, 138.1, 137.6, 133.2, 132.9, 131.0, 129.5, 129.5, 129.3, 126.8, 124.3, 118.0, 112.6, 76.3, 76.1, 70.0, 61.2, 54.7. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.20. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-(3,4-dichlorophenyl)-3-(phenylamino)-2-propiolamidopropanoate (anti-4j)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), anti-4j: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.70 Hz, 1H), 7.69–7.64 (m, 1H), 7.41 (d, J = 8.29 Hz, 1H), 7.28 (d, J = 2.55 Hz, 1H), 7.26 (d, J = 2.00 Hz, 1H), 7.20 (s, 1H), 7.16–7.10 (m, 2H), 7.03 (d, J = 2.18 Hz, 1H), 6.75 (s, 1H), 6.50 (d, J = 8.05 Hz, 2H), 5.85 (d, J = 8.86 Hz, 1H), 4.79 (s, 1H), 3.95 (s, 3H), 2.85 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.6, 150.8, 149.1, 144.5, 139.6, 137.3, 133.2, 133.0, 130.7, 129.6, 126.9, 123.4, 119.2, 113.6, 76.4, 74.5, 67.9, 56.7, 54.3. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.21. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-2-propiolamido-3-(thiophen-2-yl)-3-(p-tolylamino)propanoate (4k)

White solid, 65% yield, R_f = 0.3 (PE/EA = 3/1), syn/anti isomer (1:1) ratio. Mixture of (syn/anti) isomers of 4k: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (d, J = 2.72 Hz, 1H), 8.22 (d, J = 2.68 Hz, 1H), 7.62 (m, 2H), 7.43 (s, 1H), 7.34–7.22 (m, 6H), 7.10 (d, J = 8.33 Hz, 2H), 7.04 (d, J = 8.29 Hz, 2H), 6.98 (m, 2H), 6.90 (dd, J = 16.76, 3.60 Hz, 2H), 6.62 (d, J = 6.59 Hz, 1H), 6.53 (d, J = 8.37 Hz, 2H), 6.43 (d, J = 8.39 Hz, 2H), 6.11 (d, J = 9.47 Hz, 1H), 5.90 (d, J = 6.58 Hz, 1H), 4.75 (d, J = 9.57 Hz, 1H), 3.93 (s, 3H), 3.84 (s, 3H), 3.00 (s, 1H), 2.86 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 169.3, 152.5, 151.7, 151.5, 150.8, 149.1, 148.4, 144.4, 143.5, 141.3, 140.3, 139.4, 137.4, 129.4, 129.1, 127.2, 127.0, 126.3, 126.1, 126.0, 124.3, 123.3, 122.6, 115.0, 113.8, 76.3, 76.1, 74.4, 70.5, 68.2, 58.6, 54.7, 54.3. HRMS(ESI) calcd for C₂₃H₂₁ClN₃O₃S (M + H)⁺ 454.0987; found, 454.0980.

4.1.22. Methyl (2S,3S)-3-((4-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4l)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), anti-4l: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (d, J = 2.71 Hz, 1H), 7.58 (d, J = 2.75 Hz, 1H), 7.40–7.29 (m, 4H), 7.20–7.14 (m, 2H), 6.90 (d, J = 7.02 Hz, 1H), 6.62 (t, J = 1.75 Hz, 1H), 6.28 (d, J = 1.74 Hz, 2H), 5.59 (d, J = 7.03 Hz, 1H), 3.83 (s, 3H), 3.02 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 152.7, 151.8, 148.4, 147.2, 137.4, 135.5, 135.0, 134.8, 129.4, 129.2, 128.6, 124.4, 117.6, 110.9, 76.6, 76.0, 70.1, 61.0, 54.8. HRMS(ESI) calcd for C₂₂H₁₈Cl₂N₃O₃S (M + H)⁺ 474.0440; found, 474.0435.

4.1.23. Methyl (2S,3S)-3-((4-Bromophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4m)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), anti-4m: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (s, 1H), 7.59 (dd, J = 8.44, 2.88 Hz, 1H), 7.44 (s, 1H), 7.30 (t, J = 8.13 Hz, 2H), 7.22–7.14 (m, 2H), 6.99 (s, 1H), 6.91 (d, J = 3.63 Hz, 1H), 6.64 (d, J = 6.60 Hz, 1H), 6.38 (d, J = 8.25 Hz, 2H), 5.89 (d, J = 6.61 Hz, 1H), 3.83 (d, J = 1.85 Hz, 3H), 3.00 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 152.7, 151.8, 148.4, 147.2, 137.4, 135.5, 135.0, 134.8, 129.4, 129.2, 128.6, 124.4, 117.6, 110.9, 76.6, 76.0, 70.1, 61.0, 54.8. HRMS(ESI) calcd for C₂₂H₁₈BrClN₃O₃S (M + H)⁺ 517.9935; found, 517.9939.

4.1.24. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-((3-fluorophenyl)amino)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4n)

White solid, 31% yield, R_f = 0.3 (PE/EA = 3/1), anti-4n: ¹H NMR (400 MHz, chloroform-d): δ 8.23 (d, J = 2.73 Hz, 1H), 7.65 (dd, J = 8.56, 2.75 Hz, 1H), 7.31 (s, 1H), 7.29–7.24 (m, 2H), 7.07 (t, J = 7.60 Hz, 1H), 6.98 (dd, J = 5.06, 3.59 Hz, 1H), 6.90 (d, J = 3.54 Hz, 1H), 6.44 (td, J = 8.35, 2.34 Hz, 1H), 6.37 (dd, J = 8.10, 2.25 Hz, 1H), 6.30 (dt, J = 11.26, 2.36 Hz, 1H), 6.14 (d, J = 9.32 Hz, 1H), 4.89 (d, J = 9.36 Hz, 1H), 3.94 (s, 3H), 2.86 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 165.1, 162.7, 151.5, 150.8, 149.1, 146.8, 146.7, 140.3, 139.4, 130.7, 130.6, 130.1, 127.3, 127.1, 126.1, 123.4, 109.5, 105.9, 105.7, 100.9, 100.7, 76.6, 74.4, 68.2, 54.6, 54.4. HRMS(ESI) calcd for C₂₂H₁₈ClFN₃O₃S (M + H)⁺ 458.0736; found, 458.0731.

4.1.25. Methyl (2S,3S)-3-((3-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamido-3-(thiophen-2-yl)propanoate (4o)

White solid, 61% yield, R_f = 0.3 (PE/EA = 3/1), syn/anti isomer (3:2) ratio. Mixture of (syn/anti) isomers of 4o: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (d, J = 2.70 Hz, 2H), 8.22 (d, J = 2.65 Hz, 1H), 7.66 (dd, J = 8.48, 2.73 Hz, 1H), 7.59 (dd, J = 8.51, 2.75 Hz, 2H), 7.46 (s, 2H), 7.35 (s, 1H), 7.33–7.31 (m, 2H), 7.29–7.25 (m, 4H), 7.01 (td, J = 5.18, 3.48 Hz, 3H), 6.96–6.91 (m, 4H), 6.72 (t, J = 1.72 Hz, 1H), 6.65 (t, J = 1.74 Hz, 2H), 6.46 (d, J = 1.75 Hz, 2H), 6.38 (d, J = 1.76 Hz, 4H), 6.18 (d, J = 9.10 Hz, 1H), 5.91 (d, J = 6.74 Hz, 2H), 5.15 (d, J = 9.10 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 6H), 3.02 (s, 2H), 2.86 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.5, 169.1, 152.7, 151.8, 151.6, 150.9, 149.0, 148.3, 147.5, 146.6, 140.2, 139.6, 139.1, 137.3, 135.8, 135.5, 130.2, 129.0, 127.4, 127.3, 127.3, 126.5, 126.3, 126.3, 124.4, 123.6, 119.0, 117.8, 111.8, 110.9, 76.6, 76.4, 76.2, 74.5, 70.5, 68.1, 58.1, 54.9, 54.5, 53.7. HRMS(ESI) calcd for C₂₂H₁₈Cl₂N₃O₃S (M + H)⁺ 474.0440; found, 474.0435.

4.1.26. Methyl (2S,3S)-3-((3-Chloro-4-fluorophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4p)

White solid, 29% yield, R_f = 0.3 (PE/EA = 3/1), anti-4p: ¹H NMR (400 MHz, chloroform-d): δ 8.37 (d, J = 2.75 Hz, 1H), 7.57 (dd, J = 8.46, 2.79 Hz, 1H), 7.47 (d, J = 8.30 Hz, 1H), 7.38–7.30 (m, 3H), 7.08 (dd, J = 8.33, 2.19 Hz, 1H), 6.91 (d, J = 6.92 Hz, 1H), 6.65 (t, J = 1.79 Hz, 1H), 6.28 (d, J = 1.83 Hz, 2H), 5.57 (d, J = 6.94 Hz, 1H), 3.85 (s, 3H), 3.05 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.0, 152.8, 152.0, 148.3, 147.0, 140.2, 137.3, 136.0, 135.7, 129.5, 128.9, 125.8, 124.5, 118.1, 110.8, 77.2, 75.7, 70.1, 61.0, 54.9. HRMS(ESI) calcd for C₂₂H₁₇Cl₂FN₃O₃S (M + H)⁺ 492.0346; found, 492.0348.

4.1.27. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-((3,4-dichlorophenyl)amino)-2-propiolamido-3-(thiophen-2-yl)propanoate (syn-4q)

White solid, 40% yield, R_f = 0.3 (PE/EA = 3/1), syn-4p: ¹H NMR (400 MHz, chloroform-d): δ 8.36 (d, J = 2.75 Hz, 1H), 7.56 (dd, J = 8.53, 2.80 Hz, 1H), 7.46–7.28 (m, 3H), 7.12 (d, J = 1.87 Hz, 2H), 6.90 (d, J = 6.86 Hz, 1H), 6.68 (t, J = 1.75 Hz, 1H), 6.29 (d, J = 1.78 Hz, 2H), 5.55 (d, J = 6.90 Hz, 1H), 3.86 (s, 3H), 3.06 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.0, 152.8, 152.0, 148.3, 147.0, 140.2, 137.3, 136.0, 135.7, 129.5, 128.9, 125.8, 124.5, 118.1, 110.8, 77.2, 75.7, 70.1, 61.0, 54.9. HRMS(ESI) calcd for C₂₂H₁₇Cl₃N₃O₃S (M + H)⁺ 508.0051; found, 508.0057.

4.1.28. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-((3,4-dichlorophenyl)amino)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4q)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), anti-4q: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (d, J = 2.73 Hz, 1H), 7.60 (dd, J = 8.48, 2.75 Hz, 1H), 7.45 (s, 1H), 7.33–7.27 (m, 2H), 7.20–7.14 (m, 2H), 6.99 (dd, J = 5.08, 3.55 Hz, 1H), 6.92 (d, J = 3.61 Hz, 1H), 6.65 (d, J = 6.60 Hz, 1H), 6.46–6.33 (m, 2H), 5.90 (d, J = 6.61 Hz, 1H), 3.83 (s, 3H), 3.00 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.3, 152.6, 151.7, 148.3, 144.8, 141.2, 137.4, 132.0, 129.3, 127.2, 126.3, 126.1, 124.3, 114.3, 109.7, 76.3, 76.2, 70.5, 58.4, 54.7. HRMS(ESI) calcd for C₂₂H₁₇Cl₃N₃O₃S (M + H)⁺ 508.0051; found, 508.0057.

4.1.29. Methyl (2S,3S)-3-(Benzofuran-2-yl)-2-(6-chloropyridin-3-yl)-3-((4-methoxyphenyl)amino)-2-propiolamidopropanoate (anti-4r)

White solid, 26% yield, R_f = 0.3 (PE/EA = 3/1), anti-4r: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.76 Hz, 1H), 7.62 (dd, J = 8.53, 2.64 Hz, 1H), 7.50 (d, J = 9.49 Hz, 2H), 7.39 (d, J = 8.16 Hz, 1H), 7.29 (d, J = 8.04 Hz, 2H), 7.23 (d, J = 7.41 Hz, 1H), 6.76–6.67 (m, 3H), 6.51 (d, J = 8.35 Hz, 2H), 5.95 (d, J = 8.39 Hz, 1H), 5.82 (d, J = 8.39 Hz, 1H), 3.94 (s, 3H), 3.71 (d, J = 1.81 Hz, 3H), 2.89 (d, J = 1.73 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.7, 155.1, 154.2, 152.4, 152.2, 151.5, 148.5, 140.1, 137.8, 129.3, 127.8, 124.4, 124.0, 123.1, 121.3, 115.0, 113.9, 111.0, 106.8, 76.3, 75.6, 68.9, 58.8, 55.7, 54.8. HRMS(ESI) calcd for C₂₇H₂₃ClN₃O₅ (M + H)⁺ 504.1321; found, 504.1315.

4.1.30. Methyl (2S,3R)-3-((4-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-3-(naphthalen-1-yl)-2-propiolamidopropanoate (anti-4s)

White solid, 29% yield, R_f = 0.3 (PE/EA = 3/1), anti-4s: ¹H NMR (400 MHz, chloroform-d): δ 8.46 (d, J = 2.60 Hz, 1H), 8.23 (d, J = 8.51 Hz, 1H), 7.93 (d, J = 8.08 Hz, 1H), 7.86 (d, J = 8.15 Hz, 1H), 7.71–7.66 (m, 1H), 7.64 (d, J = 7.20 Hz, 1H), 7.55 (m, 3H), 7.33 (dd, J = 8.49, 2.10 Hz, 2H), 6.96–6.89 (m, 2H), 6.70 (d, J = 6.30 Hz, 1H), 6.34 (q, J = 6.07, 4.14 Hz, 3H), 3.49 (d, J = 2.09 Hz, 3H), 3.00 (d, J = 2.08 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 152.7, 151.4, 148.7, 144.4, 137.9, 133.8, 131.9, 131.0, 130.4, 129.7, 129.6, 129.1, 127.1, 126.6, 125.7, 125.5, 124.1, 122.3, 121.1, 113.7, 76.3, 76.1, 69.4, 54.3. HRMS(ESI) calcd for C₂₈H₂₂Cl₂N₃O₃ (M + H)⁺ 518.1033; found, 518.1037.

4.1.31. Methyl (2S,3S)-3-((4-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-3-phenyl-2-propiolamidopropanoate (syn-4t)

White solid, 41% yield, R_f = 0.3 (PE/EA = 3/1), syn-4t: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.72 Hz, 1H), 7.63 (dd, J = 8.44, 2.76 Hz, 1H), 7.32 (dd, J = 11.21, 8.35 Hz, 4H), 7.21 (d, J = 8.45 Hz, 2H), 7.06 (t, J = 7.70 Hz, 2H), 6.64 (t, J = 7.32 Hz, 1H), 6.47 (d, J = 6.80 Hz, 1H), 6.41 (d, J = 7.98 Hz, 2H), 5.61 (d, J = 6.86 Hz, 1H), 3.80 (s, 3H), 2.99 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 152.4, 151.6, 148.5, 145.6, 137.7, 136.1, 134.5, 129.7, 129.2, 129.2, 128.9, 124.2, 117.7, 112.7, 76.2, 76.1, 70.1, 61.4, 54.6. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.32. Methyl (2S,3R)-3-((4-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-3-phenyl-2-propiolamidopropanoate (anti-4t)

White solid, 39% yield, R_f = 0.3 (PE/EA = 3/1), anti-4t: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.66 Hz, 1H), 7.64 (dd, J = 8.46, 2.83 Hz, 1H), 7.32–7.28 (m, 2H), 7.27 (s, 1H), 7.24 (d, J = 8.42 Hz, 1H), 7.14–7.07 (m, 4H), 6.73 (s, 1H), 6.51 (d, J = 7.99 Hz, 2H), 5.80 (d, J = 9.25 Hz, 1H), 4.69 (d, J = 9.26 Hz, 1H), 3.93 (s, 3H), 2.84 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.5, 150.8, 149.2, 144.7, 139.7, 135.0, 134.7, 130.1, 129.6, 129.5, 129.0, 129.0, 123.3, 119.1, 113.8, 113.6, 76.5, 74.4, 67.9, 57.6, 54.2. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.33. Methyl (2S,3S)-3-(4-Chlorophenyl)-3-((4-chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamidopropanoate (syn-4u)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), syn-4u: ¹H NMR (400 MHz, chloroform-d): δ 8.39 (d, J = 2.91 Hz, 1H), 7.60 (dd, J = 8.30, 3.35 Hz, 1H), 7.36–7.28 (m, 4H), 7.21–7.09 (m, 4H), 6.61 (d, J = 6.78 Hz, 1H), 6.37–6.24 (m, 2H), 5.57 (d, J = 6.81 Hz, 1H), 3.81 (d, J = 2.26 Hz, 3H), 3.00 (d, J = 2.31 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 152.5, 151.7, 148.5, 144.6, 137.6, 135.5, 134.7, 132.0, 129.5, 129.3, 128.8, 124.3, 114.2, 109.5, 76.3, 76.1, 70.1, 61.4, 54.6. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.34. Methyl (2S,3R)-3-(4-Chlorophenyl)-3-((4-chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-2-propiolamidopropanoate (anti-4u)

White solid, 29% yield, R_f = 0.3 (PE/EA = 3/1), anti-4u: ¹H NMR (400 MHz, chloroform-d): δ 8.21 (d, J = 2.84 Hz, 1H), 7.71–7.62 (m, 1H), 7.30 (td, J = 8.63, 1.85 Hz, 3H), 7.22–7.17 (m, 2H), 7.15 (s, 1H), 7.12–7.06 (m, 2H), 6.43–6.32 (m, 2H), 5.80 (d, J = 8.87 Hz, 1H), 4.90 (d, J = 8.88 Hz, 1H), 3.93 (d, J = 1.94 Hz, 3H), 2.84 (d, J = 1.92 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 152.5, 151.7, 148.5, 144.2, 137.6, 135.5, 134.76 129.5, 129.3, 129.1, 128.8, 124.3, 122.4, 113.7, 76.3, 76.1, 70.1, 61.5, 54.7. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.35. Methyl (2S,3R)-3-((4-Bromophenyl)amino)-3-(4-chlorophenyl)-2-(6-chloropyridin-3-yl)-2-propiolamidopropanoate (anti-4v)

White solid, 29% yield, R_f = 0.3 (PE/EA = 3/1), anti-4v: ¹H NMR (400 MHz, chloroform-d): δ 8.21 (d, J = 2.84 Hz, 1H), 7.71–7.62 (m, 1H), 7.30 (td, J = 8.63, 1.85 Hz, 3H), 7.22–7.17 (m, 2H), 7.15 (s, 1H), 7.12–7.06 (m, 2H), 6.43–6.32 (m, 2H), 5.80 (d, J = 8.87 Hz, 1H), 4.90 (d, J = 8.88 Hz, 1H), 3.93 (d, J = 1.94 Hz, 3H), 2.84 (d, J = 1.92 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 152.5, 151.7, 148.5, 144.2, 137.6, 135.5, 134.76 129.5, 129.3, 129.1, 128.8, 124.3, 122.4, 113.7, 76.3, 76.1, 70.1, 61.5, 54.7. HRMS(ESI) calcd for C₂₄H₁₉BrCl₂N₃O₃ (M + H)⁺ 545.9981; found, 545.9987.

4.1.36. Methyl (2S,3R)-3-(4-Chlorophenyl)-2-(6-chloropyridin-3-yl)-3-((3,4-dichlorophenyl)amino)-2-propiolamidopropanoate (anti-4w)

White solid, 33% yield, R_f = 0.3 (PE/EA = 3/1), anti-4w: ¹H NMR (400 MHz, chloroform-d): δ 8.39 (d, J = 2.72 Hz, 1H), 7.60 (dd, J = 8.46, 2.79 Hz, 1H), 7.34 (dd, J = 16.40, 6.88 Hz, 4H), 7.17 (d, J = 8.33 Hz, 2H), 7.08 (d, J = 8.68 Hz, 1H), 6.77 (d, J = 6.97 Hz, 1H), 6.46 (d, J = 2.73 Hz, 1H), 6.25 (d, J = 2.74 Hz, 1H), 5.58 (d, J = 6.96 Hz, 1H), 3.82 (s, 3H), 3.02 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.2, 152.6, 151.8, 148.4, 145.1, 137.5, 135.0, 134.9, 132.9, 130.7, 129.4, 129.3, 128.7, 124.3, 120.4, 113.5, 112.6, 76.5, 76.0, 70.1, 61.3, 54.8. HRMS(ESI) calcd for C₂₄H₁₈Cl₄N₃O₃ (M + H)⁺ 536.0097; found, 536.0091.

4.1.37. Methyl (2S,3S)-3-((3-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-3-phenyl-2-propiolamidopropanoate (syn-4x)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), syn-4x: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.67 Hz, 1H), 7.63 (m, 1H), 7.35 (m, 4H), 7.30 (d, J = 3.22 Hz, 1H), 7.25 (t, J = 5.60 Hz, 2H), 6.89 (s, 1H), 6.71 (dd, J = 18.79, 7.47 Hz, 2H), 6.57 (d, J = 2.62 Hz, 1H), 6.35 (d, J = 8.16 Hz, 1H), 5.61 (dd, J = 7.16, 2.23 Hz, 1H), 3.81 (d, J = 2.35 Hz, 3H), 2.98 (d, J = 2.34 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.5, 152.5, 151.6, 148.5, 147.2, 137.7, 136.6, 130.5, 129.7, 129.0, 128.8, 127.4, 124.2, 123.2, 120.3, 115.1, 111.5, 76.2, 76.0, 70.2, 61.7, 54.5. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.38. Methyl (2S,3R)-3-((3-Chlorophenyl)amino)-2-(6-chloropyridin-3-yl)-3-phenyl-2-propiolamidopropanoate (anti-4x)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), anti-4x: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.70 Hz, 1H), 7.64 (dd, J = 8.49, 2.74 Hz, 1H), 7.33 (p, J = 3.90 Hz, 3H), 7.28–7.25 (m, 1H), 7.18 (s, 1H), 7.13 (dd, J = 6.71, 2.93 Hz, 2H), 6.94 (t, J = 8.00 Hz, 1H), 6.81 (dd, J = 7.79, 1.74 Hz, 1H), 6.69 (t, J = 2.05 Hz, 1H), 6.43 (dd, J = 8.11, 2.32 Hz, 1H), 5.78 (d, J = 9.24 Hz, 1H), 4.95 (d, J = 9.29 Hz, 1H), 3.91 (s, 3H), 2.82 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.4, 150.9, 149.2, 146.3, 139.6, 135.8, 130.7, 130.4, 129.0, 128.9, 127.5, 123.3, 123.3, 121.6, 116.7, 112.1, 76.6, 74.3, 67.9, 57.7, 54.2. HRMS(ESI) calcd for C₂₄H₂₀Cl₂N₃O₃ (M + H)⁺ 468.0876; found, 468.0873.

4.1.39. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenyl-2-propiolamidopropanoate (syn-4y)

White solid, 39% yield, R_f = 0.3 (PE/EA = 3/1), syn-4y: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.61 Hz, 1H), 7.62 (dd, J = 8.62, 2.75 Hz, 1H), 7.37 (dt, J = 7.38, 3.93 Hz, 3H), 7.32 (d, J = 9.85 Hz, 1H), 7.22 (d, J = 6.86 Hz, 2H), 6.91 (d, J = 7.06 Hz, 1H), 6.60 (d, J = 2.29 Hz, 1H), 6.30 (s, 2H), 5.60 (d, J = 7.13 Hz, 1H), 3.83 (d, J = 2.10 Hz, 3H), 2.99 (d, J = 2.11 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.3, 152.7, 151.7, 148.5, 147.5, 137.5, 136.1, 135.4, 129.5, 129.1, 129.0, 127.3, 124.3, 117.4, 110.9, 76.3, 76.1, 70.2, 61.6, 54.6. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.40. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenyl-2-propiolamidopropanoate (anti-4y)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), anti-4y: ¹H NMR (400 MHz, chloroform-d): δ 8.21 (s, 1H), 7.71–7.63 (m, 1H), 7.42–7.35 (m, 3H), 7.29 (dd, J = 8.85, 2.13 Hz, 1H), 7.19–7.13 (m, 2H), 7.08 (s, 1H), 6.67 (s, 1H), 6.39 (s, 2H), 5.81 (d, J = 8.85 Hz, 1H), 5.22 (d, J = 8.48 Hz, 1H), 3.93 (d, J = 2.21 Hz, 3H), 2.82 (d, J = 2.21 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.5, 150.9, 149.1, 146.8, 139.3, 135.7, 135.6, 130.6, 129.2, 129.0, 127.4, 123.4, 118.5, 111.8, 76.6, 74.2, 67.9, 57.1, 54.3. HRMS(ESI) calcd for C₂₄H₁₉Cl₃N₃O₃ (M + H)⁺ 502.0487; found, 502.0486.

4.1.41. Methyl (2S,3S)-3-(4-Chlorophenyl)-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (syn-4z)

White solid, 33% yield, R_f = 0.3 (PE/EA = 3/1), syn-4z: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (t, J = 2.23 Hz, 1H), 7.59 (dt, J = 8.47, 2.29 Hz, 1H), 7.40–7.29 (m, 4H), 7.17 (dd, J = 8.51, 1.96 Hz, 2H), 6.90 (d, J = 7.01 Hz, 1H), 6.62 (d, J = 2.18 Hz, 1H), 6.28 (s, 2H), 5.59 (d, J = 7.04 Hz, 1H), 3.83 (d, J = 1.89 Hz, 3H), 3.03 (d, J = 1.90 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 152.7, 151.8, 148.4, 147.2, 137.4, 135.5, 135.0, 134.8, 129.4, 129.2, 128.6, 124.4, 117.6, 110.9, 76.6, 76.0, 70.1, 61.0, 54.8. HRMS(ESI) calcd for C₂₄H₁₈Cl₄N₃O₃ (M + H)⁺ 536.0097; found, 536.0091.

4.1.42. Methyl (2S,3R)-3-(4-Chlorophenyl)-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (anti-4z)

White solid, 33% yield, R_f = 0.3 (PE/EA = 3/1), anti-4z: ¹H NMR (400 MHz, chloroform-d): δ 8.20 (d, J = 2.60 Hz, 1H), 7.67 (dd, J = 8.54, 2.63 Hz, 1H), 7.37–7.33 (m, 2H), 7.31 (d, J = 8.44 Hz, 1H), 7.15–7.07 (m, 3H), 6.69 (d, J = 2.00 Hz, 1H), 6.36 (d, J = 2.04 Hz, 2H), 5.86 (d, J = 8.59 Hz, 1H), 5.25 (d, J = 8.59 Hz, 1H), 3.94 (d, J = 1.30 Hz, 3H), 2.84 (d, J = 1.34 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.5, 151.7, 150.9, 149.0, 146.5, 139.1, 135.8, 135.1, 134.3, 130.5, 129.3, 128.8, 123.6, 118.7, 111.7, 76.5, 74.5, 67.9, 56.1, 54.5. HRMS(ESI) calcd for C₂₄H₁₈Cl₄N₃O₃ (M + H)⁺ 536.0097; found, 536.0091.

4.1.43. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-(3,4-dichlorophenyl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (syn-4aa)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), syn-4aa: ¹H NMR (400 MHz, chloroform-d): δ 8.37 (d, J = 2.69 Hz, 1H), 7.56 (d, J = 2.70 Hz, 1H), 7.47 (d, J = 8.30 Hz, 1H), 7.37–7.30 (m, 3H), 7.08 (d, J = 8.22 Hz, 1H), 6.90 (d, J = 6.90 Hz, 1H), 6.66 (d, J = 1.67 Hz, 1H), 6.29 (d, J = 1.65 Hz, 2H), 5.57 (d, J = 6.90 Hz, 1H), 3.85 (s, 3H), 3.05 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.0, 152.7, 152.0, 148.3, 147.0, 137.3, 136.8, 135.6, 133.6, 133.4, 131.2, 129.3, 128.9, 126.5, 124.5, 118.0, 110.8, 76.9, 75.8, 70.1, 60.8, 54.9. HRMS(ESI) calcd for C₂₄H₁₇Cl₅N₃O₃ (M + H)⁺ 569.9707; found, 569.9702.

4.1.44. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-(3,4-dichlorophenyl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (anti-4aa)

White solid, 34% yield, R_f = 0.3 (PE/EA = 3/1), anti-4aa: ¹H NMR (400 MHz, chloroform-d): δ 8.20 (d, J = 2.67 Hz, 1H), 7.66 (dd, J = 8.44, 2.71 Hz, 1H), 7.46 (d, J = 8.27 Hz, 1H), 7.33–7.27 (m, 3H), 7.11–7.04 (m, 2H), 6.72 (d, J = 1.68 Hz, 1H), 6.37 (d, J = 1.75 Hz, 2H), 5.89 (d, J = 8.38 Hz, 1H), 5.26 (d, J = 8.40 Hz, 1H), 3.96 (s, 3H), 2.86 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 177.4, 150.9, 149.0, 146.2, 139.0, 136.5, 135.9, 130.9, 130.5, 130.5, 129.1, 126.9, 123.7, 118.9, 111.5, 76.3, 74.7, 67.9, 55.4, 54.6. HRMS(ESI) calcd for C₂₄H₁₇Cl₅N₃O₃ (M + H)⁺ 569.9707; found, 569.9702.

4.1.45. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-(3,5-dichlorophenyl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (syn-4ab)

White solid, 25% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ab: ¹H NMR (400 MHz, chloroform-d): δ 8.38 (d, J = 2.70 Hz, 1H), 7.59 (dd, J = 8.55, 2.69 Hz, 1H), 7.46 (s, 1H), 7.36–7.29 (m, 2H), 7.02 (t, J = 4.38 Hz, 1H), 6.94 (dd, J = 11.93, 5.15 Hz, 2H), 6.65 (d, J = 2.18 Hz, 1H), 6.37 (d, J = 1.93 Hz, 2H), 5.91 (d, J = 6.72 Hz, 1H), 3.86 (s, 3H), 3.02 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 152.7, 151.8, 148.3, 147.4, 140.2, 137.3, 135.5, 129.0, 127.3, 126.5, 126.3, 124.4, 117.8, 110.9, 76.4, 76.1, 70.5, 58.1, 54.9. HRMS(ESI) calcd for C₂₄H₁₇Cl₅N₃O₃ (M + H)⁺ 569.9707; found, 569.9702.

4.1.46. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-(3,5-dichlorophenyl)-3-((3,5-dichlorophenyl)amino)-2-propiolamidopropanoate (anti-4ab)

White solid, 26% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ab: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.66 Hz, 1H), 7.67 (dd, J = 8.54, 2.69 Hz, 1H), 7.28 (d, J = 9.34 Hz, 3H), 7.00 (t, J = 4.33 Hz, 1H), 6.95 (s, 1H), 6.73 (s, 1H), 6.46 (s, 2H), 6.18 (d, J = 9.05 Hz, 1H), 5.14 (d, J = 9.09 Hz, 1H), 3.95 (s, 3H), 2.87 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 151.7, 150.8, 149.0, 146.6, 139.6, 139.2, 135.8, 130.2, 127.4, 127.3, 126.2, 123.6, 119.0, 111.8, 74.4, 68.1, 54.5, 53.7. HRMS(ESI) calcd for C₂₄H₁₇Cl₅N₃O₃ (M + H)⁺ 569.9707; found, 569.9702.

4.1.47. Methyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-2-propiolamido-3-(thiophen-2-yl)propanoate (syn-4ac)

White solid, 32% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ac: ¹H NMR (400 MHz, chloroform-d): δ 8.36 (d, J = 2.73 Hz, 1H), 7.56 (dd, J = 8.48, 2.69 Hz, 1H), 7.35 (dd, J = 13.94, 5.53 Hz, 3H), 7.12 (d, J = 1.94 Hz, 2H), 6.90 (d, J = 6.84 Hz, 1H), 6.68 (s, 1H), 6.29 (s, 2H), 5.55 (d, J = 6.90 Hz, 1H), 3.86 (s, 3H), 3.06 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.0, 152.8, 152.0, 148.3, 147.0, 140.2, 137.3, 136.0, 135.7, 129.5, 128.9, 125.8, 124.5, 118.1, 110.8, 75.7, 70.1, 61.0, 55.0. HRMS(ESI) calcd for C₂₂H₁₇Cl₃N₃O₃S (M + H)⁺ 508.0051; found, 508.0057.

4.1.48. Methyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-2-propiolamido-3-(thiophen-2-yl)propanoate (anti-4ac)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ac: ¹H NMR (400 MHz, chloroform-d): δ 8.23–8.13 (m, 1H), 7.65 (dt, J = 8.46, 2.30 Hz, 1H), 7.37 (d, J = 1.86 Hz, 1H), 7.32 (d, J = 8.49 Hz, 1H), 7.11 (d, J = 6.20 Hz, 3H), 6.74 (d, J = 2.00 Hz, 1H), 6.37 (s, 2H), 5.89 (d, J = 8.36 Hz, 1H), 5.25 (d, J = 8.21 Hz, 1H), 3.97 (d, J = 1.50 Hz, 3H), 2.88 (d, J = 1.46 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 151.8, 150.9, 148.9, 146.1, 139.9, 139.1, 135.9, 135.8, 130.4, 129.5, 125.9, 123.7, 119.0, 111.5, 76.2, 74.8, 68.0, 55.4, 54.7. HRMS(ESI) calcd for C₂₂H₁₇Cl₃N₃O₃S (M + H)⁺ 508.0051; found, 508.0057.

4.1.49. Methyl (2R,3R)-2-Acrylamido-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (syn-4ad)

White solid, 42% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ad: ¹H NMR (400 MHz, chloroform-d): δ 8.37 (d, J = 2.68 Hz, 1H), 7.60 (dd, J = 8.47, 2.66 Hz, 1H), 7.47 (d, J = 6.97 Hz, 1H), 7.33 (q, J = 2.74, 1.64 Hz, 3H), 7.19 (dd, J = 6.16, 2.77 Hz, 2H), 7.00 (s, 1H), 6.58 (d, J = 1.77 Hz, 1H), 6.45–6.29 (m, 3H), 6.22 (dd, J = 16.84, 10.16 Hz, 1H), 5.83 (d, J = 10.14 Hz, 1H), 5.61 (d, J = 6.97 Hz, 1H), 3.82 (d, J = 1.31 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 170.0, 166.2, 151.4, 148.5, 147.8, 137.6, 136.5, 135.4, 130.2, 129.6, 129.5, 129.0, 128.9, 127.4, 124.2, 117.0, 110.7, 69.5, 61.7, 54.5. HRMS(ESI) calcd for C₂₄H₂₁Cl₃N₃O₃ (M + H)⁺ 504.0643; found, 504.0647.

4.1.50. Methyl (2R,3S)-2-acrylamido-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (anti-4ad)

White solid, 43% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ad: ¹H NMR (400 MHz, chloroform-d): δ 8.20 (d, J = 2.63 Hz, 1H), 7.72 (dd, J = 8.48, 2.62 Hz, 1H), 7.35–7.29 (m, 3H), 7.27 (d, J = 4.80 Hz, 1H), 7.12 (q, J = 3.67, 2.93 Hz, 2H), 6.81 (s, 1H), 6.66 (d, J = 2.00 Hz, 1H), 6.41 (d, J = 1.90 Hz, 2H), 6.28 (d, J = 16.88 Hz, 1H), 6.08 (s, 1H), 5.94 (d, J = 8.72 Hz, 1H), 5.72 (d, J = 10.27 Hz, 1H), 5.34 (s, 1H), 3.93 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 170.3, 164.5, 151.2, 149.1, 147.0, 139.4, 136.0, 135.6, 131.5, 130.0, 128.9, 128.8, 128.5, 127.5, 123.4, 118.3, 111.7, 111.7, 67.5, 56.9, 54.2. HRMS(ESI) calcd for C₂₄H₂₁Cl₃N₃O₃ (M + H)⁺ 504.0643; found, 504.0647.

4.1.51. Methyl (2R,3R)-2-(2-Chloroacetamido)-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (syn-4ae)

White solid, 38% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ae: ¹H NMR (400 MHz, chloroform-d): δ 8.37 (d, J = 2.17 Hz, 1H), 8.01 (s, 1H), 7.63–7.58 (m, 1H), 7.39–7.32 (m, 4H), 7.21–7.16 (m, 2H), 7.03 (d, J = 7.06 Hz, 1H), 6.60 (d, J = 1.70 Hz, 1H), 6.31 (d, J = 1.65 Hz, 2H), 5.62 (d, J = 7.07 Hz, 1H), 4.10 (d, J = 1.40 Hz, 2H), 3.83 (d, J = 1.41 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 169.6, 167.5, 151.6, 148.4, 147.6, 137.6, 136.1, 135.4, 129.7, 129.1, 129.1, 127.1, 124.3, 117.3, 110.8, 69.5, 61.5, 54.6, 42.5. HRMS(ESI) calcd for C₂₃H₂₀Cl₄N₃O₃ (M + H)⁺ 526.0253; found, 526.0255.

4.1.52. Methyl (2R,3S)-2-(2-Chloroacetamido)-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (anti-4ae)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ae: ¹H NMR (400 MHz, chloroform-d): δ 8.24 (d, J = 2.24 Hz, 1H), 7.80 (s, 1H), 7.67 (dd, J = 8.50, 2.17 Hz, 1H), 7.39–7.32 (m, 3H), 7.29 (dd, J = 8.60, 1.66 Hz, 1H), 7.11 (dd, J = 5.67, 2.97 Hz, 2H), 6.67 (d, J = 2.13 Hz, 1H), 6.40 (d, J = 1.90 Hz, 2H), 5.74 (d, J = 8.69 Hz, 1H), 5.29 (d, J = 8.66 Hz, 1H), 3.99–3.97 (m, 1H), 3.92 (d, J = 1.74 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 169.8, 165.4, 151.5, 149.0, 146.9, 139.1, 135.6, 135.5, 130.9, 129.2, 129.0, 127.3, 123.4, 118.5, 111.8, 67.4, 57.9, 54.2, 42.5. HRMS(ESI) calcd for C₂₃H₂₀Cl₄N₃O₃ (M + H)⁺ 526.0253; found, 526.0255.

4.1.53. Methyl (2R,3R)-2-Benzamido-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (syn-4af)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), syn-4af: ¹H NMR (400 MHz, chloroform-d): δ 8.43 (t, J = 2.17 Hz, 1H), 7.71 (d, J = 7.68 Hz, 2H), 7.68–7.63 (m, 2H), 7.59 (t, J = 7.87 Hz, 2H), 7.48 (t, J = 7.20 Hz, 2H), 7.32 (dt, J = 5.78, 3.83 Hz, 4H), 7.24 (dt, J = 6.52, 3.70 Hz, 2H), 6.59 (d, J = 2.05 Hz, 1H), 6.36 (d, J = 1.92 Hz, 2H), 5.68 (d, J = 6.94 Hz, 1H), 3.85 (d, J = 1.68 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 170.3, 168.3, 151.4, 148.6, 147.9, 137.7, 136.6, 135.4, 133.1, 132.7, 130.4, 129.0, 129.0, 128.9, 127.4, 127.1, 124.2, 117.0, 110.8, 69.6, 61.7, 54.5. HRMS(ESI) calcd for C₂₈H₂₃Cl₃N₃O₃ (M + H)⁺ 554.0800; found, 554.0806.

4.1.54. Methyl (2R,3S)-2-Benzamido-2-(6-chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (anti-4af)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), anti-4af: ¹H NMR (400 MHz, chloroform-d): δ 8.27 (d, J = 2.22 Hz, 1H), 7.79 (dt, J = 8.51, 2.29 Hz, 1H), 7.64 (d, J = 7.67 Hz, 2H), 7.58–7.51 (m, 1H), 7.48–7.39 (m, 3H), 7.30 (dq, J = 7.91, 4.62 Hz, 4H), 7.16 (d, J = 6.68 Hz, 2H), 6.67 (d, J = 1.90 Hz, 1H), 6.44 (d, J = 2.12 Hz, 2H), 6.03 (d, J = 8.66 Hz, 1H), 5.39 (d, J = 8.69 Hz, 1H), 3.96 (d, J = 1.69 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 170.5, 166.4, 151.2, 149.1, 147.0, 139.4, 136.1, 135.7, 133.5, 132.3, 131.6, 129.0, 128.9, 128.8, 127.5, 126.9, 123.4, 118.3, 111.7, 67.6, 57.3, 54.2. HRMS(ESI) calcd for C₂₈H₂₃Cl₃N₃O₃ (M + H)⁺ 554.0800; found, 554.0806.

4.1.55. Methyl (2R,3R)-2-(6-Chloropyridin-3-yl)-2-(cyclopropanecarboxamido)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (syn-4ag)

White solid, 35% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ag: ¹H NMR (400 MHz, chloroform-d): δ 8.36 (d, J = 2.67 Hz, 1H), 7.61 (dd, J = 8.51, 2.66 Hz, 1H), 7.49 (d, J = 7.08 Hz, 1H), 7.36 (d, J = 6.60 Hz, 3H), 7.31 (d, J = 8.57 Hz, 1H), 7.22–7.17 (m, 2H), 7.10 (s, 1H), 6.56 (d, J = 1.88 Hz, 1H), 6.28 (d, J = 2.04 Hz, 2H), 5.56 (d, J = 7.13 Hz, 1H), 3.81 (s, 3H), 1.54 (d, J = 4.42 Hz, 1H), 1.11–0.94 (m, 2H), 0.88 (dt, J = 7.69, 4.06 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 174.8, 170.1, 151.2, 148.4, 147.9, 137.7, 136.7, 135.3, 130.7, 128.9, 128.8, 127.5, 124.1, 116.8, 110.6, 69.4, 61.6, 54.4, 15.1, 8.2, 7.7. HRMS(ESI) calcd for C₂₅H₂₃Cl₃N₃O₃ (M + H)⁺ 518.0800; found, 518.0801.

4.1.56. Methyl (2R,3S)-2-(6-Chloropyridin-3-yl)-2-(cyclopropanecarboxamido)-3-((3,5-dichlorophenyl)amino)-3-phenylpropanoate (anti-4ag)

White solid, 33% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ag: ¹H NMR (400 MHz, chloroform-d): δ 8.19 (d, J = 2.69 Hz, 1H), 7.66 (dd, J = 8.54, 2.54 Hz, 1H), 7.33 (t, J = 3.25 Hz, 3H), 7.27 (d, J = 1.99 Hz, 1H), 7.12 (q, J = 3.08, 2.51 Hz, 2H), 6.87 (s, 1H), 6.39 (d, J = 1.95 Hz, 2H), 5.85 (d, J = 8.71 Hz, 1H), 5.33 (d, J = 8.69 Hz, 1H), 3.91 (d, J = 1.67 Hz, 3H), 1.38 (d, J = 4.51 Hz, 1H), 1.00 (s, 1H), 0.86 (dd, J = 25.16, 6.74 Hz, 2H), 0.74 (d, J = 3.04 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 172.9, 170.4, 151.0, 149.0, 147.0, 139.3, 136.1, 135.6, 131.8, 128.9, 128.7, 127.6, 123.3, 118.2, 111.7, 67.4, 57.4, 54.0, 15.1, 7.8, 7.2. HRMS(ESI) calcd for C₂₅H₂₃Cl₃N₃O₃ (M + H)⁺ 518.0800; found, 518.0801.

4.1.57. Ethyl (2S,3S)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenyl-2-propiolamidopropanoate (syn-4ah)

White solid, 36% yield, R_f = 0.3 (PE/EA = 3/1), syn-4ah: ¹H NMR (400 MHz, chloroform-d): δ 8.40 (d, J = 2.65 Hz, 1H), 7.62 (dd, J = 8.56, 2.74 Hz, 1H), 7.36 (d, J = 5.40 Hz, 3H), 7.33 (d, J = 3.01 Hz, 2H), 7.24 (s, 2H), 6.92 (d, J = 7.13 Hz, 1H), 6.60 (d, J = 2.02 Hz, 1H), 6.30 (d, J = 1.78 Hz, 2H), 5.60 (d, J = 7.16 Hz, 1H), 4.26 (d, J = 7.09 Hz, 2H), 2.99 (s, 1H), 1.26 (d, J = 2.06 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 168.7, 152.7, 151.6, 148.5, 147.6, 137.5, 136.1, 135.4, 129.7, 129.1, 129.0, 127.4, 124.2, 117.3, 110.9, 76.2, 76.1, 70.2, 64.6, 61.4, 29.7, 13.8. HRMS(ESI) calcd for C₂₅H₂₁Cl₃N₃O₃ (M + H)⁺ 516.0643; found, 516.0641.

4.1.58. Ethyl (2S,3R)-2-(6-Chloropyridin-3-yl)-3-((3,5-dichlorophenyl)amino)-3-phenyl-2-propiolamidopropanoate (anti-4ah)

White solid, 32% yield, R_f = 0.3 (PE/EA = 3/1), anti-4ah: ¹H NMR (400 MHz, chloroform-d): δ 8.22 (d, J = 2.64 Hz, 1H), 7.68 (dd, J = 8.53, 2.69 Hz, 1H), 7.39–7.33 (m, 3H), 7.30 (d, J = 8.48 Hz, 1H), 7.19 (dt, J = 6.05, 3.45 Hz, 2H), 7.09 (s, 1H), 6.67 (d, J = 1.66 Hz, 1H), 6.37 (d, J = 1.77 Hz, 2H), 5.84 (d, J = 8.80 Hz, 1H), 5.34 (d, J = 8.84 Hz, 1H), 4.41 (dd, J = 7.21, 3.91 Hz, 2H), 2.81 (s, 1H), 1.39 (t, J = 7.14 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 169.1, 151.4, 150.8, 149.1, 146.8, 139.3, 135.7, 135.6, 130.9, 129.1, 128.9, 127.5, 123.5, 118.4, 111.6, 74.1, 67.7, 64.3, 56.7, 14.0. HRMS(ESI) calcd for C₂₅H₂₁Cl₃N₃O₃ (M + H)⁺ 516.0643; found, 516.0641.

4.2. Enzymatic Inhibition Assay

SARS-CoV-2 3CL^pro was expressed and purified according to our previous method.^41,42 Fluorescent substrates Dabcyl-KNSTLQSGLRK-Edans (GenScript Biotech Corporation, Nanjing, China) were synthesized to assess the activity of SARS-CoV-2 3CL^pro. In brief, the test compounds were dissolved in DMSO to obtain 10 mM stock solutions. Then, the test compounds were diluted to the indicated concentration and incubated with SARS-CoV-2 3CL^pro for 30 min at 37 °C. Upon addition of the substrate (20 μM, final concentration) for 20 min at 37 °C, the measurement was started and recorded using a Cytation 5 plate reader (BioTek, USA) with excitation and emission wavelengths of 340 and 490 nm, respectively. The IC₅₀ value was determined by plotting the initial velocity against various concentrations of the test compounds using a dose–response curve in software 8.0 (GraphPad Software, Inc., San Diego, CA, USA). All experiments were performed in triplicate, and data are presented as mean ± SD.

4.3. Surface Plasmon Resonance Assay

SPR assay was performed according to our previous reported methods.^41,42 Briefly, the binding affinities between the compounds [anti-4y, (R,S)-4y, and (S,R)-4y] and SARS-CoV-2 3CL^pro were assessed using Biacore T200 instruments (Cytiva, Sweden). The amine-coupling approach was employed to immobilize SARS-CoV-2 3CL^pro onto the CM5 chip surface by using a 10 mM sodium acetate buffer (pH 5.5) at a flow rate of 10 μL/min. 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) were mixed to activate the sensor surface for 7 min. Subsequently, 30 μg/mL of SARS-CoV-2 3CL^pro was injected at a flow rate of 10 μL/min for 10 min. Following this, 1 M ethanolamine at pH 8.5 was injected to block the surface of the chip. A series of concentrations of compounds containing 5% dimethyl sulfoxide (DMSO) were injected into the flow cell with association and dissociation times of 60 s, respectively. All binding assays were conducted in PBS with 0.05% (v/v) Tween-20 and 5% DMSO, pH 7.4, at 25 °C. Before analysis, bulk refractive index changes, injection noise, and data drift were eliminated by double reference subtractions and solvent corrections. The binding affinities were evaluated using a 1:1 Langmuir binding model in Biacore Evaluation software (Cytiva, Sweden).

4.4. Determination of the Inhibition Mode

Different concentrations of the test compounds (0, 2, 4, and 6 μM) were incubated with SARS-CoV-2 3CL^pro for 30 min at 37 °C with slow shaking, followed by different concentrations of substrates (5, 10, 20, 40, and 80 μM), which were added and incubated under the same conditions for 20 min. Fluorescence values were assayed using Cytation 5 plate reader (BioTek, USA) with excitation and emission wavelengths of 340 and 490 nm, respectively. The data analysis was performed using a Lineweaver–Burk plot by fitted the reciprocal of velocity (1/V) vs the reciprocal of the substrate concentration (1/[S]). The inhibitory constant (K_i) was obtained as described before.^41,42

4.5. Cell Viability Assay

Vero-E6 cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (YEASEN, China). The cytotoxicity of the compounds in Vero-E6 cells was determined by the CCK8 assay (Meilunbio, China). In short, the preseeded Vero-E6 cells (1 × 10⁵ cells/mL) were incubated with the test compounds at various concentrations for 24 h, and then the medium was replaced with 10% CCK-8 solution. The absorbance was detected at a wavelength of 450 nm by Cytation 5 Microplate Reader (BioTek, Winooski, VT, USA).

4.6. Cathepsin B and Cathepsin L Enzymatic Assay

Cathepsin B (CTSB) and cathepsin L (CTSL) (Sino Biological, Beijing, China) activity was assessed according to our previous method.^41,42 CTSB and CTSL were diluted to 100 nM to activate in the buffer (20 mM sodium acetate, 1 mM EDTA and 2 mM DTT, pH 5.5) at 30 °C. After 30 min, test compounds were incubated with CTSB (500 pM) or CTSL (300 pM) in the assay buffer (100 mM MES, 1 mM EDTA, 2 mM DTT, and 0.01% Tween 20, pH 6.0) for 30 min. The mixture was added with 5 μM of substrate Z-Phe-Arg-AMC (NJPeptide, Nanjing, China) to initiate the reaction. Fluorescence signals (λ_ex = 360 nm; λ_em = 460 nm) were recorded through a Cytation 5 Microplate Reader (BioTek, Winooski, VT, USA).

4.7. Human Chymotrypsin C Enzymatic Assay

The activity of human chymotrypsin C (CTRC) was measured based on our previous publication.^41,42 CTRC (5 μg/mL) (Novoprotein, Suzhou, China) was incubated with the test sample in reaction buffer (50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5) at 37 °C. After 30 min, trypsin (1 μg/mL) (Sigma, New Jersey, US) was added and incubated for 60 min. Substrate Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (NJPeptide, Nanjing, China) (100 μM) was added, and the absorbance (450 nm) was detected immediately for 1 h.

4.8. TMPRSS2 Enzymatic Assay

TMPRSS2 activity was determined as previously described.^41,42 In brief, TMPRSS2 was diluted to a final concentration of 5 nM in the reaction buffer (50 mM Tris–HCl, pH 8.0, 1 mM CaCl₂, 0.1% Tween 20) and incubated with the test compound at 37 °C. After 60 min, 45 μM of Boc-GIn-Ala-Arg-AMC (NJPeptide, Nanjing, China) was added to initiate the reaction for 90 min. Fluorescence signals at 380 nm (excitation)/460 nm (emission) were immediately measured using a Cytation 5 Microplate Reader (BioTek, Winooski, VT, USA).

4.9. Potential Binding Mode of (R,S)-4y and (S,R)-4y in SARS-CoV-2 3CL^pro

In this study, all molecular docking experiments were conducted using the AutoDock4.1 (AD4) software.⁴³ Initially, water and cofactors were removed from these protein crystal structures (7U29, 7AGA, and 7AXM), and receptor structures were hydrogenated using AutoDockTools (ADT). Gasteiger charges were calculated, and PDBQT files were generated accordingly.

For compounds (R,S)-4y and (S,R)-4y, a flexible docking approach was employed to enhance the accuracy of the results, allowing for a more precise prediction of possible binding modes between the receptor and ligand. To reduce unnecessary computational complexity, specific amino acid residues in the protein binding sites were set as flexible, while the rest remained fixed. Additionally, the ligand was hydrogenated using ADT, Gasteiger charges were computed, and torsion centers and torsion bonds were detected to construct a torsion tree, resulting in the generation of the ligand PDBQT file.

Subsequently, AutoGrid4 was utilized to generate a receptor grid with dimensions of 60 Å × 60 Å × 60 Å and a spacing of 0.375 Å, using the original ligand coordinates in the protein crystal structure as the center. The receptor interaction energy was calculated using ligand atomic types as probes. Finally, the Lamarckian Genetic Algorithm was employed for docking simulations, outputting the top 50 docking poses. The optimal docking structure was selected based on the analysis of docking poses, scoring results, and interactions between the ligand and the receptor.

4.10. Parallel Artificial Membrane Permeability Assay

The membrane permeability of selected compounds was evaluated using the Corning BioCoatTM Precoated PAMPA Plate System (Corning 353015; Glendale, AZ, USA). The precoated plate assembly, which was stored at −20 °C, was taken to thaw for 30 min at room temperature. The permeability assay was carried out in accordance with the manufacturer’s protocol. Briefly, the 96-well filter plate, precoated with lipids, was used as the permeation acceptor, and a matching 96 well receiver plate was used as the permeation donor. Compound solutions were prepared by diluting the 10 mmol/L DMSO stock solutions with 10% methanol in DPBS to a final concentration of 10 μmol/L. The compound solutions were added to the wells (300 μL/well) of the receiver plate, and DPBS with 10% methanol was added to the wells (200 μL/well) of the precoated filter plate. The filter plate was then coupled with the receiver plate, and the plate assembly was incubated at 25 °C without agitation for 5 h. At the end of the incubation, the plate was separated, and the final concentrations of compounds in both donor wells and acceptor wells were analyzed using LC–MS/MS.⁵²

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00369.

• Inhibitory effects of 4a–ah against SARS-CoV-2 3CL^pro, absolute configuration of (R,S)-4y and (S,R)-4y determined by theoretical circular dichroism analysis and experimental analysis, mass spectra of native SARS-CoV-2 3CL^pro after incubation with DMSO, anti-4y, and (S,R)-4y, ¹H, ¹³C, and ¹⁹F NMR spectra data of 4a–ah, and LC–MS analysis of the cellular permeability of the compound (S,R)-4y (PDF)

Author Contributions

^∥ Jian Xue and Hongtao Li contributed equally to this work. Shunying Liu and Lili Chen conceived and designed the experiments. Jian Xue, Hongtao Li, Ruyu Wang, Meiting Wang, Yaqi Deng, Xixiang Chen, Yexi Li, Yuheng Song, and Jianrong Xu carried out the experiments and data analysis. Jian Xue, Hongtao Li, Jiani Lu, Tong Zhu, Lili Chen, and Shunying Liu wrote the manuscript. Tong Zhu, Shunying Liu, and Lili Chen critically checked and revised the manuscript.

The authors declare no competing financial interest.