Pharmacophore-Based Virtual Screening and Structural Modification of Novel Benzamide Derivatives as HBV Capsid Assembly Modulators

Abstract

Hepatitis B virus (HBV) infection is the most common cause of death from liver disease worldwide. The use of capsid assembly modulators is considered a prominent strategy for the development of novel anti-HBV therapies. We performed a pharmacophore-based virtual screening strategy, and a benzamide scaffold hit, WAI-5, was chosen for further structural optimization. A series of novel HBV capsid assembly modulators (CAMs) were found. Compared with the lead hit, the representative compounds 11g and 11n exhibited a 10-fold increase in anti-HBV activity with 50% effective concentration (EC₅₀) values of 1.74 and 1.90 µM, respectively.

INTRODUCTION

Hepatitis B virus (HBV) is a member of the hepadnaviridae family and causes nearly one million deaths worldwide yearly.^1,2) The current treatment of HBV infection relies on nucleotide analogs (NAs) and interferons (IFNs).³⁾ However, long-term use of NAs frequently causes side effects, and IFNs-based therapies are associated with poor tolerance. In addition, the available treatments are still unable to achieve a functional cure for HBV infection.^4–6)

The HBV nucleocapsid plays a crucial role in genome packaging and maintenance during the viral replication cycle. The core protein homodimer encapsulates pregenomic RNA (pgRNA) and a polymerase to form a biologically competent HBV nuclear capsid, which protects the closed viral genome and creates an environment for reverse transcription of pgRNA into DNA.^7–12) Disruption of normal functional capsid formation prevents the replication of infectious viral particles and amplification of the covalently closed circular DNA (cccDNA) libraries.¹³⁾ Therefore, targeting the capsid protein assembly is considered as an important strategy for a novel anti-HBV therapy.

In recent years, various capsid assembly modulators (CAMs) with different structural scaffolds have been reported as anti-HBV drug candidates, including heteroaryl-dihydropyrimidines (HAPs), bis-heterocycles, sulfamoyl benzamides (SBAs), phenyl acrylamides (PPAs), pyridazinones and others.¹⁴⁾ Several of them have progressed into clinical research (Fig. 1).

Fig. 1. Drug Candidates in Clinical Trials for the Treatment of HBV

Our previous work established a virtual screening model using the Specs commercial database containing 212959 structures with HBV core protein (Protein Data Bank (PDB): 5E0I&5WRE). Ninety-three compounds were obtained through the BlockBuster filters and pan-assay interference, Glide SP docking, and the Prime MM/GBSA analysis. Subsequently, manual selection and biological evaluation led to the discovery of 2-aryl-4-quinolyl amide derivatives as novel HBV capsid modulators.¹⁵⁾ As a continuation of this work, we apply here a pharmacophore-based virtual screening strategy to obtain a new scaffold hit from these 93 compounds, followed by structural optimization for improving anti-HBV activities.

MATERIALS AND METHODS

Pharmacophore-Based Virtual Screening

The Discovery Studio 4.5 Pharmacophores function was employed to generate a pharmacophore model. The SBAs show potent anti-HBV activity in clinical trials, therefore three of the compounds are selected as active molecules during the process. Different pharmacophore hypotheses are generated by defining various features of three active compounds. The best hypothesis was selected based on the score, a combination of the vector, site, volume scores, and a term for the number of matches. This pharmacophore hypothesis was used for subsequent screening. Grounded in the pharmacophore model generated above, virtual screening was conducted by a pharmacophore search protocol in Discovery studio.

Molecular Docking

The cocrystal structure of the HBV capsid Y132A mutant (VCID 8772) in complex with NVR10-001E2 was obtained from the RCSB Protein Data Bank (PDB: 5E0I, resolution: 1.95 Å). All ligands and bound water molecules were removed from the protein structure before protein preparation. The NVR10-001E2 binding pocket of the cocrystal structure was defined as a binding site. The binding pocket was defined as all residues within 5 Å of the original ligand. The water molecules were removed, atom types were checked, and hydrogens were added. All single bonds of ligand and residue side chains inside the binding pocket were regarded as rotatable. KOLL all charges were loaded to the protein, and Gasteiger–Hückel charges were assigned to the ligand atoms. The structural optimization was performed for 100000 generations using a TRIPOS forcefield and genetic algorithm. Docked models of the ligand-receptor complex were visualized using PyMOL.

Chemistry

Chart 1 displays the synthetic route of target compounds 9a–l. Compounds 12 and 15 were commercially available and used as starting materials. Compound 12 was condensed with methyl bromoacetate to obtain 13, followed by hydrolysis of the ester moiety to obtain intermediate 14. Compound 15 was reacted with various R₁-substituted amines to yield intermediate compound 16, which was condensed with intermediate compound 14 to achieve target compounds 9a–l.

Chart 1. Synthetic Route to Obtain Compounds 9a–l

Chart 2 displayed the synthetic routes to target compounds 10a, 10b, and 11a–p. Compound 15 reacted with phenylethylamine to yield intermediate compound 17, which was condensed with chloroacetyl chloride to obtain intermediate compound 18. After condensation with m-toluidine or N-(m-tolyl)acetamide, the target compounds 10a and 10b were obtained. Different R₃-substituted amines 19a–p reacted with methanesulfonyl chloride to obtain various intermediate compounds 20a–p, which were condensed with intermediate compound 18 to obtain the final target compounds 11a–p.

Chart 2. Synthetic Route to Obtain Compounds 10a–b and 11a–p

Anti-HBV Activity

The test compound was prepared into a homogeneous 10 mM stock solution using dimethyl sulfoxide (DMSO). One hundred times dilution of 10 mM stock solution with complete culture medium (Dulbecco’s modified Eagle’s medium (DMEM) medium containing 2% fetal bovine serum (FBS) and 1‰ DMSO) to obtain 100 µM stock solution, and 10 times dilution of the stock solution to obtain 10 µM dilution of the test compound. The starting concentration of 10 µM was diluted 3 times to obtain a total of 8 concentrations of gradient concentration dilutions. HepG2.2.15 cells grown in log phase were digested, blown into a single cell suspension, inoculated with 1 × 10⁴ cells/well in a 96-well cell culture plate, and incubated at 37 °C for 24 h in a 5% CO₂ incubator. On the second day, after the cell adhesion, a gradient concentration of the test compound is added and incubated at 37 °C for 4 d in a 5% CO₂ incubator. On the 5th day, the cells were replaced with fresh drug-containing culture medium and incubated for 3 d. The cell supernatant was collected on the 8th day, and the HBV DNA copy number in the supernatant was detected by qPCR according to the operation of the hepatitis B virus nucleic acid detection kit (Shenzhen Xincheng Biotechnology Co., China), and the results were recorded. The inhibition rate was calculated using the formula “Inhibition rate = (HBV DNA copy number of blank group − HBV DNA copy number of experimental group)/HBV DNA copy number of blank group × 100%,” and lgC was used as the X value and the corresponding inhibition rate was used as the Y value, and the inhibition rate was calculated using Graphpad Prism. The 50% effective concentration (EC₅₀) values were obtained by fitting with Graphpad Prism 8.0.2.

Cytotoxicity Assays

Dilute 10 mM stock solution 10 times with complete culture medium (DMEM medium containing 2% FBS and 1‰ DMSO) to obtain 100 µM stock solution. Using 100 µM as the starting concentration, dilute 3 times to obtain 8 concentrations of gradient concentration diluted solution. The protocols of stock solution preparation, seeding plate, drug administration and drug exchange are the same as anti-HBV assays. Discard the supernatant on the 8th day, add 100 µL of medium and 10 µL of CCK-8 solution to each well, incubate for 1 h at 37 °C in an incubator, then measure the absorbance value at 450 nm with an enzyme marker and record the results. The cell survival rate was calculated according to the following formula, with lgC as the X value and the corresponding survival rate as the Y value, and the 50% cytotoxicity concentration (CC₅₀) value was obtained by fitting with Graphpad Prism 8.0.2. Cell survival rate (%) = [(As−Ab)/(Ac−Ab)] × 100%. As: absorbance of the experimental wells (culture containing the administered cells, CCK-8); Ac: absorbance of control wells (culture medium containing unadministered cells, CCK-8); Ab: absorbance of blank wells (blank culture medium without cells, CCK-8).

RESULTS AND DISCUSSION

Combination of Pharmacophore Based and Docking Based Screening

Pharmacophore modeling makes use of compound collections to generate structural patterns present in active compounds.^16–19) Pharmacophore-based virtual screening relies on molecular simulation technology, which is fast, versatile, and easy to operate. Additionally, the tandem approach is performed to obtain hits efficiently by combining structure-based virtual screening with ligand-based pharmacophore screening. For instance, a novel Pim-1 kinase inhibitor (compound 7) was discovered by employing a proposed hierarchical multistage virtual screening approach containing pharmacophore-based and docking-based virtual screening²⁰⁾ (Fig. 2A). Similarly, compound 8 was identified as a lead of Src kinase inhibitors with the same method²¹⁾ (Fig. 2B). Inspired by above results, we envisioned that combining molecular docking and pharmacophore-based virtual screening could be a feasible strategy to discover novel CAM hits.

Fig. 2. Representative Examples of Pharmacophore-Based Screening

Among all HBV CAMs clinical candidates, SBA derivatives aroused special attention due to their excellent anti-HBV potency. For our new virtual screening strategy, we determined a common SBA pharmacophore based on AB-423, JNJ-56136379, and NVR 3–778 (Fig. 3A), featuring an aromatic ring, four acceptors, a donor, and a hydrophobic group (Fig. 3B). The 93 compounds obtained by previous molecular docking virtual screening were employed as substrates. After analyzing their overlap data with the pharmacophore model, six compounds with at least four overlapping properties were selected for an anti-HBV biological evaluation (Fig. 3C).

Fig. 3. Pharmacophore Model Generated by SBAs and the Matched Six Compounds

(A) The pharmacophore properties of SBAs; (B) The pharmacophore model generated by Discovery Studio; (C) Structures of six matched compounds.

As shown in Table 1, compounds WAI-1, WAI-4, and WAI-5 could inhibit HBV DNA replication with moderate to good activities, indicating that our discovery strategy is reasonable. Especially, the compound WAI-5 showed the most potent anti-HBV activity with 63.9% inhibition at 20.0 µM EC₅₀ value of 11.17 µM against viral DNA replication, accompanied with weak in vitro cytotoxicity (CC₅₀ > 20 µM), which prompt us to carry out further structural optimization on WAI-5.

Table 1. HBV DNA Inhibition Rates (%) of Selected 6 Compounds

Compd.	Inhibition rate (%) a)	Compd.	Inhibition rate (%) a)
WAI-1	11.4 ± 3.71	WAI-4	8.0 ± 3.24
WAI-2	NAb)	WAI-5	63.9 ± 1.25 EC50 = 11.17 ± 0.97 µM CC50 > 20 µM
WAI-3	NAb)	WAI-6	NAb)

a) The concentration of compounds is 20.0 µM. b) NA = not active.

Firstly, the molecular docking study of compound WAI-5 with HBV core protein (PDB: 5E0I) was performed to get some idea for structural optimization. As shown in Fig. 4, compound WAI-5 combined with the HBV protein in a “V” shape mode (Fig. 4). The oxygens of the amides moiety in WAI-5 formed two hydrogen bonds with the nitrogens of Trp102 and Leu140, respectively. In addition, the amino moiety of benzamide formed a third hydrogen bond with the oxygen of Thr128. On the entrance of pocket, the phenyl ring of WAI-5 extends to the solvent region and formed a vertical π–π stacking with the benzene ring of Tyr118.

Fig. 4. WAI-5 Binding Model with HBV Core Protein by Molecular Docking Analysis

To improve the anti-HBV activity of WAI-5 and explore the structure–activity relationships (SARs) of the benzamide derivatives, structural modifications on WAI-5 were carried out (Fig. 5). The docking study demonstrated that the phenylethyl moiety of WAI-5 extends to the solvent region, indicating greater tolerance for modification. Therefore, we used various moieties (including methyl, aryl, benzylic, and heterocycle ethyl groups) to achieve series A compounds 9a–l. the m-methyl-phenyl methanesulfonamide moiety fitted well into the HBV core protein’s hydrophobic pocket, showing minor modification tolerance. Accordingly, the methanesulfonyl was removed or replaced by an acetyl moiety to achieve series B compounds 10a and 10b. Furthermore, the m-methylphenyl moiety was replaced by various aryl or cyclohexyl groups obtaining series C compounds 11a–p.

Fig. 5. Systematic Structural Modification on WAI-5

Structural Optimization of Compound WAI-5

SAR Analysis of Series A Compounds

As shown in Table 2, the modification of WAI-5 phenylethyl moiety affected the anti-HBV activities obviously. The replacement of phenylethyl with methyl (9a), benzyl (9c), 4-fluorobenzyl (9d), 4-chlorobenzyl (9e), and pyridine-2-yl (9i) resulted in the decrease of anti-HBV activity, with inhibition rates ranging from 23.71 to 38.57% at 10 µM. In addition, compounds with incorporated phenyl (9b), 4-bromobenzyl (9f), 4-methoxybenzyl (9g), pyridin-3-yl (9j), pyridin-4-yl (9k) and 4-methylpiperazin-1-yl (9l) moieties almost lost the anti-HBV activity with less than 15.00% inhibition at 10 µM. Accordingly, the results revealed that the phenylethyl is the optimal moiety in this position for a potent anti-HBV activity.

Table 2. The Antiviral Activity of Series A Compounds

a) Concentration is 10 µM. b) NA = not active. * GLS4 used in the test is a racemate.

SAR Analysis of Series B Compounds

As shown in Table 3, the methylsulfonyl moiety was replaced with hydrogen or acetyl group to achieve compounds 10a and 10b. Both compounds showed a dramatic decrease in anti-HBV activity, indicating the crucial role of this moiety.

Table 3. The Antiviral Activity of Series B Compounds

a) Concentration is 10 µM. * GLS4 used in the test is a racemate.

SAR Analysis of Series C Compounds

As shown in Table 4, compounds with a substituted aryl moiety showed moderate to potent anti-HBV activity with inhibition rates ranging from 19.08 to 77.68% at 10 µM. In particular, compounds 11g and 11n exhibited excellent antiviral activity with the EC₅₀ values of 1.74 and 1.90 µM, respectively, nearly 10-fold increased potency than lead compound WAI-5 (EC₅₀ = 11.17 µM). However, the replacement of m-methylphenyl with phenyl, m-methoxyphenyl, m-fluorophenyl, 2-bromophenyl, cyclohexyl, quinolin-7-yl, and thiazol-2-yl led to a dramatic reduction or loss of anti-HBV activity.

Table 4. The Antiviral Activities of Series C Compounds

a) Concentration is 10 µM. b) NA = not active. *. GLS4 used in the test is a racemate.

As shown in Table 5, the compounds over 30% inhibition at 10 µM were then tested for their in vitro EC₅₀ and CC₅₀ values. The results showed that the CC₅₀ values of tested compounds were >20 µM.

Table 5. The in Vitro EC₅₀ and CC₅₀ Values of WAI-5 Derivatives

Compd.	EC50 (µM)	CC50 (µM)
9c	9.73 ± 2.53	>20
9d	10.10 ± 2.15	>20
11g	1.74 ± 0.33	>20
11n	1.90 ± 0.27	>20
11o	5.32 ± 0.88	>20
11p	13.21 ± 2.51	>20

The EC₅₀ values of compound 11g and 11n were 1.74 and 1.90 µM, respectively, showing about 10-fold increase in activity compared to the lead compound WAI-5. The molecular docking modes of 11g and 11n with HBV core protein are shown in Fig. 6. Both 11g and 11n combined with the protein in a “V” shape mode, which were similar to that of WAI-5. Additionally, the pyridine nitrogen atom of 11n forms another hydrogen bond with the carbonyl group of the HBV capsid protein Ser106 at a distance of 2.60 Å, which may interpret the good anti-HBV activity of compound 11n.

Fig. 6. 11g (Left) and 11n (Right) Binding Model with HBV Core Protein by Molecular Docking Analysis

CONCLUSION

A tandem approach combining structure-based virtual screening and ligand-based pharmacophore screening was performed to efficiently identify novel scaffolds for HBV CAM hits. The 93 compounds obtained from a previous virtual screening were employed as substrates and matched with a pharmacophore model obtained from three SBA derivatives. Six compounds were selected for anti-HBV biological evaluation, and three of them exhibited moderate to good anti-HBV activities, demonstrating the rationality of our strategy. Especially, compound WAI-5 showed good anti-HBV activity with an EC₅₀ value of 11.17 µM. Further structural modifications on WAI-5 produced three series of compounds, and the SAR was discussed. Most of these newly designed compounds showed moderate to good anti-HBV activity. Notably, the compounds 11g and 11n exhibited a 10-fold increase in antiviral activity over WAI-5 with EC₅₀ values of 1.74 and 1.90 µM, respectively, indicating be worthy of further investigation.

CHEMISTRY

General

All reagents or solvents used were pretreated according to the requirements of the synthesis experiments. NMR spectra were obtained on a Bruker AV400, Agilent MR400 (400 MHz for ¹H; 100 MHz for ¹³C) or Bruker AV500 (500 MHz for ¹H; 126 MHz for ¹³C). ¹H-NMR and ¹³C-NMR chemical shifts were determined relative to internal (CH₃)₄Si (TMS) at δ 0.0. Mass spectra were obtained on a mass spectrometer. High-resolution mass data were recorded on a high resolution mass spectra (HR-MS) in the electrospray ionization (ESI) mode (Agilent 6546).

General Procedure for Synthesis of Compounds 9a–l

A solution of 12 (500 mg, 2.7 mmol) and potassium carbonate (559 mg, 4.05 mmol) in 10 mL of acetonitrile was added dropwise to methyl 2-bromoacetate (0.51 mL, 5.41 mmol) under nitrogen protection and stirred at room temperature for 4 h. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain 13. 13 (450 mg, 1.8 mmol) and lithium hydroxide (147 mg, 3.5 mmol) were added to a reaction vial containing 10 mL of MeOH/ tetrahydrofuran (THF)/H₂O (V_MeOH/V_THF/V_H2O = 3 : 1 : 1) and stirred at room temperature for 3 h. After complete reaction was monitored by TLC, MeOH and THF were removed under reduced pressure and 10 mL of water was added. The aqueous phase was washed with ethyl acetate, and 2N aqueous HCl was added dropwise under ice bath to adjust to acidity, and ethyl acetate was added to extract the solution, and the solvent was removed under reduced pressure to obtain 14. A solution of 15 (3.1 mmol) and different amines (3.4 mmol) in 15 mL of acetonitrile was stirred at room temperature for 12 h. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain a residue, which was subjected to silica gel column chromatography to obtain 16a–l. A solution of 16a–l (0.58 mmol), HATU (330 mg, 0.87 mmol) and N,N-diisopropylethylamine (DIPEA) (0.29 mL, 1.74 mmol) in 5 mL of dichloromethane was stirred at room temperature for 30 min. 14 (0.58 mmol) was added and the mixture was stirred at room temperature overnight. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain a residue, which was subjected to silica gel column chromatography to obtain 9a–l.

N-Methyl-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)benzamide (9a)

White solid, yield 84%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.81 (s, 1H, Ar-NH), 8.76 (q, J = 4.5 Hz, 1H, NHCH₃), 8.42 (d, J = 8.5 Hz, 1H, Ar-H), 7.70 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.48–7.45 (m, 2H, Ar-H), 7.32 (t, J = 8.0 Hz, 1H, Ar-H), 7.17–7.14 (m, 2H, Ar-H), 4.49 (s, 2H, CH₂), 3.12 (s, 3H, SCH₃), 2.87 (d, J = 4.0 Hz, 3H, NHCH₃), 2.32 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.91, 167.29, 140.73, 139.15, 138.40, 132.15, 129.42, 129.09, 129.04, 128.36, 125.48, 123.47, 121.91, 120.61, 55.43, 37.44, 26.73, 21.35; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₁₈H₂₂N₃O₄S: 376.1331, Found: 376.1340.

N-Phenyl-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)benzamide (9b)

White solid, yield 85%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.15 (s, 1H, Ar-NH), 10.53 (s, 1H, Ar-NH), 8.34 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.84–7.82 (m, 3H, Ar-H), 7.56–7.52 (m, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.43–7.38 (m, 3H, Ar-H), 7.27–7.24 (m, 1H, Ar-H), 7.19–7.15 (m, 2H, Ar-H), 7.11–7.09 (m, 1H, Ar-H), 4.50 (s, 2H, CH₂), 3.08 (s, 3H, SCH₃), 2.18 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 167.36, 167.32, 140.69, 139.17, 139.15, 137.80, 132.35, 129.36, 129.14, 128.98, 125.21, 124.66, 123.82, 123.76, 121.21, 121.12, 55.39, 37.25, 21.14; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₄N₃O₄S: 438.1488, Found: 438.1471.

N-Benzyl-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)benzamide (9c)

White solid, yield 88%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.84 (s, 1H, Ar-NH), 9.36 (t, J = 6.0 Hz, 1H, CH₂NH), 8.45 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.81 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.51–7.47 (m, 2H, Ar-H), 7.45–7.43 (m, 1H, Ar-H), 7.40–7.38 (m, 2H, Ar-H), 7.36–7.33 (m, 2H, Ar-H), 7.29–7.23 (m, 2H, Ar-H), 7.19–7.13 (m, 2H, Ar-H), 4.56 (d, J = 6.0 Hz, 2H, CH₂NH), 4.49 (s, 2H, CH₂), 3.10 (s, 3H, SCH₃), 2.28 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.48, 167.38, 140.79, 139.54, 139.17, 138.69, 132.42, 129.42, 129.02, 128.95, 128.85, 128.58, 127.88, 127.38, 125.58, 123.48, 121.55, 120.66, 55.49, 43.05, 37.43, 21.34; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₆N₃O₄S: 452.1644, Found: 452.1654.

N-(4-Fluorobenzyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9d)

White solid, yield 87%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.82 (s, 1H, Ar-NH), 9.36 (t, J = 6.0 Hz, 1H, CH₂NH), 8.44 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.79 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 7.51–7.48 (m, 2H, Ar-H), 7.45–7.42 (m, 3H, Ar-H), 7.27 (t, J = 7.5 Hz, 1H, Ar-H), 7.19–7.14 (m, 4H, Ar-H), 4.54 (d, J = 6.0 Hz, 2H, CH₂NH), 4.49 (s, 2H, CH₂), 3.10 (s, 3H, SCH₃), 2.28 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.49, 167.40, 162.68 (J = 243.2 Hz), 140.78, 139.18, 138.65, 135.74 (J = 2.9 Hz), 132.45, 129.99 (J = 7.6 Hz), 129.41, 129.04, 128.95, 128.59, 125.58, 123.49, 121.55, 120.68, 115.63 (J = 21.4 Hz), 55.51, 42.39, 37.34, 21.33; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅FN₃O₄S: 470.1550, Found: 470.1554.

N-(4-Chlorobenzyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9e)

White solid, yield 88%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.80 (s, 1H, Ar-NH), 9.38 (t, J = 6.0 Hz, 1H, CH₂NH), 8.44 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.80 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.51–7.48 (m, 2H, Ar-H), 7.44–7.38 (m, 5H, Ar-H), 7.26 (t, J = 7.5 Hz, 1H, Ar-H), 7.20–7.14 (m, 2H, Ar-H), 4.54 (d, J = 6.0 Hz, 2H, CH₂NH), 4.48 (s, 2H, CH₂), 3.10 (s, 3H, SCH₃), 2.27 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.55, 167.40, 140.78, 139.17, 138.65, 138.61, 132.49, 131.95, 129.83, 129.40, 129.03, 128.93, 128.78, 128.59, 125.57, 123.50, 121.50, 120.69, 55.50, 42.44, 37.33, 21.32; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅ClN₃O₄S: 486.1254, Found: 486.1258.

N-(4-Bromobenzyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9f)

White solid, yield 91%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.80 (s, 1H, Ar-NH), 9.36 (t, J = 6.0 Hz, 1H, CH₂NH), 8.44–8.42 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.79–7.78 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.54–7.51 (m, 2H, Ar-H), 7.50–7.48 (m, 2H, Ar-H), 7.44–7.42 (m, 1H, Ar-H), 7.37–7.35 (m, 2H, Ar-H), 7.26–7.23 (t, J = 8.0 Hz, 1H, Ar-H), 7.20–7.16 (m, 1H, Ar-H), 7.15–7.13 (m, 1H, Ar-H), 4.52 (d, J = 6.0 Hz, 2H, CH₂NH), 4.48 (s, 2H, CH₂), 3.10 (s, 3H, SCH₃), 2.27 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.55, 167.40, 140.77, 139.17, 139.04, 138.65, 132.50, 131.70, 130.19, 129.40, 129.03, 128.93, 128.59, 125.58, 123.51, 121.48, 120.69, 120.43, 55.50, 42.50, 37.32, 21.33; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅BrN₃O₄S: 530.0749, Found: 530.0753.

N-(4-Methoxybenzyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9g)

White solid, yield 89%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.86 (s, 1H, Ar-NH), 9.29 (t, J = 6.0 Hz, 1H, CH₂NH), 8.44 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.77 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.51–7.44 (m, 3H, Ar-H), 7.33–7.30 (m, 2H, Ar-H), 7.28 (t, J = 7.5 Hz, 1H, Ar-H), 7.18–7.14 (m, 2H, Ar-H), 6.91–6.88 (m, 2H, Ar-H), 4.49–4.47 (m, 4H, CH₂), 3.73 (s, 3H, OCH₃), 3.11 (s, 3H, SCH₃), 2.29 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.34, 167.37, 158.77, 140.78, 139.17, 138.66, 132.36, 131.50, 129.42, 129.30, 129.03, 128.98, 128.56, 125.59, 123.45, 121.61, 120.62, 114.23, 55.53, 55.49, 42.52, 37.40, 21.35; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₅H₂₈N₃O₅S: 482.1750, Found: 482.1757.

N-(2-(Thien-2-yl)ethyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9h)

White solid, yield 85%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.73 (s, 1H, Ar-NH), 8.95 (t, J = 5.6 Hz, 1H, CH₂NH), 8.42 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.69 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.52–7.51 (m, 1H, Ar-H), 7.50–7.46 (m, 2H, Ar-H), 7.36 (dd, J = 5.0, 1.0 Hz, 1H, Ar-H), 7.31 (t, J = 7.5 Hz, 1H, Ar-H), 7.18–7.14 (m, 2H, Ar-H), 6.97–6.94 (m, 2H, Ar-H), 4.48 (s, 2H, CH₂), 3.58–3.54 (m, 2H, CH₂), 3.14 (t, J = 7.0 Hz, 2H, CH₂CH₂NH), 3.10 (s, 3H, SCH₃), 2.31 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.55, 167.37, 141.85, 140.83, 139.17, 138.44, 132.26, 129.41, 129.04, 129.01, 128.46, 127.44, 125.77, 125.52, 124.62, 123.46, 121.98, 120.64, 55.51, 41.49, 37.28, 29.39, 21.38; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₆N₃O₄S₂: 472.1365, Found: 472.1368.

N-(2-(Pyridin-2-yl)ethyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9i)

White solid, yield 86%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.75 (s, 1H, Ar-NH), 8.89 (t, J = 5.6 Hz, 1H, CH₂NH), 8.53–8.51 (m, 1H, Ar-H), 8.41 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.73 (td, J = 7.5, 2.0 Hz, 1H, Ar-H), 7.65 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 7.52 (m, 1H, Ar-H), 7.50–7.45 (m, 2H, Ar-H), 7.32–7.29 (m, 2H, Ar-H), 7.25–7.22 (m, 1H, Ar-H), 7.16 (td, J = 7.5, 1.5 Hz, 2H, Ar-H), 4.48 (s, 2H, CH₂), 3.70–3.66 (m, 2H, CH₂), 3.11 (s, 3H, SCH₃), 3.08 (t, J = 7.5 Hz, 2H, CH₂CH₂NH), 2.30 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.49, 167.35, 159.51, 149.58, 140.83, 139.18, 138.38, 136.97, 132.17, 129.43, 129.06, 129.03, 128.45, 125.55, 123.65, 123.44, 122.12, 122.02, 120.61, 55.51, 37.46, 37.32, 21.36; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₇N₄O₄S: 467.1753, Found: 467.1758.

N-(2-(Pyridin-3-yl)ethyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-Benzamide (9j)

White solid, yield 87%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.72 (s, 1H, Ar-NH), 8.89 (t, J = 5.5 Hz, 1H, CH₂NH), 8.49 (d, J = 2.0 Hz, 1H, Ar-H), 8.43–8.39 (m, 2H, Ar-H), 7.71–7.69 (m, 1H, Ar-H), 7.63 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.50–7.45 (m,2H, Ar-H),7.33–7.29 (m, 2H, Ar-H), 7.17 (t, J = 8.0 Hz, 2H, Ar-H), 4.48 (s, 2H, CH₂), 3.59 (q, J = 6.5 Hz, 2H, CH₂CH₂NH), 3.11 (s, 3H, SCH₃), 2.96 (t, J = 7.0 Hz, 2H, CH₂CH₂NH), 2.31 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.57, 167.37, 150.40, 147.93, 140.87, 139.18, 138.37, 136.73, 135.36, 132.21, 129.42, 129.06, 128.99, 128.37, 125.54, 123.92, 123.44, 122.05, 120.63, 55.54, 40.91, 37.23, 32.24, 21.37; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₇N₄O₄S: 467.1753, Found: 467.1753.

N-(2-(Pyridin-4-yl)ethyl)-2-(2-(N-(m-tolyl)methylsulfonamido)acetamido)-benzamide (9k)

White solid, yield 88%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.66 (s, 1H, Ar-NH), 8.87 (t, J = 5.5 Hz, 1H, CH₂NH), 8.66–8.65 (m, 2H, Ar-H), 8.39 (dd, J = 8.0, 1.0 Hz, 1H, Ar-H), 7.67–7.65 (m, 2H, Ar-H), 7.62 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.53–7.46 (m, 3H, Ar-H), 7.33 (t, J = 8.0 Hz, 1H, Ar-H), 7.17–7.14 (m, 2H, Ar-H), 4.47 (s, 2H, CH₂), 3.68–3.64 (m, 2H, CH₂), 3.11–3.09 (m, 2H, CH₂), 3.11 (s, 3H, SCH₃) 2.31 (s, 3H, Ar-CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.58, 167.38, 149.80, 140.87, 139.18, 138.70, 138.35, 132.23, 129.43, 129.06, 128.98, 128.40, 127.29, 125.53, 124.80, 123.46, 122.05, 120.64, 55.54, 37.20, 34.35, 21.38; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₇N₄O₄S: 467.1753, Found: 467.1754.

N-(2-(4-Methylpiperazin-1-yl)ethyl)-2-(2-(N-(m-tolyl)methylsulfonamido)-acetamido)benzamide (9l)

White solid, yield 62%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.76 (s, 1H, Ar-NH), 8.72 (t, J = 6.0 Hz, 1H, CH₂NH), 8.42 (d, J = 8.5 Hz, 1H, Ar-H), 7.69 (d, J = 8.0 Hz, 1H, Ar-H), 7.51–7.46 (m, 3H, Ar-H), 7.32 (t, J = 8.0 Hz, 1H, Ar-H), 7.18–7.15 (m, 2H, Ar-H), 4.47 (s, 2H, CH₂), 3.45–3.41 (m, 2H, CH₂), 3.10 (s, 3H, SCH₃), 2.52 (m, 2H, CH₂), 2.45 (m, 3H, Ar-CH₃), 2.37–2.30 (m, 8H, CH₂), 2.14 (s, 3H, NCH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.48, 167.37, 140.83, 139.18, 138.43, 132.23, 129.44, 129.07, 128.94, 128.55, 125.59, 123.46, 121.99, 120.61, 56.38, 55.54, 53.72, 51.09, 45.77, 37.27, 21.41, 8.95; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₃₄N₅O₄S: 488.2332, Found: 488.2336.

General Procedure for Synthesis of Compounds 10a and 10b

A solution of 15 (3.1 mmol) and 2-phenylethylamine (0.42 mL, 3.4 mmol) in 15 mL of acetonitrile was stirred at room temperature for 12 h. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain a residue, which was subjected to silica gel column chromatography to obtain 17. To a reaction vial containing 2 mL of dichloromethane was added 17 (200 mg, 0.83 mmol), chloroacetyl chloride (0.067 mL, 0.84 mmol) dissolved in 1 mL of dichloromethane, triethylamine (0.12 mL, 0.044 mmol), and stirred at room temperature for 12 h. After complete reaction was monitored by TLC, the solvent was extracted with water and dichloromethane and removed under pressure to obtain 18. To a solution of 18 (36.8 mg, 0.12 mmol) in 4 mL of anhydrous acetone was added amide (0.12 mmol), potassium carbonate (33 mg, 0.24 mmol) and sodium iodide (1.8 mg, 0.012 mmol). Reaction mixture was heated reflux for 2 h under nitrogen protection. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain a residue, which was subjected to silica gel column chromatography to obtain 10a–b.

N-Phenethyl-2-(2-(m-tolylamino)acetamido)benzamide (10a)

Prepared from m-methylaniline. White solid, yield 88%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.61 (s, 1H, Ar-NH), 8.68 (t, J = 5.5 Hz, 1H, CH₂NH), 8.50 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.58 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 7.48–7.45 (m, 1H, Ar-H), 7.30–7.27 (m, 2H, Ar-H), 7.21–7.18 (m, 1H, Ar-H), 7.16–7.14 (m, 2H, Ar-H), 7.12 (td, J = 7.5, 1.0 Hz, 1H, Ar-H), 6.96 (t, J = 8.0 Hz, 1H, CH₂NH), 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 6.39–6.34 (m, 3H, Ar-H), 3.77 (d, J = 6.0 Hz, 2H, CH₂NH), 3.32–3.28 (m, 2H, CH₂), 2.67–2.64 (m, 2H, CH₂), 2.11 (s, 3H, CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 170.84, 168.15, 148.66, 139.85, 138.43, 138.28, 131.94, 129.20, 129.10, 128.75, 128.27, 126.53, 123.11, 122.47, 120.63, 118.41, 113.67, 110.04, 49.42, 41.23, 35.05, 21.76; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₆N₃O₂: 388.2025, Found: 388.2030.

N-Phenethyl-2-(2-(N-(m-tolyl)acetamido)acetamido)benzamide (10b)

Prepared from N-(m-tolyl)acetamide. White solid, yield 85%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.75 (s, 1H, Ar-NH), 8.96 (t, J = 5.5 Hz, 1H, CH₂NH), 8.45 (d, J = 8.5 Hz, 1H, Ar-H), 7.70 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.51–7.48 (m, 1H, Ar-H), 7.37–7.28 (m, 5H, Ar-H), 7.25–7.24 (m, 2H, Ar-H), 7.22–7.14 (m, 3H, Ar-H), 4.33 (s, 2H,CH₂), 3.53–3.49 (m, 2H, CH₂CH₂NH), 2.90 (t, J = 7.5 Hz, 2H, CH₂), 2.32 (s, 3H, Ar-CH₃), 1.95 (s, 3H, CH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 170.71, 168.72, 167.50, 143.84, 139.76, 139.67, 138.84, 132.41, 129.85, 129.08, 128.94, 128.85, 128.45, 128.34, 126.64, 124.92, 123.27, 121.36, 120.52, 54.66, 41.40, 35.25, 22.64, 21.35; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₆H₂₈N₃O₃: 430.2131, Found: 430.2141.

General Procedure for Synthesis of Compounds 11a–p

A solution of 19a–p (18.7 mmol) in 10 mL of pyridine was stirred at room temperature and gradually add methanesulfonyl chloride (1.88 mL, 24.3 mmol) dropwise with stirring at room temperature for 5 min. After complete reaction was monitored by TLC, adjust the pH to neutral by adding 2N HCl under an ice bath, add ethyl acetate and water to extract, and remove the solvent under reduced pressure to obtain 20a–p. A solution of 18 (36.8 mg, 0.12 mmol), 20a–p (0.12 mmol), potassium carbonate (33 mg, 0.24 mmol) and sodium iodide (1.8 mg, 0.012 mmol) was refluxed for 2 h under nitrogen protection. After monitoring the completion of the reaction by TLC, the solvent was removed under reduced pressure to obtain a residue, which was subjected to silica gel column chromatography to obtain 11a–p.

N-Phenethyl-2-(2-(N-phenylmethylsulfonamido)acetamido)benzamide (11a)

White solid, yield 85%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.78 (s, 1H, Ar-NH), 8.89 (t, J = 5.6 Hz, 1H, CH₂NH), 8.41 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.72–7.70 (m, 2H, Ar-H), 7.64 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.48–7.41 (m, 3H, Ar-H), 7.36–7.28 (m, 5H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.15 (td, J = 7.5, 1.0 Hz, 1H, Ar-H), 4.51 (s, 2H, CH₂), 3.57–3.53 (m, 2H, CH₂CH₂NH), 3.12 (s, 3H, SCH₃), 2.92 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.50, 167.30, 140.90, 139.88, 138.38, 132.16, 129.67, 129.17, 128.84, 128.51, 128.39, 126.61, 123.44, 122.11, 120.60, 60.22, 55.48, 41.39, 37.33, 35.28; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₆N₃O₄S: 452.1644, Found: 452.1646.

2-(2-(N-(3-Methoxyphenyl)methylsulfonamido)acetamido)-N-phenethyl-benzamide (11b)

White solid, yield 93%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.80 (s, 1H, Ar-NH), 8.89 (t, J = 5.5 Hz, 1H, CH₂NH), 8.44 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.66 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.49–7.45 (m, 1H, Ar-H), 7.37–7.36 (m, 1H, Ar-H), 7.35–7.25 (m, 6H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.17–7.14 (m, 1H, Ar-H), 6.94–6.92 (m, 1H, Ar-H), 4.49 (s, 2H, CH₂), 3.75 (s, 3H, OCH₃), 3.56–3.52 (m, 2H, CH₂CH₂NH), 3.12 (s, 3H, SCH₃), 2.93–2.90 (m, 2H, CH₂CH₂NH); ¹³C-NMR (125 MHz, DMSO-d₆) δ: 168.49, 167.36, 160.12, 142.02, 139.88, 138.46, 132.20, 130.33, 129.16, 128.82, 128.42, 126.60, 123.40, 121.99, 120.52, 120.18, 114.92, 113.84, 55.72, 55.55, 41.39, 37.23, 35.25; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₅H₂₈N₃O₅S: 482.1750, Found: 482.1758.

2-(2-(N-(3-Cyanophenyl)methylsulfonamido)acetamido)-N-phenethyl-benzamide (11c)

White solid, yield 87%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.82 (s, 1H, Ar-NH), 8.92 (t, J = 5.5 Hz, 1H, CH₂NH), 8.41 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 8.21 (t, J = 2.0 Hz, 1H, Ar-H), 8.06–8.04 (m, 1H, Ar-H), 7.84–7.82 (m, 1H, Ar-H), 7.67–7.63 (m, 2H, Ar-H), 7.49–7.46 (m, 1H, Ar-H), 7.32–7.27 (m, 4H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.17 (td, J = 7.5, 1.5 Hz, 1H, Ar-H), 4.59 (s, 2H, CH₂), 3.58–3.53 (m, 2H, CH₂CH₂NH), 3.17 (s, 3H, SCH₃), 2.93–2.90 (m, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.53, 167.00, 141.62, 139.83, 138.45, 133.33, 132.29, 131.89, 131.86, 131.00, 129.18, 128.82, 128.45, 126.60, 123.49, 121.82, 120.56, 118.58, 112.59, 54.96, 41.37, 37.58, 35.24; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₅H₂₅N₄O₄S: 477.1597, Found: 477.1604.

2-(2-(N-(3-Nitrophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11d)

White solid, yield 84%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.95(s, 1H, Ar-NH), 8.90 (t, J = 5.5 Hz, 1H, CH₂NH), 8.64–8.63 (m, 1H, Ar-H), 8.43 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 8.22–8.20 (m, 1H, Ar-H), 8.15-8.13 (m, 1H, Ar-H), 7.75–7.72 (m, 1H, Ar-H), 7.68 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H),7.50–7.46 (m, 1H, Ar-H), 7.32–7.26 (m, 4H, Ar-H), 7.22–7.19 (m, 1H, Ar-H), 7.17–7.14 (m, 1H, Ar-H), 4.65 (s, 2H, CH₂), 3.58–3.54 (m, 2H, CH₂CH₂NH), 3.20 (s, 3H, SCH₃), 2.93 (t, J = 7.5, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.49, 167.01, 148.59, 141.92, 139.86, 138.59, 134.57, 132.32, 130.94, 129.16, 128.80, 128.44, 126.58, 123.46, 123.39, 122.96, 121.63, 120.55, 55.09, 41.31, 37.66, 35.24; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅N₄O₆S: 497.1495, Found: 497.1494.

2-(2-(N-(3-Fluorophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11e)

White solid, yield 84%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.82 (s, 1H, Ar-NH), 8.92 (t, J = 5.5 Hz, 1H, CH₂NH), 8.43 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 8.67-7.64 (m, 2H, Ar-H), 7.56–7.54 (m, 1H, Ar-H), 7.50–7.45 (m, 2H, Ar-H), 7.33–7.27 (m, 4H, Ar-H), 7.24–7.20 (m, 2H, Ar-H), 7.18 (td, J = 7.5, 1.5 Hz, 1H, Ar-H), 4.55 (s, 2H, CH₂), 3.57–3.52 (m, 2H, CH₂CH₂NH), 3.15 (s, 3H, SCH₃), 2.95–2.90 (m, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.51, 167.12, 163.34 (J = 244.4 Hz), 142.39 (J = 10.8 Hz), 139.84, 138.41, 132.25, 131.17 (J = 10.8 Hz), 129.16, 128.83, 128.43, 126.61, 124.06 (J = 3.8 Hz), 123.47, 121.94, 120.56, 115.98 (J = 23.9 Hz), 115.33 (J = 20.6 Hz), 55.25, 41.40, 37.38, 35.26; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅FN₃O₄S: 470.1550, Found: 470.1552.

2-(2-(N-(3-Chlorophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11f)

White solid, yield 86%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.84 (s, 1H, Ar-NH), 8.92 (t, J = 5.5 Hz, 1H, CH₂NH), 8.43 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.89 (t, J = 2.0 Hz, 1H, Ar-H), 7.68–7.65 (m, 2H, Ar-H), 7.50–7.42 (m, 3H, Ar-H), 7.32–7.27 (m, 4H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.18 (dd, J = 8.0, 2,0 Hz, 1H, Ar-H), 4.54 (s, 2H, CH₂), 3.58–3.54 (m, 2H, CH₂CH₂NH), 3.14 (s, 3H, SCH₃), 2.93–2.90 (dd, J = 8.4, 6.6 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.51, 167.12, 142.24, 139.84, 138.48, 133.66, 132.28, 131.19, 129.17, 128.84, 128.81, 128.44, 128.35, 126.67, 126.61, 123.46, 121.82, 120.53, 55.26, 41.41, 37.37, 35.25; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅ClN₃O₄S: 486.1254, Found: 486.1255.

2-(2-(N-(3-Bromophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11g)

White solid, yield 85%, ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.85 (s, 1H, Ar-NH), 8.92 (t, J = 5.5 Hz, 1H, CH₂NH), 8.43 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 8.03 (t, J = 2.0 Hz, 1H, Ar-H), 7.71–7.69 (m, 1H, Ar-H), 7.67 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.57–7.55 (m, 1H, Ar-H), 7.50–7.46 (m, 1H, Ar-H), 7.42–7.38 (m, 1H, Ar-H), 7.32–7.27 (m, 4H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.18–7.14 (m, 1H, Ar-H), 4.54 (s, 2H, CH₂), 3.59-3.54 (m, 2H, CH₂CH₂NH), 3.14 (s, 3H, SCH₃), 2.94–2.91 (m, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.50, 167.13, 142.37, 139.85, 138.50, 132.28, 131.69, 131.46, 131.25, 129.19, 128.81, 128.44, 127.10, 126.60, 123.45, 122.00, 121.77, 120.52, 55.30, 41.42, 37.35, 35.25; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅BrN₃O₄S: 530.0749, Found: 530.0758.

2-(2-(N-(4-Bromophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11h)

White solid, yield 87%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.76 (s, 1H, Ar-NH), 8.90 (t, J = 5.5 Hz, 1H, CH₂NH), 8.40 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 7.68-7.61 (m, 5H, Ar-H), 7.49–7.45 (m, 1H, Ar-H), 7.33–7.26 (m, 4H, Ar-H), 7.24–7.20 (m, 1H, Ar-H), 7.17–7.14 (m, 1H, Ar-H), 4.52 (s, 2H, CH₂), 3.56-3.52 (m, 2H, CH₂CH₂NH), 3.14 (s, 3H, SCH₃), 2.92 (t, J = 5.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.49, 167.10, 140.17, 139.87, 138.36, 132.59, 132.21, 130.49, 129.17, 128.83, 128.41, 126.61, 123.48, 122.04, 121.35, 120.62, 55.19, 41.35, 37.45, 35.23; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅BrN₃O₄S: 530.0749, Found: 530.0745.

2-(2-(N-(2-Bromophenyl)methylsulfonamido)acetamido)-N-phenethylbenzamide (11i)

White solid, yield 90%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.28 (s, 1H, Ar-NH), 8.84 (t, J = 5.5 Hz, 1H, CH₂NH), 8.29 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.93 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.78 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.49–7.43 (m, 2H, Ar-H), 7.37–7.24 (m, 5H, Ar-H), 7.20–7.15 (m, 2H, Ar-H), 7.23–7.20 (m, 1H, Ar-H), 7.17–7.14 (m, 1H, Ar-H), 6.97–6.94 (m, 2H, Ar-H), 4.44 (s, 2H, CH₂), 3.57–3.53 (m, 2H, CH₂CH₂NH), 3.09 (s, 3H, SCH₃), 2.94–2.91 (m, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.34, 167.04, 139.83, 138.88, 138.15, 134.21, 133.78, 132.06, 131.01, 129.16, 129.04, 128.79, 128.44, 126.58, 124.55, 123.67, 122.81, 121.38, 54.64, 49.07, 41.54, 35.29; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₂₅BrN₃O₄S: 530.0749, Found: 530.0755.

2-(2-(N-Cyclohexylmethylsulfonamido)acetamido)-N-phenethylbenzamide (11j)

White solid, yield 85%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.32 (s, 1H, Ar-NH), 8.83 (t, J = 5.6 Hz, 1H, CH₂NH), 8.43 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.62 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 7.49–7.46 (m, 1H, Ar-H), 7.32–7.29 (m, 2H, Ar-H), 7.27–7.25 (m, 2H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.16–7.13 (m, 1H, Ar-H), 3.96 (s, 2H, CH₂), 3.69–3.63 (m, 1H, CH), 3.49–3.44 (m, 2H, CH₂), 3.12 (s, 3H, SCH₃), 2.87–2.84 (m, 2H, CH₂), 1.93–1.90 (m, 2H, CH₂), 1.73–1.70 (m, 2H, CH₂), 1.54–1.51 (m, 1H, CH₂), 1.45–1.23 (m, 5H, CH₂); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 169.05, 168.38, 139.89, 138.33, 132.00, 129.15, 128.82, 128.34, 126.59, 123.29, 122.59, 120.81, 57.61, 47.00, 41.36, 35.27, 30.44, 25.84, 25.05; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₄H₃₂N₃O₄S: 458.2114, Found: 458.2120.

N-Phenethyl-2-(2-(N-(quinolin-7-yl)methylsulfonamido)acetamido)-benzamide (11k)

White solid, yield 84%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.72 (s, 1H, Ar-NH), 9.03–9.02 (m, 1H, Ar-H), 8.89 (t, J = 5.5 Hz, 1H, CH₂NH), 8.51 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 8.44 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 8.38 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 8.08 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.66–7.62 (m, 3H, Ar-H), 7.47–7.44 (m, 1H, Ar-H), 7.30–7.28 (m, 4H, Ar-H), 7.21–7.17 (m, 1H, Ar-H), 7.17 (td, J = 7.5, 1.0 Hz, 1H, Ar-H), 4.73 (s, 2H, CH₂), 3.58-3.54 (m, 2H, CH₂CH₂NH), 3.20 (s, 3H, SCH₃), 2.95 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.26, 167.05, 149.81, 146.62, 139.24, 137.12, 134.80, 132.30, 131.78, 129.41, 128.78, 128.64, 128.42, 128.17, 127.37, 126.93, 126.18, 124.38, 123.54, 116.74, 111.19, 54.74, 42.26, 35.27, 31.15; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₇H₂₇N₄O₄S: 503.1753, Found: 503.1753.

N-Phenethyl-2-(2-(N-(thiazol-2-yl)methylsulfonamido)acetamido)benzamide (11l)

White solid, yield 83%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.58 (s, 1H, Ar-NH), 8.90 (t, J = 5.5 Hz, 1H, CH₂NH), 8.37–8.36 (m, 1H, Ar-H), 7.69 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.52–7.49 (m, 1H, Ar-H), 7.44 (d, J = 4.5 Hz, 1H, Ar-H), 7.33–7.30 (m, 2H, Ar-H), 7.27–7.25 (m, 2H, Ar-H), 7.24–7.21 (m, 1H, Ar-H), 7.19 (td, J = 7.5, 1.0 Hz, 1H, Ar-H), 6.96 (d, J = 5.0 Hz, 1H, Ar-H), 4.84 (s, 2H, CH₂), 3.49–3.45 (m, 2H, CH₂CH₂NH), 2.87–2.84 (m, 2H, CH₂CH₂NH), 2.82 (s, 3H, SCH₃); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.46, 167.35, 164.96, 139.78, 138.63, 132.44, 129.17, 128.92, 128.85, 128.47, 126.64, 123.63, 121.52, 120.88, 107.15, 51.25, 41.33, 40.87, 35.25; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₁H₂₃N₄O₄S₂: 459.1161, Found: 459.1167.

N-Phenethyl-2-(2-(N-(pyridin-2-yl)methylsulfonamido)acetamido)benzamide (11m)

White solid. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.66 (s, 1H, Ar-NH), 8.83 (t, J = 5.5 Hz, 1H, CH₂NH), 8.40–8.38 (m, 1H, Ar-H), 8.36 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.87–7.83 (m, 1H, Ar-H), 7.70–7.68 (m, 1H, Ar-H), 7.64 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.48–7.44 (m, 1H, Ar-H), 7.33–7.29 (m, 2H, Ar-H), 7.27–7.20 (m, 4H, Ar-H), 7.16–7.12 (m, 1H, Ar-H), 4.73 (s, 2H, CH₂), 3.51–3.46 (m, 2H, CH₂CH₂NH), 3.33 (s, 3H, SCH₃), 2.85 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.43, 167.31, 152.75, 148.45, 139.84, 138.95, 138.53, 132.18, 129.16, 128.82, 128.37, 126.60, 123.40, 121.95, 121.28, 120.77, 118.17, 51.57, 41.30, 35.21, 31.76; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₅N₄O₄S: 453.1597, Found: 453.1601.

N-Phenethyl-2-(2-(N-(pyridin-3-yl)methylsulfonamido)acetamido)benzamide (11n)

White solid, yield 78%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.80 (s, 1H, Ar-NH), 8.94 (d, J = 2.5 Hz, 1H, CH₂NH), 8.90 (t, J = 7.5 Hz, 1H, Ar-H), 8.54 (d, J = 5.0 Hz, 1H, Ar-H), 8.40 (d, J = 8.5 Hz, 1H, Ar-H), 8.13–8.11 (m, 1H, Ar-H), 7.66 (d, J = 8.0 Hz, 1H, Ar-H), 7.50–7.46 (m, 2H, Ar-H), 7.33–7.28 (m, 4H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.16 (t, J = 7.5 Hz, 1H, Ar-H), 4.59 (s, 2H, CH₂), 3.56 (q, J = 7.0 Hz, 2H, CH₂CH₂NH), 3.17 (s, 3H, SCH₃), 2.91 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.51, 167.03, 149.92, 148.99, 139.85, 138.38, 137.47, 135.43, 132.24, 129.19, 128.83, 128.42, 126.61, 124.51, 123.51, 121.97, 120.62, 54.98, 41.40, 37.68, 35.26; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₅N₄O₄S: 453.1597, Found: 453.1599.

2-(2-(N-(2-Fluoropyridin-3-yl)methylsulfonamido)acetamido)-N-phenethyl-benzamide (11o)

White solid, yield 82%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.65 (s, 1H, Ar-NH), 8. 91 (t, J = 5.5 Hz, 1H, CH₂NH), 8.50–8.46 (m, 1H, Ar-H), 8.50 (dd, J = 8.5, 1.0 Hz, 1H, Ar-H), 8.26–8.24 (m, 1H, Ar-H), 7.67 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.50–7.44 (m, 2H, Ar-H), 7.32–7.26 (m, 4H, Ar-H), 7.24–7.19 (m, 1H, Ar-H), 7.18–7.15 (m, 1H, Ar-H), 4.53 (s, 2H, CH₂), 3.55–3.50 (m, 2H, CH₂CH₂NH), 3.25 (s, 3H, SCH₃), 2.91 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (125 MHz, DMSO-d₆) δ: 168.50, 166.94, 160.55 (J = 240.8 Hz), 147.62, 147.50, 143.70, 139.85, 138.40, 132.23, 129.16, 128.83, 128.43, 126.60, 123.51 (J = 9.7 Hz), 123.19 (J = 4.0 Hz), 122.11, 120.80, 60.23, 54.25, 41.38, 35.26; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₄FN₄O₄S: 471.1502, Found: 471.1505.

2-(2-(N-(2-Fluoropyridin-5-yl)methylsulfonamido)acetamido)-N-phenethyl-benzamide (11p)

White solid, yield 80%. ¹H-NMR (500 MHz, DMSO-d₆) δ: 11.79 (s, 1H, Ar-NH), 8.91 (t, J = 5.6 Hz, 1H, CH₂NH), 8.61 (d, J = 2.5 Hz, 1H, Ar-H), 8.41 (d, J = 8.5 Hz, 1H, Ar-H), 8.33–8.29 (m, 1H, Ar-H), 7.67 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.32–7.28 (m, 1H, Ar-H), 7.33–7.26 (m, 5H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.18–7.15 (m, 1H, Ar-H), 4.57 (s, 2H, CH₂), 3.55–3.51 (m, 2H, CH₂CH₂NH), 3.18 (s, 3H, SCH₃), 2.92 (t, J = 7.5 Hz, 2H, CH₂CH₂NH); ¹³C-NMR (125 MHz, DMSO-d₆) δ: 168.54, 166.97, 162.96 (J = 237.9 Hz), 148.18 (J = 9.3 Hz), 142.04 (J = 8.9 Hz), 139.83, 138.39, 135.78 (J = 4.8 Hz), 132.28, 129.18, 128.82, 128.44, 126.61, 123.52, 121.92, 120.61, 110.69 (J = 39.8 Hz), 55.15, 41.39, 37.62, 35.24; HR-MS (ESI) m/z [M + H]⁺ Calcd for C₂₃H₂₄FN₄O₄S: 471.1502, Found: 471.1511.

Acknowledgments

This work is supported by National Natural Science Foundation of China (No. 21877097). The authors would like to thank Jianyang Pan (Research and Service Center, College of Pharmaceutical Sciences, Zhejiang University) for performing NMR spectrometry for structure elucidation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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