Tripeptide-like compounds and application thereof in preparation of anti-pseudorabies virus drugs

Abstract

The invention relates to the field of medicines, and particularly discloses tripeptide-like compounds and application thereof in preparation of anti-pseudorabies virus medicines. The tripeptide-like compound provided by the invention has a structural formula as shown in a general formula 1. The tripeptide-like compound disclosed by the invention has better inhibition on PRV (porcine reproductive and respiratory syndrome) and can be used for preparing an anti-PRV drug.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, specifically to a novel class of tripeptide-like compounds and their antiviral uses, and more specifically to a novel class of tripeptide-like compounds and their application in the field of anti-pseudorabies virus. Background Technology

[0002] Pseudorabies virus (PRV) is a DNA virus that primarily infects pigs, its natural host. It causes an acute, highly contagious disease characterized by fever and encephalomyelitis, with an extremely high mortality rate in infected piglets, reaching up to 100%. Currently, there are no reported highly active antiviral drugs against PRV DNA virus, which cannot meet the needs of pig production.

[0003] Nectin-1 (PVRL1), a member of the immunoglobulin superfamily, is a key receptor for pseudorabies virus (PRV) entry into cells, and its mechanism of action is similar to that of herpes simplex virus type 1 (HSV-1) entry into cells. Soluble Nectin-1 fragments and antibodies can inhibit PRV infection of cells. Pseudorabies virus glycoprotein D (gD) can directly bind to the IgV domain of human and porcine Nectin-1 with high affinity. This specific interaction (confirmed by structural studies, such as PDB 5X5W) is crucial for initiating membrane fusion, which enables Pseudorabies virus to enter host cells. Therefore, disrupting the gD-Nectin-1 interface is a promising antiviral strategy. Proof-of-concept studies have shown that engineered mini-binding proteins targeting Pseudorabies virus gD achieve potent inhibition (EC). 50 = 2.97 nM) (Wei L., et al.,De novodesign mini-binder proteins targeting the glycoproteins D to inhibit PRVreplication in PK15 cells, Int. J. Biol, Macromol. (2025, 315, 144403), demonstrating the therapeutic potential of blocking this key receptor-ligand interaction to combat pseudorabies virus infection. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a class of tripeptide-like compounds and their application in the preparation of anti-pseudorabies virus drugs. The tripeptide-like compounds provided by this invention exhibit good inhibition of PRV and can be used in the preparation of anti-PRV drugs.

[0005] The first aspect of this invention provides a class of tripeptide-like compounds, with general formula I being: , R1-R4 are selected from H, -OH, halogen, alkyl, alkoxy, substituted alkyl, etc., respectively, and X represents the configuration of the chiral center, which is R or S.

[0006] According to the above scheme, R1 is selected from H, -OH, halogen, alkyl; R2 and R4 are selected from H, halogen, alkyl, respectively; R3 is selected from H, halogen; X represents the configuration of the chiral center, which is R or S.

[0007] According to the above scheme, R1 is selected from H, -OH, halogen, -CH3; R2 is selected from H, Cl, -CH3; R3 is selected from H, F; R4 is selected from H, F, -CH3; X represents the configuration of the chiral center, which is R or S.

[0008] According to the above scheme, R1 is selected from H, -OH, F, and -CH3; R2 is selected from H, Cl, and -CH3; R3 is selected from H and F; R4 is selected from H, F, and -CH3; at least two of R1, R2, R3, and R4 are not H; X represents the configuration of the chiral center, which is R or S.

[0009] According to the above scheme, the compound of general formula I is selected from at least one of the target compounds in Table 2: Table 2 Tripeptide-like Compounds

[0010] In this invention, the halogens include fluorine, chlorine, bromine, and iodine.

[0011] In this invention, the alkyl group includes straight-chain alkyl groups with 1-18 carbon atoms and branched-chain alkyl groups with 3-18 carbon atoms.

[0012] In this invention, the alkoxy group includes straight-chain alkoxy groups with 1-18 carbon atoms and branched-chain alkoxy groups with 3-18 carbon atoms.

[0013] In this invention, the substituted alkyl refers to the fact that the hydrogen atom on the carbon atom of the alkyl group can be replaced by a halogen.

[0014] This invention also provides a method for preparing the tripeptide-like compounds of general formula I described above, including the reaction synthesis route:

[0015] At room temperature, compound 1, substituted aniline, first undergoes a condensation reaction with compound 2, boc-GLY, at room temperature. Then, compound 2 is hydrolyzed to remove the boc protecting group, generating intermediate 3. Compound 4, benzoyl chloride, condenses with methyl 2-aminobutyrate to generate compound 5. Compound 5 is hydrolyzed to generate intermediate 6. Intermediate 3 and intermediate 6 condense to generate the target product, compound 7.

[0016] This invention also provides the application of the tripeptide-like compounds of general formula I in the preparation of anti-pseudorabies virus (PRV) drugs.

[0017] According to the above scheme, the specific application method is as follows: the tripeptide compound described in the above general formula I is used as the active ingredient of the anti-PRV virus drug, and the content of the active ingredient in the anti-PRV virus drug is 0.1-100wt%.

[0018] According to the above scheme, the specific application method is as follows: the tripeptide compound described in the above general formula I is used as the active ingredient of the anti-PRV virus drug, and a veterinary drug acceptable carrier and surfactant are added as needed to formulate tablets, capsules, granules, drops, liquid preparations, decoctions, suppositories, gels, aerosols or patches, etc.

[0019] A third aspect of this invention provides an anti-PRV drug comprising an active ingredient of a tripeptide-like compound effective against PRV and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are selected from one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, and carriers.

[0020] According to the above scheme, the dosage form of the drug includes, but is not limited to, tablets, capsules, granules, pills, liquid preparations, decoctions, suppositories, gels, aerosols, or patches.

[0021] The beneficial effects of this invention are: This invention provides a novel class of tripeptide-like compounds represented by general formula I. The tripeptide-like compounds represented by general formula I have good inhibitory effects on PRV and can be used to prepare anti-PRV drugs, exhibiting excellent PRV activity. Attached Figure Description

[0022] Figure 1 SDS-PAGE results for biotinylated protein (0.1 mg / mL Avi-tag Nectin-1 IgV domain) Detailed Implementation Example 1 C1(WLN2)(N-[(2R)-1-({2-[(2-hydroxy-3-methylphenyl)amino]-2-oxomethyleneethyl}amino)-1-oxomethylenebutyl-2-yl]benzamide):

[0023] N,N,N',N'-Tetramethylchloromethanemida hexafluorophosphate (1.99 g, 7.10 mmol) was added to a solution of 2-amino-6-methylphenol (1 g, 8.12 mmol), N-tert-butoxycarbonylglycine (1.19 g, 1.77 mmol), and N-methylimidazolium (1.97 g, 23.68 mmol) in acetonitrile (20 mL). The mixture was stirred at room temperature for 5 hours, and the reaction progress was monitored by thin-layer chromatography. The reaction mixture was diluted with saturated sodium bicarbonate (NaHCO3) aqueous solution, and the aqueous phase was extracted three times with ethyl acetate (EtOAc). The organic layers were combined, washed with brine, dried over anhydrous magnesium sulfate (Mg2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give ({2-[(2-hydroxy-3-methylphenyl)amino]-2-oxonylethyl}amino)methane-2-methylpropyl-2-yl ester).

[0024] Trifluoroacetic acid (10 mL) was added to a 15 mL solution of ({2-[(2-hydroxy-3-methylphenyl)amino]-2-oxonylethyl}amino)methane-2-methylpropyl-2-yl ester) in dichloromethane. The reaction mixture was stirred at room temperature for 3 hours, and the solvent was removed under reduced pressure. The resulting residue was neutralized with a saturated aqueous solution of sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3 × 15 mL). The combined organic layers were washed with brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-amino-N-(2-hydroxy-3-methylphenyl)acetamide.

[0025] Triethylamine (1.98 g, 19.53 mmol) was added to a 20 mL solution of (R)-2-aminobutyrate methyl hydrochloride (1 g, 6.51 mmol) in dichloromethane. Benzoyl chloride (1.37 g, 9.77 mmol) was then added dropwise to the mixture at room temperature. The reaction was stirred at room temperature until complete (monitored by thin-layer chromatography). After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give methyl(R)-2-benzoylaminobutyrate.

[0026] 1.2 g (5.45 mmol) of methyl(R)-2-benzoamide butyrate was dissolved in 20 mL of acetonitrile, followed by the addition of water (400 μL), lithium bromide (4.71 g, 54.24 mmol), and triethylamine (1.65 g, 16.27 mmol). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was acidified to pH 2–3 with 1 M hydrochloric acid and extracted with dichloromethane (3 × 15 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give (R)-2-benzoamide butyrate.

[0027] N,N,N',N'-Tetramethylchloromethanemid hexafluorophosphate (1.07 g, 3.80 mmol) was added to a solution of 2-amino-N-(2-hydroxy-3-methylphenyl)acetamide (0.791 g, 4.34 mmol), (R)-2-benzamide butyric acid (0.75 g, 3.62 mmol), and N-methylimidazole (0.631 g, 7.60 mmol) in 15 mL acetonitrile. The mixture was stirred at room temperature for 5 hours, and the reaction progress was monitored by thin-layer chromatography. The reaction mixture was diluted with saturated sodium bicarbonate (NaHCO3) aqueous solution, and the aqueous phase was extracted three times with ethyl acetate (EtOAc). The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain N-[(2R)-1-({2-[(2-hydroxy-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebut-2-yl]benzamide.

[0028]

[0029] 1 H NMR (400 MHz, DMSO- d6, δ) 9.49 (s, 1H), 8.96 (s, 1H), 8.54 (d, J =7.5 Hz, 1H), 8.45 (t, J = 5.8 Hz, 1H), 7.96 – 7.87 (m, 2H), 7.60 – 7.51 (m,1H), 7.48 (dd, J = 8.2, 6.6 Hz, 2H), 7.39 – 7.31 (m, 1H), 6.92 (d, J = 7.3Hz, 1H), 6.73 (t, J = 7.7 Hz, 1H), 4.40 (ddd, J = 9.0, 7.5, 5.4 Hz, 1H), 4.09– 3.89 (m, 2H), 2.18 (s, 3H), 1.97 – 1.68 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). MS m / z (ESI): 370.17742. [M+H] + . Example 2 C2(WLN1) (N-[(2S)-1-({2-[(2-hydroxy-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebut-2-yl]benzamide): synthesized from 2-amino-6-methylphenol, N-tert-butoxycarbonylglycine, (S)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0030]

[0031] 1 H NMR (400 MHz, DMSO- d6, δ) 9.48 (s, 1H), 8.97 (s, 1H), 8.54 (d, J =7.5 Hz, 1H), 8.44 (t, J = 5.8 Hz, 1H), 7.96 – 7.87 (m, 2H), 7.60 – 7.51 (m,1H), 7.48 (dd, J = 8.2, 6.6 Hz, 2H), 7.39 – 7.31 (m, 1H), 6.92 (d, J = 7.3Hz, 1H), 6.73 (t, J = 7.7 Hz, 1H), 4.41 (ddd, J = 9.0, 7.5, 5.4 Hz, 1H), 4.10– 3.89 (m, 2H), 2.18 (s, 3H), 1.97 – 1.68 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). MS m / z (ESI): 370.17693. [M+H] + . Example 3 C3(WLN3)(N-[(2R)-1-({2-[(3-chloro-2-hydroxyphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 2-amino-6-chlorophenol, N-tert-butoxycarbonylglycine, (R)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0032]

[0033] 1 H NMR (400 MHz, DMSO- d6, δ) 9.74 (s, 1H), 9.49 (s, 1H), 8.45 (d, J =7.6 Hz, 1H), 8.36 (t, J = 5.8 Hz, 1H), 7.94 – 7.87 (m, 2H), 7.62 – 7.50 (m,2H), 7.47 (dd, J = 8.2, 6.6 Hz, 2H), 7.16 (dd, J = 8.1, 1.5 Hz, 1H), 6.84 (t,J = 8.1 Hz, 1H), 4.41 (td, J = 8.4, 5.5 Hz, 1H), 4.07 – 3.90 (m, 2H), 1.89(ddd, J = 13.2, 7.4, 5.6 Hz, 1H), 1.76 (ddd, J = 13.6, 8.9, 7.1 Hz, 1H), 0.95(t, J = 7.4 Hz, 3H). MS m / z(ESI): 390.1219. Example 4 C4(WLN7)(N-[(2R)-1-({2-[(2-fluoro-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 2-fluoro-3-methylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyric acid methyl ester hydrochloride, and benzoyl chloride as per C1.

[0034]

[0035] 1 H NMR (400 MHz, DMSO- d6, δ) 9.54 (s, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.33 (t, J = 5.9 Hz, 1H), 7.92 (d, J = 7.7 Hz, 2H), 7.66 (d, J = 7.5 Hz, 1H), 7.59 – 7.50 (m, 2H), 7.48 (d, J = 7.5 Hz, 1H), 7.11 (dq, J = 13.0, 4.2 Hz,1H), 6.96 (d, J = 7.1 Hz, 1H), 4.41 (td, J = 8.3, 5.4 Hz, 1H), 3.98 (q, J =8.0 Hz, 2H), 2.26 (s, 3H), 1.87 (dq, J = 13.9, 7.1 Hz, 1H), 1.82 – 1.69 (m,1H), 0.96 (t, J = 7.4 Hz, 3H). MS m / z(ESI): 372.1722 [M+H] + . Example 5 C5(WLN8)(N-[(2R)-1-({2-[(4-fluoro-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebut-2-yl]benzamide): synthesized from 2-fluoro-5-aminotoluene, N-tert-butoxycarbonylglycine, (R)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0036]

[0037] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.71 (s, 1H), 7.97 – 7.90 (m, 1H), 7.80(t, J = 6.0 Hz, 2H), 7.67 (dt, J = 12.7, 1.8 Hz, 2H), 7.53 – 7.47 (m, 2H), 7.39(ddd, J = 8.8, 4.6, 2.7 Hz, 2H), 7.10 (t, J = 9.2 Hz, 1H), 4.32 (dt, J = 8.5, 6.2 Hz, 1H), 3.92 (dt, J= 7.4, 3.5 Hz, 2H), 2.21 (t, J = 3.2 Hz, 3H), 1.81(ddd, J = 26.8, 14.2, 6.9 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). MS m / z(ESI): 372.05 [M+H] + . Example 6 C6(WLN9)(N-[(2R)-1-({2-[(5-fluoro-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 3-fluoro-5-methylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0038]

[0039] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.84 (s, 1H), 8.61 (d, J = 6.9 Hz, 1H), 8.46 (t, J = 5.9 Hz, 1H), 7.99 (dt, J = 7.0, 1.4 Hz, 2H), 7.65 – 7.45 (m,4H), 7.28 (s, 1H), 6.84 – 6.73 (m, 1H), 4.38 (dt, J = 8.4, 6.2 Hz, 1H), 4.02– 3.87 (m, 2H), 2.33 (s, 3H), 1.98 – 1.75 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). MS m / z(ESI): 372.1726 [M+H] + . Example 7 C7(WLN10)(N-[(2R)-1-({2-[(2-fluoro-5-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebut-2-yl]benzamide): synthesized from 2-fluoro-5-methylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyric acid methyl ester hydrochloride, and benzoyl chloride as per C1.

[0040]

[0041] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.52 (s, 1H), 8.44 (d, J = 7.6 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 7.94 – 7.86 (m, 2H), 7.67 (dd, J = 8.1, 3.6 Hz, 1H),7.58 – 7.50 (m, 1H), 7.50 – 7.42 (m, 2H), 7.04 – 6.97 (m, 2H), 4.40 (ddd, J =8.8, 7.6, 5.5 Hz, 1H), 4.04 – 3.89 (m, 2H), 2.23 (d, J = 2.2 Hz, 3H), 1.87(ddd, J = 13.3, 7.1, 5.6 Hz, 1H), 1.75 (ddd, J = 13.6, 8.7, 7.1 Hz, 1H), 0.94(t, J = 7.4 Hz, 3H). MS m / z(ESI): 372.05 [M+H] + . Example 8 C8(WLN11)(N-[(2R)-1-({2-[(2,3-dimethylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 2,3-dimethylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0042]

[0043] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.22 (s, 1H), 8.54 (d, J = 7.3 Hz, 1H), 8.41 (t, J = 5.9 Hz, 1H), 7.92 – 7.85 (m, 2H), 7.57 – 7.50 (m, 1H), 7.50 –7.36 (m, 2H), 7.12 (dd, J= 7.5, 1.9 Hz, 1H), 7.08 – 6.90 (m, 2H), 4.37 (ddd, J = 8.7, 7.3, 5.6 Hz, 1H), 3.92 (dd, J = 5.8, 3.6 Hz, 2H), 2.24 (s, 3H), 2.04(s, 3H), 1.87 (ddd, J = 13.4, 7.6, 5.8 Hz, 1H), 1.76 (ddd, J = 13.5, 8.6, 7.0Hz, 1H), 0.95 (t, J = 7.3 Hz, 3H). MS m / z(ESI): 368.10 [M+H] + . Example 9 C9(WLN12)(N-[(2R)-1-({2-[(2-chloro-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 2-chloro-3-methylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyrate methyl hydrochloride, and benzoyl chloride as per C1.

[0044]

[0045] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.33 (s, 1H), 8.51 (d, J = 7.7 Hz, 1H), 8.46 (t, J = 5.8 Hz, 1H), 7.95 – 7.86 (m, 2H), 7.65 (dd, J = 8.1, 1.7 Hz,1H), 7.60 – 7.51 (m, 1H), 7.51 – 7.39 (m, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.18– 7.05 (m, 1H), 4.43 (ddd, J = 9.0, 7.6, 5.4 Hz, 1H), 3.98 (dd, J = 5.8, 2.1Hz, 2H), 2.34 (s, 3H), 1.96 – 1.68 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). MS m / z(ESI): 388.1438 [M+H] + . Example 10 C10(WLN13)(N-[(2R)-1-({2-[(2-bromo-3-methylphenyl)amino]-2-oxylideneethyl}amino)-1-oxylidenebutyl-2-yl]benzamide): synthesized from 2-bromo-3-methylaniline, N-tert-butoxycarbonylglycine, (R)-2-aminobutyric acid methyl ester hydrochloride, and benzoyl chloride as per C1.

[0046]

[0047] 1 H NMR (400 MHz, DMSO- d 6, δ) 9.28 (s, 1H), 8.54 – 8.43 (m, 2H), 7.95– 7.86 (m, 2H), 7.54 (ddt, J = 8.8, 6.4, 1.7 Hz, 2H), 7.50 – 7.40 (m, 2H),7.26 (t, J = 7.8 Hz, 1H), 7.21 – 7.14 (m, 1H), 4.45 (ddd, J = 9.1, 7.8, 5.3 Hz,1H), 3.96 (d, J = 5.8 Hz, 2H), 2.37 (s, 3H), 1.88 (tq, J = 12.8, 6.4 Hz, 1H),1.76 (ddq, J = 14.4, 9.0, 7.3 Hz, 1H), 0.94 (t, J = 7.4 Hz, 3H). MS m / z(ESI): 432.0937 [M+H] + . Taking the C1 compound as an example, the anti-PRV activity experiment is as follows: The porcine kidney cell line (PK15) was cultured in fresh Dublin Modified Eagle Medium (DMEM) (No. 11965092, Gibco). TM ), 10% fetal bovine serum (FBS) (Code: P30-3306, PAN) TM ) and 1% penicillin-streptomycin solution (100X, catalog number: BL505A, Biosharp) TMCells were cultured at 37 °C. PK15 cells at 80% confluence were cultured in 96-well plates. After 24 hours, the culture medium was discarded, and the cells were washed three times with PBS. 1 mL of fresh DMEM containing 2% FBS and 1% penicillin-streptomycin solution was added, and the cells were cultured at 37 °C for 4 hours. Cells were then treated with different concentrations (1 μM, 0.5 μM, 0.25 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM) of C1 compound for 2 hours. Cells were then infected with PRV (10 MOI), and total DNA was extracted with phenol-chloroform (Sorapio Biotechnology Co., Ltd.) after 48 hours. The target gene was amplified by SYBR green-based quantitative PCR (qPCR) using 2 × Taq Master Mix (Vazyme, Nanjing, China). Table 3 lists the primers used in this study. The inhibition rate of the experimental groups was calculated using the formula...

[0048] In Origin software, the EC curve is obtained by performing a nonlinear regression fitting using the logistic equation. 50 . Table 3

[0049] The anti-PRV activity of the compounds in Examples 1-10 is shown in Table 4. Table 4. Activity of compounds against PRV virus

[0050] A: <0.1nM B: 0.1-1nM C: 1-10 nM Example 11 The biotinylated protein (Avi-tag porcine Nectin-1 IgV domain) gene was cloned into the pET-22b vector via fusion cloning. The construct was transformed into *E. coli* BL21(DE3) competent cells, and protein expression was induced by IPTG at 18°C ​​for 20 hours. *E. coli* cultures were centrifuged at 10,000 × g for 20 min, and the precipitate was collected. The precipitate was resuspended in lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, pH 8.0) at a 9:1 (v / w / w) ratio and subjected to two pass-through homogenization lysis processes at 300 kPa and 700 kPa to achieve >95% lysis. The lysis buffer was centrifuged (10,000 × g, 30 min) to collect crude inclusion bodies. Inclusion bodies were washed sequentially with the following solutions: (1) 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2% sodium deoxycholate, pH 8.0; (2) the same buffer with 2 M urea added; (3) the detergent was removed. The purified inclusion bodies were dissolved in denaturing buffer (50 mM Tris, 8 M urea, 80 mM Cys, pH 9.7) at a ratio of 9:1 (volume / mass). After centrifugation (10,000 × g, 10 min), the supernatant concentration was adjusted to 7 mg / mL and diluted 40-fold to refolding buffer (50 mM Tris, 0.78 M urea, 10 mM MgSO4, pH 9.7), and refolded at 4°C for 40 h with stirring. The refolded protein was acid-precipitated (pH 5.0), centrifuged, and the supernatant was purified by SPFF cation exchange chromatography. Purity was confirmed by SDS-PAGE. Figure 1 ).

[0051] Biotinylated protein (0.1 mg / mL Avi-tag Nectin-1 IgV domain) was immobilized on a streptavidin-coated sensor tip in buffer I (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, and 1% BSA) for 30 minutes. The analyte compound C1 was diluted to the specified concentration in buffer II (PBS, 0.05% Tween 20). The sensor tip was sequentially immersed in wells containing buffer II to run the background, then immersed in the binding agent solution for binding. The tip was then returned to the buffer wells for dissociation. Data were analyzed and processed using Octet Analysis Studio v.13.0.1.35, and the equilibrium dissociation constant K was calculated. D .

[0052] The affinity K between compound C1 and Nectin-1 D As shown in Table 5.

[0053] Table 5. Affinity K for the binding of compound C1 to Nectin-1 D

[0054] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The tripeptide-like compound of general formula I, characterized in that: General formula I is: General Formula I R1-R4 are selected from H, -OH, halogen, alkyl, alkoxy, and substituted alkyl groups, respectively; X represents the configuration of the chiral center, which is R or S.

2. The tripeptide-like compound according to claim 1, characterized in that: R1 is selected from H, -OH, halogen, and alkyl; R2 and R4 are selected from H, halogen, and alkyl, respectively; R3 is selected from H and halogen; X represents the configuration of the chiral center, which is R or S.

3. The tripeptide-like compound according to claim 1, characterized in that: R1 is selected from H, -OH, halogen, -CH3; R2 is selected from H, Cl, -CH3; R3 is selected from H, F; R4 is selected from H, F, -CH3; X represents the configuration of the chiral center, which is R or S.

4. The tripeptide-like compound according to claim 1, characterized in that: R1 is selected from H, -OH, F, -CH3; R2 is selected from H, Cl, -CH3; R3 is selected from H, F; R4 is selected from H, F, -CH3; at least two of R1, R2, R3, and R4 are not H; X represents the configuration of the chiral center, which is R or S.

5. The tripeptide-like compound according to claim 1, characterized in that: The compounds described in Formula I are selected from the compounds in Table 1: Table 1 。 6. The tripeptide-like compound according to any one of claims 1-4, characterized in that: The halogens include fluorine, chlorine, bromine, and iodine; the alkyl groups include straight-chain alkyl groups with 1-18 carbon atoms and branched alkyl groups with 3-18 carbon atoms; the alkoxy groups include straight-chain alkoxy groups with 1-18 carbon atoms and branched alkoxy groups with 3-18 carbon atoms; and the substituted alkyl groups refer to alkyl groups in which the hydrogen atom on the carbon atom is replaced by a halogen.

7. The use of the tripeptide compound according to any one of claims 1-6 in the preparation of anti-PRV virus drugs.

8. The application according to claim 7, characterized in that: The specific application method is as follows: the tripeptide-like compound according to any one of claims 1-6 is used as the active ingredient against PRV virus, and the content of the active ingredient in the anti-PRV virus drug is 0.1-100 wt%; Alternatively, the tripeptide compound of any one of claims 1-6 may be used as the active ingredient against PRV virus, and a veterinary-acceptable carrier and surfactant may be added as needed to formulate it into tablets, capsules, granules, pellets, liquid preparations, decoctions, suppositories, gels, aerosols or patches.

9. An anti-PRV drug, characterized in that: The active ingredient comprising any one of claims 1-6 and pharmaceutically acceptable excipients, including an effective amount of the tripeptide compound according to any one of claims 1-6 against PRV.

10. The anti-PRV virus drug according to claim 10, characterized in that: The dosage forms of the drug include, but are not limited to, tablets, capsules, granules, pills, liquid preparations, decoctions, suppositories, gels, aerosols, or patches.

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