Coronavirus plpro fluorescent ligand probe and uses thereof

Abstract

This invention discloses a coronavirus PLpro fluorescent ligand probe represented by formula (1): This invention also provides the use of the compound of formula (1) in screening PLpro ligands or in preparing products for screening PLpro ligands.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a coronavirus PLpro fluorescent ligand probe and its applications. Background Technology

[0002] Coronaviruses are a class of enveloped, single-stranded, positive-sense RNA viruses whose primary hosts are vertebrates, including humans, mice, pigs, cattle, cats, dogs, and birds. Canine coronaviru (CCV) typically causes mild to severe gastroenteritis in dogs, and its variants pose a risk of zoonotic transmission. Porcine acute diarrhea syndrome coronavirus (SADA-CoV) causes acute diarrhea, vomiting, and dehydration in newborn piglets, with a mortality rate as high as 90%, posing a significant threat to the livestock industry. Human coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 generally cause mild, seasonal respiratory infections, while highly pathogenic coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe and even fatal respiratory diseases, becoming a major challenge to global public health security.

[0003] Currently, the main drug targets for coronavirus therapy include RNA-dependent RNA polymerase (RdRp), major protease (Mpro), and papain-like protease (PLpro). Among these, PLpro, as a viral protease, plays a crucial role in the cleavage of viral polyproteins. It recognizes and cleaves the conserved LXGG sequence in the polyprotein, forming a functional replicase complex that participates in viral replication. Notably, PLpro also participates in the deubiquitination and ISG (ISGylation) processes of host antiviral proteins, enabling viral immune evasion by interfering with host immune signaling pathways. Therefore, drugs targeting PLpro may possess both antiviral and anti-inflammatory effects.

[0004] In antiviral drug development, high-throughput screening (HTS) is a key strategy for efficiently identifying lead compounds. It often uses recombinant target proteins as a basis, rapidly screening molecules with strong binding affinity or excellent enzyme activity inhibition through biochemical methods. Biochemical screening typically employs microplate systems, utilizing trace amounts of chemical reagents to achieve initial screening of ligand molecule activity, offering advantages such as high throughput, low cost, and high efficiency. Currently commonly used biochemical screening techniques include fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and time-resolved fluorescence resonance energy transfer (TR-FRET). FP technology monitors changes in intermolecular binding or dissociation by tracking changes in the fluorescence polarization of fluorescent tracers. It is simple to operate and highly sensitive for measuring interactions between small and large molecules, but it is insensitive to interactions between large molecules and is easily affected by background fluorescence. FRET (Fluorescent Transient Emission Spectrometry) technology infers the donor-acceptor distance by measuring the energy transfer efficiency between the fluorescent donor and acceptor, making it suitable for studying interactions between small molecules and macromolecules, and between macromolecules. TR-FRET, a variant of FRET, uses a lanthanide element with long-lasting fluorescence as the donor and a fluorescent protein or organic fluorophore as the acceptor. It avoids interference from short-lived background fluorescence by delaying the fluorescence emission time of the acceptor. TR-FRET can measure intermolecular interactions on sub-nanosecond to microsecond timescales, offering advantages such as high signal-to-noise ratio, high sensitivity, fast detection speed, and strong anti-interference capabilities, making it ideal for high-throughput drug screening. Competitive TR-FRET introduces a fluorescent ligand probe (i.e., a tracer) into the traditional TR-FRET system. In a competitive TR-FRET system, the unlabeled analyte (competitor) competes with the fluorescent ligand probe for binding to the target molecule, and the binding affinity between the competitor and the target is quantified based on the degree of FRET signal reduction. The advantage of competitive TR-FRET over traditional TR-FRET lies in its ability to reduce the risk of altering the molecular structure, activity, or accessibility of binding sites caused by labeling the analyte, and to simplify experimental design by labeling only the target molecule. However, there is currently a lack of highly sensitive coronavirus PLpro fluorescent ligand probes, which makes it difficult to meet the detection needs of high-throughput drug screening. Summary of the Invention

[0005] This invention provides a class of compounds that can be used as fluorescent ligand probes for coronavirus PLpro. These compounds have a strong binding affinity to coronavirus PLpro, are suitable for competitive TR-FRET systems, have high detection sensitivity, and can be used for high-throughput screening of PLpro ligands, especially PLpro inhibitors.

[0006] Specifically, the first aspect of the present invention relates to the compound shown in formula (1):

[0007]

[0008] Where L is selected from the following structure:

[0009] A is selected from -O-, -NH-, -NCH3-, or -CONH-;

[0010] n, m, and q are each independently selected from integers from 0 to 10;

[0011] k is an integer from 2 to 10.

[0012] According to some embodiments of the present invention, n, m, and q are each independently selected from integers from 1 to 10, for example, selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0013] According to some preferred embodiments of the present invention, n is selected from an integer from 1 to 8.

[0014] According to some preferred embodiments of the present invention, m and q are each independently selected from integers from 1 to 8, and further selected from integers from 1 to 5.

[0015] According to some embodiments of the present invention, k is selected from an integer from 2 to 8, for example, selected from 2, 3, 4, 5, 6, 7 or 8.

[0016] According to some embodiments of the present invention, L is selected from...

[0017] According to some embodiments of the present invention, A is selected from -O-, -NH- or -NCH3-, and further selected from -O-.

[0018] Specific examples of L include, but are not limited to, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -CH2OCH2-, -CH2O(CH2)2-, or -(CH2)2O(CH2)2-.

[0019] The second aspect of the present invention provides applications of the said compound, including at least one of the following:

[0020] a1) as a fluorescent probe for screening PLpro ligands;

[0021] a2) Prepare products for screening PLpro ligands.

[0022] According to some embodiments of the present invention, the PLpro ligand has PLpro inhibitory activity, for example, and is used as a PLpro inhibitor.

[0023] Further, the PLpro is a protease derived from alpha coronavirus and / or beta coronavirus. As an example, the alpha coronavirus is selected from canine coronavirus CCV and swine coronavirus SADS-CoV; the beta coronavirus is selected from human coronavirus SARS-CoV-2, HCoV-OC43, HCoV-HKU1 and swine coronavirus PHEV, and more specifically from SARS-CoV-2.

[0024] According to some embodiments of the present invention, the product includes at least one of reagents, reagent kits, and detection systems.

[0025] A third aspect of the present invention provides a reagent comprising the aforementioned compound. The reagent can be used to screen PLpro ligands, such as PLpro inhibitors.

[0026] A fourth aspect of this invention provides a kit comprising the described compound and / or the described reagent. The kit can be used to screen PLpro ligands, such as PLpro inhibitors.

[0027] According to some embodiments of the present invention, the kit further comprises: a protein containing a PLpro ligand-binding domain, the protein being linked to a tagged protein.

[0028] According to some embodiments of the present invention, the kit further comprises rare earth element-labeled antibodies that specifically recognize the tagged protein.

[0029] Furthermore, the rare earth element comprises at least one of La, Sc, Y, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu; furthermore, the rare earth element comprises lanthanide elements, and even more specifically, Tb.

[0030] Furthermore, the tag protein comprises at least one of c-Myc, His, GST, HA, and Flag; and even further comprises His.

[0031] According to some embodiments of the present invention, the kit further comprises a buffer solution.

[0032] According to some embodiments of the invention, the kit also includes a container, such as a bottle, ampoule, syringe and / or dispenser, or other suitable container.

[0033] A fifth aspect of the present invention provides a detection system comprising at least one of b1) to b3):

[0034] The compound described in b1);

[0035] The reagent described in b2);

[0036] The kit described in b3;

[0037] And optionally, include an ELISA reader.

[0038] The detection system can be used to screen PLpro ligands, such as PLpro inhibitors.

[0039] A sixth aspect of the present invention provides a method for screening PLpro ligands, comprising at least one of the following steps:

[0040] c1) Use the compound described above;

[0041] c2) Use the reagents described above;

[0042] c3) Use the kit described above;

[0043] c4) Use the detection system described above.

[0044] According to some embodiments of the present invention, the method for screening PLpro ligands includes the following steps:

[0045] S1. Mix the compound, protein A containing a PLpro ligand-binding domain and linked to a tagged protein, a rare earth element-labeled antibody that specifically recognizes the tagged protein, and the test substance to obtain a mixture;

[0046] S2. Measure the signal value of the mixture using time-resolved fluorescence resonance energy transfer (RTE) technology, and determine whether the analyte is a PLpro ligand based on the signal value.

[0047] Wherein, the signal value refers to the ratio of the fluorescence signal intensity of the receptor to the fluorescence signal intensity of the donor, the receptor is the fluorescent group carried by the compound (i.e., the fluorescent ligand probe), and the donor is the rare earth element fluorescent group labeled by protein A, which is linked to the tag protein and recognized by the antibody.

[0048] Furthermore, the mixture also contains a buffer solution.

[0049] Further, in the mixture, the concentration of the compound is 10-50 nM, more specifically 20-30 nM.

[0050] Furthermore, in the mixture, the concentration of protein A is 5–20 nM, more specifically 5–15 nM.

[0051] Furthermore, the concentration of the antibody in the mixture is 0.5 nM to 1.5 nM.

[0052] The structure of the compound is as follows Figure 1 As shown, the orange area on the left represents the original ligand group, which is used to bind to the PLpro protein; the blue area on the right is the fluorescent group; and the green area in the middle is the linker group, which is used to connect the original ligand and the fluorescent group and adjust the overall spatial configuration of the molecule to optimize target binding ability and fluorescence signal transmission efficiency.

[0053] The compounds described herein can be prepared using common organic synthesis methods, with mature synthetic routes and easy availability. As fluorescent ligand probes, they exhibit strong binding affinity to the PLpro ligand binding domains of various coronaviruses (including human coronaviruses SARS-CoV-2, HCoV-OC43, HCoV-HKU1, canine coronavirus CCV, and porcine coronaviruses SADS-CoV and PHEV), demonstrating broad spectral adaptability. Furthermore, these probes exhibit high sensitivity and a clear signal response window in competitive TR-FRET detection systems for PLpro and its inhibitors, making them suitable for high-throughput screening of PLpro ligands and providing effective support for the development of drugs such as coronavirus PLpro inhibitors.

[0054] In this article, the numerical ranges mentioned all include the endpoint values ​​and cover any subranges within that range, such as the ranges obtained by arbitrarily combining the specifically listed numerical values. Attached Figure Description

[0055] Figure 1 Here is the structural diagram of the compound of formula (1).

[0056] Figure 2 The binding curves of PL1 to PL7 fluorescent probes to SARS-CoV-2 PLpro are shown.

[0057] Figure 3 The binding curve of the PL3 fluorescent probe to HCoV-OC43 PLpro is shown.

[0058] Figure 4 The binding curves of the PL3 fluorescent probe and HCoV-HKU1 PLpro are shown.

[0059] Figure 5The binding curves of the PL3 fluorescent probe and CCV PLpro are shown.

[0060] Figure 6 The binding curves of the PL3 fluorescent probe and SADS-CoV PLpro are shown.

[0061] Figure 7 The binding curves of the PL3 fluorescent probe and PHEV PLpro are shown.

[0062] Figure 8 The curves show the competitive binding of PL1 to PL7 fluorescent probes to SARS-CoV-2 PLpro. Detailed Implementation

[0063] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0064] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0065] Example 1: Synthesis of compound PL1

[0066] 1. Synthesis of intermediates 1-10

[0067]

[0068]

[0069] Step 1.1: 1,8-Naphtholactamimide (10 g, 59.1 mmol, 1.0 eq.) and acetonitrile (ACN, 50 mL) were added to a 250 mL round-bottom flask. N-bromosuccinimide (NBS, 11.6 g, 65.0 mmol, 1.1 eq.) was added at 0 °C, followed by stirring at 0 °C for 4 hours. After the reaction was complete, H2O (50 mL) was added, and the mixture was stirred for 5 minutes. The mixture was then filtered, washed, and the filter cake was dried to obtain a yellow powdery intermediate 1 (11.7 g, 80%).

[0070] 1 H NMR(500MHz, CDCl3)δ8.12(s,1H),7.97(s,1H),7.67(s,1H),7.61(s,1H),7.44(s,1H).MS(ESI)247.96[M+H] + .

[0071] Step 1.2: In a 250 mL round-bottom flask, add intermediate 1 (11.7 g, 47.4 mmol, 1.0 eq.) and DMF (100 mL). Add NaH (2.8 g, 71.1 mmol, 1.5 eq.) at 0 °C, stir for 30 minutes, then add iodomethane (8.1 g, 56.9 mmol, 1.2 eq.) and stir at room temperature for 1 hour. Monitor the reaction with LC-MS. After the reaction is complete, add H2O (50 mL), stir for 5 minutes, filter, wash, and dry the filter cake to obtain a yellow powder intermediate 2 (8.0 g, 65%).

[0072] 1 H NMR(500MHz, CDCl3)δ8.07(s,1H),7.97(s,1H),7.67(s,1H),7.51(d,J=11.5Hz,2H),3.65(s,3H).MS(ESI)261.97[M+H] + .

[0073] Step 1.3: In a 250 mL round-bottom flask, add intermediate 2 (8.0 g, 30.8 mmol, 1.0 eq.), pinacol diborate (9.4 g, 36.9 mmol, 1.2 eq.), PdCl2 (dppf) (1.1 g, 1.54 mmol, 0.05 eq.), potassium acetate (7.5 g, 77 mmol, 2.5 eq.), and 1,4-dioxane (100 mL). Under N2 protection, stir at 100 °C for 12 hours. After the reaction is complete, add 100 mL of water and extract three times (100 mL × 3) with ethyl acetate (EA). Combine the organic phases, dry under anhydrous Na2SO4, evaporate to dryness under reduced pressure, and purify by column chromatography (petroleum ether:ethyl acetate = 5:1, v / v) to obtain intermediate 3 (7.1 g, 75%) as a yellow powder.

[0074] 1 H NMR(500MHz, CDCl3)δ8.11(s,1H),7.76(s,1H),7.68(s,1H),7.54(s,1H),3.65(s,1H),1.30(s,5H).MS(ESI)310.15[M+H] + .

[0075] Step 1.4: In a 250 mL round-bottom flask, add intermediate 3 (7.1 g, 23.0 mmol, 1.0 eq.), 1-bromo-cyclopropaneformonitrile (4.0 g, 27.6 mmol, 1.2 eq.), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 1.3 g, 1.1 mmol, 0.05 eq.), potassium carbonate (7.9 g, 57.5 mmol, 2.5 eq.), and a 1,4-dioxane / H2O mixed solution (3:1, total 80 mL, v / v). Stir at 100 °C for 12 hours under N2 protection. After the reaction is complete, add 100 mL of water and extract three times with EA (100 mL × 3). Combine the organic phases, dry under anhydrous Na2SO4, evaporate to dryness under reduced pressure, and purify by column chromatography (petroleum ether:ethyl acetate = 5:1, v / v) to obtain a yellow oily intermediate 4 (4.0 g, 71%).

[0076] 1 H NMR(500MHz, CDCl3)δ8.07(s,1H),7.99(s,1H),7.66(s,1H),7.34(s,1H),3.65(s,3H),1.97(d,J=4.9Hz,2H),1.73(s,2H).MS(ESI)249.09[M+H] + .

[0077] Step 1.5: In a 250 mL round-bottom flask, add intermediate 4 (4.0 g, 16.1 mmol, 1.0 eq.) and DMSO (100 mL), then add potassium carbonate (2.3 g, 17.7 mmol, 1.1 eq.) at 0 °C, followed by 30 wt% H₂O₂ (80.6 mmol, 5 eq.). Stir for 30 minutes, then transfer to room temperature and react for 1 hour. After the reaction is complete, add H₂O (100 mL) and stir for 5 minutes. Filter, wash, and dry the filter cake to obtain a yellow powder, intermediate 5 (2.6 g, 61%).

[0078] 1 H NMR(500MHz, CDCl3)δ8.07(s,1H),7.89(s,1H),7.66(s,1H),7.24(s,1H),6.60 (s,2H),3.65(s,2H),1.64(s,1H),1.40(d,J=4.9Hz,2H).MS(ESI)267.10[M+H] + .

[0079] Step 1.6: In a 250 mL round-bottom flask, add intermediate 5 (2.6 g, 10.0 mmol, 1.0 eq.) and tert-butanol (50 mL), and add sodium hydroxide (1.1 g, 28.0 mmol, 2.8 eq.) at 0 °C, followed by 10 wt%–15 wt% NaClO solution (20 mL). React at room temperature for 12 hours. After the reaction is complete, evaporate the solvent to dryness, add 100 mL of water and extract three times with EA (100 mL × 3). Combine the organic phases, dry under anhydrous Na2SO4, evaporate to dryness under reduced pressure, and purify by column chromatography (petroleum ether: ethyl acetate = 3:1, v / v) to obtain a yellow oily intermediate 6 (2.0 g, 86%).

[0080] 1 H NMR (500MHz, CDCl3) δ8.07 (d, J = 0.8Hz, 2H), 7.66 (s, 1H), 7.37 (s, 1H), 3.65 ( s,2H),3.55(s,1H),1.71(s,1H),1.46(d,J=4.9Hz,2H).MS(ESI)239.11[M+H] + .

[0081] Step 1.7: Add methyl 2-methyl-5-bromobenzoate (5 g, 22.0 mmol, 1.0 eq.), 6-(tert-butoxycarbonyl)-3,6-diazabicyclo[3.1.1]heptane (6.5 g, 32.9 mmol, 1.5 eq.), Pd2(dba)3 (0.4 g, 0.44 mmol, 0.2 eq.), X-Phos (0.42 g, 0.88 mmol, 0.4 eq.), cesium carbonate (14.3 g, 44.0 mmol, 2 eq.), and toluene (100 mL) to a 250 mL round-bottom flask, and react at 110 °C for 12 hours under N2 protection. After the reaction was complete, the solvent was evaporated to dryness, 100 mL of water was added, and the mixture was extracted three times with EA (100 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, evaporated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 6:1, v / v) to give intermediate 7 (3.1 g, 42.6%) as a yellow powder.

[0082] 1 H NMR (500MHz, CDCl3) δ7.18 (dq, J=7.5, 1.0Hz, 1H), 6.80 (dd, J=7.5, 2.0Hz, 1H), 4.14 (s, 1H), 3. 89(s,2H),3.82(s,1H),3.57(s,1H),2.43(d,J=0.9Hz,2H),2.14(s,1H).MS(ESI)347.18[M+H] + .

[0083] Step 1.8: Add intermediate 7 (3.1 g, 9.3 mmol, 1.0 eq.), sodium hydroxide (1.9 g, 46.5 mmol, 5 eq.), and a mixed solution of MeOH / THF / H2O (1:3:1, v / v / v, 50 mL) to a 100 mL round-bottom flask. React at 60 °C for 1 hour. After the reaction is complete, adjust the pH to 6-7 with 1 M HCl. A solid precipitates out. Filter the solid and dry the filter cake to obtain a gray powder intermediate 8 (2.8 g, 95%).

[0084] 1 H NMR(500MHz, CDCl3) δ7.49(d,J=2.0Hz,1H),7.17–7.11(m,1H),6.80(dd,J=7.5,2.0Hz,1H),4. 14(s,1H),3.82(s,1H),3.57(s,1H),2.43(d,J=1.1Hz,2H),2.14(s,1H).MS(ESI)333.17[M+H] + .

[0085] Step 1.9: In a 250 mL round-bottom flask, add intermediate 6 (2.0 g, 8.4 mmol, 1.0 eq.), intermediate 8 (2.7 g, 8.4 mmol, 1.0 eq.), HATU (4.8 g, 12.6 mmol, 1.5 eq.), DIPEA (3.2 g, 25.2 mmol, 3.0 eq.), and DMF (50 mL). React at 50 °C for 2 hours. After the reaction is complete, add 100 mL of water and extract three times with EA (100 mL × 3). Combine the organic phases, dry under anhydrous Na2SO4, evaporate to dryness under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 6:1, v / v) to obtain a yellow oily intermediate 9 (3.5 g, 75%).

[0086] 1 H NMR(500MHz, CDCl3)δ8.07(s,1H),7.91(s,1H),7.84(s,1H),7.66(s,1H),7.42(s, 1H),7.33(s,1H),7.19(dq,J=7.4,0.9Hz,1H),6.80(s,1H),6.80(dd,J=7.5,2.0Hz ,1H),4.14(s,2H),3.82(s,2H),3.65(s,2H),3.57(s,2H),2.41(d,J=1.1Hz,3H),2 .14(s,2H),1.95(d,J=5.1Hz,2H),1.70(s,1H),1.45(s,7H).MS(ESI)553.27[M+H] + .

[0087] Step 1.10: Add intermediate 9 (3.5 g, 6.3 mmol, 1.0 eq.), TFA (1.4 g, 12.6 mmol, 2.0 eq.), and DCM (40 mL) to a 100 mL round-bottom flask, react at room temperature for 2 hours, evaporate the solvent, add methyl tert-butyl ether (20 mL), stir for 5 minutes, filter, and obtain yellow powdery solid intermediate 10 (2.5 g, 87%).

[0088] 1 H NMR (500MHz, CDCl3) δ8.07(s,1H),7.91(s,1H),7.84(s,1H),7.66(s,1H),7.42(s,1H),7.31(s,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.75(m,2 H),3.82(s,2H),3.65(s,2H),3.57(s,2H),3.46(s,2H),2.41(d,J=1.1Hz ,3H),2.32(s,1H),1.97–1.90(m,4H),1.70(s,1H).MS(ESI)453.22[M+H] + .

[0089] 2. Synthesis of compound PL1

[0090]

[0091] Step 2.1: In a 100 mL round-bottom flask, N-(tert-butyloxycarbonyl)ethanolamine (200 mg, 1.2 mmol, 1.0 eq.) and anhydrous dichloromethane (DCM, 20 mL) were added. At 0 °C, methanesulfonic anhydride (324 mg, 1.8 mmol, 1.5 eq.) and 4-dimethylaminopyridine (1.5 mg, 0.012 mmol, 0.01 eq.) were added. After stirring for 10 minutes, the mixture was transferred to room temperature and reacted for 2 hours. After the reaction was complete, 100 mL of water was added and the mixture was extracted three times with EA (100 mL × 3). The organic phases were combined, dried over anhydrous Na₂SO₄, and evaporated under reduced pressure to obtain a colorless oily intermediate 11 (237 mg, 80%), which could be used directly in the next reaction without further purification.

[0092] Step 2.2: In a 100 mL round-bottom flask, add intermediate 11 (80 mg, 0.15 mmol, 1.5 eq.), intermediate 10 (100 mg, 0.22 mmol, 1.0 eq.), triethylamine (67 mg, 0.66 mmol, 3.0 eq.), and DMF (50 mL), and stir at 90 °C for 12 hours. After the reaction is complete, add 100 mL of water and extract three times with EA (100 mL × 3). Combine the organic phases, dry to anhydrous Na2SO4, evaporate to dryness under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 50:1, v / v) to obtain a yellow powder, intermediate 12 (110 mg, 84%).

[0093] Step 2.3: Add intermediate 12 (110 mg, 0.18 mmol, 1.0 eq.), TFA (42 mg, 0.36 mmol, 2.0 eq.), and DCM (10 mL) to a 100 mL round-bottom flask, react at room temperature for 2 hours, and evaporate the solvent to obtain a yellow oily intermediate 13-1, which can be used directly in the next step of the reaction without further purification.

[0094] Step 2.4: Add intermediate 13-1 (79.2 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), triethylamine (TEA, 48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL1, 5-(6-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)ethyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (31 mg, 21%).

[0095] 1H NMR (500MHz, CDCl3) δ9.74(s,1H),8.07(s,0H),8.02(d,J=1.9Hz,1H),7.91(s,0H),7.85(d,J=9.0Hz,1H),7 .74–7.65(m,1H),7.36–7.29(m,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,1H),6.76–6.69(m,2H),6.59 (dd,J=16.6,1.7Hz,1H),3.70(s,1H),3.70–3.60(m,1H),3.65(s,2H),3.45(s,1H),3.18(s,1H),2.93(d,J= 12.5Hz,0H),2.80(d,J=12.5Hz,0H),2.41(d,J=1.1Hz,2H),1.99(s,1H),1.95(d,J=5.1Hz,1H),1.70(s,1H).

[0096] 13 C NMR (125MHz, CDCl3) δ179.64,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,14 4.23,138.44,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,12 7.86,127.17,126.25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,1 01.57,83.31,65.16,62.44,61.58,57.64,43.77,36.68,30.16,22.56,21.77.MS(ESI)885.29[M+H] + .

[0097] Example 2: Synthesis of compound PL2

[0098]

[0099] The synthesis of intermediate 13-2 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of 2-(2-BOC-aminoethoxy)ethanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-2.

[0100] Add intermediate 13-2 (86.3 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL2,5-(6-(2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)ethoxy)ethyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (24 mg, 16%).

[0101] 1 H NMR (500MHz, CDCl3) δ9.74 (s, 2H), 8.22 (s, 1H), 8.07 (s, 1H), 8.02 (d, J = 1.9Hz, 1H), 7.91 (s, 1H), 7.84 (s, 1H) ,7.74–7.65(m,2H),7.36–7.29(m,2H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,2H),6.76–6.69(m,4H),6. 59(dd,J=16.6,1.7Hz,2H),3.90(s,2H),3.72(s,2H),3.69–3.56(m,7H),3.47(s,2H),3.16(s,2H),2.72(d,J =12.5Hz,1H),2.59(d,J=12.5Hz,1H),2.41(d,J=1.1Hz,3H),1.99(s,2H),1.95(d,J=5.1Hz,2H),1.70(s,1H).

[0102] 13C NMR (125MHz, CDCl3) δ180.77,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.2 3,138.44,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,1 27.17,126.25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83. 31,71.48,69.46,65.16,61.90,61.58,56.03,46.48,36.33,30.16,22.56,21.77.MS(ESI)929.32[M+H] + .

[0103] Example 3: Synthesis of compound PL3

[0104]

[0105] The synthesis of intermediate 13-3 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of (N-tert-butoxycarbonylamino)-1-butanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-3.

[0106] Add intermediate 13-3 (84 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL3,5-(6-(4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)butyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (20.4 mg, 14%).

[0107] 1H NMR (500MHz, CDCl3) δ9.74 (s, 1H), 8.02 (d, J = 1.9Hz, 0H), 7.84 (s, 1H), 7.74–7.65 (m, 1H), 7. 46(s,0H),7.36–7.29(m,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,1H),6.76–6.69(m, 2H),6.59(dd,J=16.6,1.7Hz,1H),3.70(s,1H),3.65(s,1H),3.61(s,1H),3.45(s,1H),3.19 (s,1H),2.41(d,J=1.1Hz,2H),1.95(d,J=5.1Hz,1H),1.74–1.67(m,2H),1.67–1.57(m,1H).

[0108] 13 C NMR (125MHz, CDCl3) δ181.13,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.2 3,139.09,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,1 27.17,126.25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83. 31,65.16,61.88,61.51,56.67,45.29,36.67,30.16,28.53,27.08,22.56,21.77.MS(ESI)913.33[M+H] + .

[0109] Example 4: Synthesis of compound PL4

[0110]

[0111] The synthesis of intermediate 13-4 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of (N-tert-butoxycarbonylamino)-1-pentanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-4.

[0112] Add intermediate 13-4 (86 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL4,5-(6-(5-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)pentyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (22 mg, 15%).

[0113] 1 H NMR(500MHz, CDCl3)δ9.74(s,1H),8.02(d,J=1.9Hz,0H),7.84(s,1H),7.74–7.65(m,1H),7.51(s,0 H),7.36–7.29(m,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,1H),6.76–6.69(m,2H),6.59(dd, J=16.6,1.7Hz,1H),3.70(s,1H),3.65(s,1H),3.60(s,1H),3.45(s,1H),3.19(s,1H),2.41(d,J=1. 1Hz,2H),1.99(s,1H),1.95(d,J=5.1Hz,1H),1.70(d,J=4.9Hz,1H),1.59–1.49(m,1H),1.37(s,1H).

[0114] 13C NMR (125MHz, CDCl3) δ181.16,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.23, 139.09,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,127. 17,126.25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83.31,65 .16,61.88,61.51,55.22,45.46,36.67,30.51,30.16,28.82,27.13,22.56,21.77.MS(ESI)927.34[M+H] + .

[0115] Example 5: Synthesis of compound PL5

[0116]

[0117] The synthesis of intermediate 13-5 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of 7-(BOC-amino)-1-heptanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-5.

[0118] Add intermediate 13-5 (90 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL5, 5-(6-(7-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiouretyl)heptyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (19.8 mg, 13%).

[0119] 1H NMR(500MHz, CDCl3)δ9.74(s,1H),8.02(d,J=1.9Hz,0H),7.84(s,0H),7.74–7.65(m,1H),7.51(s,0H),7. 36–7.29(m,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,1H),6.76–6.69(m,2H),6.59(dd,J=16.6,1.7 Hz,1H),3.70(s,1H),3.65(s,1H),3.60(s,1H),3.45(s,1H),3.19(s,1H),2.41(d,J=1.1Hz,1H),1.99(s, 1H),1.95(d,J=5.1Hz,1H),1.70(d,J=4.9Hz,1H),1.59–1.49(m,1H),1.34(s,1H),1.28(d,J=1.0Hz,2H).

[0120] 13 C NMR (125MHz, CDCl3) δ181.23,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.23,139 .09,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,127.17,126 .25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83.31,65.16,61.88 ,61.51,55.42,45.46,36.67,31.72,30.79,30.16,29.30,29.13,27.94,22.56,21.77.MS(ESI)955.37[M+H] + .

[0121] Example 6: Synthesis of compound PL6

[0122]

[0123] The synthesis of intermediate 13-6 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of (N-tert-butoxycarbonylamino)-1-propanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-6.

[0124] Add intermediate 13-6 (81.5 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL6,5-(6-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)propyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (15.8 mg, 11%).

[0125] 1 H NMR(500MHz,Chloroform-d)δ9.74(s,1H),8.02(d,J=1.9Hz,0H),7.84(s,1H),7.74–7.65(m,1 H),7.36–7.29(m,1H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,1H),6.76–6.69(m,2H),6.59( dd,J=16.6,1.7Hz,1H),3.70(s,1H),3.65(s,1H),3.45(s,1H),3.40(d,J=0.9Hz,1H),3.18(s, 1H),2.41(d,J=1.1Hz,2H),1.99(s,1H),1.95(d,J=5.1Hz,1H),1.92–1.82(m,1H),1.70(s,1H).

[0126] 13C NMR (125MHz, Common NMR Solvents)δ181.11,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.23,139 .09,136.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,127 .17,126.25,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83 .31,65.16,61.93,61.51,53.04,44.20,36.67,30.16,29.28,22.56,21.77.MS(ESI)899.31[M+H]+.

[0127] Example 7: Synthesis of compound PL7

[0128]

[0129] The synthesis of intermediate 13-7 is the same as that of intermediate 13-1, except that the N-(tert-butoxycarbonyl)ethanolamine used in step 2.1 is replaced with an equimolar amount of (N-tert-butoxycarbonylamino)-1-hexanol, while other reaction conditions remain unchanged, thus obtaining intermediate 13-7.

[0130] Add intermediate 13-7 (88.2 mg, 0.16 mmol, 1.0 eq.), intermediate 14 (65 mg, 0.16 mmol, 1.0 eq.), TEA (48.6 mg, 0.48 mmol, 3.0 eq.), and THF / EtOH (volume ratio 1:1, total 20 mL) to a 100 mL round-bottom flask, and react overnight at room temperature in the dark. Purification was performed using preparative liquid chromatography (elution buffer A was acetonitrile, eluent B was 0.1% formic acid aqueous solution; gradient: from 30% A to 45% A within 20 min) to obtain a yellow powdery product (PL7, 5-(6-(6-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthanene]-5-yl)thiourea)hexyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-2-methyl-N-(1-(1-methyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclopropyl)benzamide) (18.1 mg, 12%).

[0131] 1H NMR(500MHz,Chloroform-d)δ9.74(s,1H),8.07(s,1H),8.02(d,J=1.9Hz,1H),7.91(s,1H),7.84(s,1H),7.74–7.65(m,2H) ,7.51(s,1H),7.36–7.29(m,2H),7.18(dq,J=7.5,1.0Hz,1H),6.82–6.76(m,2H),6.76–6.69(m,3H),6.59(dd,J=16.6,1.7H z,2H),3.70(s,1H),3.65(s,2H),3.60(s,1H),3.45(s,1H),3.19(s,1H),2.67(d,J=12.3Hz,1H),2.54(d,J=12.3Hz,1H),2. 41(d,J=1.1Hz,3H),1.99(s,1H),1.95(d,J=5.1Hz,2H),1.70(d,J=4.9Hz,2H),1.59–1.49(m,2H),1.34(s,1H),1.30(s,2H).

[0132] 13 C NMR (125MHz, Common NMR Solvents)δ181.23,169.69,169.46,168.51,164.41,154.50,153.80,147.80,147.06,144.23,139.09,1 36.66,134.28,132.51,131.24,129.90,129.56,128.99,128.91,128.55,128.41,127.86,127.17,126.2 5,125.97,123.07,119.15,117.91,117.48,115.46,113.97,113.23,102.20,101.57,83.31,65.16,61.8 8,61.51,55.42,45.46,36.67,30.96,30.16,29.53,29.28,27.99,22.56,21.77.MS(ESI)941.36[M+H]+.

[0133] Test case

[0134] The PLpro protein sequence involved in the test is as follows:

[0135] (1) The PLpro protein sequence of human coronavirus SARS-CoV-2:

[0136] EVRTIKVFTTVDNINLHTQVVDMSMTYGQQFGPTYLDGADVTKIKPHNSHEGKTFYVLPN

[0137] DDTLRVEAFEYYHTTDPSFLGRYMSALNHTKKWKYPQVNGLTSIKWADNNCYLATALLT

[0138] LQQIELKFNPPALQDAYYRARAGEAANFCALILAYCNKTVGELGDVRETMSYLFQHANLD

[0139] [[ID=�]]SCKRVLNVVCKTCGQQQTTLKGVEAVMYMGTLSYEQFKKGVQIPCTCGKQATKYLVQQE

[0140] SPFVMMSAPPAQYELKHGTFTCASEYTGNYQCGHYKHITSKETLYCIDGALLTKSSEYKGPITDVFYKENSYTTTIK (SEQ ID NO.1).

[0141] (2) Amino acid sequence of the PLpro protein of human coronavirus HCoV-OC43:

[0142] ILLTVDGVNFTNRFVPVGESFGKSLGNVFCDGVNVTKHKCDINYKGKVFFQFDNLSSEDLK

[0143] AVRSSFNFDQKELLAYYNMLVNCFKWQVVVNGKYFTFKQANNNCFVNVSCLMLQSLHL

[0144] TFKIVQWQEAWLEFRSGRPARFVALVLAKGGFKFGDPADSRDFLRVVFSQVDLTGAICDFE

[0145] IACKCGVKQEQRTGLDAVMHFGTLSREDLEIGYTVDCSCGKKLIHCVRFDVPFLICSNTPAS

[0146] VKLPKGVGSANIFIGDNVGHYVHVKCEQSYQLYDASNVKKVTDVTGKLSDCLYLKNLKQT (SEQ ID NO.2). [[ID=͟2]]

[0147] (3) Amino acid sequence of the PLpro protein of human coronavirus HCoV-HKU1:

[0148] AKKIDVLLTVDGVNFKSISLTVGEVFGKILGNVFCDGIDVTKLKCSDFYADKILYQYENLSL

[0149] ADISAVQSSFGFDQQQLLAYYNFLTVCKWSVVVNGPFFSFEQSHNNCYVNVACLMLQHIN

[0150] LKFNKWQWQEAWYEFRAGRPHRLVALVLAKGHFKFDEPSDATDFIRVVLKQADLSGAIC

[0151] ELELICDCGIKQESRVGVDAVMHFGTLAKTDLFNGYKIGCNCAGRIVHCTKLNVPFLICSNT

[0152] PLSKDLPDDVVAANMFMGVGVGHYTHLKCGSPYQHYDACSVKKYTGVSGCLTDCLYLKNLTQTFTSMLTNY(SEQ ID NO.3).

[0153] (4) Amino acid sequence of PLpro protein of canine coronavirus CCV:

[0154] SIPVKVTEDNVNHERVSVSFDKTYGEQLKGTVVIKDKDVTNQLPSAFDVGQKVVKAIDLD <0000s74>WQAHYGFHDAAAFSASSHDAYKFEVVTHSNFIVHKQTDNNCWINAICLALQRLKPQWKF <000037s>PGVRGLWNDFLERKTQGFVHMLYHISGVKKGEPGDAELTLHKLGDLMDNDCEIIVTHTTA

[0157] CDKCAKVEKFTGPVVAAPLAIHGTDETCVHGVSVNVKVTQIKGTVAITSLSGPVIGEVLEATGYICYRGSNKNGHYTYYDNRNGLMIDAEKAYHFNKDLLQVTTVI(SEQ ID NO.4).

[0158] (5) Amino acid sequence of PLpro protein of porcine coronavirus SADS-CoV:

[0159] KDVKVKVTADGRNINDVIVTTAETFDAQLGPSANGAESLVGVVPTPTDGGKVVNTVPDVN

[0160] WSKHFGFSDAAAFAVLDHSKFAFDSEVVDGKRALADSDNNCWVNATCLALQFLKPTFKY

[0161] VGWEDLWNKFVTGDVAGFVHLLYYIEGVDKGAKGDVESTLSKLDKYIVSSGSVTVERSTL

[0162] CDRCNSTVKTVTGAIAEASVILNGHTDGHCPHNFEWRVQVIGVKGDIILLHSGSLLNGPYVYGDAYVAFSGNTDNGHYTVFDNKLSKMYDGIKCVKTTLDTLVASSVVI (SEQ ID NO. 5).

[0163] (6) PLpro protein sequence of porcine coronavirus PHEV:

[0164] DKVDVLLTVDGVNFTNRFIPVSESFGKILGNVFCDGVNVTKHKCDIKYKGKVFFQFDNLSS

[0165] EELKAVRSSFNFDQKELLAYYNMLVNCNKWQVVVNGKYFTFKQANNNCFVNVSCLMLQ

[0166] SLTLKFKIVQWQEAWLEFRSGRPARFVALVMAKGGFKFGDPADSRDFLRVVFSQVDLTGA

[0167] TCDFEIACKCGVKQEQRTGLDAVMHFGTLSREELEVGYTVDCSCGKKLIHCVRFDVPFLIC

[0168] SNTPSSVKLPKGVVCANIFIGDKVGHYVHVKCEQAYQLYDASNVKKVADVTGNLSDCLY (SEQ ID NO. 6).

[0169] Test Example 1: Fluorescence polarization experiment of PL series fluorescent probes binding to the PLpro ligand binding domain of SARS-CoV-2

[0170] (1) Dilute 50 μM SARS-CoV-2PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a 1:3 ratio to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0171] (2) Add 0.8 μL of fluorescent probe (PL1-PL5) solution with a final concentration of 200 nM to each well of the protein solution in step (1), mix well, and then transfer to a 384-well plate with two replicates, each well containing 22.5 μL.

[0172] (3) Dilute 17 μM SARS-CoV-2PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a 1:3 ratio to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0173] (4) Add 0.8 μL of fluorescent probe (PL6 and PL7) solution with a final concentration of 200 nM to each well of the protein solution in step (3), mix well, and then transfer to a 384-well plate with two replicates, each well containing 22.5 μL.

[0174] (5) Incubate the mixture in each well of steps (2) and (4) at room temperature for 5 minutes, and read the values ​​on the multi-functional microplate reader. The excitation wavelength is 485 nm and the emission wavelength is 528 nm.

[0175] (6) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) of the fluorescent probe to SARS-CoV-2 PLpro was calculated. For example... Figure 2 As shown, the Kd values ​​of the fluorescent probes PL1 to PL7 are 94.62 nM, 11.94 nM, 20.87 nM, 16.85 nM, 25.10 nM, 29.52 nM and 30.59 nM, respectively, indicating that these probes all have strong binding affinity and are suitable for screening high-affinity ligands of SARS-CoV-2 PLpro.

[0176] Test Example 2: Fluorescence polarization experiment of PL3 fluorescent probe binding to the PLpro ligand binding domain of HCoV-OC43

[0177] (1) Dilute 7.15 μM HCoV-OC43 PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a ratio of 1:3 to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0178] (2) Add 0.8 μL of PL3 fluorescent probe solution with a final concentration of 200 nM to each well of the protein solution above, mix well, and then transfer to a 384-well plate, 2 replicates, 22.5 μL per well;

[0179] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 528 nm.

[0180] (4) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) of the fluorescent probe to HCoV-OC43 PLpro was calculated. For example... Figure 3 As shown, the Kd value of the PL3 fluorescent probe is 5.655 μM, which is suitable for screening high-affinity ligands of HCoV-OC43 PLpro.

[0181] Test Example 3: Fluorescence polarization experiment of PL3 fluorescent probe binding to the PLpro ligand binding domain of HCoV-HKU1

[0182] (1) Dilute 11.81 μM HCoV-HKU1 PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a ratio of 1:3 to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0183] (2) Add 0.8 μL of PL3 fluorescent probe solution with a final concentration of 200 nM to each well of the protein solution above, mix well, and then transfer to a 384-well plate, 2 replicates, 22.5 μL per well;

[0184] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 528 nm.

[0185] (4) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) of the fluorescent probe to HCoV-HKU1 PLpro was calculated. For example... Figure 4 As shown, the Kd value of the PL3 fluorescent probe is 2.216 μM, indicating that this type of probe has good binding affinity and is suitable for screening high-affinity ligands for HCoV-HKU1 PLpro.

[0186] Test Example 4: Fluorescence polarization experiment of PL3 fluorescent probe binding to the PLpro ligand binding domain of CCV

[0187] (1) Dilute the 14.29 μM PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a 1:3 ratio to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0188] (2) Add 0.8 μL of PL3 fluorescent probe solution with a final concentration of 200 nM to each well of the protein solution above, mix well, and then transfer to a 384-well plate, 2 replicates, 22.5 μL per well;

[0189] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 528 nm.

[0190] (4) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) between the fluorescent probe and CCV PLpro was calculated. For example... Figure 5 As shown, the Kd value of the PL3 fluorescent probe is 36.45 μM, indicating that this type of probe has good binding affinity and is suitable for screening high-affinity ligands for CCV PLpro.

[0191] Test Example 5: Fluorescence polarization experiment of PL3 fluorescent probe binding to the PLpro ligand binding domain of SADS-CoV

[0192] (1) Dilute the 53.97 μM PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a 1:3 ratio to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0193] (2) Add 0.8 μL of PL3 fluorescent probe solution with a final concentration of 200 nM to each well of the protein solution above, mix well, and then transfer to a 384-well plate, 2 replicates, 22.5 μL per well;

[0194] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 528 nm.

[0195] (4) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) of the fluorescent probe to SADS-CoV PLpro was calculated. For example... Figure 6 As shown, the Kd value of the PL3 fluorescent probe is 106.9 μM, indicating that this type of probe has good binding affinity and is suitable for screening high-affinity ligands for SADS-CoVPLpro.

[0196] Test Example 6: Fluorescence polarization experiment of PL3 fluorescent probe binding to the PLpro ligand binding domain of PHEV

[0197] (1) Dilute 19.05 μM PLpro-His protein solution with 0.005 wt% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) at a ratio of 1:3 to obtain 12 concentrations, and add 79.2 μL of each solution to a 96-well plate;

[0198] (2) Add 0.8 μL of PL3 fluorescent probe solution with a final concentration of 200 nM to each well of the protein solution above, mix well, and then transfer to a 384-well plate, 2 replicates, 22.5 μL per well;

[0199] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 528 nm.

[0200] (4) Data analysis was performed using Graphpad Prism software. A nonlinear regression model was used to fit the relationship curve of PLpro-His protein concentration changes, and the binding dissociation constant (Kd) of the fluorescent probe and PHEV PLpro was calculated. For example... Figure 7 As shown, the Kd value of the PL3 fluorescent probe is 9.730 μM, indicating that this type of probe has good binding affinity and is suitable for screening high-affinity ligands for PHEV PLpro.

[0201] Test Example 7: Competitive TR-FRET Binding Experiment of PL Series Fluorescent Probes to the PLpro Ligand Binding Domain of SARS-CoV-2

[0202] (1) Prepare a mixture containing 10 nM SARS-CoV-2PLpro-His protein, 1 nM terbium-labeled anti-His tag antibody, 25 nM fluorescent probes (PL1-PL7) and 0.005% polysorbate-20 test buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4). Take 79.2 μL of the above mixture and place it in a 96-well plate.

[0203] (2) The PLpro protein inhibitor GRL0617 was serially diluted from 2mM to 8 concentration points at a ratio of 1:4. 0.8 μL of each solution was added to the protein solution of the 96-well plate and mixed thoroughly. The solution was then transferred to a 384-well plate with 22.5 μL in each of the three replicate wells.

[0204] (3) The mixture was incubated at room temperature for 5 minutes, and the readings were taken with a multi-functional microplate reader. The excitation wavelength was 495 nm and the emission wavelength was 520 nm.

[0205] (4) Data analysis was performed using Graphpad Prism software, and a nonlinear regression model was used to fit the relationship curve between inhibitor concentration and fluorescence signal ratio. The vertical axis represents the energy transfer ratio (the ratio of emission intensity of the fluorescent receiver to that of the donor), reflecting the relative degree of binding between the fluorescent probe and the protein under different inhibitor concentrations.

[0206] The results are as follows Figure 8 As shown, in the TR-FRET detection systems established using PL1–PL7 as fluorescent tracers, the dissociation constants (Ki) of GRL0617 and the PLpro protein ligand-binding domain of SARS-CoV-2 were not significantly different, with Ki values ​​of 1.228 μM, 0.408 μM, 0.932 μM, 0.844 μM, 0.882 μM, 0.719 μM, and 0.995 μM, respectively. This indicates that the above probes can all be used for affinity detection of PLpro inhibitors. Notably, the competitive TR-FRET system using PL3 as a tracer exhibited a signal ratio change window of approximately 2-fold, indicating that PL3 ​​has higher detection sensitivity compared to other probes.

Claims

1. The compound shown in formula (1): Equation (1); in, L is selected from the following structure: or ; A is selected from -O-, -NH-, or -NCH3; n, m, and q are each independently selected from integers from 1 to 10.

2. Use of the compound as claimed in claim 1, comprising at least one of a1) to a2): a1) as a fluorescent probe for screening PLpro ligands; a2) Prepare products for screening PLpro ligands.

3. The use according to claim 2, characterized in that, The PLpro ligand includes a PLpro inhibitor.

4. A reagent, characterized in that, The reagent comprises the compound as described in claim 1.

5. A reagent kit, characterized in that, The kit contains the compound as described in claim 1, and / or the reagent as described in claim 4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes a protein containing a PLpro ligand-binding domain, the protein being linked to a tagged protein.

7. The reagent kit according to claim 6, characterized in that, The kit also contains rare earth element-labeled antibodies that specifically recognize the tagged protein.

8. A detection system, characterized in that, The detection system includes at least one of b1) to b3): b1) The compound according to claim 1; b2) The reagent according to claim 4; b3) The kit according to any one of claims 5-7.

9. The detection system according to claim 8, characterized in that, The detection system includes an enzyme-linked immunosorbent assay (ELISA) reader.

10. Use of the reagent as claimed in claim 4, or the kit as claimed in any one of claims 5-7, or the detection system as claimed in claim 8 or 9, for screening PLpro ligands.

11. The use according to claim 10, characterized in that, The PLpro ligand includes a PLpro inhibitor.

12. A method for screening PLpro ligands, characterized in that, Includes at least one of the following steps: c1) Using the compound as described in claim 1; c2) Using the reagent as described in claim 4; c3) Using the kit as described in any one of claims 5-7; c4) Use the detection system as described in claim 8 or 9.

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