PROTACs Targeting Coronavirus 3CL Protease, Preparation Method and Application Thereof

Abstract

The present invention discloses a preparation method and application of PROTACs targeting coronavirus 3CL protease, belonging to the field of medicinal chemistry. The PROTACs targeting coronavirus 3CL protease have the structures shown in Formula I or Formula II. The present invention selects lenalidomide and pomalidomide, which are ligands of Cereblon protein (CRBN), as E3 ligase ligands, and couples coronavirus 3CL protease inhibitors with E3 ligase through linkers of different types and different chain lengths, successfully preparing PROTACs targeting coronavirus 3CL protease, which can effectively target the target protein. The present invention overcomes the defects of the existing coronavirus 3CL protease inhibitors, such as a single structural type and limited ways of exerting drug effects (only having inhibitory effects), has good inhibitory and degradation activities against coronavirus 3CL protease, and can be developed and studied as an anti-coronavirus candidate drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to PROTACs targeting coronavirus 3CL protease, a preparation method thereof, and an application thereof. Background Art

[0002] COVID-19 (novel coronavirus pneumonia) is highly contagious and pathogenic, and its mutant strains such as Delta and Omicron have stronger transmission capabilities. The emerging mutant strains have made the global epidemic situation more complex, and the novel coronavirus has posed a serious threat to human health, social stability, and economic development.

[0003] 3CL pro (3C-like protease, also known as the main protease M pro ) As an important non-structural protein in coronaviruses, it has a cleavage site specificity similar to that of 3C protease of microRNA viruses and plays an extremely crucial role in the replication and transcription processes of progeny viruses. 3CL pro is a cysteine protease of about 33 kDa composed of 306 amino acids (much smaller than the S protein), which can specifically recognize and cleave 11 cleavage sites of non-structural proteins NSP4-NSP16, thereby releasing other non-structural proteins of coronaviruses. The non-structural proteins NSP4-NSP16 released by self-hydrolysis and cleavage by 3CL pro are carriers of viral genome replication and transcription, and are responsible for important life processes such as post-translational cleavage, modification, and nucleic acid synthesis of proteins. Inhibiting 3CL pro can effectively block the processes of RNA replication and transcription, thereby blocking the proliferation of the virus. Therefore, 3CL pro is considered to be one of the most attractive targets for the development of coronavirus-targeted drugs.

[0004] Proteolysis-targeting chimeric molecules (PROTACs) are one of the most subversive technologies in the field of drug development in recent years. Proteolysis-targeting chimera (PROTAC) is a technology for chemically degrading proteins. It can simultaneously bind to the target protein and the E3 ubiquitin ligase, bringing the target protein close to the E3 ubiquitin ligase, ubiquitinating the target protein, and then degrading the target protein through the degradation action of the ubiquitin-proteasome system (UPS). It is worth mentioning that regardless of the function of the target protein, the PROTAC technology can degrade it. The research on degrading exogenous proteins (such as viral proteins, etc.) using the PROTAC technology is currently in its infancy. The HCV NS3 / 4A protease degrader DGY-08-097 has significantly better antiviral activity and anti-drug resistance characteristics than the traditional drug Telaprevir, which confirms that PROTACs can degrade virus-related proteins. Summary of the Invention

[0005] In order to overcome the disadvantages of the above-mentioned prior art, the purpose of the present invention is to provide PROTACs targeting coronavirus 3CL protease, their preparation methods and applications, for treating diseases caused by coronaviruses.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions to be realized:

[0007] The present invention discloses PROTACs targeting coronavirus 3CL protease, which are compounds shown by formula I or formula II or pharmaceutically acceptable salts thereof, as well as solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion of the compounds shown by formula I or formula II or pharmaceutically acceptable salts thereof, including racemic mixtures;

[0008] The structural formulas of the compounds shown by formula I or formula II are:

[0009]

[0010] or

[0011]

[0012] Among them, Linker is one of the following linking groups:

[0013]

[0014] Among them, n = 1 - 6.

[0015] Preferably, the representative compounds are selected from the following compounds:

[0016]

[0017]

[0018] Preferably, the pharmaceutically acceptable salts are salts formed by PROTACs targeting coronavirus 3CL protease and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0019] The present invention also discloses a preparation method of the above-mentioned PROTACs targeting coronavirus 3CL protease, including the following operation steps:

[0020] (1) Using 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione as a raw material, undergoing an alkylation reaction with 2,4,5-trifluorobenzyl bromide to obtain compound a1; then introducing a 6-chloro-2-methyl-2H-indazole unit at the 6-position of the triazine nucleus to obtain compound a2; removing the tert-butyl group at the 3-position of the triazine nucleus of compound a2 in an acidic solvent to obtain compound a3; subsequently introducing a 3-propynyl group at the 3-position of the triazine nucleus of compound a3 to finally obtain compound a4;

[0021] Among them, a1 is a2 is a3 is a4 is

[0022] (2) Using lenalidomide as a raw material, undergoing an acid amide condensation reaction with bromoalkanoic acids of different lengths to obtain the corresponding compounds b1-b6, and then reacting with sodium azide under the catalysis of potassium iodide to obtain the corresponding compounds c1-c6;

[0023] Alternatively, using pomalidomide as a raw material, undergoing an acid amide condensation reaction with bromoalkanoic acids of different lengths to obtain the corresponding compounds b7-b12, and then reacting with sodium azide under the catalysis of potassium iodide to obtain the corresponding compounds c7-c12;

[0024] Among them, b1-b6 are n = 1 - 6; c1-c6 are n = 1 - 6; b7-b12 are n = 1 - 6; c7-c12 are n = 1 - 6;

[0025] Alternatively, using lenalidomide as a raw material, undergoing an acid amide condensation reaction with azido-polyethylene glycol-acetic acid compounds to obtain the corresponding compounds d1-d3;

[0026] Alternatively, using pomalidomide as a raw material, undergoing an acid amide condensation reaction with azido-polyethylene glycol-acetic acid compounds to obtain the corresponding compounds d4-d6;

[0027] Among them, d1-d3 are n = 1 - 6; d4-d6 are

[0028] n = 1 - 6;

[0029] (3) Reacting compound a4 with compounds c1-c6 or d1-d3 to generate PROTACs targeting the coronavirus 3CL protease with the structure of formula I;

[0030] Alternatively, compound a4 reacts with c7-c12 or d4-d6 to generate PROTACs targeting coronavirus 3CL protease with the structure of Formula II.

[0031] Preferably, in step (1), the molar ratio of 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione to 2,4,5-trifluorobenzyl bromide is 1:1.1, the molar ratio of compound a1 to 6-chloro-2-methyl-2H-indazol-5-amine is 1:1.3, and the molar ratio of compound a3 to 3-bromopropyne is 1:1.2; in step (2), the molar ratio of pomalidomide or lenalidomide to bromoalkanoic acids with different lengths is 1:2, and the molar ratio of pomalidomide or lenalidomide to azido-polyethylene glycol-acetic acid compounds is 1:2; in step (3), the molar ratio of compound a4 to c1-c12 or d1-d6 is 1:1.2.

[0032] Preferably, in step (1), during the synthesis of compound a2, the reaction temperature is 0 °C and the catalyst used is lithium bis(trimethylsilyl)amide; in step (2), during the synthesis of compounds b1-b6 and b7-b12, acyl chlorination reagents are used to dissolve bromoalkanoic acids with different lengths, and during the synthesis of compounds d1-d3 and d4-d6, the condensing agent used is thionyl chloride and the solvent used is tetrahydrofuran; in step (3), the solvent used is a mixed solvent of tetrahydrofuran and water, and in the mixed solvent, the volume ratio of tetrahydrofuran to water is 10:1.

[0033] Preferably, in step (1), the solvent used during the synthesis of compound a1 is acetonitrile, and the reaction is carried out under potassium carbonate and heating under reflux; the solvent used during the synthesis of compound a2 is tetrahydrofuran; the solvent used during the synthesis of compound a4 is DMF and the reaction temperature is 60 °C; during the synthesis of compounds b1-b6 and b7-b12 described in step (2), the acyl chlorination reagent used is thionyl chloride; the solvent used for the synthesis of compounds c1-c6 and c7-c12 is DMF; in step (3), the catalysts are copper sulfate pentahydrate and sodium ascorbate; the reaction condition is to react at 45 °C under argon protection.

[0034] More preferably, in step (2), the acyl chlorination reagent used is thionyl chloride.

[0035] The present invention also discloses the application of the above-mentioned PROTACs targeting coronavirus 3CL protease in the preparation of anti-coronavirus drug preparations.

[0036] Preferably, the coronavirus is novel coronavirus SARS-CoV-2.

[0037] Preferably, the preparation is used alone or in combination with other anti-coronavirus drugs, or mixed with pharmaceutically acceptable excipients and diluents to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or liniments or injections for non-oral administration.

[0038] The present invention also discloses a pharmaceutical composition, which contains the above-mentioned PROTACs targeting coronavirus 3CL protease as an active ingredient.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The PROTACs targeting coronavirus 3CL protease provided by the present invention select lenalidomide, a ligand of Cereblon protein (CRBN), and pomalidomide as E3 ligase ligands, and couple coronavirus 3CL protease inhibitors with E3 ligase through linkers of different types and different chain lengths, and successfully prepare PROTACs targeting coronavirus 3CL protease, which can effectively target the target protein. 3CL pro The results of the inhibitory activity test show that the compounds synthesized by the present invention have a strong inhibitory effect on 3CLpro. Preferably, compounds 3, 4, 8, 9, 10, 14 and 17 have an IC pro value of 3CL 50 below 100 nM; the results of the 3CL pro degradation activity experiment show that the compounds synthesized by the present invention have degradation activity on 3CL pro . Preferably, the DC pro values of compounds 2, 3, 4, 5, 8, 9, 10, 13, 14, 15, 16 and 17 on 3CL 50 are all below 100 nM. The compounds provided by the present invention overcome the defects of the existing coronavirus 3CL protease inhibitors, such as single structural type and limited ways to exert pharmacological effects (only having inhibitory effects), have good inhibitory and degradation activities on coronavirus 3CL protease, and can be developed and studied as anti-coronavirus candidate drugs.

[0041] The preparation method of the PROTACs targeting coronavirus 3CL protease provided by the present invention uses various commercially available low-cost synthetic building blocks as raw materials, and obtains the target product with a high yield through common chemical reactions. All reactions avoid the use of high temperature, high pressure and highly toxic reagents, can be carried out under relatively mild conditions, have low requirements for reaction equipment, and cause little environmental pollution; at the same time, it has high atom economy and is suitable for industrial production. Detailed implementation manners

[0042] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention are used to distinguish similar objects, and do not necessarily need to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described here can be implemented in an order other than those described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0044] The present invention will be further described in detail below:

[0045] The present invention provides a kind of PROTACs targeting coronavirus 3CL protease, which is a compound shown in formula I or formula II or a pharmaceutically acceptable salt thereof, as well as a solvate, enantiomer, diastereomer, tautomer or any mixture in any ratio of the compound shown in formula I or formula II or a pharmaceutically acceptable salt thereof, including a racemic mixture;

[0046] The structural formula of the compound shown in formula I or formula II is:

[0047]

[0048] Among them, Linker is one of the following linking groups:

[0049] Among them, n = 1 - 6;

[0050] The pharmaceutically acceptable salt is a salt formed by the PROTACs targeting coronavirus 3CL protease and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0051] The preparation method of the PROTACs targeting coronavirus 3CL protease provided by the present invention is as follows:

[0052] (1) Using 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione as a raw material, it undergoes an alkylation reaction with 2,4,5-trifluorobenzyl bromide to obtain compound a1; then a 6-chloro-2-methyl-2H-indazole unit is introduced at the 6-position of the triazine nucleus to obtain compound a2; the tert-butyl group at the 3-position of the triazine nucleus of compound a2 is removed in an acidic solvent to obtain compound a3; subsequently, a 3-propynyl group is introduced at the 3-position of the triazine nucleus of compound a3, and finally compound a4 is obtained. The reaction formula is as follows:

[0053]

[0054] Among them, the solvent used in the synthesis of compound a1 is acetonitrile, the molar ratio of 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione to 2,4,5-trifluorobenzyl bromide is 1:1.1, and the reaction is carried out under the conditions of potassium carbonate and heating under reflux; in the synthesis of compound a2, the molar ratio of the raw material compound a1 to 6-chloro-2-methyl-2H-indazol-5-amine is 1:1.3, the reaction temperature is 0 °C, the solvent used is tetrahydrofuran, and the catalyst used is lithium bis(trimethylsilyl)amide (LiHMDS); the acidic solvent used in the synthesis of compound a3 is trifluoroacetic acid (TFA); in the synthesis of compound a4, the molar ratio of the raw material compound a3 to 3-bromopropyne is 1:1.2, the solvent used is N,N-dimethylformamide (DMF), and the reaction temperature is 60 °C;

[0055] (2) Using lenalidomide (purchased from Shanghai Macklin Biochemical Co., Ltd.) as a raw material, it undergoes an acid amide condensation reaction with bromoalkanoic acids of different lengths to obtain the corresponding compounds b1-b6, and then reacts with sodium azide under the catalysis of potassium iodide to obtain the corresponding compounds c1-c6;

[0056] Alternatively, using pomalidomide (purchased from Shanghai Macklin Biochemical Co., Ltd.) as a raw material, it undergoes an acid amide condensation reaction with bromoalkanoic acids of different lengths to obtain the corresponding compounds b7-b12, and then reacts with sodium azide under the catalysis of potassium iodide to obtain the corresponding compounds c7-c12. The reaction formula is as follows:

[0057]

[0058] Alternatively, using lenalidomide as a raw material, it undergoes an acid amide condensation reaction with azido-polyethylene glycol-acetic acid compounds to obtain the corresponding compounds d1-d3;

[0059] Alternatively, using pomalidomide as a raw material, it undergoes an acid amide condensation reaction with azido-polyethylene glycol-acetic acid compounds to obtain the corresponding compounds d4-d6. The reaction formula is as follows:

[0060]

[0061] Among them, in the synthesis of the compounds b1-b6 and b7-b12, the molar ratio of pomalidomide or lenalidomide to bromoalkanoic acids with different lengths is 1:2. The bromoalkanoic acids with different lengths are dissolved with an acyl chlorination reagent, and the acyl chlorination reagent used is further preferably thionyl chloride; the solvent used in the synthesis of the compounds c1-c6 and c7-c12 is DMF, the molar ratio of pomalidomide or lenalidomide to azido-polyethylene glycol-acetic acid compounds is 1:2, and the catalyst used is potassium iodide; in the synthesis of the compounds d1-d3 and d4-d6, the molar ratio of pomalidomide or lenalidomide to azido-polyethylene glycol-acetic acid compounds is 1:2, the condensing agent used is thionyl chloride, and the solvent used is tetrahydrofuran;

[0062] (3) Compound a4 reacts with compounds c1-c6 or d1-d3 to generate PROTACs targeting coronavirus 3CL protease with the structure of Formula I;

[0063] Alternatively, compound a4 reacts with c7-c12 or d4-d6 to generate PROTACs targeting coronavirus 3CL protease with the structure of Formula II, and the reaction formula is as follows:

[0064]

[0065] Among them, the molar ratio of compound a4 to c1-c12 or d1-d6 in the reaction is 1:1.2. The solvent used in the reaction is a mixed solvent of tetrahydrofuran and water, and the volume ratio is tetrahydrofuran:water = 10:1; the catalysts are copper sulfate pentahydrate and sodium ascorbate; the reaction conditions are reaction at 45 °C under argon protection.

[0066] 1. Specific examples for synthesizing compounds 1-18

[0067] The structural formulas of the representative compounds of the present invention are shown as follows:

[0068]

[0069] The following are examples for synthesizing the above compounds.

[0070] Example 1

[0071] Compound 1: Preparation of (E)-3-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)propanamide

[0072] (1) Preparation of Compound a4

[0073]

[0074] Step 1: Synthesis of Compound a1

[0075] Put 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione (91.72 mg, 0.4 mmol), 2,4,5-trifluorobenzyl bromide (99.0 mg, 0.44 mmol) and potassium carbonate (66.3 mg, 0.48 mmol) into a reactor, dissolve them with 10 mL of acetonitrile, heat under reflux, stir and react for 3 hours, monitored by TLC. After the reaction, concentrate the reaction solution under reduced pressure to remove the solvent. The obtained solid residue is washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phase is collected, and purified by column chromatography (n-hexane:ethyl acetate (V:V) = 8:2 as the mobile phase), and dried to obtain 133.3 mg of a1, with a yield of 89.25%.

[0076] Step 2: Synthesis of Compound a2

[0077] Put Compound a1 (186.7 mg, 0.5 mmol) and 6-chloro-2-methyl-2H-indazol-5-amine (118.1 mg, 0.65 mmol) into a reactor, dissolve them with 5 mL of tetrahydrofuran. Slowly add a solution of 1 mmol of lithium bis(trimethylsilyl)amide (LiHMDS) (0.2 mL, 1 mmol) in tetrahydrofuran to the reactor at 0 °C, stir and react for 3 hours, monitored by TLC. After the reaction, cool to room temperature, quench with ammonium chloride aqueous solution, concentrate the reaction solution under reduced pressure to remove tetrahydrofuran. The obtained residual solution is washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phase is collected, and purified by column chromatography (methylene chloride:methanol (V:V) = 10:1 as the mobile phase), and dried to obtain 60.7 mg of a2, with a yield of 24.63%.

[0078] Step 3: Synthesis of Compound a3

[0079] Put the obtained Compound a2 (246.4 mg, 0.5 mmol) into a reactor, add 3 mL of trifluoroacetic acid (TFA), stir at room temperature overnight, then co-distill with toluene under reduced pressure to remove the solvent, and dry to obtain 201.0 mg of a3, with a yield of 92.03%.

[0080] Step 4: Synthesis of Compound a4

[0081] Compound a3 (436.8 mg, 1 mmol), 3-bromopropyne (0.1 mL, 1.2 mmol) and potassium carbonate (165.9 mg, 1.2 mmol) were placed in a reactor, dissolved in 10 mL of N,N-dimethylformamide, heated and stirred at 60 °C for 5 hours, monitored by TLC. After the reaction, the resulting reaction solution was washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phase was collected, and purified by column chromatography (n-hexane:ethyl acetate (V:V) = 6:4 as the mobile phase), dried to obtain 312.0 mg of compound a4, with a yield of 65.71%.

[0082] (2) Preparation of compound c1

[0083]

[0084] Step 1: Synthesis of compound b1

[0085] 3-Bromopropionic acid (305.9 mg, 2 mmol) was dissolved in 5 mL of thionyl chloride, heated under reflux for 2 hours. After the reaction, the solvent was removed by concentration under reduced pressure. Subsequently, lenalidomide (1 mmol, 259.3 mg) was added, using 10 mL of tetrahydrofuran as the solvent, heated under reflux for 5 hours, monitored by TLC. After the reaction, it was cooled to room temperature, 2 mL of methanol was added and stirred for 1 hour. The solvent was removed by concentration under reduced pressure, and b1 362.1 mg was obtained by column chromatography (eluent: dichloromethane:methanol (V:V) = 20:1), with a yield of 91.85%.

[0086] Step 2: Synthesis of compound c1

[0087] Compound b1 (197.1 mg, 0.5 mmol), sodium azide (97.5 mg, 1.5 mmol) and potassium iodide (8.3 mg, 0.05 mmol) were placed in a reactor, dissolved in 10 mL of N,N-dimethylformamide, heated and stirred at 70 °C for 5 hours, monitored by TLC. After the reaction, the resulting reaction solution was washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phase was collected, and dried to obtain 151.6 mg of compound c1, with a yield of 85.16%.

[0088] (4) Preparation of compound 1

[0089]

[0090] Compound a4 (142.4 mg, 0.3 mmol), compound c1 (128.3 mg, 0.36 mmol), copper(II) sulfate pentahydrate (30.0 mg, 0.12 mmol), and sodium ascorbate (23.8 mg, 0.12 mmol) were placed in a reactor, dissolved in a mixed solution of 10 mL of tetrahydrofuran and 1 mL of water, protected by argon, heated and stirred at 45 °C overnight, and monitored by TLC. After the reaction was completed, the resulting reaction solution was concentrated under reduced pressure to remove the solvent, and purified by column chromatography (dichloromethane:methanol (V:V) = 15:1 as the mobile phase), and dried to obtain 95.7 mg of compound 1, with a yield of 38.41%.

[0091] 1 H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 9.79 (s, 1H), 9.33 (s, 1H), 8.32 (s, 1H), 7.85 (d, J = 7.1 Hz, 1H), 7.75 (s, 1H), 7.62 - 7.50 (m, 2H), 7.44–7.37 (m, 2H), 7.30 (s, 1H), 7.28 - 7.21 (m, 1H), 5.22 (s, 2H), 5.12 (dd, J = 13.3, 4.8 Hz, 1H), 5.03 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.45–4.23 (m, 2H), 4.12 (3H, s), 3.04–2.86 (m, 1H), 2.70 (d, J = 17.3 Hz, 1H), 2.33 - 2.12 (m, 2H), 1.95–1.78 (m, 2H).

[0092] 13 C NMR (101 MHz, DMSO) δ 173.34, 171.42, 171.25, 168.38, 155.61, 155.17, 150.54, 150.41, 148.55, 146.65, 146.31, 145.74, 143.88, 134.34, 133.12, 132.13, 129.10, 129.02, 127.24, 125.89, 125.82, 120.70, 120.41, 119.44, 118.11, 116.74, 116.36, 106.12, 52.08, 46.94, 40.28, 40.13, 38.05, 36.15, 35.60, 31.65, 28.68.

[0093] Example 2

[0094] Preparation of Compound 2: (E)-4-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)butyramide

[0095]

[0096] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 2: 35.23%.

[0097] 1 H NMR(400MHz,DMSO)δ11.12(s,1H),9.77(s,1H),9.32(s,1H),8.29(s,1H),7.88(d,J=7.1Hz,1H),7.71(s,1H),7.65-7.53(m,2H),7.48–7.40(m,2H),7.35(s,1H),7.32-7.21(m,1H),5.28(s,2H),5.21(dd,J=13.3,4.8Hz,1H),5.05(s,2H),4.69(t,J=6.8Hz,2H),4.43–4.29(m,2H),4.16(3H,s),3.02–2.81(m,1H),2.60(d,J=17.3Hz,1H),2.50–2.38(m,3H),2.09–2.01(m,1H),1.92–1.80(m,2H).

[0098] 13 C NMR(101MHz,DMSO)δ173.35,171.46,171.26,168.39,155.65,155.15,150.56,150.42,148.57,146.69,146.34,145.77,143.89,134.36,133.15,132.16,129.12,129.00,127.23,125.91,125.80,120.71,120.43,119.46,118.10,116.79,116.32,106.11,52.06,46.84,40.21,40.10,38.06,36.12,35.59,31.61,27.58,24.65.

[0099] Example 3

[0100] Preparation of Compound 3: (E)-5-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)pentanamide

[0101]

[0102] The preparation method was the same as that of Example 1, only the corresponding raw materials needed to be replaced. The yield of the obtained Compound 3: 30.76%.

[0103] 1 H NMR (400 MHz, DMSO) δ 11.11 (s, 1H), 9.79 (s, 1H), 9.28 (s, 1H), 8.36 (s, 1H), 7.84 (d, J = 7.1 Hz, 1H), 7.75 (s, 1H), 7.63 - 7.52 (m, 2H), 7.47–7.41 (m, 2H), 7.36 (s, 1H), 7.32 - 7.22 (m, 1H), 5.24 (s, 2H), 5.20 (dd, J = 13.3, 4.8 Hz, 1H), 5.07 (s, 2H), 4.72 (t, J = 6.8 Hz, 2H), 4.44–4.30 (m, 2H), 4.18 (3H, s), 3.06–2.88 (m, 1H), 2.61 (d, J = 17.3 Hz, 1H), 2.51–2.40 (m, 3H), 2.09–2.01 (m, 1H), 1.82–1.70 (m, 2H), 1.57–1.41 (m, 2H).

[0104] 13 C NMR (101 MHz, DMSO) δ 173.45, 171.56, 171.34, 168.43, 155.62, 155.11, 150.53, 150.41, 148.54, 146.67, 146.33, 145.88, 143.81, 134.34, 133.17, 132.19, 129.10, 129.08, 127.22, 125.90, 125.75, 120.72, 120.44, 119.49, 118.12, 116.80, 116.34, 106.12, 52.00, 46.87, 40.22, 40.11, 38.08, 36.04, 35.62, 32.37, 31.65, 27.54, 24.64.

[0105] Example 4

[0106] Compound 4: (E)-6-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-

[0107] (2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)hexanamide Preparation

[0108]

[0109] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 4: 36.55%.

[0110] 1 H NMR(400MHz,DMSO)δ11.05(s,1H),9.81(s,1H),9.31(s,1H),8.40(s,1H),7.83(d,J=7.1Hz,1H),7.73(s,1H),7.65-7.54(m,2H),7.50–7.41(m,2H),7.38(s,1H),7.32-7.25(m,1H),5.26(s,2H),5.18(dd,J=13.3,4.8Hz,1H),5.04(s,2H),4.67(t,J=6.8Hz,2H),4.47–4.31(m,2H),4.15(3H,s),3.02–2.88(m,1H),2.64(d,J=17.3Hz,1H),2.44–2.30(m,3H),2.10–2.02(m,1H),1.92–1.81(m,2H),1.72–1.60(m,2H),1.57–1.41(m,2H).

[0111] 13 C NMR(101MHz,DMSO)δ173.35,171.68,171.56,168.33,155.58,155.22,150.50,150.43,148.52,146.60,146.38,145.98,143.84,134.26,133.17,132.35,129.12,129.10,127.21,125.93,125.79,120.76,120.54,119.53,118.12,116.70,116.46,106.16,52.00,46.96,40.29,40.06,38.04,36.08,35.61,32.47,31.69,27.66,24.71,23.15.

[0112] Example 5

[0113] Compound 5: Preparation of (E)-7-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)heptanamide

[0114]

[0115] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 5: 21.18%.

[0116] 1 H NMR(400MHz,DMSO)δ11.08(s,1H),9.79(s,1H),9.34(s,1H),8.42(s,1H),7.85(d,J=7.1Hz,1H),7.71(s,1H),7.67-7.54(m,2H),7.49–7.41(m,2H),7.37(s,1H),7.32-7.25(m,1H),5.24(s,2H),5.17(dd,J=13.3,4.8Hz,1H),5.02(s,2H),4.63(t,J=6.8Hz,2H),4.49–4.31(m,2H),4.16(3H,s),3.02–2.88(m,1H),2.65(d,J=17.3Hz,1H),2.42–2.29(m,2H),2.20–2.03(m,4H),1.90–1.79(m,2H),1.70–1.61(m,2H),1.54–1.39(m,2H).

[0117] 1313C NMR (101 MHz, DMSO) δ 173.39, 171.62, 171.49, 168.36, 155.49, 155.23, 150.51, 150.43, 148.54, 146.62, 146.39, 145.90, 143.81, 134.22, 133.19, 132.29, 129.09, 129.03, 127.26, 125.90, 125.75, 120.72, 120.54, 119.52, 118.11, 116.76, 116.41, 106.14, 52.08, 46.95, 40.30, 40.05, 38.03, 36.12, 35.59, 32.45, 31.70, 28.90, 27.62, 24.74, 23.35.

[0118] Example 6

[0119] Compound 6: Preparation of (E)-8-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)octanamide

[0120]

[0121] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained compound 2: 17.83%.

[0122] 1 1H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 9.76 (s, 1H), 9.33 (s, 1H), 8.41 (s, 1H), 7.88 (d, J = 7.1 Hz, 1H), 7.73 (s, 1H), 7.67 - 7.54 (m, 2H), 7.48 – 7.42 (m, 2H), 7.36 (s, 1H), 7.31 - 7.25 (m, 1H), 5.25 (s, 2H), 5.16 (dd, J = 13.3, 4.8 Hz, 1H), 5.04 (s, 2H), 4.69 (t, J = 6.8 Hz, 2H), 4.45 – 4.30 (m, 2H), 4.17 (3H, s), 3.08 – 2.80 (m, 1H), 2.69 (d, J = 17.3 Hz, 1H), 2.44 – 2.29 (m, 2H), 2.24 – 2.05 (m, 4H), 1.95 – 1.76 (m, 4H), 1.69 – 1.58 (m, 2H), 1.49 – 1.38 (m, 2H).

[0123] 13 C NMR (101 MHz, DMSO) δ 173.38, 171.58, 171.46, 168.35, 155.43, 155.24, 150.50, 150.41, 148.53, 146.61, 146.36, 145.91, 143.83, 134.23, 133.18, 132.29, 129.12, 129.02, 127.24, 125.95, 125.78, 120.71, 120.56, 119.52, 118.14, 116.72, 116.44, 106.18, 52.12, 46.98, 40.33, 40.10, 38.08, 36.16, 35.61, 32.46, 31.77, 30.15, 28.96, 27.65, 24.76, 23.42.

[0124] Example 7

[0125] Compound 7: Preparation of (E)-3-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)propanamide

[0126]

[0127] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 7: 37.54%.

[0128] 1 H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 9.84 (s, 1H), 9.72 (s, 1H), 8.42 (d, J = 8.3 Hz, 1H), 8.33 (s, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.64 - 7.53 (m, 2H), 7.42 (s, 1H), 7.32 - 7.22 (m, 1H), 5.23 (s, 2H), 5.14 (dd, J = 12.8, 5.3 Hz, 1H), 5.11 (s, 2H), 4.68 (t, J = 6.5 Hz, 2H), 4.21 (s, 3H), 2.56–2.44 (m, 2H), 2.33–2.21 (m, 2H), 1.95–1.83 (m, 2H).

[0129] 13 C NMR (101 MHz, DMSO) δ 173.32, 172.23, 170.40, 168.21, 167.23, 155.78, 155.33, 150.89, 150.55, 148.63, 146.66, 146.34, 145.86, 143.82, 136.91, 136.62, 132.32, 131.89, 129.43, 127.32, 126.87, 125.89, 120.82, 120.43, 118.82, 117.78, 116.71, 116.38, 106.21, 48.34, 40.45, 39.89, 37.05, 35.29, 34.22, 32.29, 28.78.

[0130] Example 8

[0131] Compound 8: Preparation of (E)-4-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)butyramide

[0132]

[0133] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained compound 8: 33.77%.

[0134] 1 H NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 9.82 (s, 1H), 9.74 (s, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.35 (s, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.63 - 7.53 (m, 2H), 7.33 (s, 1H), 7.29 - 7.22 (m, 1H), 5.20 (s, 2H), 5.16 (dd, J = 12.8, 5.3 Hz, 1H), 5.09 (s, 2H), 4.58 (t, J = 6.5 Hz, 2H), 4.16 (s, 3H), 2.95–2.88 (m, 1H), 2.66–2.54 (m, 2H), 2.43–2.31 (m, 1H), 2.25–2.14 (m, 2H), 1.98–1.84 (m, 2H).

[0135] 1313C NMR (101 MHz, DMSO) δ 173.29, 172.19, 170.29, 168.12, 167.21, 155.45, 155.23, 150.55, 150.44, 148.55, 146.62, 146.31, 145.90, 143.85, 136.89, 136.57, 132.34, 131.95, 129.12, 127.31, 126.92, 125.91, 120.75, 120.52, 118.80, 117.69, 116.69, 116.42, 106.17, 49.33, 40.31, 40.12, 38.03, 35.73, 35.20, 32.09, 30.41, 24.45.

[0136] Example 9

[0137] Compound 9: Preparation of (E)-5-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)pentanamide

[0138]

[0139] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 9: 27.64%.

[0140] 1 1H NMR (400 MHz, DMSO) δ 11.13 (s, 1H), 9.84 (s, 1H), 9.73 (s, 1H), 8.46 (d, J = 8.3 Hz, 1H), 8.42 (s, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.72 (s, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.64 - 7.53 (m, 2H), 7.36 (s, 1H), 7.30 - 7.22 (m, 1H), 5.24 (s, 2H), 5.18 (dd, J = 12.8, 5.3 Hz, 1H), 5.06 (s, 2H), 4.61 (t, J = 6.5 Hz, 2H), 4.14 (s, 3H), 2.98–2.87 (m, 1H), 2.68–2.52 (m, 4H), 2.13–2.01 (m, 1H), 1.95–1.84 (m, 2H), 1.81–1.64 (m, 2H).

[0141] 1313C NMR (101 MHz, DMSO) δ 173.24, 172.17, 170.26, 168.07, 167.14, 155.57, 155.20, 150.51, 150.42, 148.51, 146.64, 146.37, 145.97, 143.81, 136.91, 136.56, 132.35, 131.97, 129.10, 127.36, 126.94, 125.93, 120.76, 120.54, 118.86, 117.65, 116.70, 116.46, 106.16, 49.38, 40.29, 40.06, 38.04, 35.78, 35.17, 32.02, 31.41, 23.81, 22.45.

[0142] Example 10

[0143] Compound 10: Preparation of (E)-6-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)hexanamide

[0144]

[0145] The preparation method was the same as that of Example 1, only the corresponding raw materials needed to be replaced. The yield of the obtained Compound 10: 22.19%.

[0146] 1 1H NMR (400 MHz, DMSO) δ 11.14 (s, 1H), 9.90 (s, 1H), 9.83 (s, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.45 (s, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.74 (s, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.62 - 7.50 (m, 2H), 7.39 (s, 1H), 7.32 - 7.21 (m, 1H), 5.25 (s, 2H), 5.19 (dd, J = 12.8, 5.3 Hz, 1H), 5.16 (s, 2H), 4.71 (t, J = 6.5 Hz, 2H), 4.24 (s, 3H), 3.02–2.87 (m, 2H), 2.69–2.50 (m, 4H), 2.23–2.11 (m, 2H), 1.91–1.83 (m, 2H), 1.72–1.65 (m, 2H).

[0147] 1313C NMR (101 MHz, DMSO) δ 173.19, 172.09, 170.32, 168.12, 167.10, 155.63, 155.22, 150.49, 150.43, 148.55, 146.67, 146.39, 146.02, 143.79, 136.88, 136.52, 132.38, 132.01, 129.14, 127.38, 126.96, 125.95, 120.78, 120.55, 118.88, 117.66, 116.74, 116.42, 106.14, 50.02, 40.39, 40.16, 38.24, 35.92, 35.27, 32.32, 31.44, 25.72, 23.89, 22.55.

[0148] Example 11

[0149] Compound 11: Preparation of (E)-7-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)heptanamide

[0150]

[0151] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 11: 19.53%.

[0152] 1 1H NMR (400 MHz, DMSO) δ 11.13 (s, 1H), 9.88 (s, 1H), 9.82 (s, 1H), 8.44 (d, J = 8.3 Hz, 1H), 8.41 (s, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.61 - 7.52 (m, 2H), 7.36 (s, 1H), 7.31 - 7.20 (m, 1H), 5.27 (s, 2H), 5.21 (dd, J = 12.8, 5.3 Hz, 1H), 5.18 (s, 2H), 4.74 (t, J = 6.5 Hz, 2H), 4.22 (s, 3H), 3.04–2.85 (m, 2H), 2.72–2.55 (m, 4H), 2.24–1.88 (m, 6H), 1.82–1.68 (m, 2H).

[0153] 1313C NMR (101 MHz, DMSO) δ 173.20, 172.12, 170.34, 168.16, 167.12, 155.67, 155.34, 150.83, 150.52, 148.67, 146.80, 146.42, 146.12, 143.80, 136.81, 136.12, 132.42, 132.10, 129.21, 127.42, 126.93, 125.91, 120.92, 120.51, 118.82, 117.76, 116.77, 116.38, 106.16, 50.09, 40.42, 40.18, 38.32, 35.88, 35.42, 32.43, 31.54, 27.34, 25.56, 23.92, 22.45.

[0154] Example 12

[0155] Compound 12: Preparation of (E)-8-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)octanamide

[0156]

[0157] The preparation method is the same as that of Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 12: 15.87%.

[0158] 1 1H NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 9.91 (s, 1H), 9.79 (s, 1H), 8.46 (d, J = 8.3 Hz, 1H), 8.39 (s, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.79 (s, 1H), 7.71 (d, J = 7.2 Hz, 1H), 7.64 - 7.50 (m, 2H), 7.39 (s, 1H), 7.32 - 7.23 (m, 1H), 5.31 (s, 2H), 5.24 (dd, J = 12.8, 5.3 Hz, 1H), 5.21 (s, 2H), 4.78 (t, J = 6.5 Hz, 2H), 4.25 (s, 3H), 3.01–2.82 (m, 2H), 2.68–2.35 (m, 4H), 2.14–1.82 (m, 6H), 1.74–1.54 (m, 4H).

[0159] 1313C NMR (101 MHz, DMSO) δ 173.22, 172.15, 170.42, 168.23, 167.17, 155.71, 155.39, 150.97, 150.58, 148.71, 146.89, 146.56, 146.09, 143.85, 136.79, 136.09, 132.45, 132.14, 129.19, 127.45, 126.88, 125.89, 120.89, 120.49, 118.79, 117.81, 116.79, 116.41, 106.21, 50.11, 40.52, 40.21, 38.41, 35.91, 35.32, 32.63, 31.44, 28.21, 27.34, 25.56, 22.92, 21.65.

[0160] Example 13

[0161] Compound 13: Preparation of (E)-2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)acetamide

[0162] (1) Preparation of Compound a4

[0163] The preparation method was the same as that in Example 1.

[0164] (2) Preparation of Compound d1

[0165]

[0166] Dissolve 2-(2-azidoethoxy)acetic acid (290.2 mg, 2 mmol) in 5 mL of thionyl chloride and reflux for 2 hours. After the reaction, concentrate under reduced pressure to remove the solvent, then add lenalidomide (1 mmol, 259.3 mg), use 10 mL of tetrahydrofuran as the solvent, reflux for 5 hours, and monitor by TLC. After the reaction, cool to room temperature, add 2 mL of methanol and continue stirring for 1 hour. Concentrate under reduced pressure to remove the solvent, and separate by column chromatography to obtain 296.8 mg of d1 (the eluent is dichloromethane:methanol (V:V) = 10:1), with a yield of 76.83%.

[0167] (3) Preparation of Compound 13

[0168]

[0169] Compound a4 (142.4 mg, 0.3 mmol), compound d1 (139.1 mg, 0.36 mmol), copper(II) sulfate pentahydrate (30.0 mg, 0.12 mmol), and sodium ascorbate (23.8 mg, 0.12 mmol) were placed in a reactor, dissolved in a mixed solution of 10 mL of tetrahydrofuran and 1 mL of water, protected by argon, heated and stirred at 45 °C overnight, and monitored by TLC. After the reaction was completed, the resulting reaction solution was concentrated under reduced pressure to remove the solvent, and purified by column chromatography (dichloromethane:methanol (V:V) = 20:1 as the mobile phase), and dried to obtain 66.2 mg of compound 13, with a yield of 25.63%.

[0170] 1 H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 9.83 (s, 1H), 9.30 (s, 1H), 8.42 (s, 1H), 7.95 (d, J = 7.1 Hz, 1H), 7.75 (s, 1H), 7.65 - 7.53 (m, 2H), 7.46–7.35 (m, 2H), 7.31 (s, 1H), 7.26 - 7.13 (m, 1H), 5.24 (s, 2H), 5.17 (dd, J = 13.3, 4.8 Hz, 1H), 5.04 (s, 2H), 4.58 (s, 2H), 4.31 (s, 2H), 4.12 (3H, s), 3.95–3.82 (m, 2H), 3.75 (t, J = 7.1 Hz, 2H), 2.80–2.65 (m, 2H), 2.43 - 2.24 (m, 2H).

[0171] 13 C NMR (101 MHz, DMSO) δ 173.52, 171.32, 171.12, 168.42, 155.69, 155.31, 150.56, 150.48, 148.60, 146.61, 146.34, 145.70, 143.89, 134.38, 133.11, 132.21, 129.14, 129.06, 127.28, 125.92, 125.73, 120.69, 120.38, 119.46, 118.09, 116.81, 116.36, 106.09, 69.10, 68.24, 52.08, 51.73, 46.92, 40.31, 40.10, 38.07, 31.55, 29.18.

[0172] Example 14

[0173] Preparation of Compound 14: (E)-2-(2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

[0174]

[0175] The preparation method is the same as that of Example 13, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 14: 21.45%;

[0176] 1 H NMR(400MHz,DMSO)δ11.23(s,1H),9.86(s,1H),9.33(s,1H),8.38(s,1H),7.89(d,J=7.1Hz,1H),7.73(s,1H),7.60-7.51(m,2H),7.41–7.32(m,2H),7.28(s,1H),7.22-7.14(m,1H),5.22(s,2H),5.16(dd,J=13.3,4.8Hz,1H),5.01(s,2H),4.49(s,2H),4.28(s,2H),4.09(3H,s),3.85–3.74(m,2H),3.64(t,J=7.1Hz,2H),3.35(s,4H),2.76–2.59(m,2H),2.23-2.04(m,2H).

[0177] 13 C NMR(101MHz,DMSO)δ173.68,171.42,171.23,168.38,155.71,155.34,150.61,150.52,148.63,146.66,146.31,145.72,143.91,134.42,133.12,132.24,129.16,129.01,127.32,125.91,125.77,120.71,120.42,119.51,118.11,116.84,116.32,106.14,69.16,68.45,68.34,68.16,52.12,51.75,46.96,40.34,40.09,38.12,31.56,29.23.

[0178] Example 15

[0179] Preparation of Compound 15: (E)-2-(2-(2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

[0180]

[0181] The preparation method is the same as that of Example 13, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 15: 16.36%.

[0182] 1 H NMR (400 MHz, DMSO) δ 11.18 (s, 1H), 9.82 (s, 1H), 9.41 (s, 1H), 8.43 (s, 1H), 7.92 (d, J = 7.1 Hz, 1H), 7.71 (s, 1H), 7.65 - 7.54 (m, 2H), 7.44–7.33 (m, 2H), 7.29 (s, 1H), 7.24 - 7.16 (m, 1H), 5.19 (s, 2H), 5.12 (dd, J = 13.3, 4.8 Hz, 1H), 5.03 (s, 2H), 4.51 (s, 2H), 4.32 (s, 2H), 4.08 (3H, s), 3.82–3.71 (m, 2H), 3.63 (t, J = 7.1 Hz, 2H), 3.39 (s, 8H), 2.67–2.53 (m, 2H), 2.24 - 2.07 (m, 2H).

[0183] 13 C NMR (101 MHz, DMSO) δ 173.71, 171.46, 171.25, 168.42, 155.78, 155.41, 150.69, 150.58, 148.67, 146.72, 146.35, 145.71, 144.02, 134.46, 133.21, 132.25, 129.22, 129.08, 127.34, 126.02, 125.81, 120.77, 120.45, 119.53, 118.17, 116.84, 116.36, 106.16, 70.41, 70.12, 69.21, 68.46, 68.37, 68.21, 52.18, 51.82, 47.03, 40.42, 40.19, 38.17, 31.62, 29.27.

[0184] Example 16

[0185] Preparation of Compound 16: (E)-2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide

[0186]

[0187] The preparation method is the same as that of Example 13, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 16: 26.44%.

[0188] 1 H NMR(400MHz,DMSO)δ11.11(s,1H),9.82(s,1H),9.75(s,1H),8.44(d,J=8.3Hz,1H),8.36(s,1H),7.81(t,J=7.8Hz,1H),7.72(s,1H),7.65(d,J=7.2Hz,1H),7.60 - 7.51(m,2H),7.43(s,1H),7.31 - 7.19(m,1H),5.26(s,2H),5.12(dd,J=12.8,5.3Hz,1H),5.16(s,2H),4.48(s,2H),4.19(s,3H),3.73–3.58(m,2H),3.51(t,J=6.8Hz,2H),2.32–2.19(m,2H),2.10–1.93(m,2H).

[0189] 13 C NMR(101MHz,DMSO)δ173.42,172.31,170.48,168.19,167.26,155.82,155.34,150.91,150.58,148.66,146.71,146.38,145.92,143.79,136.89,136.67,132.34,131.91,129.48,127.29,126.91,125.95,120.81,120.47,118.90,117.83,116.71,116.42,106.25,68.91,67.23,52.14,48.37,40.41,39.92,37.06,32.32,28.83.

[0190] Example 17

[0191] Preparation of Compound 17: (E)-2-(2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoquinolin-4-yl)acetamide

[0192]

[0193] The preparation method is the same as that in Example 13, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 17: 20.81%.

[0194] 1 H NMR(400MHz,DMSO)δ11.16(s,1H),9.79(s,1H),9.70(s,1H),8.41(d,J = 8.3Hz,1H),8.33(s,1H),7.82(t,J = 7.8Hz,1H),7.71(s,1H),7.63(d,J = 7.2Hz,1H),7.59 - 7.47(m,2H),7.42(s,1H),7.32 - 7.21(m,1H),5.28(s,2H),5.14(dd,J = 12.8,5.3Hz,1H),5.19(s,2H),4.52(s,2H),4.21(s,3H),3.76–3.61(m,2H),3.48(t,J = 6.8Hz,2H),3.39(s,4H),2.28–2.17(m,2H),2.11–1.95(m,2H).

[0195] 13 C NMR(101MHz,DMSO)δ173.38,172.28,170.51,168.21,167.22,155.84,155.36,150.89,150.62,148.65,146.73,146.42,145.91,143.80,136.94,136.72,132.36,131.88,129.54,127.34,126.85,125.88,120.79,120.52,118.93,117.80,116.74,116.42,106.21,69.74,69.12,68.85,67.22,52.16,48.39,40.38,39.91,37.10,32.24,28.90.

[0196] Example 18

[0197] Preparation of Compound 18: (E)-2-(2-(2-(2-(4-((4-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-2,6-dioxo-3-(2,4,5-trifluorobenzyl)-1,3,5-triazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoquinolin-4-yl)acetamide

[0198]

[0199] The preparation method is the same as that of Example 13, only the corresponding raw materials need to be replaced. The yield of the obtained Compound 18: 17.26%.

[0200] 1 H NMR (400 MHz, DMSO) δ 11.15 (s, 1H), 9.82 (s, 1H), 9.75 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 8.37 (s, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.60 - 7.49 (m, 2H), 7.42 (s, 1H), 7.34 - 7.20 (m, 1H), 5.33 (s, 2H), 5.16 (dd, J = 12.8, 5.3 Hz, 1H), 5.21 (s, 2H), 4.49 (s, 2H), 4.18 (s, 3H), 3.73–3.59 (m, 2H), 3.53 (t, J = 6.8 Hz, 2H), 3.42 (s, 8H), 2.23–2.14 (m, 2H), 2.06–1.87 (m, 2H).

[0201] 13 C NMR (101 MHz, DMSO) δ 173.42, 172.33, 170.49, 168.28, 167.23, 155.92, 155.34, 150.90, 150.63, 148.67, 146.76, 146.44, 145.96, 143.83, 136.92, 136.71, 132.38, 131.90, 129.51, 127.36, 126.87, 125.91, 120.83, 120.54, 118.91, 117.87, 116.72, 116.48, 106.25, 71.01, 70.87, 69.68, 69.32, 68.90, 67.43, 52.25, 48.46, 40.52, 39.98, 37.11, 32.27, 28.96.

[0202] 2. Bioactivity assay

[0203] (1) 3CL pro Inhibitory activity test

[0204] The inhibitory activity of the compound against SARS-CoV-2 3CL was determined using fluorescence resonance energy transfer technology pro .

[0205] Mix 10 μL of the above-prepared compound solutions at different concentrations (final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95 nM, prepared in DMSO) and 40 μL of SARS-CoV-2 3CL pro (purchased from Shanghai Beyotime Biotechnology Co., Ltd., final concentration of 0.5 μM, diluted with Tris-HCl buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4)), add to a black 96-well plate, and incubate at 37 °C for 10 min. Add 50 μL of the fluorescent substrate Dabcyl-KTSAVLQSGFRKME-Edans (purchased from Shanghai Beyotime Biotechnology Co., Ltd., final concentration of 20 μM) to initiate the reaction. After incubating for 10 min, perform fluorescence detection on a multifunctional microplate reader (Thermo Fisher Scientific Inc., Varioskan Flash). The excitation wavelength is 340 nm, and the emission wavelength is 490 nm. Record the fluorescence values and calculate the inhibition percentage of the samples. Use DMSO without the compound as the enzyme activity control, and Tris-HCl buffer without SARS-CoV-2 3CL pro as the blank control, and the remaining treatment methods are the same. Use GraphPad Prism software to perform nonlinear regression analysis to calculate the IC 50 values of the samples (compounds 1 - 18 synthesized in the present invention).

[0206]

[0207] The experimental results are shown in Table 1 (in Table 1, the column where IC 50 is located, A: IC 50 < 100 nM, B: IC 50 = 100 - 1000 nM). The compounds in the examples all have inhibitory activity against 3CL pro , and among them, compounds 3, 4, 8, 9, 10, 14, and 17 have stronger inhibitory effects on 3CL pro , and the IC 50 values are all below 100 nM.

[0208] (2) Western Blot determination of 3CLpro Degradation activity

[0209] HEK293E cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded in a 6-well plate at a density of 6.0×10 5 cells / well and incubated in an incubator at 37°C with 5% CO2 for 8 - 24 h. When the cells grew to a density of 70% confluence, they were switched to 2 mL of pre-warmed serum-free medium (purchased from Shanghai Opum Biosciences Co., Ltd.). SARS-CoV-2 3CL was transfected with a 10 μM PEI (polyethyleneimine, purchased from Shanghai Macklin Biochemical Co., Ltd.) solution at a mass-to-volume ratio of 3:4 pro expression plasmid (2 μg / well, prepared with 1×HBS, purchased from Beijing Sino Biological Inc.). The PEI-plasmid mixture was added dropwise to the above serum-free medium, gently shaken and mixed, and incubated in an incubator at 37°C with 5% CO2 for 10 h. Then, the medium containing the transfection reagent was removed and replaced with a medium containing the test samples at gradient concentrations (the final concentrations of the samples were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95 nM). After culturing for 24 h at 37°C and 5% CO2, the supernatant was discarded, the cells were collected, and RIPA cell lysate (purchased from Shanghai Macklin Biochemical Co., Ltd.) was added to lyse the cells on ice for 30 min. The expression of 3CL was detected by Western Blot, and the relative expression level of 3CL was analyzed by Image J pro to calculate the protein degradation rate. The medium without the test samples was used as the control group, and the other treatment methods were the same. Nonlinear regression analysis was performed using GraphPad Prism software to calculate the protein degradation activity (DC pro ) of the samples. 50 )

[0210]

[0211] The experimental results are shown in Table 1 (in Table 1, the column where DC 50 is located, A: DC 50 <100 nM, B: DC 50 =100 - 1000 nM). The compounds in the examples all had degradation activity against 3CL pro , and among them, compounds 2, 3, 4, 5, 8, 9, 10, 13, 14, 15, 16, and 17 had relatively strong degradation activity against 3CL pro , and the DC 50 values were all below 100 nM.

[0212] Table 1 Inhibitory and degradation activities of compounds 1 - 18 against 3CL pro ​

[0213]

[0214] Table 1 data shows that compounds 1-18 have inhibitory and degradative effects on 3CL pro to varying degrees. Among them, compounds 3, 4, 8, 9, 10, 14, and 17 have IC pro values and DC 50 values both less than 100 nM. This indicates that the PROTACs targeting the coronavirus 3CL protease in the present invention have both inhibitory activity and good degradative activity against 3CL 50 and can be developed and studied as anti-coronavirus candidate drugs. pro

[0215] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.​

Claims

1. A kind of PROTACs targeting coronavirus 3CL protease, characterized in that, A compound represented by Formula I or Formula II, or a pharmaceutically acceptable salt thereof, and a tautomer of the compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof; The structural formula of the compound of Formula I or Formula II is: Wherein, Linker is one of the following linking groups: Wherein, n = 1 - 6.

2. The PROTACs targeting coronavirus 3CL protease according to claim 1, characterized in that, The representative compounds are selected from the following compounds:

3. A kind of PROTACs targeting coronavirus 3CL protease according to claim 1, characterized in that, The pharmaceutically acceptable salts are salts formed by PROTACs targeting coronavirus 3CL protease and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

4. A method for preparing PROTACs targeting coronavirus 3CL protease according to any one of claims 1-3, characterized in that, Including the following operation steps: (1) Using 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione as a raw material, reacting with 2,4,5-trifluorobenzyl bromide for alkylation reaction to obtain compound a1; then introducing a 6-chloro-2-methyl-2H-indazole unit at the 6-position of the triazine nucleus to obtain compound a2; The tert-butyl group at the 3-position of the triazine nucleus of compound a2 is removed in an acidic solvent to obtain compound a3; subsequently, a 3-propynyl group is introduced at the 3-position of the triazine nucleus of compound a3 to finally obtain compound a4; where a1 is a2 is a3 is a4 is (2) Using lenalidomide as a raw material, reacting with bromoalkanoic acids of different lengths for acid amide condensation reaction to obtain corresponding compounds b1 - b6, and then reacting with sodium azide under the catalysis of potassium iodide to obtain corresponding compounds c1 - c6; Or, using pomalidomide as a raw material, reacting with bromoalkanoic acids of different lengths for acid amide condensation reaction to obtain corresponding compounds b7 - b12, and then reacting with sodium azide under the catalysis of potassium iodide to obtain corresponding compounds c7 - c12; Among them, b1 - b6 are c1 - c6 are b7 - b12 are c7 - c12 are Or, using lenalidomide as a raw material, reacting with azido-polyethylene glycol-acetic acid compounds for acid amide condensation reaction to obtain corresponding compounds d1 - d3; Or, using pomalidomide as a raw material, reacting with azido-polyethylene glycol-acetic acid compounds for acid amide condensation reaction to obtain corresponding compounds d4 - d6; Among them, d1-d3 are d4-d6 are (3) Compound a4 reacts with compounds c1 - c6 or d1 - d3 to generate PROTACs targeting coronavirus 3CL protease with the structure of Formula I; Or, compound a4 reacts with c7 - c12 or d4 - d6 to generate PROTACs targeting coronavirus 3CL protease with the structure of Formula II.

5. The method according to claim 4, wherein In step (1), the molar ratio of 3-tert-butyl-6-(ethylthio)-1,3,5-triazine-2,4(1H,3H)-dione to 2,4,5-trifluorobenzyl bromide is 1:1.1, the molar ratio of compound a1 to 6-chloro-2-methyl-2H-indazol-5-amine is 1:1.3, and the molar ratio of compound a3 to 3-bromopropyne is 1:1.2; in step (2), the molar ratio of pomalidomide or lenalidomide to bromoalkanoic acids of different lengths is 1:2, and the molar ratio of pomalidomide or lenalidomide to azido-polyethylene glycol-acetic acid compounds is 1:2; in step (3), the molar ratio of compound a4 to c1 - c12 or d1 - d6 is 1:1.

2.

6. The method according to claim 4, wherein In step (1), the reaction temperature during the synthesis of compound a2 is 0 °C, and the catalyst used is lithium bis(trimethylsilyl)amide; in step (2), during the synthesis of compounds b1-b6 and b7-b12, an acyl chloride reagent is used to dissolve bromoalkanoic acids of different lengths, and during the synthesis of compounds d1-d3 and d4-d6, the condensing agent used is thionyl chloride, and the solvent used is tetrahydrofuran; in step (3), the solvent used is a mixed solvent of tetrahydrofuran and water, and in the mixed solvent, the volume ratio of tetrahydrofuran to water is 10:

1.

7. Use of any one of the PROTACs targeting coronavirus 3CL protease according to claims 1-3 in the preparation of an anti-coronavirus drug preparation, characterized in that, The coronavirus is the novel coronavirus SARS-CoV-2.

8. The application according to claim 7, wherein The preparation is used alone or in combination with other anti-coronavirus drugs, or mixed with pharmaceutically acceptable excipients and diluents to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or inhalants, liniments or injections for non-oral administration.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the PROTACs targeting coronavirus 3CL protease described in any one of claims 1-3 as the active ingredient.

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