Novel coronavirus 3CL protease degradation agent as well as application and preparation method thereof

Abstract

The invention provides a compound shown in a general formula (I) or a pharmaceutically acceptable salt, a prodrug, a solvate, a polymorphic substance or an isotope derivative thereof and application of the compound or the pharmaceutically acceptable salt, the prodrug, the solvate, the polymorphic substance or the isotope derivative thereof in preparation of a coronavirus degradation medicine, in the # imgabs0 # formula, R1 is selected from-H or = O; r2 is selected from a straight chain or branched chain alkyl group of # imgabs 1 # or C1-C22; r3 and R4 are independently selected from hydrogen, isopropyl, isobutyl, benzyl or cyclohexyl; and R5 is selected from t-butyloxycarbonyl, carbobenzoxy or 9-fluorenyl oxycarbonyl.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a PROTAC (proteolysis targeting chimeras) compound, or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative thereof, which is a degrader targeting the key target 3CL protease of the new coronavirus (SARS-CoV-2), and also relates to a preparation method of the above-mentioned compound and its use as a 3CL protease degrader or ligand, especially an anti-coronavirus drug. Background Art

[0002] The new coronavirus seriously threatens human health and life safety, and has brought huge negative impacts to the world. The new coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) mainly attacks the human lower respiratory system and causes viral pneumonia. In addition to affecting the respiratory system, the new coronavirus also affects multiple organs such as the heart, liver, esophagus, kidney, bladder, and ileum. Severe patients often experience dyspnea and / or hypoxemia one week after onset. Severe cases can quickly progress to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, coagulation disorders, and multiple organ failure. Very few patients may also have manifestations such as central nervous system involvement and ischemic necrosis of the extremities. There are also sequelae after infection with the new coronavirus, such as swollen toes (new coronavirus feet), abnormal taste, abnormal tongue (new coronavirus tongue), dyspnea, fatigue, olfactory disorders, etc. The SARS-CoV-2 virus has a long incubation period, high concealment (asymptomatic transmission, latent transmission), and strong infectiousness, which brings great difficulties to prevention and treatment. The pneumonia pandemic caused by the novel coronavirus has not only caused huge losses in social economy, life and health, but the novel coronavirus is also likely to coexist with humans for a long time like influenza, and may become an endemic epidemic among the population. Therefore, now and in the future, all mankind is in urgent need of highly effective drugs to treat SARS-CoV-2 infection.

[0003] Although vaccines against the new coronavirus can significantly reduce severe illness and mortality, in the face of the high mutation of the new coronavirus, vaccines face the dilemma of breaking through infection and declining protective efficacy. The epidemic caused by the new coronavirus can no longer completely prevent the spread of the virus by relying solely on vaccines. Oral special medicines may be the key to finally freeing people from the fear of new coronavirus infection. Looking back at the history of influenza for a hundred years (1918-2020), the real means to free human society from the impact of influenza is the "prevention + treatment" combination of "influenza vaccine + oral special medicine", which allows people to maintain normal social activities after being infected with the virus by taking medicine and simple protection. From the perspective of prevention and treatment paradigm, "new coronavirus vaccine + oral anti-new coronavirus drug" is also a better choice, more convenient, and lower social cost, similar to "influenza vaccine + oseltamivir".

[0004] The non-structural proteins of coronavirus are highly conserved components in its life cycle and can mediate viral replication, including 3-chymotrypsin-like protease 3CL pro (3CL protease, or main protease Mpro), papain like protease (papain like protease, PLpro) and RNA-dependent RNA polymerase (RdRp), which are recognized antiviral targets. After the new coronavirus invades the host cell, it first synthesizes two ultra-long polyprotein precursors (pp1a and pp1ab) required for its own replication using the substances in the cell. Subsequently, these two polyprotein precursors are cut by 3CLpro and papain-like protease PLpro to produce non-structural proteins such as RdRp and helicase required for viral replication. RdRp and other auxiliary factors (such as nsp7, nsp8, etc.) are assembled into an efficient RNA synthesis machine (transcription / replication complex) to replicate itself and produce the viral genome.

[0005] At present, 3CLpro, PLpro and RdRp are the key drug target proteins against the new coronavirus that have been verified in large quantities. Among them, 3CL protease is a key enzyme essential in the viral replication cycle. It can release the protease itself by self-cleaving polyproteins, and then cut more than 11 sites on the polyprotein peptide segment to produce various proteins essential for the viral life cycle such as viral replication and transcription. In addition, since 3CL protease has no obvious homology with human proteins, it is an important target for the development of anti-coronavirus drugs. Among the currently marketed drugs, Pfizer's Paxlovid, Japan's Shionogi's Xocova (Ensitrelvir, Ensitrelvir), and Xiannuoxin from Xiansheng Pharmaceutical all target 3CL protease. Nirmatrelvir, Simnotrelvir, Leritrelvir, etc., which target 3CL protease, are all 3CL protease inhibitors.

[0006] These existing small molecule drugs are mainly based on traditional inhibitor principles and have pharmacological limitations. There are two types of small molecule inhibitors for the novel coronavirus 3CL protease: ① Peptidomimetic inhibitors that mimic natural peptide substrates, mainly covalent inhibitors; ② Non-peptidomimetic small molecule inhibitors. However, small molecule inhibitors are difficult to completely block the replication of the virus, cannot completely inhibit the virus's infection process, and have limited efficacy. For example, namatevir and sennotevir still need to be used in combination with ritonavir, which is only effective for patients with mild to moderate novel coronavirus and cannot completely eliminate the virus.

[0007] After the emergence of the technology strategy of targeted protein degradation (TPD), degradation agent molecules represented by PROTACs (Proteolysis-targeting chimera) have made great progress in the treatment of anti-tumor, anti-viral, neurodegenerative diseases, and the development of new influenza vaccines, and have gained advantages over small molecule inhibitors. For target proteins, degrader molecules no longer use the occupancy-driven pharmacological action mode of traditional inhibitors, but can adopt an event-driven pharmacological action mode. Targeted protein degradation (TPD) technology controls the degradation of target proteins by selectively binding bifunctional small molecules to target proteins (POIs) and combining with protein homeostasis control mechanisms. Its target protein degradation-inducing activity is more thorough than the inhibitory effect of inhibitors on target proteins. The TPD strategy no longer relies on the high affinity of compounds to target proteins. It not only makes non-drugable targets druggable, but also greatly expands the chemical space of candidate drug molecules for druggable targets.

[0008] In the field of antiviral drugs, especially anti-coronavirus and novel coronavirus, there are no small molecule drugs based on degraders on the market, and the advantages of small molecule antiviral drugs based on targeted protein degradation (TPD) technology in terms of efficacy and anti-mutation have not yet been brought into play. Recently, patent publication number CN 114874204 A discloses PROTAC (proteolysis targeting chimeras) molecules prepared based on 3CL protease inhibitor GC376 and its derivatives, which can not only achieve 3CL protease inhibitory activity equivalent to or stronger than GC376, but also inhibit the viral infection process. Patent CN 114874204 A discloses the preparation method of its PROTAC molecules and the activity of drug molecules against viral infection. However, the patent does not disclose evidence or experimental data that the so-called PROTAC molecules do work mainly by degrading target proteins through degraders. Therefore, it is still questionable whether the effective anti-novel coronavirus "PROTAC" molecules disclosed in the patent are true "PROTAC" active molecules, or just another form of "inhibitor".

[0009] The inventors have designed and prepared a library of novel coronavirus 3CL protease inhibitors and conducted systematic screening to obtain a large number of novel molecular structures with novel coronavirus 3CL protease inhibitor activity. Different from the previously reported GC376, there are derivatives based on thiadiazole dione and thiazolidine dione inhibitors, as well as novel 3CL protease inhibitors based on peptidomimetics. Based on the many advantages of protein degraders, especially PROTACs, over inhibitors, PROTAC molecules based on novel inhibitors were designed, prepared and screened. The active PROTAC molecules disclosed in the present invention do have the activity of degrading the novel coronavirus 3CL protease in cells, and are true 3CL protease degraders. Summary of the invention

[0010] Technical problem to be solved by the invention

[0011] The purpose of the present invention is to provide a novel compound having the function of a 3CL protease degrader, wherein the compound is a thalidomide derivative (or analog) or a lenalidomide derivative (or analog), or a pharmaceutically acceptable salt, solvate, prodrug, polymorph or isotope derivative thereof. The present invention also provides a preparation method, medical use and pharmaceutical composition of the compound. The present invention provides a new option for treating coronavirus.

[0012] Technical solutions to technical problems

[0013] The 3CL protease degrading agent of the present invention is a compound represented by the general formula (I) or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, or isotope derivative thereof:

[0014]

[0015] In the formula, R1 is selected from hydrogen or carbonyl;

[0016] R2 is selected from a C1-C22 straight chain or branched alkyl group, or a group represented by the following structural formula

[0017] Wherein, n represents an integer from 2 to 14;

[0018] R3 and R4 are independently selected from hydrogen, isopropyl, isobutyl, benzyl or cyclohexyl;

[0019] R5 is selected from tert-butyloxycarbonyl, benzyloxycarbonyl or 9-fluorenyloxycarbonyl.

[0020] More preferably, R1 is H and R2 is

[0021] wherein n=an integer from 2 to 12.

[0022] Better yet, R2 is wherein n=1-5; or R2 is a C2-C12 straight chain alkyl group.

[0023] More preferred are any of the following compounds:

[0024] 2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)

[0025] 2-(tert-butyloxycarbonyl)amino)-2-isobutylacetyl)phenylalanine;

[0026] 2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethyl

[0027] 2-(tert-butyloxycarbonyl)amino)-2-cyclohexylacetyl)valine;

[0028] (6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)hexanoyl)leucine methyl ester;

[0029] (1-cyclohexyl-2-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)

[0030] amino)-4-oxobutyramido)butyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxo

[0031] tert-butyl ethyl)carbamate;

[0032] (1-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)

[0033] tert-butyl-4-oxobutyramido)hexyl)amino)4-methyl-1-oxopentan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl)carbamate;

[0034] Tert-butyl (1-cyclohexyl-2-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutanamido)hexyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate.

[0035] More preferably, the pharmaceutically acceptable salt is selected from an inorganic acid addition salt or an organic acid addition salt, an inorganic acid salt such as hydrochloride, an organic acid salt such as benzenesulfonate, toluenesulfonate.

[0036] More preferably, the solvate is selected from hydrates, ethanolates and methanolates.

[0037] The present invention also relates to the use of the compound of formula (I) or its pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative in the preparation of anti-coronavirus drugs.

[0038] Wherein, the coronavirus is any one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, and CCoV-HuPn-2018.

[0039] The present invention also relates to the use of the above-mentioned compound of formula (I) or its pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative in the preparation of coronavirus 3CL protease degrading agent.

[0040] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative thereof; and a pharmaceutically acceptable carrier.

[0041] The present invention also relates to a method for preparing the compound of formula (I), comprising:

[0042] Preparation of thalidomide derivatives or lenalidomide derivatives;

[0043] Preparation of dipeptides;

[0044] The thalidomide derivative or the lenalidomide derivative is reacted with the dipeptide.

[0045] Beneficial technical effects

[0046] The compounds provided by the present invention can effectively hijack the ubiquitin proteasome system, thereby effectively achieving the conservative target protein 3CL of coronavirus pro Degradation of protein level, regulating protein target level. Therefore, it is expected that the compound represented by the general formula (I) will have a broad spectrum activity against many types of coronaviruses, providing a new option for treating and preventing diseases caused by coronaviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The results show that different concentrations of compound 1 (Ws-1-186) prepared in Example 1 have an effect on 3CL pro Protein degradation rate.

[0048] Figure 2 The results show that different concentrations of compound 2 (Ws-1-208) prepared in Example 2 have an effect on 3CL pro Protein degradation rate.

[0049] Figure 3 The results show that different concentrations of compound 3 (Zry-2-185) prepared in Example 3 have an effect on 3CL pro Protein degradation rate.

[0050] Figure 4 The results show that different concentrations of compound 4 (WS-1-200) prepared in Example 4 have an effect on 3CL pro Protein degradation rate.

[0051] Figure 5 The results show that different concentrations of compound 5 (Ws-1-216) prepared in Example 5 have an effect on 3CL pro Protein degradation rate.

[0052] Figure 6 The results show that different concentrations of compound 6 (Ws-1-218) prepared in Example 6 have an effect on 3CL pro Protein degradation rate. DETAILED DESCRIPTION

[0053] To facilitate understanding of the present invention, certain terms and phrases are defined and explained below.

[0054] As used herein, the term "inhibitor" refers to any molecule that partially or completely blocks, inhibits, reduces or neutralizes a target biological activity and / or signaling pathway.

[0055] The term "pharmaceutically acceptable" refers to products or compounds approved by the national drug regulatory authorities or listed in the Chinese Pharmacopoeia or other recognized pharmacopoeias for use in animals including humans.

[0056] The term "treat" refers to therapeutic measures that cure, alleviate, lessen the symptoms of and / or halt the progression of the diagnosed condition or disease, and therefore subjects / patients in need of treatment include those already suffering from the disease.

[0057] The term "degradation" refers to the biochemical process by which proteins are broken down into smaller molecules such as peptides or amino acids.

[0058] As used in the specification and claims, the singular forms "a," "an," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0059] The terms "above", "below" and "within" herein include the number unless the context clearly dictates otherwise.

[0060] When the embodiments are described herein as "comprising" or "containing", other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that when the embodiments are described herein as "consisting essentially of", other similar embodiments described as "consisting of" are also provided.

[0061] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, which participates in carrying or transporting the active compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin; proteins, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG); and other nontoxic compatible substances used in pharmaceutical formulations.

[0063] As used herein, "pharmaceutically acceptable salts" are pharmaceutically acceptable organic or inorganic acid or base salts of the compounds described herein. Pharmaceutically acceptable salts of the compounds of the present invention may be, for example, acid addition salts of compounds of the present invention carrying nitrogen atoms in the chain or ring, for example, acid addition salts of sufficiently basic compounds of the present invention, for example, acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, bisulfuric acid, phosphoric acid or nitric acid, or acid addition salts formed with organic acids, for example, benzenesulfonic acid, toluenesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphor Cerebral acid, cinnamic acid, cyclopentanoic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid, or thiocyanic acid.

[0064] As used herein, "solvate" refers to a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. Such physical associations typically include hydrogen bonding. In some cases, the solvate is capable of separation, for example, when one or more solvate molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution phase and separable solvates. Exemplary solvates include hydrates, ethanolates, and methanolates.

[0065] As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to result in improvement of one or more symptoms of a disorder, or to prevent progression of a disorder, or to cause regression of a disorder.

[0066] Herein, "prodrug", also referred to as prodrug, refers to a pharmacologically inactive derivative of the parent "drug" molecule that needs to be biotransformed (e.g., spontaneously or enzymatically) in the target physiological system to release the active drug or convert the prodrug (e.g., enzymatically, physiologically, mechanically, electromagnetically) to the active drug. Prodrugs are intended to overcome problems associated with stability, water solubility, toxicity, lack of specificity or limited bioavailability. Exemplary prodrugs include the active drug molecule itself and a chemical masking group (e.g., a group that reversibly inhibits the activity of the drug). Some prodrugs are modifications or derivatives of compounds with groups that are cleavable under metabolic conditions. Prodrugs can be easily prepared from parent compounds using methods known in the art.

[0067] As used herein, to "inhibit" a function or activity is to reduce the function or activity when compared to otherwise identical conditions except for the target condition or parameter, or alternatively, when compared to another condition.

[0068] "C m-n " means that the part modified by the term has mn carbon atoms (n is greater than m, and both are integers). For example, C1-22 means that the part modified by the term has 1-22 carbon atoms.

[0069] Abbreviations

[0070] DMAP: 4-Dimethylaminopyridine

[0071] DIPEA: N,N-diisopropylethylamine

[0072] DMF: N,N-dimethylformamide

[0073] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0074] o-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tramethyluronium

[0075] hexafluorophosphate

[0076] PE:EA petroleum ether:ethyl acetate

[0077] BOC: t-butyloxycarbonyl

[0078] HOAT: 1-Hydroxy-7-azabenzotriazole

[0079] EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0080] Synthesis process steps

[0081] Step 1: Synthesis of thalidomide intermediate or lenalidomide intermediate

[0082] 1. Synthesis of thalidomide intermediates

[0083] In a 50mL round-bottom flask, add 3mmol 2-fluorothalidomide, 6mmol substrate 1 (such as 4-aminobutyric acid, diglycolamine, etc.), 1mL DIPEA, and 10mL DMF as solvent, and heat to reflux at 145℃ for 4h. During this period, use TLC (CH2Cl2:CH3OH=94:6, V:V) to detect the progress of the reaction. After the reaction is completed, it is naturally cooled to room temperature, and then the solvent is concentrated under reduced pressure to dry. The obtained oil is dissolved in 80mL ethyl acetate, and washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively, and the ethyl acetate phase is separated, and anhydrous sodium sulfate is added to dry, and the solvent is recovered. The crude product is purified by column chromatography, eluted with eluent (PE:EA=4:6, V:V), and the thalidomide intermediate is separated.

[0084] 2. Synthesis of Lenalidomide Intermediates

[0085] In a 250 mL round-bottom flask, add 20 mmol of lenalidomide, 24 mmol of succinic anhydride, 60 mL of acetonitrile, and 20 mL of DMF as a mixed solvent, and react at 80°C for 4 h, and a large amount of solid precipitates. Cool naturally to room temperature, and directly filter to obtain a white solid.

[0086] Add 3mmol of the above product to a 100mL round-bottom flask, 15mL DMF as solvent, and stir under ice bath until completely dissolved. Then add 1.5mmol DMAP, stir for 10min, then add 1.5mL DIPEA, add 3mmol substrate 2 (such as mono-Boc diamine, etc.) after a period of time, and finally add 4.5mmol EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, use TLC (CH2Cl2:CH3OH=94:6, V:V) to monitor the progress of the reaction. After the reaction is completed, add 1.5mL formic acid to neutralize DIPEA, then dissolve the resulting mixture in 80mL ethyl acetate, and then wash twice with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (CH2Cl2:CH3OH=96:4, V:V), and the lenalidomide intermediate product is separated.

[0087] Step 2: Synthesis of dipeptide intermediates

[0088] Add 10mmol of a BOC-protected amino acid and 40mL of dichloromethane as solvent to a 100mL round-bottom flask and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol of another amino acid methyl ester, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL of formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL of ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid dipeptide intermediate is isolated.

[0089] Step 3: Synthesize PROTAC molecules

[0090] 1. Synthesis of PROTAC molecules through thalidomide intermediates and dipeptides

[0091] In a 50mL round-bottom flask, add 1mmol of thalidomide intermediate, 5mL of dichloromethane and 5mL of DMSO as a mixed solvent, and stir under an ice bath until completely dissolved. Then add 1mmol of HOAT, stir for 10min, add 0.5mL of DIPEA, then add 0.8mmol of dipeptide intermediate, and finally add 1.5mmol of EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (CH2Cl2:CH3OH=96:4, V:V). After the reaction is completed, add 0.5mL of formic acid to neutralize DIPEA, then concentrate the solvent under reduced pressure, dissolve the resulting mixture in 40mL of ethyl acetate, and then wash once with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=5:5, V:V), and an oily product is separated.

[0092] 2. Synthesis of PROTAC molecules through lenalidomide intermediates and dipeptides

[0093] Add 0.75mmol of lenalidomide intermediate to a 50mL round-bottom flask, add 10mL of dichloromethane and 2mL of isopropanol to dissolve, then add 6mL of hydrochloric acid (1,4-dioxane 4M solution) dropwise, and stir at room temperature for 4h. After the reaction is completed, no purification is required, and the solvent is concentrated under reduced pressure to dryness and directly used for the next reaction. Add 0.8mmol of dipeptide intermediate to a 100mL round-bottom flask, add 8mL of DMF to dissolve, add 1mmol of diphenylphosphine chloride dropwise under an ice bath, then add 0.3mL of DIPEA dropwise, stir for a while, and add the product of the previous step. Remove the ice bath after 30min of reaction and react overnight at room temperature. Use TLC during the period

[0094] (CH2Cl2:CH3OH=94:6, V:V) monitor the reaction progress. After the reaction is completed, add 0.3mL formic acid to neutralize DIPEA, dissolve the resulting mixture in 40mL ethyl acetate, and then wash once with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (CH2Cl2:CH3OH=29:1, V:V), and separate the solid product.

[0095] Example

[0096] The present invention is described in detail below with reference to the examples. The examples provide detailed implementation methods and processes to fully disclose and demonstrate that the invention is implementable, and the examples do not represent limitations on the invention.

[0097] The compounds, instruments, reagents, materials, experimental animals, etc., whose sources are not indicated in the embodiments of the specification, are purchased through general commercial channels. The experimental methods for which specific conditions are not indicated in the embodiments of the specification are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer. The progress of the reaction of the present invention can be monitored by conventional monitoring methods in the art (such as TLC, HPLC, LCMS or NMR), and the reaction endpoint is generally when the reaction substrate disappears.

[0098] Example 1: Preparation of 2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethoxy)ethyl(2-(tert-butyloxycarbonyl)amino)-2-isobutylacetyl)phenylalanine

[0099] In a 50 mL round-bottom flask, 3 mmol 2-fluorothalidomide, 6 mmol triglycolamine, 1 mL DIPEA and 10 mL DMF as solvent were added and heated to reflux at 145° C. for 4 h.

[0100] During the reaction, TLC (CH2Cl2:CH3OH=94:6, V:V) was used to monitor the progress of the reaction. After the reaction was completed, the mixture was naturally cooled to room temperature, and then the solvent was concentrated under reduced pressure to dry. The obtained oil was dissolved in 80 mL of ethyl acetate, and washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively. The ethyl acetate phase was separated, dried with anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography, eluted with eluent (PE:EA=4:6, V:V), and the product 1 (thalidomide derivative) was isolated.

[0101] Add 10mmol Boc-L-leucine and 40mL dichloromethane as solvent to a 100mL round-bottom flask and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-phenylalanine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0102] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly dropwise add to the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6, V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust to pH=1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and concentrate under reduced pressure to drain the solvent to obtain a solid product 2.

[0103] In a 50mL round-bottom flask, add 1mmol of product 1, 5mL of dichloromethane and 5mL of DMSO as a mixed solvent, and stir under an ice bath until completely dissolved. Then add 1mmol of HOAT, stir for 10min, then add 0.5mL of DIPEA, then add 0.8mmol of product 2, and finally add 1.5mmol of EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (CH2Cl2:CH3OH=96:4, V:V). After the reaction is completed, add 0.5mL of formic acid to neutralize DIPEA, then concentrate the solvent under reduced pressure, dissolve the resulting mixture in 40mL of ethyl acetate, and then wash once with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=5:5, V:V), and a yellow-green oily product (compound 1: code Ws-1-186) is isolated, with a yield of 31%.

[0104] 1H NMR(600MHz,DMSO)δ11.10(s,1H),8.67(d,J=6.2Hz,0.5H),8.66(d,J=6.2Hz,0.5H),8.18(d,J=7.7Hz,0.5H),8.17(d,J=7.7Hz,0.5H),7.59–7.55(t,J=4.9Hz,1H),7.52(d,J=4.4Hz,1H),7.26–7.17(m,5H),7.12(d,J=8.6Hz,1H),7.04(d,J=7.0Hz,1H),5.06(d,J=5.4Hz,1H),4.55–4.43(t,J=4.9Hz,1H),4.21–4.06(t,J=4.4Hz,2H),3.97(t,J=8.9Hz,1H),3.64–3.44(m,10H),3.07–2.88(t,J=4.4Hz,2H),2.04–1.99(m,1H),1.58–1.41(m,1H),1.36–1.10(m,14H),0.85–0.76(m,6H).

[0105] 13 C NMR(151MHz,DMSO)δ172.82(s),172.60(s),171.44(s),170.09(s),168.93(s),167.29(s),151.08(s),146.38(s),137.03(s),136.21(s),132.08(s),129.20(s),128.85(s),128.14(s),126.49(s),120.71(s),117.41(s),110.67(s),109.23(s),77.89(s),69.72(s),68.92(s),68.17(s),63.90(s),53.28(s),53.28(s),52.40(s),48.55(s),41.68(s),36.62(s),31.00(s),28.18(s),24.10(s),22.88(s),22.88(s),22.15(s),21.55(s).HRESIMS:m / z calcd for C 39 H 51 N5O 11 [M+Na]+

[0106] 788.3483,found 788.3486.

[0107] Example 2: Preparation of 2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethyl(2-(tert-butyloxycarbonyl)amino)-2-cyclohexylacetyl)valine

[0108] In a 50 mL round-bottom flask, 3 mmol 2-fluorothalidomide, 6 mmol diglycolamine, 1 mL DIPEA and 10 mL DMF as solvent were added and heated to reflux at 145° C. for 4 h.

[0109] During the reaction, TLC (CH2Cl2:CH3OH=94:6, V:V) was used to monitor the progress of the reaction. After the reaction was completed, the mixture was naturally cooled to room temperature, and then the solvent was concentrated under reduced pressure to dry. The obtained oil was dissolved in 80 mL of ethyl acetate, and washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively. The ethyl acetate phase was separated, dried with anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography, eluted with eluent (PE:EA=4:6, V:V), and the product 1 (thalidomide derivative) was isolated.

[0110] Add 10mmol Boc-L-cyclohexylglycine and 40mL dichloromethane as solvent to a 100mL round-bottom flask and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-valine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0111] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly dropwise add to the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6, V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust to pH=1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and concentrate under reduced pressure to drain the solvent to obtain a solid product 2.

[0112] Add 1mmol product 1, 5mL dichloromethane and 5mL DMSO as mixed solvents to a 50mL round-bottom flask and stir under ice bath until completely dissolved. Then add 1mmol HOAT, stir for 10min, add 0.5mL DIPEA, then add 0.8mmol product 2, and finally add 1.5mmol EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (CH2Cl2:CH3OH=96:4, V:V). After the reaction is completed, add 0.5mL formic acid to neutralize DIPEA, then concentrate the solvent under reduced pressure, dissolve the resulting mixture in 40mL ethyl acetate, and then wash once with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product was purified by column chromatography with an eluent (PE:EA=5:5, V:V) to separate a yellow-green oily product (Compound 2: codename Ws-1-208) with a yield of 37.1%.

[0113] 1 H NMR (600MHz, DMSO) δ11.11(s,1H),8.22(s,1H),7.78(d,J=8.4Hz,1H),7.59(t,J=7.8Hz,1H),7.15 (d,J=5.0Hz,1H),7.05(d,J=7.0Hz,1H),6.62(s,1H),5.06(d,J=5.4Hz,1H),4.27–4.17(m,2H),4. 15(d,J=5.8Hz,1H),4.03(q,J=7.1Hz,1H),3.70–3.60(m,4H),3.52–3.43(t,J=5.8Hz,2H),2.58(m ,2H),2.03(m,2H),1.65(m,2H),1.37(d,J=6.4Hz,9H),1.32–1.05(m,10H),0.86(d,J=5.5Hz,6H).

[0114] 13C NMR(151MHz,DMSO)δ172.80(s),171.45(s),170.09(s),168.91(s),167.27(s),162.10(s),155. 32(s),146.37(s),136.21(s),132.08(s),117.46(s),110.70(s),109.27(s),77.95(s),68.79(s) ),68.05(s),62.57(s),59.75(s),59.06(s),56.97(s),48.54(s),41.58(s),30.98(s),29.95(s) ,29.81(s),29.08(s),28.17(s),25.83(s),25.55(s),22.13(s),20.76(s),19.03(s),17.97(s).

[0115] HRESIMS: m / z calcd for C 35 H 49 N5O 10 [M+Na]+722.3377,found 722.3378.

[0116] Example 3: (6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)

[0117] Preparation of leucine methyl ester

[0118] In a 50 mL round-bottom flask, 3 mmol 2-fluorothalidomide, 6 mmol 6-aminocaproic acid, 1 mL DIPEA and 10 mL DMF as solvent were added and heated to reflux at 145° C. for 4 h.

[0119] During the reaction, TLC (CH2Cl2:CH3OH=94:6, V:V) was used to monitor the progress of the reaction. After the reaction was completed, the mixture was naturally cooled to room temperature, and then the solvent was concentrated under reduced pressure to dry. The obtained oil was dissolved in 80 mL of ethyl acetate, and washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively. The ethyl acetate phase was separated, dried with anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography, eluted with eluent (PE:EA=4:6, V:V), and the product 1 (thalidomide derivative) was isolated.

[0120] Add 10mmol Boc-L-leucine and 40mL dichloromethane as solvent to a 100mL round-bottom flask and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-phenylalanine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0121] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly drop it into the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6,V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust the pH to 1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, concentrate under reduced pressure to drain the solvent, dissolve it in 6mL of dichloromethane, add 2.5mL of HCl (1,4-dioxane 4M solution, 10mmol), and react at room temperature overnight. After the reaction is completed by TLC monitoring (PE:EA=2:8), spin dry to a white solid to obtain solid product 2.

[0122] In a 50mL round-bottom flask, add 1mmol of product 1, 5mL of dichloromethane and 5mL of DMSO as a mixed solvent, and stir under an ice bath until completely dissolved. Then add 1mmol of HOAT, stir for 10min, then add 0.5mL of DIPEA, then add 0.8mmol of product 2, and finally add 1.5mmol of EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (CH2Cl2:CH3OH=96:4, V:V). After the reaction is completed, add 0.5mL of formic acid to neutralize DIPEA, then concentrate the solvent under reduced pressure, dissolve the resulting mixture in 40mL of ethyl acetate, and then wash once with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=5:5, V:V), and a yellow solid product (compound 3: code Zry-2-185) is isolated with a yield of 65.2%.

[0123] 1 H NMR (600MHz, DMSO) δ11.11(s,1H),8.79–8.51(m,1H),8.06(d,J=7.9Hz,1H),7.95(d,J=8.0Hz, 1H),7.57(t,J=7.6Hz,1H),7.08(d,J=8.5Hz,1H),7.03(d,J=6.8Hz,1H),5.07–5.05(m,1H),4.4 3–4.39(m,1H),4.15(t,J=7.0Hz,1H),3.62(s,3H),3.27(d,J=5.6Hz,2H),2.91–2.87(m,1H),2. 61–2.57(m,1H),2.17–2.10(m,2H),2.06–2.00(m,2H),1.27–1.58(m,10H),0.88–0.83(m,12H).

[0124] 13 C NMR (151MHz, DMSO) δ173.28,173.12,172.49,172.34,170.55,169.40,167.76,151.57,146.85,136.72,132.63,129.30,121.18,117.57,110 .84,109.45,57.74,52.08,51.05,49.00,42.25,41.07,35.50,31.45, 30.32,28.90,26.32,25.53,24.64,23.46,22.63,22.04,19.34,18.68.

[0125] HR-MS(ESI)m / z:Calcd for C 31 H 43 N5O8Na[M+Na+]636.3009, found 636.3013.

[0126] Example 4: Preparation of tert-butyl (1-cyclohexyl-2-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutyramido)butyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate

[0127] In a 250mL round-bottom flask, add 20mmol lenalidomide, 24mmol succinic anhydride, 60mL acetonitrile and 20mL DMF as a mixed solvent, and react at 80°C for 4h. A large amount of solid precipitates. Naturally cool to room temperature and directly filter to obtain a white solid. In a 100mL round-bottom flask, add 3mmol of the above product and 15mL DMF as solvent, and stir in an ice bath until completely dissolved. Then add 1.5mmol DMAP, stir for 10min, then add 1.5mL DIPEA, add 3mmol mono-Boc-butylene diamine after a while, and finally add 4.5mmol EDCI. After reacting for 30min, remove the ice bath and react at room temperature overnight. Use TLC during the period

[0128] (CH2Cl2:CH3OH=94:6, V:V) monitor the reaction progress, add 1.5mL formic acid to neutralize DIPEA after the reaction is completed, then dissolve the resulting mixture in 80mL ethyl acetate, and then wash twice with saturated NaHCO3 solution and saturated NaCl solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (CH2Cl2:CH3OH=96:4, V:V), and 1 (lenalidomide derivative) is isolated.

[0129] In a 100mL round-bottom flask, add 10mmol Boc-L-cyclohexylglycine and 40mL dichloromethane as solvent, and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-phenylalanine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0130] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly drop it into the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6,V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust the pH to 1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, concentrate under reduced pressure to drain the solvent, dissolve it in 6mL of dichloromethane, add 2.5mL of HCl (1,4-dioxane 4M solution, 10mmol), and react at room temperature overnight. After the reaction is completed by TLC monitoring (PE:EA=2:8), spin dry to a white solid to obtain solid product 2.

[0131] 0.75mmol of product 1 was added to a 50mL round-bottom flask, and 10mL of dichloromethane and 2mL of isopropanol were added to dissolve it. Then 6mL of hydrochloric acid (1,4-dioxane 4M solution) was added dropwise, and stirred at room temperature for 4h. After the reaction was completed, no purification was required, and the solvent was concentrated under reduced pressure to dry it, and it was directly used for the next step reaction. 0.8mmol of product 2 was added to a 100mL round-bottom flask, and 8mL of DMF was added to dissolve it. 1mmol of diphenylphosphine chloride was added dropwise under an ice bath, and then 0.3mL of DIPEA was added dropwise. After stirring for a period of time, the product of the previous step was added. After reacting for 30min, the ice bath was removed and the reaction was allowed to proceed overnight at room temperature. During this period, the reaction progress was monitored by TLC (CH2Cl2:CH3OH=94:6, V:V). After the reaction was completed, 0.3mL of formic acid was added to neutralize DIPEA, and the resulting mixture was dissolved in 40mL of ethyl acetate, and then washed once with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated NaCl solution. The ethyl acetate phase was separated, dried over anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography with an eluent (CH2Cl2:CH3OH=29:1, V:V) to separate a white solid product (Compound 4: codename Ws-1-200) in a yield of 40.9%.

[0132] 1 H NMR (600MHz, DMSO) δ11.04(s,1H),9.92(s,1H),9.24(s,1H),8.34(d,J=8.5Hz,1H),7.95– 7.81(m,3H),7.48(q,J=7.4Hz,2H),7.24–7.12(m,5H),5.15(d,J=4.9Hz,1H),4.40(d,J=7. 5Hz,1H),4.32(d,J=7.5Hz,1H),3.69(t,J=7.6Hz,1H),3.59(d,J=7.0Hz,2H),3.18–2.75(m ,8H),2.65–2.54(m,3H),2.42(s,2H),2.32(m,1H),2.09–1.96(m,1H),1.43–1.21(m,23H).

[0133] 13C NMR(151MHz,DMSO)δ172.86(s),171.53(s),171.05(s),170.96(s),170.73(s),170.65(s),167.84(s),155.38(s) ,137.65(s),133.84(s),133.47(s),132.62(s),129.15(s),128.60(s),127.94(s),126.16(s),124.96(s),118.84 (s),78.17(s),78.13(s),59.76(s),53.69(s),51.51(s),46.46(s),41.59(s),38.17(s),31.34(s),31.20(s),30 .43(s),28.88(s),28.15(s),26.56(s),26.41(s),25.71(s),25.56(s),22.70(s),17.98(s),16.70(s),12.24(s).

[0134] HRESIMS: m / z calcd for C 43 H 57 N7O9[M+Na]+838.4115,found 838.4118.

[0135] Example 5: Preparation of tert-butyl 1-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutanamido)hexyl)amino)4-methyl-1-oxopentan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl)carbamate

[0136] In a 250mL round-bottom flask, 20mmol lenalidomide, 24mmol succinic anhydride, 60mL acetonitrile and 20mL DMF were added as mixed solvents. The mixture was reacted at 80°C for 4h, and a large amount of solid precipitated. The mixture was naturally cooled to room temperature and directly filtered to obtain a white solid. In a 100mL round-bottom flask, 3mmol of the above product and 15mL DMF were added as solvent, and the mixture was stirred under an ice bath until it was completely dissolved. Then 1.5mmol DMAP was added, and 1.5mL DIPEA was added after stirring for 10min. After a period of time, 3mmol (6-aminohexyl) tert-butyl carbamate was added, and finally 4.5mmol EDCI was added. After reacting for 30min, the ice bath was removed and the mixture was reacted at room temperature overnight. During the reaction, TLC (CH2Cl2:CH3OH=94:6, V:V) was used to monitor the progress of the reaction. After the reaction was completed, 1.5mL formic acid was added to neutralize DIPEA, and then the resulting mixture was dissolved in 80mL ethyl acetate, and then washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively. The ethyl acetate phase was separated, dried over anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography using an eluent (CH2Cl2:CH3OH=96:4, V:V) to separate a solid product 1 (lenalidomide derivative 2).

[0137] Add 10mmol Boc-L-phenylalanine and 40mL dichloromethane as solvent to a 100mL round-bottom flask and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-leucine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0138] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly drop it into the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6,V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust the pH to 1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, concentrate under reduced pressure to drain the solvent, dissolve it in 6mL of dichloromethane, add 2.5mL of HCl (1,4-dioxane 4M solution, 10mmol), and react at room temperature overnight. After the reaction is completed by TLC monitoring (PE:EA=2:8), spin dry to a white solid to obtain solid product 2.

[0139] 0.75mmol of product 1 was added to a 50mL round-bottom flask, and 10mL of dichloromethane and 2mL of isopropanol were added to dissolve it. Then 6mL of hydrochloric acid (1,4-dioxane 4M solution) was added dropwise, and stirred at room temperature for 4h. After the reaction was completed, no purification was required, and the solvent was concentrated under reduced pressure to dry it, and it was directly used for the next step reaction. 0.8mmol of product 2 was added to a 100mL round-bottom flask, and 8mL of DMF was added to dissolve it. 1mmol of diphenylphosphine chloride was added dropwise under an ice bath, and then 0.3mL of DIPEA was added dropwise. After stirring for a period of time, the product of the previous step was added. After reacting for 30min, the ice bath was removed and the reaction was allowed to proceed overnight at room temperature. During this period, the reaction progress was monitored by TLC (CH2Cl2:CH3OH=94:6, V:V). After the reaction was completed, 0.3mL of formic acid was added to neutralize DIPEA, and the resulting mixture was dissolved in 40mL of ethyl acetate, and then washed once with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated NaCl solution. The ethyl acetate phase was separated, dried over anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography with an eluent (CH2Cl2:CH3OH=29:1, V:V) to separate a white solid product (Compound 5: codename Ws-1-216) in a yield of 79.5%.

[0140] 1H NMR(600MHz,DMSO)δ11.04(s,1H),9.90(s,1H),8.03–7.77(m,4H),7.49(t,J=8.1Hz,2H),7.24(d,J=3.3Hz,4H),7.18(d,J=4.4Hz,1H),6.96(d,J=8.4Hz,1H),5.15(d,J=5.0Hz,1H),4.36(q,J=8.7Hz,2H),4.27(d,J=8.6Hz,1H),4.16(d,J=4.7Hz,1H),3.63–3.55(m,1H),3.11(d,J=4.1Hz,1H),2.98(m,6H),2.73(d,J=9.4Hz,1H),2.59(d,J=6.7Hz,3H),2.41(t,J=7.2Hz,2H),2.32(d,J=4.2Hz,1H),2.06–1.99(m,1H),1.38–1.25(m,18H),0.85(d,J=6.4Hz,6H).

[0141] 13 C NMR(151MHz,DMSO)δ172.84(s),171.53(s),171.24(s),171.04(s),170.89(s),170.71(s),167.83(s),155.23(s),138.09(s),133.83(s),133.47(s),132.61(s),129.18(s),128.59(s),127.97(s),126.14(s),124.95(s),118.84(s),78.09(s),55.77(s),53.33(s),51.25(s),51.25(s),46.43(s),41.60(s),41.41(s),38.45(s),37.11(s),31.20(s),30.44(s),29.13(s),28.07(s),26.08(s),24.04(s),22.99(s),22.69(s),21.75(s),17.98(s),16.69(s),12.25(s).

[0142] HRESIMS:m / z calcd for C 43 H 59 N7O9[M+Na]+840.4272,found 840.4280.

[0143] Example 6: Preparation of tert-butyl (1-cyclohexyl-2-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutyramido)hexyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate

[0144] In a 250mL round-bottom flask, 20mmol lenalidomide, 24mmol succinic anhydride, 60mL acetonitrile and 20mL DMF were added as mixed solvents. The mixture was reacted at 80°C for 4h, and a large amount of solid precipitated. The mixture was naturally cooled to room temperature and directly filtered to obtain a white solid. In a 100mL round-bottom flask, 3mmol of the above product and 15mL DMF were added as solvent, and the mixture was stirred under an ice bath until it was completely dissolved. Then 1.5mmol DMAP was added, and 1.5mL DIPEA was added after stirring for 10min. After a period of time, 3mmol (6-aminohexyl) tert-butyl carbamate was added, and finally 4.5mmol EDCI was added. After reacting for 30min, the ice bath was removed and the mixture was reacted at room temperature overnight. During the reaction, TLC (CH2Cl2:CH3OH=94:6, V:V) was used to monitor the progress of the reaction. After the reaction was completed, 1.5mL formic acid was added to neutralize DIPEA, and then the resulting mixture was dissolved in 80mL ethyl acetate, and then washed twice with saturated NaHCO3 solution and saturated NaCl solution respectively. The ethyl acetate phase was separated, dried over anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography using an eluent (CH2Cl2:CH3OH=96:4, V:V) to separate a solid product 1 (lenalidomide derivative 3).

[0145] In a 100mL round-bottom flask, add 10mmol Boc-L-cyclohexylglycine and 40mL dichloromethane as solvent, and stir under ice bath until completely dissolved. Add 14mmol HATU in batches, stir for 10min, then add 10mmol L-phenylalanine methyl ester hydrochloride, continue stirring, and finally add 5mL DIPEA. After reacting for 30min, remove the ice bath and react at room temperature overnight. During this period, monitor the progress of the reaction by TLC (PE:EA=8:2, V:V). After the reaction is completed, add 5mL formic acid to neutralize DIPEA, then concentrate under reduced pressure to drain the solvent, dissolve the resulting mixture in 150mL ethyl acetate, and then wash three times with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated Na2SO4 solution respectively. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, and recover the solvent. The crude product is purified by column chromatography, eluted with eluent (PE:EA=8:2, V:V), and a solid (peptoid intermediate) is separated.

[0146] In a 100mL round-bottom flask, add 18mmol of lithium hydroxide monohydrate, 36mmol of hydrogen peroxide solution (30%), and 5mL of H2O. Dissolve 4.5mmol of the intermediate obtained in the previous step in 15mL of tetrahydrofuran, slowly drop it into the flask under ice bath, stir for 2h, remove the ice bath, and react at room temperature for 6h. During this period, monitor the progress of the reaction by TLC (PE:EA=4:6,V:V). After the reaction is completed, add 30mL of saturated Na2SO3 solution to neutralize hydrogen peroxide, then add 30mL of H2O and 80mL of ethyl acetate to dilute, and add hydrochloric acid to adjust the pH to 1. Separate the ethyl acetate phase, dry it with anhydrous sodium sulfate, concentrate under reduced pressure to drain the solvent, dissolve it in 6mL of dichloromethane, add 2.5mL of HCl (1,4-dioxane 4M solution, 10mmol), and react at room temperature overnight. After the reaction is completed by TLC monitoring (PE:EA=2:8), spin dry to a white solid to obtain solid product 2.

[0147] 0.75mmol of product 1 was added to a 50mL round-bottom flask, and 10mL of dichloromethane and 2mL of isopropanol were added to dissolve it. Then 6mL of hydrochloric acid (1,4-dioxane 4M solution) was added dropwise, and stirred at room temperature for 4h. After the reaction was completed, no purification was required, and the solvent was concentrated under reduced pressure to dry it, and it was directly used for the next step reaction. 0.8mmol of product 2 was added to a 100mL round-bottom flask, and 8mL of DMF was added to dissolve it. 1mmol of diphenylphosphine chloride was added dropwise under an ice bath, and then 0.3mL of DIPEA was added dropwise. After stirring for a period of time, the product of the previous step was added. After reacting for 30min, the ice bath was removed and the reaction was allowed to proceed overnight at room temperature. During this period, the reaction progress was monitored by TLC (CH2Cl2:CH3OH=94:6, V:V). After the reaction was completed, 0.3mL of formic acid was added to neutralize DIPEA, and the resulting mixture was dissolved in 40mL of ethyl acetate, and then washed once with saturated NH4Cl solution, saturated NaHCO3 solution, and saturated NaCl solution. The ethyl acetate phase was separated, dried over anhydrous sodium sulfate, and the solvent was recovered. The crude product was purified by column chromatography with an eluent (CH2Cl2:CH3OH=29:1, V:V) to separate a light green solid product (Compound 6: codename Ws-1-218) in a yield of 44.4%.

[0148] 1H NMR (600MHz, DMSO) δ11.04(s,1H),9.93(s,1H),7.88(s,3H),7.83(d,J=7.1Hz,1H),7.49(t,J=8.0Hz,2H),7.25–7 .18(m,4H),7.15(t,J=6.8Hz,1H),6.75(d,J=8.7Hz,1H),5.15(d,J=5.1Hz,1H),4.36(q,J=8.7Hz,2H),3.73–3.55( m,2H),3.11(d,J=4.0Hz,1H),3.01(d,J=6.0Hz,3H),2.94(d,J=8.7Hz,3H),2.79(d,J=9.3Hz,1H),2.65–2.56(m,3H ),2.42(t,J=7.2Hz,2H),2.32(d,J=4.2Hz,1H),2.07–1.98(m,1H),1.51(m,5H),1.37(s,10H),1.32–1.25(m,12H).

[0149] 13 C NMR(151MHz,DMSO)δ172.84(s),171.04(s),170.89(s),170.72(s),170.54(s),167.84(s),155.38(s),137.64( s),133.84(s),133.48(s),132.61(s),129.14(s),128.58(s),127.92(s),126.16(s),124.95(s),118.83(s),7 8.11(s),59.75(s),54.92(s),53.70(s),53.29(s),51.49(s),46.46(s),41.57(s),38.47(s),37.89(s),31.20 (s),30.46(s),28.87(s),28.16(s),26.05(s),26.05(s),25.70(s),22.70(s),17.96(s),16.69(s),12.21(s).

[0150] HRESIMS: m / z calcd for C 45 H 61 N7O9[M+Na]+866.4428,found 866.4434.

[0151] Biological activity test

[0152] PROTAC compounds prepared in Examples 1-6 of the present invention have an effect on 3CLpro Protein degradation activity

[0153] In this embodiment, 3CL pro Protein is the key target of the new coronavirus (SARS-CoV-2) 3CL pro (three-chymotrypsin-like protease) protease.

[0154] 1. Experimental methods

[0155] Cell culture and compound treatment: First, culture cells that stably express 3CL of the novel coronavirus pro HEK293 cell line with protease (with FLAG tag) was inoculated with 100,000 cells per well in a 24-well plate, and appropriate concentrations of the compounds prepared in Examples 1-6 and control DMSO were added to each well, and culture was continued in a constant temperature incubator at 37° C. and 5% CO 2 for 48 h to 72 h.

[0156] 2. Sample preparation

[0157] Remove the culture medium and wash the cells with PBS; add the corresponding volume of RIPA according to the cell amount to lyse the cells, and sonicate until the cells are no longer viscous; centrifuge at 12000g for 5 minutes, take the supernatant, add protein loading buffer, boil for 5 minutes, and centrifuge for later use.

[0158] 3. Western Blot Detection of 3CL pro -flag protein expression and semi-quantitative analysis

[0159] The protein samples were separated by SDS-PAGE gel, and then the proteins were transferred to PVDF membrane by electrotransfer. The membrane was blocked with 5% skim milk powder and incubated with mouse anti-FLAG-HRP antibody and β-actin-HRP antibody for 1 hour respectively. After washing the membrane, ECL luminescent solution was added for exposure.

[0160] Analysis of actin and 3CL using Gelpro32 software pro The optical density (IOD) of the bands was compared with that of the DMSO control group. pro Degradation rate.

[0161] The calculation formula is as follows:

[0162] Degradation rate = 100% - (3CL pro Experimental group / β-actin experimental group) / (3CL pro DMSO group / β-actin DMSO group)×100%.

[0163] The experimental results are as attached Figure 1-6 As shown, Figure 1-Figure 6 The compounds prepared in Examples 1-6 respectively show the effects of 3CL pro The degradation rate of Figure 1-Figure 6 It can be seen that the compounds prepared in Examples 1-6 of the present invention have an effect on 3CL pro Has good degradation effect.

[0164] It should be noted that the present invention is not limited to the above implementation modes and examples. The above implementation modes and examples are only examples, and implementation modes that have substantially the same structure as the technical idea and exert the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications and substitutions of the implementation modes / examples that can be thought of by those skilled in the art, and other methods formed by combining some of the constituent elements in the implementation modes are also included in the scope of the present application, and the protection scope of the present invention is defined by the attached claims.

Claims

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, or isotope derivative thereof: In the formula, R1 is selected from hydrogen or carbonyl; R2 is selected from a C1-C22 straight chain or branched alkyl group, or a group represented by the following structural formula in, n represents an integer from 2 to 14; R3 and R4 are independently selected from hydrogen, isopropyl, isobutyl, benzyl or cyclohexyl; R5 is selected from tert-butyloxycarbonyl, benzyloxycarbonyl or 9-fluorenyloxycarbonyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, or isotope derivative thereof, characterized in that: R1 is H, R2 is Here, n is an integer from 2 to 12.

3. The compound according to claim 1 or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, or isotope derivative thereof, characterized in that: R2 is Where n is an integer from 1 to 5; or R2 is a C2-C12 straight chain alkyl group.

4. The compound according to claim 1, selected from: 2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethoxy)ethyl(2-(tert-butoxycarbonyl)amino)-2-isobutylacetyl)phenylalanine; 2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethyl(2-(tert-butoxycarbonyl)amino)-2-cyclohexylacetyl)valine; (6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)hexanoyl)leucine methyl ester; tert-butyl (1-cyclohexyl-2-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutanamido)butyl)amino)-1-oxo-3-phenylpropan-2-yl)aminoamino)-2-oxoethyl)carbamate; tert-butyl (1-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutanamido)hexyl)amino)4-methyl-1-oxopentan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl)carbamate; Tert-butyl (1-cyclohexyl-2-(1-(6-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutanamido)hexyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate.

5. A pharmaceutically acceptable salt or solvate of the compound according to claim 1, wherein the pharmaceutically acceptable salt is selected from an inorganic acid addition salt or an organic acid addition salt; and / or The solvate is selected from the group consisting of hydrates, ethanolates and methanolates.

6. Use of the compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative in the preparation of anti-coronavirus drugs.

7. The use according to claim 6, characterized in that The coronavirus is any one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, and CCoV-HuPn-2018.

8. Use of the compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotope derivative in the preparation of a coronavirus 3CL protease degrader.

9. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, prodrug, solvate, polymorph, isotopic derivative thereof; and a pharmaceutically acceptable carrier.

10. A method for preparing the compound according to claim 1, comprising: Preparation of thalidomide derivatives or lenalidomide derivatives; Preparation of dipeptides; The thalidomide derivative or the lenalidomide derivative is reacted with the dipeptide.

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