Compound targeting ubiquitination degradation of TMPRSS2 protein and preparation method and application thereof

Abstract

The present invention discloses compounds that target ubiquitination degradation of TMPRSS2 protein, as well as preparation methods and applications thereof. The compounds of the present invention are represented by general formula (I) or (II), wherein the E3 ligand is a ligand of the E3 ligase VHL or CRBN, and Linker is a linking group. The compounds of the present invention can target and degrade TMPRSS2 protein, exhibit good inhibitory effects on various tumor cell lines, and have low toxicity to normal cells. The preparation method of the compounds of the present invention is simple to operate and requires mild conditions. The resulting compounds have TMPRSS2 protein degradation activity and significant anti-tumor effects. Pharmacologically or physiologically acceptable salts thereof can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a compound for targeting ubiquitination degradation of TMPRSS2 protein, and a preparation method and application thereof. Background Art

[0002] Transmembrane serine protease 2 (TMPRSS2) is a trypsin-like serine protease that catalyzes the hydrolysis of peptide bonds at specific locations in proteins and plays an important role in normal physiological processes in the human body. TMPRSS2 is primarily composed of three parts: an intramembrane domain, a unidirectional transmembrane domain, and an extramembrane domain that catalyzes hydrolysis.

[0003] Research on the anti-tumor effects of TMPRSS2 began in the field of prostate cancer. Studies have shown that both TMPRSS2 and TMPRSS4 promote prostate cancer metastasis. TMPRSS2 expression in prostate cancer cells is several times higher than in normal prostate cells. Furthermore, TMPRSS2 undergoes chromosomal rearrangements with the ERG, ETV1, or ETV4 genes of the E26 recombination factor (ETS) family, which contain androgen-responsive elements, making its expression regulated by androgen levels. Clinically, fusions of the ERG, ETV1, and TMPRSS2 genes occur in approximately 50-79% of prostate cancer patients, and TMPRSS2-ETS fusions are directly associated with poor prognosis in prostate cancer patients. In 2014, Lucas et al. demonstrated that knocking out TMPRSS2 in a mouse model of prostate cancer inhibited prostate cancer invasion and metastasis. Furthermore, potent TMPRSS2 inhibitors identified through compound library screening inhibited prostate cancer metastasis in vivo.

[0004] In addition to its crucial role in anti-tumor applications, TMPRSS2 also holds unique potential for development and application in the antiviral field. In 2020, Hoffmann et al. discovered that the novel coronavirus (SARS-CoV-2) utilizes the same receptor, ACE2, as the SARS-CoV to recognize host cells. TMPRSS2 on the host cell membrane hydrolyzes the S1 portion of the SARS-CoV-2 spike protein, exposing the S2 portion for membrane fusion. The same team also discovered that camostat mesylate can hinder SARS-CoV-2 from invading lung host cells by inhibiting TMPRSS2 activity. Since then, TMPRSS2 inhibitors, such as camostat mesylate and nafamostat mesylate, have garnered significant attention from both academia and industry. Therefore, TMPRSS2 is a potential new target for the development of anti-tumor and antiviral drugs. Intensifying research on this target and developing compounds related to it have potential applications in both personalized cancer treatment and antiviral treatment.

[0005] Compared with traditional small molecule inhibitors that inhibit the function of target proteins by binding to the active sites of target proteins, PROteolysis TArgeting Chimeras (PROTACs) can directly degrade target proteins through the ubiquitin proteasome system, thereby achieving the effect of treating or preventing diseases. The core of PROTAC technology is to recruit E3 ubiquitin ligases to target proteins through bifunctional molecules and tag them with ubiquitination, and then degrade the target proteins through the ubiquitin-proteasome pathway. Precisely because PROTAC molecules exert their therapeutic effects by degrading rather than inhibiting target proteins, they have the potential to break through the problems of drug resistance and poor target selectivity caused by existing traditional small molecule inhibitor treatments.

[0006] Therefore, by designing and synthesizing PROTAC small molecule degraders that can target and degrade TMPRSS2, we can provide important references for using TMPRSS2 as a drug development target to achieve anti-tumor and antiviral related applications. Summary of the Invention

[0007] In view of the above-mentioned problems existing in the prior art, the primary purpose of the present invention is to overcome the shortcomings of the prior art and provide compounds represented by the following general formula (I) or (II) for targeted ubiquitination and degradation of TMPRSS2 protein and their applications. The compounds for targeted ubiquitination and degradation of TMPRSS2 protein have the ability to target and degrade TMPRSS2 protein, have potential antiviral application prospects, and exhibit good inhibitory effects on various tumor cell lines, and can be developed as new anti-tumor drugs.

[0008] Another object of the present invention is to provide a method for preparing a compound represented by the following general formula (I) or (II) for targeting ubiquitination and degradation of TMPRSS2 protein.

[0009] In order to achieve the above object, the technical solutions adopted by the present invention are as follows:

[0010] In a first aspect, an oseltamivir PROTAC compound represented by formula (I) or (II) or a pharmacologically or physiologically acceptable salt thereof is provided.

[0011]

[0012] In the general formula, E3 ligand is VHL or CRBN ligand; Linker is a connecting group;

[0013] R is H, halogen, C1-C6 alkyl, C1-C6 alkoxy, nitro, amino, hydroxyl or cyano, R 1 is substituted H, halogen, C1-C6 alkyl, C1-C6 alkoxy, nitro, amino, hydroxyl or cyano; R 2 is H or halogen.

[0014] E3 ligase ligands are small molecule ligands that can interact with the protease encoded by the VHL (von Hippel-Lindau) or CRBN (Cereblon) gene with ubiquitination function. The specific structure is shown in one of the following formulas:

[0015]

[0016] Linker is a connecting group, represented by -alkylene, -alkoxy or -piperazinyl and -piperidinyl and any combination thereof, wherein the linker is a straight chain or branched -alkylene, -alkoxy or -aromatic ring or -aromatic heterocycle or -aliphatic ring and any combination thereof, which is optionally interrupted once or multiple times by one or more groups selected from the following groups, such as: -(CH2) n -、-(CH2) n CO-、-NR 3 (CH2) n CO-、-NR 4 (CH2) n -、-(OCH2CH2O) n -、-(CH2CH2O) n -、-(OCH2CH2OCH2) n -、-(CH2CH2OCH2) n -、-(CH2CH2OCH2CH2) n-, -alkenylene, -alkynylene, -cycloalkylene, -heteroarene or any combination thereof, wherein n represents a natural number from 1 to 20, R 3 、R 4 Each independently selects H or C 1-10 alkyl.

[0017] Preferably, the compounds provided by the present invention for targeting ubiquitination degradation of TMPRSS2 protein include but are not limited to the compounds shown in Table 1 below:

[0018] Table 1

[0019]

[0020]

[0021]

[0022]

[0023]

[0024] Furthermore, the compounds provided by the present invention for targeting ubiquitination and degradation of TMPRSS2 protein are the following compounds: compounds numbered LPOT4, PPOT8, PPOT16, PPOT20, PPOT23, PPOT25, PPOT26, VPOT57, VPOT58, VPOT59, VPOT62, VPOT64, VPOT65, VPOT66 or VPOT67 in Table 1 above.

[0025] In a second aspect, the invention provides the use of any one of the above compounds targeting ubiquitination degradation of TMPRSS2 protein or a pharmacologically or physiologically acceptable salt thereof as a TMPRSS2 degrader in the preparation of an anti-tumor drug.

[0026] In a third aspect, the invention provides the use of any one of the above-mentioned compounds targeting ubiquitination degradation of TMPRSS2 protein or a pharmacologically or physiologically acceptable salt thereof as a TMPRSS2 degrading agent in the preparation of an antiviral drug.

[0027] In a fourth aspect, a method for preparing a compound of formula (I) or (II) for targeting ubiquitination and degradation of TMPRSS2 protein is provided, wherein the compound of formula (I) or (II) for targeting ubiquitination and degradation of TMPRSS2 protein is divided into series I compounds for targeting ubiquitination and degradation of TMPRSS2 protein and series II compounds for targeting ubiquitination and degradation of TMPRSS2 protein. The compounds of series I for targeting ubiquitination and degradation of TMPRSS2 protein are compounds of formula (I), and the compounds of series II for targeting ubiquitination and degradation of TMPRSS2 protein are compounds of formula (II).

[0028] The preparation method for Series I compounds targeting ubiquitination and degradation of TMPRSS2 proteins comprises the following steps: a conjugate of a linker with a terminal Boc-protected amino group and a CRBN or VHL ligand is prepared, followed by removal of the Boc protecting group in anhydrous dioxane / dichloromethane under acidic conditions, preferably at room temperature. The conjugate is then reacted with Boc-(4-aminophenyl)acetic acid in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) in anhydrous DMF to yield a CRBN or VHL ligand derivative containing an aniline structure and a terminal Boc-protected group, preferably at room temperature. The conjugate is then further removed in anhydrous dioxane / dichloromethane under acidic conditions, preferably at room temperature. The compounds are then reacted with di-Boc-protected guanidinobenzoic acid derivatives containing various substituents in the presence of HATU and DIPEA in anhydrous DMF to yield Boc-protected compounds that ubiquitinate and degrade TMPRSS2 protein. The reaction is preferably carried out at room temperature. Finally, the Boc protecting group is removed in anhydrous dioxane / dichloromethane under acidic conditions to yield Series I compounds that target ubiquitination and degradation of TMPRSS2 protein. The reaction is preferably carried out at room temperature.

[0029]

[0030] Synthetic route of series I representative compounds targeting ubiquitination degradation of TMPRSS2 protein.

[0031] The preparation method of a series II compound targeting ubiquitination and degradation of TMPRSS2 protein comprises the following steps: a conjugate of a terminal nitro-substituted linker and a VHL ligand is reacted in water and ethanol under reflux in the presence of iron powder and ammonium chloride to obtain a terminal amino-substituted VHL derivative; the reaction is preferably carried out at 80°C for 3 hours. The conjugate is then reacted with a bis-Boc-protected guanidinobenzoic acid derivative containing different substituents in anhydrous DMF under the presence of HATU and DIPEA to obtain a Boc-protected compound that ubiquitinates and degrades TMPRSS2 protein; the reaction is preferably carried out at room temperature for 16 hours. Finally, the conjugate is reacted in anhydrous dioxane / dichloromethane under acidic conditions to obtain a series II compound targeting ubiquitination and degradation of TMPRSS2 protein; the reaction is preferably carried out at room temperature for 24 hours.

[0032]

[0033] Synthetic route of series II representative compounds targeting ubiquitination degradation of TMPRSS2 protein.

[0034] Preferably, in the preparation method of the above-mentioned series I compound for targeting ubiquitination and degradation of TMPRSS2 protein, the amount ratio of the derivative of the VHL or CRBN ligand with an amino group substitution at the end, Boc-(4-aminophenyl)acetic acid, HATU and DIPEA is 1.0:1.2:1.2~1.5:4.0-6.0. The amount ratio of the derivative of the VHL or CRBN ligand with an amino group substitution at the end, the double Boc-protected guanidinobenzoic acid derivative, HATU and DIPEA is 1.0:1.2:1.2~1.5:4.0-6.0. Wherein, the substituents on the phenyl ring of the double Boc-protected guanidinobenzoic acid derivative containing different substituents are the same as R, R in the compound structure shown in general formula (I). 1 、R 2 Corresponding.

[0035] Preferably, in the preparation method of the above series II compound targeting ubiquitination degradation of TMPRSS2 protein, the amount ratio of the derivative of the VHL ligand with a nitro group at the end, iron powder and ammonium chloride is 1.0:10.0:10.0; the amount ratio of the VHL derivative with an amino group at the end, the double Boc-protected guanidinobenzoic acid derivative, HATU and DIPEA is 1.0:1.2:1.2~1.5:4.0-6.0. Wherein, the substituents on the benzene ring of the double Boc-protected guanidinobenzoic acid derivative containing different substituents are the same as R, R in the compound structure shown in general formula (II). 1 、R 2 Corresponding.

[0036] The beneficial effects achieved by the present invention are as follows: the compound of the present invention that targets ubiquitination degradation of TMPRSS2 protein can effectively degrade TMPRSS2, thereby exerting the activity of inhibiting tumor cell growth. It shows good inhibitory effects on various tumor cell lines including prostate cancer cells, and has low toxicity to normal cells. It can be used to prepare anti-tumor drugs and also has potential antiviral application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the test result of the present invention on the targeted degradation of TMPRSS2 protein by the synthesized compound in Calu-3 tumor cells;

[0038] Figure 2 This is the protein quantification result of the synthesized compound of the present invention for targeted degradation of TMPRSS2 protein in Calu-3 tumor cells;

[0039] Figure 3 These are the experimental results of detecting the colony-forming ability of the compound VPOT76 synthesized in the present invention in HT-29 tumor cells;

[0040] Figure 4 These are the experimental results of detecting the scratch healing ability of the compound VPOT76 synthesized in the present invention in HT-29 tumor cells. DETAILED DESCRIPTION

[0041] The features and advantages of the present invention can be further understood through the following detailed description. The examples provided are merely illustrative of the method of the present invention and are not intended to limit the remainder of the present invention in any way.

[0042] Example 1: Preparation of 4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoic acid

[0043]

[0044] Step 1: Synthesis of methyl 4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoate

[0045]

[0046] To a 50 mL single-necked flask, methyl p-aminobenzoate (6.67 mmol), 1,3-di-Boc-2-methylisothiourea (6.95 mmol), and silver nitrate (9.92 mmol) were added in sequence, followed by the addition of 18 mL of dichloromethane (DCM). The reaction was stirred at room temperature for 12 hours. After the reaction was completed, the filtrate was filtered off with diatomaceous earth and the filter cake was washed three times with DCM. An appropriate amount of water was then added to the filtrate, and the mixture was extracted three times with DCM. The organic phases were combined and washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, then filtered to obtain the organic phase, and the solvent was evaporated under reduced pressure using a rotary evaporator. The residue was separated by column chromatography (eluent: V 石油醚(PE) :V 乙酸乙酯(EA) =10:1) to give the intermediate 2G1 as a white solid in a yield of 76%. 1 H NMR (400 MHz, chloroform-d) δ 11.62 (s, 1H), 10.57 (s, 1H), 8.05-7.98 (m, 2H), 7.77-7.69 (m, 2H), 3.90 (s, 3H), 1.53 (d, J = 8.4 Hz, 18H).

[0047] Step 2: Synthesis of 4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoic acid

[0048]

[0049] To a 100 mL single-necked flask, intermediate 2G1 (3.56 mmol) and lithium hydroxide (17.79 mmol) were added in sequence, followed by 23 mL of tetrahydrofuran (THF) and 11 mL of water. The reaction system was heated to 50°C and stirred at this temperature for 5 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure on a rotary evaporator when the reaction system was cooled to room temperature. An appropriate amount of water was added, and the pH of the solution was adjusted to 6 with a 2 M dilute hydrochloric acid solution. The solution was then extracted three times with ethyl acetate (EA). The organic phases were combined and washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and then filtered to obtain the organic phase. The solvent was evaporated under reduced pressure on a rotary evaporator. The residue was separated by column chromatography (eluent: V DCM :V 甲醇(MeOH) =50:1) to give 168.00 mg of a white solid with a yield of 12%. 1 H NMR (400 MHz, chloroform-d) δ 11.60 (s, 1H), 10.60 (s, 1H), 8.11-8.03 (m, 2H), 7.77-7.73 (m, 2H), 1.54 (s, 18H).

[0050] Example 2: Preparation of 3-methoxy-4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoic acid

[0051]

[0052] The target compound was prepared by the method of Example 1, except that the methyl p-aminobenzoate in Example 1 was replaced with an equal molar amount of a substituted methyl p-aminobenzoate. All other conditions remained unchanged (the same below). The product was a white solid in a 55% yield. The substituted methyl p-aminobenzoate in Example 2 was methyl 3-methoxy-4-aminobenzoate.

[0053] 1 H NMR (400 MHz, chloroform-d) δ 11.59 (s, 1H), 10.97 (s, 1H), 8.68 (d, J = 8.5 Hz, 1H), 7.80 (dd, J = 8.6, 1.8 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 3.99 (s, 3H), 1.54 (s, 18H).

[0054] Example 3: Preparation of 2-fluoro-4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoic acid

[0055]

[0056] The target compound was prepared by the method of Reference Example 1. The product was a white solid with a yield of 25%.

[0057] 1 H NMR (400 MHz, chloroform-d) δ 11.54 (s, 1H), 10.67 (s, 1H), 7.97 (t, J = 8.4 Hz, 1H), 7.83 (dd, J = 13.1, 2.1 Hz, 1H), 7.31 (dd, J = 8.6, 2.1 Hz, 1H), 1.54 (s, 18H).

[0058] Example 4: Preparation of 2,5-difluoro-4-((2,2,10,10-tetramethyl-4,8-dioxo-3,9-dioxa-5,7-diazadecane-6-ylidene)amino)benzoic acid

[0059]

[0060] The target compound was prepared by the method of Reference Example 1. The product was a white solid with a yield of 15%.

[0061] 1 H NMR (400 MHz, chloroform-d) δ 8.50–8.35 (m, 1H), 7.68–7.56 (m, 1H), 1.47 (s, 18H).

[0062] Example 5: Preparation of N-(4-(2-((6-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)amino)-2-oxoethyl)phenyl)-4-guanidinobenzamide hydrochloride

[0063]

[0064] Step 1: Synthesis of 2-(4-aminophenyl)-N-(6-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)carbamate hydrochloride

[0065]

[0066] To a 10 mL single-necked flask, tert-butyl (6-(4-(2-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)carbamate (269.96 μmol) was added in sequence, followed by 2 mL of DCM. The reaction system was cooled to 0°C in an ice-ethanol bath, and 1 mL of dioxane hydrochloride solution (4 M) was slowly added dropwise with stirring. After the addition was complete, the ice bath was removed, the temperature was raised to room temperature, and the reaction was stirred overnight. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure on a rotary evaporator, a small amount of methanol was added, and the solvent was evaporated under reduced pressure on a rotary evaporator again. The above operation was repeated three times and then evaporated to dryness under reduced pressure using an oil pump. The resulting residue was stirred and slurried with a small amount of EA for 30 minutes, and then filtered under reduced pressure to obtain intermediate 1: de-tert-butyl (Boc)-protected tert-butyl (6-(4-(2-(dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)carbamate as a bright yellow solid in a yield of 98%.

[0067] Then, the intermediate 1 (206.36 μmol) prepared above and Boc-(4-aminophenyl)acetic acid (247.63 μmol) were added to a 10 mL single-necked flask in sequence, followed by 2 mL of anhydrous DMF. The reaction system was cooled to 0°C in an ice-ethanol bath, and DIPEA (825.44 μmol) was added under stirring. HATU (247.63 μmol) was then added under low temperature. After removing the ice bath, the temperature was raised to room temperature and stirred to react overnight. After TLC monitoring, 8 mL of water was added to the reaction solution, and then extracted four times with EA. The organic phases were combined and washed twice with saturated brine. The organic phases were dried over anhydrous sodium sulfate, then filtered to obtain the organic phase, and the solvent was evaporated under reduced pressure using a rotary evaporator. The residue was separated by column chromatography (eluent: V DCM :V MeOH =90:1-30:1) to give 126.00 mg of a light yellow solid with a yield of 88%. 1 H NMR (400 MHz, chloroform-d) δ 8.18 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 7.07 (dd, J = 8.6, 2.4 Hz, 1H), 6.62 (s, 14H), 5.63 (t, J = 5.8 Hz, 1H), 4.95 (dd, J = 12.2, 5.2 Hz, 1H). z,1H),3.83-3.76(m,2H),3.51(s,2H),3.49-3.40(m,4H),3.21(q,J=6.7Hz,2H),2.93-2.72(m ,3H),2.38-2.33(m,2H),2.17-2.11(m,1H),1.62-1.59(m,4H),1.51(s,9H),1.48-1.46(m,2H).

[0068] Finally, the above-mentioned operation of removing the Boc protecting group under acidic conditions was repeated to obtain 110.50 mg of 2-(4-aminophenyl)-N-(6-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)carbamate hydrochloride as a bright yellow solid in a yield of 98%.

[0069] Step 2: Synthesis of N-(4-(2-((6-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)amino)-2-oxoethyl)phenyl)-4-guanidinobenzamide hydrochloride

[0070]

[0071] The target compound was prepared by referring to the method of step 1 above (except replacing the corresponding starting materials, other conditions remained unchanged, the same below). The product was a yellow-green solid with a yield of 95%.

[0072] 1 H NMR (600MHz, DMSO-d6) δ11.07(s,1H),10.37(s,1H),10.26(s,1H),8.08-8.03(m,3H),7.74(s,3H),7.69(t,J=8.2Hz, 3H),7.35(d,J=8.2Hz,2H),7.32(d,J=2.1Hz,1H),7.22(d,J=8.2Hz,3H),5.07(dd,J=12.8,5.4Hz,1H),3.61-3.58(m, 4H),3.51-3.48(m,2H),3.46-3.43(m,2H),3.36(s,2H),3.06-3.00(m,2H),2.91-2.85(m,1H),2.62-2.57(m,1H),2.5 6-2.51(m,1H),2.32(t,J=7.4Hz,2H),2.04-2.00(m,1H),1.50(t,J=7.5Hz,2H),1.43-1.39(m,2H),1.30-1.24(m,4H). 13 C NMR (151 MHz, DMSO-d6) δ 172.82, 172.25, 170.89, 170.08, 167.54, 166.99, 164.36, 155.83, 154.87, 138.63, 137.38, 133.86, 132.00, 131.77, 129.23, 129.06, 124.95, 122.93, 120.45, 118.47, 117.78, 107.90, 62.82, 56.50, 48.81, 41.94, 38.49, 32.21, 31.00, 28.95, 26.13, 24.38, 22.20. HPLC: 96% purity.

[0073] Example 6: Preparation of N-(4-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)phenyl)-4-guanidino-3-methoxybenzamide hydrochloride

[0074]

[0075] The target compound was prepared by following the method of Example 5, except that the conjugate of the linker with a terminal Boc-protected amino group and the E3 ligand, and the di-Boc-protected guanidinobenzoic acid containing different substituents were replaced by equal molar amounts of other structural raw materials (the same below). The product was a yellow solid with a yield of 96%.

[0076] The structures of the linker and E3 ligand in the conjugate of the linker with a Boc-protected amino group at the end and the E3 ligand refer to the structures of the corresponding positions in the target compound. The substituents on the benzene ring of the double Boc-protected guanidinobenzoic acid containing different substituents correspond to R and R in the general formula (I) of the target compound. 1 、R 2 Corresponding.

[0077] 1 H NMR (400MHz, methanol-d4) δ7.76-7.63(m,4H),7.62-7.55(m,2H),7.52-7.36(m,2 H),7.35-7.25(m,2H),5.20-5.09(m,1H),4.65-4.36(m,2H),3.98(s,3H),3 .77-3.48(m,4H),3.28-3.18(m,2H),3.00-2.65(m,2H),2.36-2.28(m,1H), 2.26-2.11(m,1H),1.80-1.66(m,2H),1.63-1.54(m,2H),1.46-1.37(m,2H). 13 C NMR(101MHz,Methanol-d4)δ174.58,172.06,170.86,167.40,165.16,158.04, 155.23,138.44,136.46,136.16,134.49,133.80,133.18,130.47,130.02,129 .82,127.85,127.77,127.48,122.61,121.51,121.26,112.68,56.95,53.74,4 3.31,40.57,37.31,32.54,30.65,30.02,27.49,26.37,24.29.HRMS(ESI):m / z calcd for C 36 H 41 N8O7:697.3092[M+H] + ; found: 697.3075. HPLC: 96% purity.

[0078] Example 7: Preparation of N-(4-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-2-oxoethyl)phenyl)-4-guanidino-3-methoxybenzamide hydrochloride

[0079]

[0080] The target compound was prepared by referring to the method in Example 5. The product was a yellow-green solid with a yield of 96%.

[0081] 1 H NMR (400MHz, methanol-d4) δ7.76-7.53(m,5H),7.53-7.47(m,1H),7.40-7.34(m, 1H),7.33-7.28(m,2H),6.98(s,0H),5.06-5.02(m,1H),3.97(s,3H),3.54 -3.48(m,2H),3.27-3.17(m,4H),2.87-2.77(m,1H),2.76-2.63(m,2H),2. 14-2.03(m,1H),1.64-1.57(m,2H),1.55-1.48(m,2H),1.42-1.33(m,4H). 13 C NMR(101MHz,Methanol-d4)δ174.61,171.68,170.61,169.24,167.35,158.06,1 55.23,147.84,138.57,137.31,136.48,133.78,133.22,132.37,130.46,127.7 8,127.50,122.48,121.48,118.35,112.68,112.03,111.14,56.85,50.16,49.8 8,43.54,43.33,40.56,32.25,30.13,30.09,27.49,23.82.HRMS(ESI):m / zcalcd for C 36 H 41 N8O7:697.3092[M+H] + ; found: 697.3091. HPLC: 97% purity.

[0082] Example 8: Preparation of N-(4-(2-(4-((4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)propyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-oxoethyl)phenyl)-4-guanidinobenzamide hydrochloride

[0083]

[0084] The target compound was prepared by referring to the method in Example 5. The product was a yellow-green solid with a yield of 93%.

[0085] 1 H NMR (600MHz, DMSO-d6) δ11.05(s,1H),10.40-10.30(m,1H),10.28(s,1H),8.06(d,J=6.9Hz,2H),7.75-7.72(m,2H),7.68-7 .64(m,4H),7.58(d,J=8.0Hz,1H),7.36(d,J=7.7Hz,2H),7.20(d,J=7.9Hz,2H),7.00(s,1H),6.95-6.85(m,1H),5.37-5.25( m,1H),5.08-4.97(m,1H),4.36(d,J=13.2Hz,1H),3.99-3.92(m,1H),3.67(s,4H),3.31-3.25(m,4H),3.00(t,J=12.5Hz,1H) ,2.86(t,J=14.8Hz,1H),2.63-2.54(m,2H),2.04-1.95(m,5H),1.87-1.83(m,1H),1.63(t,J=7.2Hz,8H),1.54-1.41(m,2H). 13 C NMR(151MHz,DMSO-d6)δ172.77,170.10,168.66,167.62,167.11,166.92,16 4.35,155.76,154.14,138.61,137.36,131.66,131.49,129.60,129.18,128. 92,128.62,125.04,122.89,120.48,116.32,64.99,62.87,48.62,48.57,45 .01,35.12,31.24,28.98,28.53,26.51,25.07,22.21,22.04.HRMS(ESI):m / z calcd for C 42 H 51 N 10 O6:791.3987[M+H] + ; found:791.3983.HPLC:95% purity.

[0086] Example 9: Preparation of N-(4-(2-((4-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-2-oxoethyl)phenyl)amino)-2-oxoethyl)phenyl)-4-guanidino-3-methoxybenzamide hydrochloride

[0087]

[0088] The target compound was prepared by referring to the method in Example 5. The product was a yellow-green solid with a yield of 96%.

[0089] 1 H NMR (600MHz, DMSO-d6) δ11.08(s,1H),10.33(s,1H),10.24(s,1H),9.57(s,1H),7.98(t,J=6.0Hz,1H),7.75-7.73(m,2H),7.73-7.65( m,4H),7.58-7.53(m,3H),7.36(d,J=8.1Hz,2H),7.32(d,J=7.9Hz,2H),7.24(d,J=8.0Hz,1H),7.16(d,J=8.1Hz,2H),7.07(d,J=8.4Hz, 1H),7.01(d,J=7.0Hz,1H),5.04(dd,J=13.0,5.3Hz,1H),3.62(s,2H),3.40-3.34(m,3H),3.32(s,2H),3.26(t,J=7.2Hz,2H),3.03-2. 99(m,2H),2.88(t,J=15.8Hz,1H),2.58(d,J=17.2Hz,1H),2.04-2.00(m,1H),1.57-1.51(m,2H),1.41-1.36(m,2H),1.35-1.27(m,5H). 13CNMR(151MHz,DMSO-d6)δ172.81,172.38,170.10,170.05,169.09,168.96,167.32,164.33,156.24 ,153.33,146.43,137.60,137.46,137.26,136.30,134.40,132.20,132.02,131.59,131.36,129.24 ,129.17,129.11,126.43,126.23,120.70,120.63,120.42,119.05,117.18,111.77,110.39,109.03,62.81,56.09,48.60,42.70,41.86,41.78,38.54,30.99,29.00,28.63,26.10,26.00.HPLC: 95% purity.

[0090] Example 10: Preparation of N-(4-(2-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-oxoethyl)phenyl)-4-guanidino-3-methoxybenzamide hydrochloride

[0091]

[0092] The target compound was prepared by referring to the method in Example 5. The product was a yellow-green solid with a yield of 95%.

[0093] 1H NMR (600MHz, DMSO-d6) δ11.08(s,1H),10.40(s,1H),9.63(s,1H),7.77-7.73(m,4H),7.67(d,J=8.0Hz,1H),7.59-7.52( m,3H),7.46(s,1H),7.37-7.35(m,1H),7.35-7.32(m,1H),7.21(d,J=8.1Hz,2H),5.08(dd,J=12.8,5.4Hz,1H),4.36(d,J =12.7Hz,1H),4.15(d,J=13.4Hz,2H),3.99-3.96(m,1H),3.95(s,3H),3.64-3.48(m,6H),3.39(s,1H),3.13-3.06(m,2H) ,3.06-2.99(m,3H),2.91-2.85(m,1H),2.65-2.50(m,4H),2.16-2.07(m,1H),2.05-2.00(m,1H),1.87(d,J=12.0Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ172.80,170.02,168.71,167.41,166.90,164.37,15 6.29,154.13,153.31,137.31,134.36,133.83,131.38,128.96,126.40,126. 24,125.01,120.79,120.47,119.71,118.62,111.80,108.86,62.81,60.46,56.13,50.64,48.86,44.91,43.75,40.65,30.98,30.25,22.15. HPLC: 95% purity.

[0094] Example 11: Preparation of N-(4-(2-((6-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-6-oxohexyl)amino)-2-oxoethyl)phenyl)-4-guanidino-3-methoxybenzamide hydrochloride

[0095]

[0096] The target compound was prepared by referring to the method in Example 5. The product was a yellow-green solid with a yield of 96%.

[0097] 1H NMR (600MHz, DMSO-d6) δ11.08(s,1H),10.34(s,1H),9.60(s,1H),8.07(t,J=5.7Hz,1H),7.76-7.74(m,1H),7.72(d,J=8 .1Hz,2H),7.70-7.67(m,2H),7.58-7.51(m,3H),7.36(d,J=8.1Hz,1H),7.35-7.33(m,1H),7.26-7.22(m,3H),5.08(dd, J=12.8,5.3Hz,1H),3.94(s,3H),3.61(s,4H),3.48(d,J=31.4Hz,4H),3.38(s,2H),3.06-3.02(m,2H),2.93-2.85(m,1H ),2.62-2.52(m,2H),2.33(t,J=7.4Hz,2H),2.05-2.00(m,1H),1.54-1.48(m,2H),1.45-1.39(m,2H),1.31-1.27(m,2H). 13 C NMR(151MHz,DMSO-d6)δ172.78,170.84,170.05,170.01,167.51,166.96,164.2 8,156.26,154.85,153.29,137.26,134.35,133.84,132.12,129.03,126.37,126 .22,124.91,120.65,120.41,118.45,117.75,111.76,107.89,70.60,62.80,56.09,48.79,41.91,38.46,32.19,30.98,28.94,26.12,24.36,22.18. HPLC: 95% purity.

[0098] Example 12: Preparation of (2S,4R)-1-((S)-2-(6-(2-(4-(4-guanidino-3-methoxybenzamido)phenyl)acetamido)hexylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0099]

[0100] The target compound was prepared by referring to the method in Example 5. The product was a light yellow solid with a yield of 95%.

[0101] 1H NMR (400MHz, methanol-d4) δ9.79 (s, 1H), 7.64 (d, J = 25.6Hz, 4H), 7.47 (d, J = 41.8 Hz,5H),7.31(s,2H),4.64-4.35(m,5H),4.00(s,3H),3.84-3.75(m,2H),3 .64(d,J=15.3Hz,2H),3.49(s,1H),3.22-3.05(m,2H),2.64-2.48(m,3H), 2.34-2.19(m,2H),2.18-1.94(m,2H),1.57(d,J=36.4Hz,2H),1.29(s,9H). 13 C NMR(101MHz,Methanol-d4)δ174.76,172.88,169.72,167.61,165.35,158.05,156.5 4,155.36,142.18,138.48,136.72,130.79,130.66,130.52,129.36,128.44,127.92, 127.57, 122.54, 122.45, 121.52, 118.40, 112.70, 70.98, 60.82, 60.03, 59.03, 56.87, 43.51, 41.93, 39.29, 36.46, 35.79, 32.95, 30.71, 27.11, 23.66, 13.89. HPLC: 95% purity.

[0102] Example 13: Preparation of (2S,4R)-1-((S)-2-(6-(2-(4-(4-guanidino-3-methoxybenzamido)phenyl)acetamido)heptylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0103]

[0104] The target compound was prepared by referring to the method in Example 5. The product was a light yellow solid with a yield of 94%.

[0105] 1H NMR (600MHz, DMSO-d6) δ10.35(s,1H),9.63(s,1H),9.17(s,1H),8.60(t,J=6.1H z,1H),8.06(t,J=5.7Hz,1H),7.83(d,J=9.3Hz,1H),7.75(d,J=1.9Hz,1H),7.71 (d,J=8.2Hz,2H),7.67(d,J=8.1Hz,1H),7.59-7.53(m,3H),7.44-7.41(m,3H),7 .41-7.38(m,2H),7.36-7.34(m,1H),7.23(d,J=8.2Hz,2H),4.54-4.52(m,1H),4 .45-4.43(m,1H),4.42-4.40(m,1H),4.36-4.33(m,1H),4.22(dd,J=15.8,5.5Hz ,1H),3.94(s,3H),3.72-3.66(m,1H),3.66-3.60(m,2H),3.52-3.43(m,1H),3.3 7(s,2H),3.04-2.98(m,2H),2.46(d,J=4.0Hz,3H),2.27-2.22(m,1H),2.16-2.0 8(m,1H),2.06-2.01(m,1H),1.92-1.87(m,1H),1.57-1.29(m,6H),0.93(s,9H). 13 C NMR(151MHz,DMSO-d6)δ172.17,172.01,170.06,169.73,164.33,156.33,153.34,152. 21,146.54,139.89,137.28,134.40,132.13,131.87,129.40,129.05,128.68,127.51,1 26.42, 126.23, 120.70, 120.46, 111.81, 68.87, 62.83, 58.72, 56.34, 56.13, 41.89, 41.67, 38.67, 37.97, 35.20, 34.86, 29.01, 28.38, 26.41, 26.19, 25.41, 15.44. HPLC: 96% purity.

[0106] Example 14: Preparation of (2S,4R)-1-((S)-2-(6-(2-(4-(4-guanidino-3-methoxybenzamido)phenyl)acetamido)octylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0107]

[0108] The target compound was prepared by referring to the method in Example 5. The product was a light yellow solid with a yield of 94%.

[0109] 1 H NMR (600MHz, DMSO-d6) δ10.38(s,1H),9.68(s,1H),9.31(t,J=4.0Hz,1H),8.62(t ,J=6.1Hz,1H),8.09(t,J=5.5Hz,1H),7.85-7.80(m,1H),7.76(s,1H),7.72(d,J=8 .1Hz,2H),7.67(d,J=8.3Hz,1H),7.63-7.56(m,3H),7.44-7.39(m,4H),7.36-7.3 3(m,1H),7.24-7.20(m,2H),4.53(d,J=9.3Hz,1H),4.45-4.42(m,1H),4.42-4.39( m,1H),4.36-4.33(m,1H),4.22(dd,J=15.9,5.3Hz,1H),3.94(s,3H),3.73-3.66( m,1H),3.66-3.59(m,2H),3.53-3.42(m,1H),3.37(s,2H),3.04-2.98(m,2H),2.47 (d,J=4.0Hz,4H),2.28-2.20(m,1H),2.15-2.07(m,1H),2.07-2.01(m,1H),1.93-1 .86(m,1H),1.52-1.42(m,2H),1.40-1.34(m,2H),1.26-1.22(m,4H),0.92(s,9H). 13 C NMR (151MHz, DMSO-d6) δ172.20,172.03,170.09,169.74,164.34,156.40,153.36,152. 68,145.75,140.13,137.33,134.38,132.31,132.13,129.03,128.84,128.71,127.56,1 26.41, 126.24, 120.72, 120.50, 111.86, 68.87, 62.85, 58.73, 56.35, 56.16, 41.68, 38.67, 37.99, 35.22, 34.88, 29.09, 28.63, 28.49, 26.73, 26.43, 25.42, 15.11. HPLC: 96% purity.

[0110] Example 15: Preparation of (2S,4R)-1-((S)-2-(2-(4-((4-(4-guanidinobenzamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0111]

[0112] Step 1: Synthesis of (2S,4R)-1-((S)-2-(2-(4-((4-(4-aminophenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0113]

[0114] To a 100 mL single-necked flask were added (2S,4R)-1-((S)-2-(2-(4-((4-(4-nitrophenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (736.79 μmol), reduced iron powder (7.37 mmol), and ammonium chloride (7.37 mmol) in sequence, followed by 15 mL of water and 15 mL of anhydrous ethanol. The reaction system was replaced with nitrogen three times with stirring at room temperature, and then the reaction system was heated to reflux temperature (about 80°C) and stirred for 3 hours. After the reaction was completed by TLC monitoring, diatomaceous earth was added and filtered while hot, the filter cake was washed three times with ethanol, the filtrate was evaporated to remove the solvent under reduced pressure on a rotary evaporator, and EA was added to the residue for extraction three times. The organic phases were combined and washed once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and then filtered to obtain the organic phase, and the solvent was evaporated under reduced pressure on a rotary evaporator. The residue was separated by column chromatography (eluent: V DCM :V MeOH =20:1-10:1+0.01% NH3·H2O) to give 211.00 mg of a light yellow solid with a yield of 38%. 1H NMR (400MHz, methanol-d4) δ8.88(s,1H),7.50-7.46(m,2H),7.43(d,J=8.3Hz,2H),6.78(dd,J=51.5,8.3Hz,4H),4.63(s,1H ),4.61-4.58(m,1H),4.58-4.54(m,1H),4.52(s,1H),4.51-4.49(m,1H),4.37(d,J=15.5Hz,1H),3.92-3.86(m,1H),3. 80(dd,J=11.0,3.7Hz,1H),3.10-3.02(m,5H),2.91(t,J=12.3Hz,2H),2.74-2.62(m,4H),2.47(s,3H),2.42-2.33(m, 2H),2.31-2.24(m,2H),2.24-2.20(m,1H),1.82(t,J=15.6Hz,2H),1.71-1.62(m,0H),1.43-1.33(m,2H),1.05(s,9H).

[0115] Step 2: Synthesis of (2S,4R)-1-((S)-2-(2-(4-((4-(4-guanidinobenzamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0116]

[0117] The target compound was prepared by referring to the method in Example 5 (except replacing the corresponding starting materials, other conditions remained unchanged, the same below). The product was a light yellow solid with a yield of 96%.

[0118] 1H NMR(600MHz,DMSO-d6)δ11.35-11.13(m,1H),10.53(s,1H),10.24(s,1H),10.2 0-10.07(m,1H),9.16(s,1H),8.76(d,J=8.7Hz,1H),8.71-8.62(m,1H),8.06(d ,J=8.2Hz,2H),7.71(d,J=8.4Hz,2H),7.45-7.37(m,4H),7.34(d,J=8.3Hz,2H) ,7.04(d,J=8.6Hz,2H),4.57-4.55(m,1H),4.46-4.44(m,2H),4.44-4.42(m,2H ),4.41-4.40(m,2H),4.22-4.21(m,1H),4.05-3.98(m,2H),3.76-3.71(m,2H), 3.70-3.68(m,1H),3.64-3.58(m,3H),3.50-3.45(m,2H),3.37-3.34(m,1H),3. 29-3.25(m,1H),3.14-3.00(m,4H),2.46(s,3H),2.18-2.11(m,2H),2.09-2.05 (m,1H),2.04-1.94(m,1H),1.93-1.88(m,1H),1.68-1.54(m,2H),0.97(s,9H). 13 CNMR(151MHz,DMSO-d6)δ171.88,168.75,164.11,163.99,155.94,152.16,146. 66,145.47,139.84,138.52,132.44,131.79,130.97,129.18,128.69,128.06,1 27.51,122.82,121.71,116.42,72.18,70.55,68.86,62.83,60.19,58.84,57.13,50.98,45.57,41.67,38.01,35.41,28.22,27.27,26.34,15.50. HPLC: 95% purity.

[0119] Example 16: Preparation of (2S,4R)-1-((S)-2-(2-(4-((4-(4-(2-fluoro-4-guanidinobenzamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0120]

[0121] The target compound was prepared by referring to the method in Example 15. The product was a light yellow solid with a yield of 95%.

[0122] 1 H NMR(600MHz,DMSO-d6)δ11.29(s,1H),10.71(s,1H),10.24(s,1H),10.16(s,1H),9. 18(s,1H),8.76(d,J=8.8Hz,1H),8.67(d,J=5.9Hz,1H),7.95(s,2H),7.71(t,J=8.1 Hz,1H),7.63(d,J=8.0Hz,2H),7.41(q,J=7.8Hz,3H),7.22(d,J=11.3Hz,1H),7.16( d,J=8.9Hz,1H),7.03(d,J=8.5Hz,2H),4.57-4.52(m,3H),4.47-4.42(m,2H),4.42-4 .39(m,1H),4.38-4.35(m,1H),4.23(dd,J=16.2,5.6Hz,1H),4.10-3.93(m,2H),3.7 7-3.70(m,2H),3.70-3.68(m,1H),3.65-3.56(m,3H),3.50-3.44(m,2H),3.37-3.34 (m,1H),3.29-3.24(m,1H),3.14-2.99(m,4H),2.46(s,3H),2.19-2.10(m,2H),2.09 -2.05(m,1H),2.03-1.93(m,1H),1.92-1.87(m,1H),1.69-1.52(m,2H),0.97(s,9H). 13 C NMR(151MHz,DMSO-d6)δ171.83,168.69,163.94,161.60,155.79,152.19,146.51,1 45.57,139.82,139.30,132.09,131.80,131.08,129.08,128.65,127.47,121.55,1 21.03,116.47,110.78,110.61,68.81,62.79,59.71,58.79,57.08,56.50,51.81,5 0.93,45.48,41.63,37.96,35.35,28.18,27.24,26.30,15.42.HRMS(ESI):m / zcalcd for C 48 H 63FN 11 O5S:924.4712[M+H] + ; found: 924.4712. HPLC: 96% purity.

[0123] Example 17: Preparation of (2S,4R)-1-((S)-2-(2-(4-((4-(4-(2-(4-(4-guanidinobenzamido)phenyl)acetamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0124]

[0125] The target compound was prepared by referring to the method in Example 15. The product was a light yellow solid with a yield of 93%.

[0126] 1 H NMR(600MHz,DMSO-d6)δ11.23(s,1H),10.51(s,1H),10.30(s,1H),10.25(s,1H),10.13( s,1H),9.09(s,1H),8.76(d,J=8.8Hz,1H),8.65(t,J=6.0Hz,1H),8.07(d,J=8.2Hz,2H), 7.73(d,J=8.1Hz,2H),7.54(d,J=8.6Hz,2H),7.46-7.37(m,5H),7.35(d,J=8.3Hz,2H),7 .31(d,J=8.4Hz,2H),6.97(d,J=8.7Hz,2H),4.55(d,J=9.0Hz,1H),4.47-4.40(m,2H),4. 37(s,1H),4.23(dd,J=15.9,5.5Hz,1H),4.08-3.95(m,2H),3.70-3.66(m,3H),3.65-3.6 2(m,1H),3.61-3.56(m,4H),3.50-3.44(m,2H),3.36-3.30(m,2H),3.28-3.24(m,1H),3. 15-3.07(m,3H),3.07-3.02(m,2H),2.45(s,3H),2.21-2.14(m,1H),2.14-2.08(m,2H),2 .07-2.04(m,1H),2.03-1.94(m,1H),1.93-1.88(m,1H),1.68-1.51(m,2H),0.97(s,9H). 13CNMR(151MHz,DMSO-d6)δ171.83,168.78,168.70,164.35,163.96,155.92,151.88,147.06,13 9.69,138.63,137.56,135.79,131.69,131.58,131.52,129.56,129.34,129.23,129.20,128. 65,127.45,123.93,122.82,120.48,120.25,116.52,68.83,62.81,58.80,57.07,56.52,56.38,51.82,50.96,45.56,42.65,41.64,37.99,35.39,28.19,27.24,26.30,15.66. HPLC: 95% purity.

[0127] Example 18: Preparation of (2S,4R)-1-((S)-2-(2-(4-((4-(4-(2-(4-(4-guanidino-3-methoxybenzamido)phenyl)acetamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0128]

[0129] The target compound was prepared by referring to the method in Example 15. The product was a light yellow solid with a yield of 95%.

[0130] 11H NMR (600 MHz, DMSO-d6) δ 11.17 (s, 1H), 10.39 (s, 1H), 10.26 (s, 1H), 10.08 (s, 1H), 9.64 (s, 1H), 9.10 (s, 1H), 8.77 (d, J = 8.7 Hz, 1H), 8.66 (t, J = 6.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 3H), 7.67 (d, J = 9.9 Hz, 1H), 7.54 (d, J = 8.6 Hz, 3H), 7.43 - 7.38 (m, 4H), 7.36 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.56 (d, J = 9.0 Hz, 1H), 4.47 - 4.42 (m, 2H), 4.37 (s, 1H), 4.25 - 4.23 (m, 1H), 4.03 - 3.98 (m, 2H), 3.94 (s, 3H), 3.72 - 3.65 (m, 3H), 3.64 - 3.62 (m, 1H), 3.62 - 3.56 (m, 4H), 3.50 - 3.44 (m, 2H), 3.32 (t, J = 7.3 Hz, 2H), 3.28 - 3.23 (m, 1H), 3.16 - 3.08 (m, 3H), 3.07 - 3.03 (m, 2H), 2.45 (s, 3H), 2.21 - 2.14 (m, 1H), 2.14 - 2.08 (m, 2H), 2.06 (d, J = 8.5 Hz, 0H), 2.04 - 1.94 (m, 1H), 1.93 - 1.88 (m, 1H), 1.66 - 1.52 (m, 2H), 0.97 (s, 9H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.80, 168.76, 168.70, 164.31, 163.93, 156.32, 153.32, 151.82, 147.09, 145.14, 139.66, 137.43, 134.35, 132.68, 131.70, 131.49, 129.36, 129.18, 128.64, 127.45, 126.38, 126.22, 120.70, 120.43, 120.26, 116.50, 111.81, 68.82, 62.79, 58.79, 56.49, 56.39, 56.10, 51.82, 50.98, 45.54, 42.63, 41.64, 37.96, 35.37, 28.17, 27.23, 26.29, 15.64. HPLC: 96% purity.

[0131] Example 19: Preparation of (2S,4R)-1-((S)-2-(2-(4-((4-(4-(2-(4-(2-fluoro-4-guanidinobenzamido)phenyl)acetamido)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride

[0132]

[0133] The target compound was prepared by referring to the method in Example 15. The product was a light yellow solid with a yield of 95%.

[0134] 1 H NMR(600MHz,DMSO-d6)δ11.18(s,1H),10.66(s,1H),10.35(s,1H),10.25(s,1H),10.11(s, 1H),9.08(s,1H),8.76(d,J=8.7Hz,1H),8.66(t,J=3.9Hz,1H),7.97-7.91(m,1H),7.71(t, J=8.0Hz,1H),7.65(d,J=7.8Hz,2H),7.53(d,J=8.2Hz,2H),7.43-7.37(m,4H),7.31(d,J=7 .9Hz,2H),7.23(d,J=11.4Hz,1H),7.16(d,J=8.3Hz,1H),6.96(d,J=8.4Hz,2H),4.55(d,J= 8.9Hz,1H),4.46-4.41(m,2H),4.37(s,1H),4.25-4.22(m,1H),4.02-4.00(m,2H),3.71-3. 65(m,3H),3.64-3.62(m,1H),3.62-3.56(m,4H),3.50-3.44(m,2H),3.35-3.29(m,2H),3.2 7-3.24(m,1H),3.14-3.08(m,3H),3.07-3.01(m,2H),2.20-2.14(m,1H),2.14-2.08(m,2H) ,2.08-2.04(m,1H),2.04-1.95(m,1H),1.93-1.87(m,1H),1.65-1.53(m,2H),0.97(s,9H). 13C NMR(151MHz,DMSO-d6)δ171.77,168.66,163.89,161.90,160.11,158.45,155.76,151.75,147.14,145.1 5,139.33(d,J=10.8Hz),137.21,132.58,131.75,131.40,131.06,129.36,129.31,128.60,127.41,121.5 4(d,J=14.8Hz),120.23,119.83,118.80,116.43,110.80,110.63,68.78,62.77,59.70,58.76,57.05,56 .46,51.79,50.97,48.54,45.48,42.59,41.61,37.93,35.32,28.93,27.55,26.26,15.66.HRMS(ESI):m / z calcd for C 56 H 70 FN 12 O6S:1057.5240[M+H] + ; found: 1057.5245. HPLC: 96% purity.

[0135] Example 20: Biological Activity Test of Compounds Targeting Ubiquitination and Degradation of TMPRSS2 Protein

[0136] (1) Cytotoxicity test of compounds targeting ubiquitination degradation of TMPRSS2 protein

[0137] Experimental materials and methods: Calu-3 (human lung adenocarcinoma cells), HT-29 (colon cancer cells), MDA-MB-231 (human breast cancer cells), DU-145 (prostate cancer cells), A549 (human non-small cell lung cancer cells), AGS (human gastric adenocarcinoma cells), PC-3 (human prostate cancer cells), MRC-5 (human normal embryonic lung cells), HepG2 (liver cancer cells), SK-N-AS (human neuroblastoma cells), A375 (human melanoma cell line), 786-O (renal cancer cells), H1299 (human non-small cell lung cancer cells), SH-SY5Y (human neuroblastoma cells), K562 (human chronic myeloid leukemia cells), MCF-7 (human breast cancer cells), 22Rv1 (human prostate cancer cells), VCaP (human prostate cancer cells).

[0138] BPMI-1640 culture medium (Gibco), DMEM medium (Gibco), CCK-8 kit (Tongren Chemical), nafamostat mesylate (MCE), paclitaxel (MCE).

[0139] Principle: The CCK-8 kit is used for rapid and sensitive detection of cell proliferation and cytotoxicity. The working principle is: In the presence of an electron coupling reagent, WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt) is reduced by mitochondrial dehydrogenases to produce a highly water-soluble orange-yellow formazan product. The intensity of this color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. For the same cells, the color intensity and cell number are linearly correlated. The OD value is measured at a wavelength of 450nm using a microplate reader, which indirectly reflects the number of viable cells.

[0140] Tumor cells were plated at 1×10 4 Cells were seeded into a 96-well plate at a density of 10 μL / well and cultured at 37°C for 12 hours. The culture medium was aspirated and cell culture medium containing various concentration gradient compounds was added to each well. After 48 hours, 10 μL / well of CCK-8 reagent was added and the cell plate was cultured in a 37°C CO2 incubator for 0.5-2 hours. The OD value at a wavelength of 450 nm was then measured using a microplate reader. The half-maximal inhibitory concentration (IC) of the compound was then calculated. 50 ) was used as an indicator of the cytotoxicity of the compound.

[0141] (a) A series of PROTAC compounds were tested for their tumor suppressive activity against Calu-3 cell lines:

[0142] The present invention uses nafamostat mesylate and paclitaxel as positive control drugs to perform cytotoxicity test on Calu-3 tumor cells. The results are shown in Table 2 (IC 50 ,μM).

[0143] Table 2. Biological activity results of some compounds synthesized in this invention (IC 50 ,μM)

[0144] Compound <![CDATA[Calu-3(IC 50 ,μM)]]> LPOT4 >40 PPOT8 >40 PPOT16 >40 PPOT20 >40 PPOT21 >40 PPOT23 >40 PPOT25 >40 PPOT26 >40 VPOT6 >40 VPOT10 >40 VPOT14 >40 VPOT22 >40 VPOT48 >40 VPOT49 >40 VPOT50 >40 VPOT57 >40 VPOT58 >40 VPOT59 >40 VPOT62 >40 VPOT63 >40 VPOT64 21.37 VPOT65 >40 VPOT66 >40 VPOT67 >40 VPOT71 34.22 VPOT72 22.72 VPOT76 1.62 VPOT77 12.93 Paclitaxel 37.24 Nafamostat 37.87

[0145] Through CCK-8 experiment, with nafamostat mesylate and paclitaxel as positive control groups, a series of PROTAC molecular compounds were incubated with Calu-3 cells for 48 hours, and then CCK-8 detection reagent was added. The corresponding cell survival rate was calculated by detection and the corresponding IC 50 The results are shown in Table 2. Among a series of PROTAC molecules, compounds VPOT64, VPOT71, VPOT72, VPOT76 and VPOT77 showed significant anti-tumor activity, and the corresponding IC 50 The values ​​were 21.37 μM, 34.22 μM, 22.72 μM, 1.62 μM and 12.93 μM, respectively.

[0146] (b) Screening of tumor suppressor activity of PROTAC compounds VPOT64 and VPOT76 in multiple tumor cell lines:

[0147] The present invention uses nafamostat mesylate and paclitaxel as positive control drugs to conduct cytotoxicity tests on 18 tumor cell lines. The results are shown in Table 3 (IC 50 ,μM).

[0148] Table 3. Biological activity results of compounds VPOT64 and VPOT76 synthesized in the present invention (IC 50 ,μM)

[0149]

[0150] Note: ND (not detected); NSC (no significant change)

[0151] Through CCK-8 experiment, compound VPOT76 was co-incubated with tumor cells for 48 hours, and then CCK-8 detection reagent was added. The corresponding cell survival rate was calculated through detection, and the cell inhibition rate of the corresponding tumor cells was calculated under different concentration gradients to obtain the corresponding IC 50 The results are shown in Table 3. Among 18 tumor cell lines, compound VPOT76 was found to exhibit good tumor inhibitory activity, and it had the strongest tumor inhibitory activity in colon cancer HT-29 cells. The corresponding IC 50 The value was 0.39±0.01μM.

[0152] (2) Compound ubiquitination targeting degradation of TMPRSS2 protein activity test:

[0153] Cell line: Calu-3 cells (human lung adenocarcinoma cells)

[0154] The TMPRSS2 protein content was detected by Western Blot. When the cells were in good growth condition and the growth density reached 80%-90%, 3×10 6Seed cells / well into a 6-well plate and transfer the plate to a 37°C incubator for 24 hours. Aspirate the culture medium from the 6-well plate and rinse once or twice with PBS buffer. Add 10 μL / well of the corresponding compound solution to 2 mL of complete culture medium and mix thoroughly. For the control group, add 10 μL of DMSO to 2 mL of complete culture medium and mix thoroughly. Then, add the solution to the 6-well plate and incubate for 24 hours. After lysis with lysis buffer, centrifuge, aspirate the supernatant, and extract cellular proteins for later protein sample preparation. Prepare gel preparation tools for gel running. Incubate the protein bands with the corresponding TMPRSS2 and GAPDH primary and secondary antibodies for 2 hours. Rinse the protein bands with TBST detergent and expose the protein bands on a gel imaging system for detection. Quantify the protein bands using Image J software.

[0155] The present invention is to test the synthesis of compounds in Calu-3 tumor cells to target degradation of TMPRSS2 protein, some of the results are shown in Table 4, the specific results are as follows Figure 1 and Figure 2 All activity results were calculated and compared with the negative control group.

[0156] Table 4

[0157]

[0158]

[0159] Note: ND (not detected); NSC (no significant change)

[0160] Western Blot experiments were performed to detect the ability of a series of PROTAC molecules to target and degrade TMPRSS2 protein in the Calu-3 cell line. The compound concentration gradient was 0.01μM, 0.1μM, 0.5μM, 2.5μM, 10μM, and 20μM. The results are shown in Figure 2. Figure 1 , protein quantification results are as follows Figure 2 As shown in Table 4, some of the results indicate that compounds PPOT21, PPOT25, VPOT57, and VPOT76 have some degradation activity against TMPRSS2 in the Calu-3 cell line. At a concentration of 2.5 μM, compounds VPOT57 and PPOT25 exhibited degradation rates of 16.23% and 16.56%, respectively. Compound PPOT21 exhibited a degradation rate of 19.61% at a concentration of 0.5 μM. Compound VPOT76 exhibited the best degradation activity, achieving a degradation rate of 27.19% at a concentration of 2.5 μM.

[0161] (3) Cell characterization experiment on colon cancer HT-29 cell line using the compound VPOT76, which ubiquitinates and targets TMPRSS2 protein for degradation:

[0162] (a) Plate cloning assay to detect the ability of compounds to inhibit colony formation of HT-29 cancer cells:

[0163] HT-29 cells that are growing well and reaching a density of approximately 90% were digested and centrifuged. The supernatant was discarded, 3 mL of complete culture medium was added, and the cells were gently pipetted. 10 μL of the resuspended solution was collected. The cells were adsorbed onto a hemocytometer and counted under a microscope. 1000 cells were seeded into a 12-well plate and the plate was transferred to a 37°C incubator. After the cells adhered, the compound solution was added. For the negative control group, an equal volume of DMSO was added instead. After 24 hours of compound treatment, the culture medium was discarded, the cell surface was washed with PBS, and fresh complete culture medium was added. Culture was continued for 2-3 weeks, with the medium changed every 3 days, until cell clusters formed, at which point the culture was discontinued. The culture medium was discarded, the cell surface was washed with PBS, and 4% paraformaldehyde was added for 10 minutes, which was then discarded. Crystal violet was added for 20 minutes. After staining, the cells were washed 3-5 times with PBS until the background was clear and transparent, and then placed in a fume hood to air dry. The plate was inverted and photographed from the bottom against a white background.

[0164] The compound VPOT76 synthesized in the present invention was tested for its colony-forming ability in HT-29 tumor cells. The experimental results are as follows: Figure 3 shown.

[0165] The negative group was used as the control group, and the compound VPOT76 was treated with a concentration gradient of 0.1μM, 0.2μM and 0.4μM for 24h. Figure 3 The results showed that the proliferation ability of HT-29 cells was significantly reduced, and the number of tumor cell colonies decreased significantly with the increase of the concentration of compound VPOT76. The corresponding tumor proliferation inhibition rates at concentrations of 0.1μM, 0.2μM and 0.4μM were 18.66%, 29.10% and 48.72%, respectively.

[0166] (b) Wound healing assay to detect the effect of compound VPOT76 on wound healing of colon cancer cells HT-29:

[0167] HT-29 cells that are growing well and have reached a density of approximately 90% are digested and centrifuged. Discard the supernatant, add 3 mL of complete culture medium, and gently pipette the cell suspension. Take 10 μL of the resuspended solution. Adsorb it onto a hemocytometer and count it under a microscope. The cell number is 3 × 10 6Seed cells / well into a 12-well plate and transfer the plate to a 37°C incubator. Wait until the cells have grown to 80-90% of their volume before removing them and proceeding to the next step. Discard the original culture medium and rinse the cell surface with PBS. Strike a line at a 45-degree angle with a 10μL pipette tip, applying consistent pressure to ensure a straight line of uniform thickness across the bottom of the plate, ensuring it is clearly visible to the naked eye. Remove floating cell clumps with PBS and add 1mL of complete culture medium mixed with a specific concentration of compound. For the negative control group, replace the compound with an equal volume of DMSO and incubate the plate in a 37°C incubator. Take photos at 0, 24, and 48 hours after drug addition.

[0168] The compound VPOT76 synthesized in the present invention was tested for its ability to heal wounds in HT-29 tumor cells. The experimental results are as follows: Figure 4 shown.

[0169] The negative group was used as the control group, and the compound VPOT76 was treated with a concentration gradient of 0.2μM, 0.4μM and 0.8μM for 24h and 48h. Figure 4 As shown, the compound VPOT76 significantly inhibited HT-29 cell migration. At 24 and 48 hours, the inhibition rates were 22.11% and 38.40% at 0.2 μM, and 60.72% and 71.63% at 0.4 μM, respectively. When the compound concentration was increased to 0.8 μM, the inhibition rates reached 71.88% and 80.94%, respectively. These results demonstrate that the compound significantly inhibits tumor cell migration in a time- and concentration-dependent manner.

[0170] As shown in the tables and figures in this article, first, compound VPOT76 has been shown to have broad anti-tumor activity in anti-tumor experiments and can effectively inhibit tumor cell proliferation, with an effect superior to the control drugs paclitaxel and nafamostat mesylate. Secondly, in an in vitro ubiquitination-targeted degradation of TMPRSS2 experiment, it was detected that the PROTAC series compounds PPOT21, PPOT25, VPOT57, and VPOT76 can target and degrade TMPRSS2 protein in the Calu-3 cell model, thereby effectively inhibiting viral invasion of host cells and achieving an antiviral effect. In summary, compounds that target ubiquitination degradation of TMPRSS2 protein can be used to prepare anti-tumor drugs and potential antiviral drugs.

[0171] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound for targeting ubiquitination degradation of TMPRSS2 protein, characterized in that: The structure of the compound targeting ubiquitination degradation of TMPRSS2 protein is shown in one of the following: 、 、 、 。 2. A stereoisomer of the compound targeting ubiquitination and degradation of TMPRSS2 protein according to claim 1.

3. Use of the compound for targeting ubiquitination degradation of TMPRSS2 protein according to claim 1 or the stereoisomer according to claim 2 in the preparation of antiviral drugs or antitumor drugs, characterized in that: The tumor is one or more of: malignant melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, breast cancer, cervical cancer, lung cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer or multiple myeloma, lymphoma, leukemia.

4. An anti-tumor drug, characterized in that: Comprising the compound for targeting ubiquitination and degradation of TMPRSS2 protein according to claim 1 or the stereoisomer according to claim 2.

5. Use of the compound for targeting ubiquitination degradation of TMPRSS2 protein according to claim 1 or the stereoisomer according to claim 2 in the preparation of a drug for preventing or treating diseases associated with abnormal expression of TMPRSS2 protein activity.

Images