A bifunctional molecular compound that induces MMP-7 protein degradation and its preparation and application

Abstract

The present invention provides a bifunctional molecular compound that induces MMP-7 protein degradation or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein the structure of the compound is shown in Formula I or II: #imgabs0#Formula I; #imgabs1#Formula II. The present invention also provides a method for preparing the above-mentioned compound and a pharmaceutical composition. In addition, the present invention also provides the use of the above-mentioned compound, its pharmaceutically acceptable salt, hydrate, prodrug, or pharmaceutical composition in the preparation of a drug for treating biliary atresia. The present invention is supported by PROTAC technology and uses MMP-7 inhibitors as raw materials to synthesize bifunctional molecular compounds with different linker lengths that induce MMP-7 protein degradation; by using the bifunctional molecular compound after biliary atresia surgery, effective degradation of MMP-7 protein can be achieved, inflammatory bile duct damage and liver fibrosis progression can be inhibited, and a new treatment strategy for biliary atresia can be provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a bifunctional molecular compound for inducing MMP-7 protein degradation, and the preparation and application thereof. Background Art

[0002] MMP-7, also known as matrilysin, is a member of the matrix metalloproteinase (MMP) family. A significant structural difference from other MMP family members is the lack of a C-terminal heme domain, present in other MMPs and involved in substrate recognition and enzyme specificity. This characteristic of MMP-7 allows it to exhibit broad substrate specificity, primarily targeting elastin, laminin, fibronectin, type IV collagen, and other important components of the extracellular matrix and basement membrane. It can also cleave various bioactive extracellular non-matrix factors, such as Fas, tumor necrosis factor, and insulin-like growth factor-connexin-3. MMP-7 expression levels are typically low in adults and normal tissues, but it plays an important role in the pathophysiology of various diseases.

[0003] The role of MMP-7 in hepatobiliary diseases is receiving increasing attention. As a highly sensitive and specific biomarker, it facilitates the early diagnosis of biliary atresia. Furthermore, changes in its serum levels in children are closely correlated with the degree of liver fibrosis and postoperative prognosis, making it a non-invasive marker for assessing liver fibrosis and predicting autologous liver survival. However, studies have found that MMP7 may contribute to the pathogenesis and progression of biliary atresia by promoting inflammatory-induced bile duct epithelial cell damage and liver fibrosis, making it a potential therapeutic target. Specifically, degradation of MMP-7 after biliary atresia surgery could further inhibit inflammatory bile duct injury and liver fibrosis progression, providing a new therapeutic strategy for biliary atresia.

[0004] Since its introduction, PROTAC technology has been widely used to target a variety of disease targets. Using PROTAC to degrade various disease-related proteins has become a very promising therapeutic strategy. PROTAC molecules can address the drug resistance problem of small molecule inhibitors. Because PROTACs mainly play a catalytic role, the dosage concentration and frequency are low, which can reduce the possibility of toxic side effects and off-target effects.

[0005] Based on this, the present invention attempts to use PROTAC technology to design a compound that can effectively degrade MMP-7 protein, in order to provide a basis for the treatment of MMP7-related diseases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a bifunctional molecular compound that induces the degradation of MMP-7 protein, and its preparation and application. It is supported by PROTAC technology and uses MMP-7 inhibitors as raw materials to synthesize bifunctional molecular compounds with different linker lengths that induce the degradation of MMP-7 protein. By using this bifunctional molecular compound after biliary atresia surgery, the effective degradation of MMP-7 protein can be achieved, inflammatory bile duct damage and the progression of liver fibrosis can be inhibited, and a new treatment strategy for biliary atresia can be provided.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A bifunctional molecular compound that induces MMP-7 protein degradation, whose structural formula is shown in Formula I or II:

[0009] Formula I;

[0010] Formula II;

[0011] Wherein, B is the ubiquitin ligase E3 ligand;

[0012] L is one of the following structural formulas:

[0013] ;

[0014] Wherein, m is selected from an integer between 1 and 10.

[0015] As one of the preferred embodiments of the present invention, the ubiquitin ligase E3 ligand is selected from one of CRBN, VHL, MDM2, cIAP, UBR7, RNF114, CBLB, and KEAP1.

[0016] As one of the preferred embodiments of the present invention, the CRBN is one of the following structural formulas:

[0017] ;

[0018] Wherein, W is selected from one of CH2, C=O, SO2, NH, and N-alkyl; the alkyl is C1-C4 alkyl;

[0019] X is selected from one or both of O and S;

[0020] Z is selected from one of hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, and halogen;

[0021] G and G' are each independently selected from H, C1-C4 alkyl, -OH, and a 5-10 membered heterocyclic group substituted with C1-C4 alkyl; the heterocyclic group contains 1-3 heteroatoms of N, O or S;

[0022] R 1 One selected from H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, and deuterated C1-C4 alkyl;

[0023] The structural formula of the VHL is:

[0024] ;

[0025] Among them, R 2 One selected from CH3, H;

[0026] The structural formula of the MDM2 is:

[0027] ;

[0028] Among them, R 3 It is a piperazinyl group, a piperidinyl group, a heterocyclic group, or one of the following linking groups:

[0029] ;

[0030] In the above linking group, n is an integer from 0 to 3;

[0031] R 3 The heterocyclic group is one of piperazinyl, pyrrolyl, pyrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl;

[0032] The cIAP is one of the following structural formulas:

[0033] ;

[0034] Among them, R 4 is H or Boc.

[0035] As one of the preferred embodiments of the present invention, the compound represented by the following formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII or formula XIV is specifically selected:

[0036] Formula III;

[0037] Formula IV;

[0038] Formula V;

[0039] Formula VI;

[0040] Formula VII;

[0041] Formula VIII;

[0042] Formula IX;

[0043] Formula X;

[0044] Formula XI;

[0045] Formula XII;

[0046] Formula XIII;

[0047] Formula XIV;

[0048] In the above formulas III-XIV, L is one of the following structures:

[0049] ;

[0050] Wherein, m is selected from an integer between 1-7.

[0051] As one of the preferred embodiments of the present invention, the bifunctional molecular compound that induces MMP-7 protein degradation is specifically MMP7-T-1P-C, MMP7-T-3P-C, MMP7-T-5P-C, MMP7-T-3C-V1, MMP7-T-5C-V1, MMP7-T-7C-V1, MMP7-T-3C-V2, MMP7-T-5C-V2, MMP7-T-7C-V2, MMP7-T-1P-M, MMP7-T-3P-M, MMP7-T-5P-M, MMP7-T-3C-B4, MMP7-T-5C-B4, MMP7-T-7C-B4 ...7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7C-B4, MMP7-T-7 P7-T-1P-B5, MMP7-T-3P-B5, MMP7-T-5P-B5, MMP7-1P-C, MMP7-3P-C, MMP7-5P-C, MMP7-3C-V1, MMP7-5C-V1, MMP7-7C-V1, MMP7-3C-V2, MMP 7-5C-V2, MMP7-7C-V2, MMP7-1P-M, MMP7-3P-M, MMP7-5P-M, MMP7-3C-B4, MMP7-5C-B4, MMP7-7C-B4, MMP7-1P-B5, MMP7-3P-B5, or MMP7-5P-B5.

[0052] A method for preparing the above-mentioned bifunctional molecular compound that induces MMP-7 protein degradation:

[0053] (1) For the MMP-7 protein degrader shown in Formula I, L is or , when m is selected from an integer between 1 and 7, the synthetic route is as follows:

[0054] ;

[0055] The details are as follows:

[0056] Step 1-1: Synthesis of compound 3

[0057] 4-Chloro-3-trifluoromethylbenzenesulfonyl chloride (1.2 equiv) was dissolved in acetonitrile, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (2.2 equiv) and 1-tert-butyl-L-glutamic acid (1.0 equiv) were added sequentially. The mixture was allowed to react at room temperature for 14 hours. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to obtain compound 3.

[0058] Step 1-2: Synthesis of compound 5

[0059] Glycine methyl ester (1.2 equiv) was added to N,N-dimethylformamide (DMF), followed by HATU (1.2 equiv), compound 3 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 h. After completion, the reaction was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to yield compound 5.

[0060] Steps 1-3: Synthesis of compound 6

[0061] Compound 5 (1.0 equiv) was dissolved in methanol, and then NaOH (4 equiv) and H2O were added. After the reaction, the methanol was dried by rotary evaporation, and the pH of the aqueous solution was adjusted to 2 with 4 M HCl. The aqueous solution was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 6.

[0062] Steps 1-4: Synthesis of compound 8

[0063] Leucine methyl ester (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), compound 6 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 h. After completion, the product was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to afford compound 8.

[0064] Steps 1-5: Synthesis of compound 9

[0065] Compound 8 (1.0 equiv) was dissolved in methanol, and then NaOH (4 equiv) and H2O were added. After the reaction, the methanol was dried by rotary evaporation, and the pH of the aqueous solution was adjusted to 2 with 4 M HCl. The aqueous solution was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 9.

[0066] Steps 1-6: Synthesis of compound 12

[0067] Propargylamine (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), N-(tert-butyloxycarbonyl)-D-valine (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 h. After completion, the product was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to provide compound 12.

[0068] Steps 1-7: Synthesis of compound 13

[0069] Compound 12 (1.0 equiv) was added to DCM, followed by TFA (5.0 equiv) and allowed to react at room temperature for 2 h. After the reaction was complete, the reaction system was dried to afford compound 13.

[0070] Step 1-8: Synthesis of compound of formula 14

[0071] Compound 13 (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), compound 9 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 h. After completion, the reaction was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol gradient elution to obtain compound 14.

[0072] Step 1-9: Synthesis of compound of formula I

[0073] Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. HO was added, followed by CuSO (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, followed by extraction with HO and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to yield the compound of Formula I.

[0074] (2) For the MMP-7 protein degrader shown in Formula II, and L is or , when m is selected from an integer between 1 and 7, the synthetic route is as follows: ;

[0075] The details are as follows:

[0076] Step 2-1: Synthesis of compound 15

[0077] Compound 14 (1.0 equiv) was dissolved in dichloromethane, and then trifluoroacetic acid (4 equiv) was added. After the reaction was completed, the dichloromethane was dried to obtain compound 15;

[0078] Step 2-2: Synthesis of compound of formula II

[0079] Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. HO was added, followed by CuSO (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, followed by extraction with HO and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to yield the compound of Formula II.

[0080] In addition, the synthetic routes of ubiquitin ligase E3 ligand and L (linker) are as follows:

[0081] (1) When the ubiquitin ligase E3 ligand is CRBN, Tha (thalidomide derivative) is preferred, and the synthesis method is:

[0082] ;

[0083] Compound Tha (thalidomide derivative) was dissolved in DMF, and DIPEA and 1.2 equiv of Linker were added to the reaction system. The mixture was reacted at 90°C for 2 h, and then extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound Tha-L.

[0084] (2) When the ubiquitin ligase E3 ligand is MDM2, the synthesis method is:

[0085] ;

[0086] MDM2 was dissolved in DCM, and then 2 equiv of EDCI, 2 equiv of HOBt, and 4 equiv of DIPEA were added under ice-cooling, followed by 1.2 equiv of Linker. After the reaction was complete, DCM was added to dilute the reaction system, and the mixture was extracted with water and DCM. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound MDM2-L.

[0087] (3) When the ubiquitin ligase E3 ligand is VHL, the synthesis method is:

[0088] ;

[0089] Its synthesis method is the same as that of MDM2-L, except that MDM2 is replaced by VHL, and VHL-L is obtained accordingly;

[0090] (4) When the ubiquitin ligase E3 ligand is cIAP, its synthesis method is:

[0091] ;

[0092] Its synthesis method is the same as that of MDM2-L, except that VHL is replaced by cIAP, and cIAP-L is prepared accordingly.

[0093] A pharmaceutically acceptable salt, hydrate or prodrug of the above-mentioned bifunctional molecular compound that induces MMP-7 protein degradation.

[0094] As one of the preferred embodiments of the present invention, the pharmaceutically acceptable salts of the bifunctional molecular compound that induces MMP-7 protein degradation include addition salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc.

[0095] As one of the preferred aspects of the present invention, prodrugs of the derivatives of the present invention are also included; they themselves may have weak activity or even no activity, but after administration, they are converted into corresponding biologically active forms under physiological conditions (such as through metabolism, solvent decomposition or other means).

[0096] A pharmaceutical composition comprises the above-mentioned bifunctional molecular compound that induces MMP-7 protein degradation, or a pharmaceutically acceptable salt, hydrate, or prodrug of the compound; and also comprises a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or a combination thereof.

[0097] As one of the preferred embodiments of the present invention, the dosage form of the pharmaceutical composition is one of injection, tablet, and capsule.

[0098] A bifunctional molecular compound that induces MMP-7 protein degradation, or a pharmaceutically acceptable salt, hydrate or prodrug of the bifunctional molecular compound that induces MMP-7 protein degradation, or use of the pharmaceutical composition in the preparation of a drug for treating biliary atresia.

[0099] As one of the preferred embodiments of the present invention, the treatment of biliary atresia includes: early diagnosis of biliary atresia, prognosis assessment and resistance to bile duct injury.

[0100] The advantages of the present invention over the prior art are:

[0101] The present invention is supported by PROTAC technology and uses MMP-7 inhibitors as raw materials to synthesize bifunctional molecular compounds with different linker lengths that induce MMP-7 protein degradation.

[0102] The bifunctional molecular compound that induces MMP-7 protein degradation provided by the present invention effectively targets and degrades MMP-7 protein. Similar to a catalytic reaction, the drug has a low onset dose. It provides only binding activity, an event-driven approach that differs from traditional occupancy-driven approaches and does not require direct inhibition of the functional activity of the target protein. Furthermore, the drug does not require prolonged or high-intensity binding to the target protein. By using the bifunctional molecular compound that induces MMP-7 protein degradation after biliary atresia surgery, effective degradation of MMP-7 protein can be achieved, inhibiting inflammatory bile duct damage and the progression of liver fibrosis, providing a new therapeutic strategy for biliary atresia. (Currently, drug treatment for biliary atresia is unresolved, with a lack of effective drugs to slow or halt the progression of liver fibrosis and cirrhosis. While some drugs, such as hormones, antibiotics, and choleretics, have been used for postoperative treatment, their effectiveness and optimal treatment options remain controversial.)

[0103] The preparation method of the bifunctional molecular compound for inducing MMP-7 protein degradation provided by the present invention has the advantages of being simple, easy to operate, low in cost, and suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 This is the result of the inhibition rate of MMP7-T-5C-V1 at different concentrations on MMP7 in vitro;

[0105] Figure 2 is the jaundice rate of biliary atresia mice in the “positive model + compound group” and “positive model control group”;

[0106] Figure 3 Serum MMP7 concentration and liver MMP7 expression level in the "positive model + compound group" and "positive model control group" mice with biliary atresia (Figure a shows serum MMP7 concentration and b shows liver MMP7 expression level);

[0107] Figure 4 These are HE staining images of the extrahepatic bile ducts of biliary atresia mice in the "positive model + compound group", "positive model control group" and blank control mice (in the figure, Figure a is the extrahepatic bile duct of the "blank control group" mice, Figure b is the extrahepatic bile duct of the biliary atresia mice in the "positive model control group", and Figure c is the extrahepatic bile duct of the biliary atresia mice in the "positive model + compound group" treated with MMP7-T-5C-V1). DETAILED DESCRIPTION

[0108] The following examples of the present invention are described in detail. These examples are based on the technical solutions of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following examples. At the same time, the materials and reagents used in the following examples of the present invention are all commercially available unless otherwise specified. The experimental methods used are conventional methods unless otherwise specified and will not be described in detail.

[0109] Example 1. Synthesis of Intermediate Compound 14

[0110]

[0111] (1) Synthesis of compound 3:

[0112]

[0113] Specific operation procedure: 4-Chloro-3-trifluoromethylbenzenesulfonyl chloride (1.2 equiv) was dissolved in acetonitrile, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (2.2 equiv) and 1-tert-butyl-L-glutamic acid (1.0 equiv) were added sequentially. The reaction was allowed to proceed at room temperature for 14 hours. After completion, the reaction was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to obtain compound 3 as a yellow oil in 85% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 12.20 (s, 1H), 8.66 (s, 1H), 8.10 (d, J = 2.2 Hz,1H), 8.03 (dd, J = 8.4, 2.2 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 3.82 (s, 1H), 2.31– 2.20 (m, 2H), 1.88 – 1.82 (m, 1H), 1.70 – 1.62 (m, 1H), 1.17 (s, 9H).

[0114] (2) Synthesis of compound 5:

[0115]

[0116] Specific operation procedure: Glycine methyl ester (1.2 equiv) was added to N,N-dimethylformamide (DMF), followed by HATU (1.2 equiv), compound 3 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 hours. After completion of the reaction, the mixture was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to obtain compound 5 as a white solid in a 79% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 8.64 (s, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.04 (dd, J =8.4, 2.2 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 3.86 – 3.75 (m, 3H), 3.62 (s, 3H), 2.69 (s, 1H), 2.22 (t, J = 7.9 Hz, 2H), 1.87 (dtd, J = 16.0, 8.1, 7.5, 5.4 Hz,1H), 1.74 – 1.64 (m, 1H), 1.18 (s, 8H).

[0117] (3) Synthesis of compound 6:

[0118]

[0119] Specific operation process: Compound 5 (1.0 equiv) was dissolved in methanol, and then NaOH (4 equiv) and H2O were added. After the reaction, the methanol was spin-dried and the pH of the aqueous solution was adjusted to 2 with 4 M HCl. The aqueous solution was extracted with water and ethyl acetate; the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 6 as a white solid with a yield of 95%.

[0120] (4) Synthesis of compound 8:

[0121]

[0122] Specific operation procedure: Leucine methyl ester (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), compound 6 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 hours. After completion, the reaction was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to obtain compound 8 as a white solid in a 67% yield.

[0123] (5) Synthesis of compound 9:

[0124]

[0125] Specific operation process: Compound 8 (1.0 equiv) was dissolved in methanol, and then NaOH (4 equiv) and H2O were added. After the reaction, the methanol was spin-dried and the pH of the aqueous solution was adjusted to 2 with 4 M HCl. The aqueous solution was extracted with water and ethyl acetate; the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 9 as a white solid in a yield of 93%.

[0126] (6) Synthesis of compound 12:

[0127]

[0128] Specific operation procedure: Propargylamine (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), N-(tert-butyloxycarbonyl)-D-valine (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 hours. After completion, the reaction was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient elution to obtain compound 12 as a white solid in a yield of 78%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.29(t, J = 5.5 Hz, 1H), 6.66 (d, J = 9.0 Hz, 1H), 3.92 – 3.77 (m, 2H), 3.74 (dd, J =9.0, 7.2 Hz, 1H), 3.08 (t, J = 2.5 Hz, 1H),1.92 - 1.89 (m, 1H), 1.37 (s, 9H),0.82 (dd, J = 8.4, 6.7 Hz, 6H).

[0129] (7) Synthesis of compound 13:

[0130]

[0131] Specific operation process: Compound 12 (1.0 equiv) was added to DCM, followed by TFA (5.0 equiv), and the reaction was allowed to react at room temperature for 2 hours. After the reaction was complete, the reaction system was spin-dried to dryness to obtain compound 13 as a white solid in a yield of 99%.

[0132] (8) Synthesis of compound 14:

[0133]

[0134] Specific operation procedure: Compound 13 (1.2 equiv) was added to DMF, followed by HATU (1.2 equiv), compound 9 (1.0 equiv), and DIPEA (3.0 equiv). The reaction was allowed to react at room temperature for 6 hours. After completion of the reaction, the mixture was extracted with water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol gradient elution to obtain compound 14 as a pale yellow solid in a 65% yield.

[0135] Example 2: Synthesis of Intermediate Compound 15

[0136]

[0137] Compound 14 (1.0 equiv) of Example 1 was dissolved in dichloromethane, and trifluoroacetic acid (4 equiv) was added. After the reaction, the dichloromethane was dried to obtain compound 15 as a white solid. The yield was 95%.

[0138] Example 3. Synthesis of Compound MMP7-nP-C (n=1, 3, 5, taking n=1 as an example)

[0139]

[0140] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a yellow-green solid in a yield of 52%. 1 H NMR (600 MHz, DMSO-d 6) δ 11.10 (s, 1H), 8.67 (d, J = 7.6 Hz, 1H), 8.35 (t, J = 5.6 Hz,1H), 8.16 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 8.4, 2.2 Hz,1H), 7.98 (dd, J = 8.5, 5.0 Hz, 2H), 7.85 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.58(dd, J = 8.6, 7.1 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.1 Hz, 1H), 6.63– 6.58 (m, 1H), 5.07 (ddd, J = 12.8, 5.5, 1.2 Hz, 1H), 4.51 (t, J = 5.2 Hz, 2H),4.34 (dt, J = 14.5, 6.7 Hz, 2H), 4.25 (dd, J = 15.1, 5.4 Hz, 1H), 4.14 – 4.07 (m,1H), 3.82 (t, J = 5.2 Hz, 2H), 3.78 (d, J = 6.4 Hz, 1H),3.72 – 3.69 (m, 2H), 3.60(t, J = 5.5 Hz, 2H), 3.45 (q, J = 5.7 Hz, 2H), 2.89 (ddd, J = 17.8, 13.7, 5.4 Hz,1H), 2.62 – 2.56 (m, 1H), 2.53 (dd, J = 13.8, 4.9 Hz, 1H), 2.20 (tt, J = 15.3,7.1 Hz, 2H), 2.03 (dtd, J= 13.6, 6.5, 5.7, 2.6 Hz, 1H), 1.93 (q, J = 6.8 Hz,1H), 1.86 (ddt, J = 14.5, 9.1, 5.9 Hz, 1H), 1.74 – 1.64 (m, 1H), 1.55 (dq, J =12.9, 6.7 Hz, 1H), 1.47 – 1.43 (m, 2H), 1.17 (s, 9H), 0.84 (s, 3H), 0.81 (d, J = 6.5 Hz, 3H), 0.79 (dd, J = 6.9, 3.9 Hz, 6H).

[0141] Example 4. Synthesis of Compound MMP7-nC-V1 (n=3, 5, 7, taking n=5 as an example)

[0142]

[0143] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a pale yellow solid in a yield of 69%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.98 (s, 1H), 8.66 (s, 1H), 8.58 (t, J = 6.1 Hz, 1H), 8.37(t, J = 5.7 Hz, 1H), 8.16 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 8.4, 2.2 Hz, 1H), 7.98 (t, J = 9.1 Hz, 2H), 7.89 (d, J= 9.4 Hz, 1H), 7.84 (s,1H), 7.77 – 7.70 (m, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H),5.14 (d, J = 3.5 Hz, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.47 – 4.39 (m, 2H), 4.34(dt, J = 8.6, 4.3 Hz, 2H), 4.32 – 4.27 (m, 3H), 4.24 – 4.18 (m, 3H), 4.08 (dd, J = 8.8, 7.1 Hz, 1H), 3.78 (dd, J = 8.9, 5.4 Hz, 1H), 3.69 (d, J = 5.8 Hz, 1H),3.67 (d, J = 5.7 Hz, 1H), 3.66 – 3.60 (m, 2H), 2.44 (s, 3H), 2.28 – 2.15 (m,3H), 2.10 (ddd, J = 14.4, 8.1, 6.3 Hz, 1H), 2.06 – 2.00 (m, 1H), 1.95 – 1.91(m, 1H), 1.90 (d, J = 4.3 Hz, 1H), 1.88 – 1.83 (m, 1H), 1.81 – 1.73 (m, 1H),1.72 – 1.66 (m, 1H), 1.66 – 1.60 (m, 1H), 1.56 (dt, J = 14.1, 7.1 Hz, 1H), 1.52– 1.46 (m, 2H), 1.43 (dt, J = 9.0, 4.7 Hz, 2H), 1.40 – 1.34 (m, 1H), 1.29 (s,1H), 1.25 (s, 1H), 1.23 (d, J = 5.6 Hz, 4H), 1.16 (s, 9H), 0.92 (s, 9H), 0.87 –0.84 (m, 3H), 0.82 (d, J = 6.5 Hz, 3H), 0.79 (dd,J = 6.8, 5.1 Hz, 6H).

[0144] Example 5. Synthesis of Compound MMP7-nC-V2 (n=3, 5, 7, taking n=3 as an example)

[0145]

[0146] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 63.7%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.98 (d, J = 3.6 Hz, 1H), 8.66 (s, 1H), 8.48 (d, J = 7.6 Hz,1H), 8.42 – 8.34 (m, 1H), 8.19 – 8.15 (m, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.07 –8.02 (m, 1H), 8.00 (d, J = 3.0 Hz, 1H), 7.98 (d, J = 5.1 Hz, 1H), 7.95 (d, J = 17.6Hz, 1H), 7.87 (s, 1H), 7.78 – 7.73 (m, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.41 –7.33 (m, 2H), 5.16 – 5.10 (m, 1H), 4.89 – 4.86 (m, 1H), 4.52 (dd, J = 9.4, 5.9Hz, 1H), 4.48 (s, 1H), 4.37 – 4.33 (m, 2H), 4.33 – 4.29 (m, 3H), 4.27 (d, J =5.3 Hz, 1H), 4.09 (td, J= 8.2, 7.7, 2.0 Hz, 1H), 3.78 (dd, J = 9.0, 5.4 Hz, 1H),3.69 (tt, J = 12.4, 6.1 Hz, 2H), 3.61 (d, J = 4.8 Hz, 2H), 2.45 (d, J = 7.0 Hz,3H), 2.27 (dt, J = 15.0, 7.6 Hz, 1H), 2.21 (q, J = 7.5 Hz, 2H), 2.16 (dd, J = 14.5,7.5 Hz, 1H), 2.00 (ddd, J = 17.2, 11.4, 7.2 Hz, 3H), 1.94 – 1.91 (m, 1H), 1.88– 1.82 (m, 1H), 1.69 (dt, J = 14.5, 7.4 Hz, 1H), 1.56 (dt, J = 13.6, 6.6 Hz, 1H),1.43 (q, J = 9.0, 6.5 Hz, 2H), 1.38 (d, J = 7.3 Hz, 3H), 1.37 – 1.33 (m, 1H), 1.29 (s, 1H), 1.25 (d, J = 1.3 Hz, 1H), 1.24 (d, J = 8.9 Hz, 2H), 1.16 (s, 9H), 0.93 (s, 2H), 0.91 (d, J = 7.3 Hz, 1H), 0.88 (s, 6H), 0.85 (dd, J = 6.6, 2.1 Hz,3H), 0.82 (dd, J = 6.7, 1.8 Hz, 3H), 0.79 (dd, J = 6.8, 3.9 Hz, 6H).

[0147] Example 6. Synthesis of Compound MMP7-T-nP-M (n=1, 3, 5, taking n=1 as an example)

[0148]

[0149] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 69%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.67 (d, J = 9.3 Hz, 1H), 8.38 (t, J = 5.7 Hz, 1H), 8.16 (t, J =5.8 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 8.4, 2.2 Hz, 1H), 7.98 (t, J =8.8 Hz, 2H), 7.93 (t, J = 5.7 Hz, 1H), 7.83 (s, 1H), 7.76 – 7.70 (m, 1H), 7.53(d, J = 8.6 Hz, 1H), 7.18 – 7.13 (m, 2H), 7.13 – 7.09 (m, 2H), 7.04 (d, J = 8.4Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 6.61 (d, J = 7.4 Hz, 2H), 5.65 (d, J = 9.8 Hz,1H), 5.58 (d, J = 9.7 Hz, 1H), 4.72 (p, J = 6.0 Hz, 1H), 4.47 (t, J = 5.2 Hz, 2H),4.34 (ddd, J = 12.2, 9.1, 5.6 Hz, 2H), 4.28 – 4.19 (m, 1H), 4.14 – 4.06 (m,1H), 3.83 (s, 3H), 3.74 (q, J= 5.5 Hz, 2H), 3.69 (dd, J = 12.5, 6.7 Hz, 2H),3.59 (d, J = 17.5 Hz, 1H), 3.37 (t, J = 6.0 Hz, 2H), 3.23 – 3.13 (m, 4H), 2.99(s, 2H), 2.26 – 2.16 (m, 2H), 2.02 – 1.91 (m, 2H), 1.90 – 1.81 (m, 1H), 1.73– 1.60 (m, 1H), 1.59 – 1.55 (m, 1H), 1.43 (dt, J = 9.0, 4.8 Hz, 2H), 1.29 (s,1H), 1.26 (d, J = 6.0 Hz, 2H), 1.23 (s, 3H), 1.21 (d, J = 6.0 Hz, 3H), 1.16 (s,9H), 0.85 (dd, J = 6.7, 3.1 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H), 0.79 (dd, J = 6.8,4.7 Hz, 6H).

[0150] Example 7. Synthesis of Compound MMP7-nC-B4 (n=3, 5, 7, taking n=3 as an example)

[0151] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 50.3%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.66 (s, 1H), 8.36 (t, J = 5.6 Hz, 1H), 8.16 (t, J = 5.8 Hz, 1H),8.11 (d, J= 2.2 Hz, 1H), 8.04 (dd, J = 8.4, 2.2 Hz, 1H), 7.98 (t, J = 8.5 Hz, 2H),7.85 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.73 (dd, J = 16.1, 7.3 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.26 (t, J = 7.5 Hz, 2H), 7.23 – 7.19 (m, 2H), 7.19 – 7.14 (m,1H), 6.19 (d, J = 6.1 Hz, 1H), 4.37 – 4.32 (m, 2H), 4.32 – 4.28 (m, 1H), 4.25(dd, J = 14.8, 5.1 Hz, 1H), 4.21 (d, J = 6.6 Hz, 1H), 4.17 (td, J = 7.0, 3.6 Hz,2H), 4.09 (dd, J = 8.8, 7.0 Hz, 1H), 3.87 (dd, J = 6.1, 2.7 Hz, 1H), 3.78 (dd, J =8.9, 5.4 Hz, 1H), 3.74 – 3.63 (m, 2H), 3.60 (s, 3H), 2.81 (dd, J = 13.4, 7.0Hz, 1H), 2.65 (dd, J = 13.4, 8.0 Hz, 1H), 2.20 (tq, J = 15.6, 8.0, 6.7 Hz, 2H),2.05 (dq, J = 13.7, 6.9, 6.2 Hz, 1H), 2.01 (s, 1H), 1.94 (q, J = 6.9 Hz, 1H),1.87 (dt, J= 13.9, 6.8 Hz, 2H), 1.69 (m, 1H), 1.66 – 1.59 (m, 1H), 1.58 – 1.49(m, 2H), 1.44 (m, 3H), 1.30 (s, 1H), 1.26 (s, 1H), 1.24 (d, J = 8.9 Hz, 3H), 1.16 (s, 9H), 0.85 (d, J = 6.6 Hz, 3H), 0.82 (dd, J = 6.5, 3.2 Hz, 6H), 0.79 (dd, J = 6.8, 4.6 Hz, 6H), 0.76 (d, J = 6.5 Hz, 3H).

[0152] Example 8. Synthesis of Compound MMP7-nP-B5 (n=1, 3, 5, taking n=1 as an example)

[0153]

[0154] Specific operation procedure: Compound 14 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 73%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.67 (d, J = 8.4 Hz, 1H), 8.38 (t, J = 5.7 Hz, 1H), 8.16 (t, J =5.8 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 8.09 – 8.02 (m, 2H), 7.98 (dd, J = 8.5,5.9 Hz, 2H), 7.83 (s, 1H), 7.76 – 7.71 (m, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.27(t, J= 7.5 Hz, 2H), 7.23 – 7.15 (m, 3H), 6.19 (d, J = 9.4 Hz, 1H), 5.97 (d, J =6.2 Hz, 1H), 4.47 (t, J = 5.2 Hz, 2H), 4.38 – 4.30 (m, 3H), 4.28 – 4.20 (m,1H), 4.14 – 4.08 (m, 1H), 3.95 (dtd, J = 10.0, 7.5, 2.5 Hz, 1H), 3.82 – 3.80(m, 1H), 3.78 (d, J = 5.5 Hz, 1H), 3.74 (t, J = 5.3 Hz, 2H), 3.72 – 3.62 (m, 2H), 3.38 (t, J = 6.0 Hz, 2H), 2.78 (dd, J = 13.3, 7.3 Hz, 1H), 2.66 (dd, J = 13.3, 7.7Hz, 1H), 2.27 – 2.15 (m, 2H), 1.93 (p, J = 6.9 Hz, 1H), 1.86 (ddd, J = 14.4, 8.7,5.8 Hz, 1H), 1.74 – 1.61 (m, 1H), 1.55 (dp, J = 14.2, 6.8 Hz, 2H), 1.49 – 1.42(m, 3H), 1.41 – 1.36 (m, 1H), 1.28 (s, 9H), 1.17 (s, 9H), 0.86 (dd, J = 6.7,1.3 Hz, 6H), 0.83 – 0.81 (m, 6H), 0.80 (s, 6H).

[0155] Example 9. Synthesis of Compound MMP7-T-nP-C (n=1, 3, 5, taking n=1 as an example)

[0156] Specific operation procedure: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a yellow-green solid in a yield of 71%. 1 H NMR (600 MHz, DMSO- d 6) δ 11.11 (s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 8.16 (d, J = 2.2 Hz, 1H),8.09 (dd, J = 19.1, 7.2 Hz, 2H), 7.90 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.57(dd, J = 8.6, 7.1 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 7.1 Hz, 1H), 6.60(t, J = 6.0 Hz, 1H), 5.07 (dd, J = 12.8, 5.5 Hz, 1H), 4.51 (t, J = 5.3 Hz, 2H),4.34 (dt, J = 16.8, 8.4 Hz, 2H), 4.24 (dd, J = 15.1, 5.5 Hz, 1H), 4.05 (d, J = 9.6Hz, 1H), 3.82 (t, J = 5.3 Hz, 2H), 3.71 (dd, J = 16.8, 6.1 Hz, 1H), 3.59 (t, J =5.6 Hz, 2H), 3.50 (d, J = 10.6 Hz, 0H), 3.44 (q, J = 5.7 Hz, 2H), 2.89 (ddd, J=16.8, 13.8, 5.4 Hz, 1H), 2.61 – 2.55 (m, 1H), 2.55 – 2.51 (m, 1H), 2.24 (dt, J = 14.9, 7.4 Hz, 1H), 2.11 (d, J = 25.7 Hz, 1H), 2.05 – 2.01 (m, 1H), 1.98 (dd, J = 12.9, 6.8 Hz, 1H), 1.94 – 1.91 (m, 1H), 1.83 (s, 1H), 1.55 (dd, J = 12.5, 7.0Hz, 2H), 1.47 – 1.41 (m, 1H), 1.40 – 1.32 (m, 1H), 1.31 – 1.27 (m, 1H), 1.26(s, 1H), 1.25 – 1.20 (m, 9H), 0.85 (d, J = 5.9 Hz, 4H), 0.81 (d, J = 6.4 Hz, 3H),0.75 (d, J = 6.8 Hz, 3H), 0.72 (d, J = 6.3 Hz, 3H).

[0157] Example 10. Synthesis of Compound MMP7-T-nC-V1 (n=3, 5, 7, taking n=5 as an example)

[0158]

[0159] Specific operation procedure: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 69%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.98 (s, 1H), 8.61 (td, J = 6.2, 2.5 Hz, 1H), 8.47 (t, J= 5.5Hz, 1H), 8.33 (s, 3H), 8.17 (d, J = 2.2 Hz, 1H), 8.09 (ddd, J = 10.2, 6.7, 3.7Hz, 2H), 7.90 (d, J = 8.8 Hz, 2H), 7.85 (s, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.38(d, J = 8.2 Hz, 2H), 5.18 (s, 1H), 4.53 (d, J = 9.4 Hz, 1H), 4.46 – 4.40 (m, 2H), 4.36 – 4.33 (m, 1H), 4.35 – 4.30 (m, 2H), 4.22 (ddd, J = 13.9, 9.9, 7.5 Hz,2H), 4.07 – 4.01 (m, 1H), 3.73 – 3.69 (m, 1H), 3.68 – 3.61 (m, 2H), 3.60 –3.52 (m, 1H), 2.44 (s, 3H), 2.24 (dd, J = 15.3, 8.5 Hz, 2H), 2.17 – 2.07 (m,2H), 2.06 – 2.01 (m, 1H), 1.97 – 1.88 (m, 2H), 1.84 (t, J = 6.6 Hz, 0H), 1.81–1.77 (m, 2H), 1.56 (dq, J = 12.0, 6.0, 5.5 Hz, 2H),1.52 – 1.49 (m, 1H), 1.46 –1.37 (m, 1H), 1.34 – 1.24 (m, 1H), 1.24 (s, 1H), 1.21 – 1.19 (m, 2H), 0.92(s, 11H), 0.85(d, J = 6.2 Hz, 4H), 0.81 (d, J = 6.4 Hz, 4H), 0.77 – 0.70 (m,7H).

[0160] Example 11. Synthesis of Compound MMP7-T-nC-V2 (n=3, 5, 7, taking n=3 as an example)

[0161]

[0162] Specific operation procedure: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in an 83% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 8.98 (d, J = 3.7 Hz, 1H), 8.49 (d, J = 7.5 Hz, 1H), 8.40 (d, J =6.0 Hz, 1H), 8.16 – 8.11 (m, 2H), 8.06 – 8.00 (m, 3H), 7.93 (d, J = 8.5 Hz,1H), 7.87 (s, 2H), 7.54 – 7.49 (m, 2H), 7.43 – 7.37 (m, 1H), 7.36 – 7.32 (m,2H), 5.16 (s, 1H), 4.89 – 4.85 (m, 1H), 4.52 (dd, J = 9.3, 6.0 Hz, 1H), 4.48(t, J = 8.0 Hz, 1H), 4.36 (s, 1H), 4.33 – 4.29 (m, 3H), 4.29 – 4.25 (m, 1H), 4.08 (dd, J = 8.9, 7.1 Hz, 1H), 3.78 (s, 1H), 3.69 (td, J = 19.1, 17.9, 4.9 Hz,1H), 3.65 – 3.57 (m, 2H), 2.45 (d, J = 7.2 Hz, 3H), 2.26 (ddd, J = 14.9, 8.2, 6.6Hz, 1H), 2.23 – 2.13 (m, 3H), 1.98 (td, J= 14.2, 6.8 Hz, 3H), 1.95 – 1.85 (m,3H), 1.74 (dt, J = 14.7, 7.6 Hz, 1H), 1.55 (ddt, J = 15.3, 12.9, 6.5 Hz, 1H),1.48 (d, J = 14.3 Hz, 2H), 1.37 (t, J = 8.0 Hz, 3H), 1.27 – 1.21 (m, 2H), 0.93(s, 1H), 0.91 (t, J = 7.5 Hz, 1H), 0.88 (s, 7H), 0.85 (dd, J = 6.7, 2.1 Hz, 3H),0.81 (dd, J = 6.6, 2.0 Hz, 3H), 0.78 (dd, J = 6.7, 2.5 Hz, 6H).

[0163] Example 12. Synthesis of Compound MMP7-T-nP-M (n=1, 3, 5, taking n=1 as an example)

[0164]

[0165] Specific operation procedure: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a 70% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 12.87 (s, 1H), 8.59 (dd, J = 9.0, 1.8 Hz, 1H), 8.41 (t, J =5.8 Hz, 1H), 8.15 (t, J = 5.8 Hz, 1H), 8.13 (d, J = 2.2 Hz, 1H), 8.03 (dd, J= 8.5,2.1 Hz, 1H), 7.99 (t, J = 5.7 Hz, 2H), 7.94 (dd, J = 8.5, 2.3 Hz, 1H), 7.84 (d, J =1.7 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 7.9Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 8.0 Hz,2H), 6.69 (s, 2H), 5.89 (s, 1H), 5.78 (d, J = 22.6 Hz, 1H), 4.80 (s, 1H), 4.47(t, J = 5.3 Hz, 2H), 4.34 (dt, J = 15.6, 5.6 Hz, 2H), 4.25 (dd, J = 15.1, 5.5 Hz,1H), 4.09 (dd, J = 8.9, 7.1 Hz, 1H), 3.86 – 3.82 (m, 4H), 3.75 (t, J = 5.4 Hz,2H), 3.73 – 3.61 (m, 2H), 3.51 – 3.48 (m, 1H), 3.38 (t, J = 5.9 Hz, 2H), 3.01(s, 1H), 2.19 (m, 2H), 1.98 (dq, J = 21.5, 6.4, 5.4 Hz, 2H), 1.94 – 1.87 (m,2H), 1.74 – 1.65 (m, 1H), 1.55 (q, J = 7.2, 6.7 Hz, 1H), 1.46 – 1.41 (m, 2H),1.29 (d, J = 5.9 Hz, 4H), 1.25 (s, 2H), 1.23 (s, 6H), 0.84 (dd, J= 7.1, 1.7 Hz,3H), 0.81 (d, J = 6.5 Hz, 3H), 0.80 – 0.77 (m, 6H).

[0166] Example 13. Synthesis of Compound MMP7-T-nC-B4 (n=3, 5, 7, taking n=3 as an example)

[0167]

[0168] Specific operation procedure: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF. H₂O was added, followed by CuSO₄ (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and the mixture was extracted with H₂O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a pale yellow solid in a yield of 71%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.47 (t, J = 5.7 Hz, 1H), 8.27 (s, 1H), 8.24 – 8.21 (m, 1H), 8.17 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4Hz, 1H), 7.87 (s, 1H), 7.73 (d, J = 9.0 Hz, 1H), 7.32 (t, J = 7.4 Hz, 2H), 7.28(d, J = 7.4 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 6.33 (s, 1H), 4.41 (d, J = 5.5 Hz,1H), 4.40 – 4.35 (m, 2H), 4.34 – 4.26 (m, 2H), 4.23 (tt, J = 8.9, 4.6 Hz, 2H),4.11 (t, J = 8.1 Hz, 1H), 3.92 (d, J= 2.7 Hz, 1H), 3.76 (dd, J = 16.8, 6.1 Hz,1H), 3.66 (s, 3H), 3.64 (s, 1H), 2.87 (dd, J = 13.4, 7.0 Hz, 1H), 2.71 (dd, J =13.3, 7.9 Hz, 1H), 2.25 (ddt, J = 48.4, 15.0, 7.3 Hz, 2H), 2.11 (dt, J = 14.9,7.4 Hz, 1H), 2.06 – 2.01 (m, 1H), 1.99 – 1.95 (m, 2H), 1.91 (dt, J = 14.0, 7.1Hz, 2H), 1.68 (ddd, J = 13.1, 10.3, 4.7 Hz, 1H), 1.62 (d, J = 6.4 Hz, 1H), 1.61 –1.55 (m, 2H), 1.50 (ddt, J = 14.1, 10.1, 5.2 Hz, 2H), 1.30 (d, J = 4.4 Hz, 1H),0.97 (s, 1H), 0.91 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.5 Hz, 6H), 0.86 – 0.73 (m,9H).

[0169] Example 14. Synthesis of Compound MMP7-T-nP-B5 (n=1, 3, 5, taking n=1 as an example)

[0170] Specific operation process: Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF, and H2O was added, followed by CuSO4 (1.2 equiv) and sodium ascorbate (3.0 equiv). The mixture was stirred at room temperature until the reaction was complete. The THF was removed by concentration under reduced pressure, and then extracted with H2O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the title compound as a white solid in a yield of 72%. 1H NMR (600 MHz, DMSO- d 6) δ 8.43 (t,J = 5.7 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.20 (t, J = 5.7Hz, 1H), 8.16 (d, J = 2.1 Hz, 2H), 8.11 (t, J = 5.5 Hz, 1H), 8.06 (d, J = 8.5 Hz,1H), 7.97 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 7.32 (t, J =7.5 Hz, 2H), 7.25 (dd, J = 7.9, 2.4 Hz, 3H), 4.48 (t, J = 5.3 Hz, 2H), 4.34 (dt, J = 12.0, 6.0 Hz, 2H), 4.29 – 4.23 (m, 2H), 4.06 (dd, J = 9.0, 7.1 Hz, 1H), 3.90(d, J = 3.2 Hz, 1H), 3.78 (t, J = 5.3 Hz, 2H), 3.70 (dd, J = 16.8, 6.0 Hz, 1H),3.58 (dd, J = 16.8, 5.5 Hz, 2H), 3.51 (d, J = 4.0 Hz, 2H), 3.49 (s, 14H), 3.36(d, J = 6.0 Hz, 3H), 3.24 – 3.11 (m, 2H), 2.87 (dd, J = 13.6, 7.5 Hz, 1H), 2.71(dd, J = 13.6, 6.8 Hz, 1H), 2.23 (dt, J = 14.7, 7.4 Hz, 1H), 2.14 (dt, J = 14.6,7.1 Hz, 1H), 1.92 (dt, J = 13.8, 6.9 Hz, 1H), 1.85 (q,J = 6.9 Hz, 2H), 1.61 –1.52 (m, 3H), 1.52 – 1.48 (m, 1H), 1.47 – 1.41 (m, 2H), 1.27 – 1.22 (m, 2H), 0.89 – 0.83 (m, 9H), 0.82 (d, J = 6.4 Hz, 3H), 0.76 (dd, J = 14.4, 6.7 Hz, 6H).

[0171] Experimental Example 1

[0172] This experimental example is used to verify the application effect of the bifunctional molecular compound of the present invention for inducing MMP-7 protein degradation (taking the compound MMP7-T-5C-V1 of Example 4 as an example).

[0173] 1. Degradation effect on MMP-7 and half-inhibitory concentration:

[0174] (1) Experimental methods

[0175] Compound pretreatment: Prepare dilutions of the compound MMP7-T-5C-V1 at concentrations ranging from 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. For the control group, add an equal volume of DMSO instead of the compound.

[0176] Reaction system preparation: Prepare a reaction system to a final volume of 100 μL per well containing the following: 50 μL MMP-7 enzyme solution (final concentration 0.5 μg / mL), 10 μL MMP7-T-5C-V1 dilution (or DMSO control), and 40 μL substrate solution. Add enzyme reaction buffer to a final volume of 150 μL. Mix thoroughly and add to a 96-well black plate.

[0177] Start the reaction: Incubate the reaction system at 37°C, protected from light, for 2 hours. Detect the fluorescence signal in real time or at the endpoint using a fluorescence detector (e.g., excitation wavelength 320 nm, emission wavelength 400 nm).

[0178] (2) Experimental results

[0179] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the compound MMP7-T-5C-V1 can effectively inhibit MMP7, and the half-inhibitory concentration was measured to be 2.966uM.

[0180] 2. Effect on jaundice rate in mice with biliary atresia:

[0181] (1) Experimental methods

[0182] Animal model: A biliary atresia mouse model was established by intraperitoneal injection of 20 μl of rhesus rotavirus into Balb / c newborn mice within 24 hours of birth.

[0183] Experimental groups: blank control group (normal newborn mice without intervention), positive model control group (mice were induced with rhesus monkey rotavirus on Day 1, and DMSO solvent was intraperitoneally administered on Days 1, 3, 5, and 7), positive model + compound group (mice were induced with rhesus monkey rotavirus on Day 1, and 10 μl of compound MMP7-T-5C-V1 was intraperitoneally administered on Days 1, 3, 5, and 7);

[0184] Jaundice assessment: Jaundice was defined as the presence of yellowing in the skin, sclera, auricle, or limbs of mice in each group.

[0185] Jaundice rate = (total number of mice / number of mice with jaundice) × 100%

[0186] (2) Experimental results

[0187] The results are as follows Figure 2 As shown. Figure 2 It can be seen that compared with the "positive model control group", the jaundice rate of mice in the "positive model + compound group" was reduced and the onset of jaundice was delayed.

[0188] 3. Effect on the degradation of MMP7 in serum and liver of mice with biliary atresia:

[0189] (1) Experimental methods

[0190] Animal model: A biliary atresia mouse model was established by intraperitoneal injection of 20 μl of rhesus rotavirus into Balb / c newborn mice within 24 hours of birth.

[0191] Experimental groups: blank control group (normal newborn mice without intervention), positive model control group (mice were induced with rhesus monkey rotavirus on Day 1, and DMSO solvent was intraperitoneally administered on Days 1, 3, 5, and 7), positive model + compound group (mice were induced with rhesus monkey rotavirus on Day 1, and 10 μl of compound MMP7-T-5C-V1 was intraperitoneally administered on Days 1, 3, 5, and 7);

[0192] Sample collection and analysis: Blood and liver tissue samples were collected from each group of mice on Day 7 and Day 14 after birth. The level of MMP7 in serum was detected by ELISA, and the expression of MMP7 in the liver was detected by qPCR.

[0193] (2) Experimental results

[0194] The results are as follows Figure 3 As shown. Figure 3It can be seen that compared with the "positive model control group", the expression level of MMP7 in the serum and liver tissue of mice in the "positive model + compound group" was reduced.

[0195] 4. Effects on bile duct patency in mice with biliary atresia:

[0196] (1) Experimental methods

[0197] Animal model: A biliary atresia mouse model was established by intraperitoneal injection of 20 μl of rhesus rotavirus into Balb / c newborn mice within 24 hours of birth.

[0198] Experimental groups: blank control group (normal newborn mice without intervention), positive model control group (mice were induced with rhesus monkey rotavirus on Day 1, and DMSO solvent was intraperitoneally administered on Days 1, 3, 5, and 7), positive model + compound group (mice were induced with rhesus monkey rotavirus on Day 1, and 10 μl of compound MMP7-T-5C-V1 was intraperitoneally administered on Days 1, 3, 5, and 7);

[0199] Specimen collection and analysis: Extrahepatic bile duct specimens were collected from mice on Day 7 and Day 14 after birth and stained with hematoxylin and eosin (H&E) to observe the bile duct structure and patency.

[0200] (2) Experimental results

[0201] The results are as follows Figure 4 As shown. Figure 4 It can be seen that compared with the "positive model control group", the degree of bile duct obstruction in mice in the "positive model + compound group" was alleviated and the patency was better.

[0202] In summary, the bifunctional molecular compound that induces MMP-7 protein degradation provided by the present invention can effectively degrade MMP7. In the biliary atresia mouse model, the compound can reduce the jaundice rate of the model mice, delay the onset of jaundice, effectively reduce the MMP7 levels in the serum and liver of the model mice, and improve the patency of the bile duct in biliary atresia mice.

[0203] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bifunctional molecular compound that induces MMP-7 protein degradation, characterized in that: Its structural formula is shown in Formula I or II: Formula I; Formula II.

2. A pharmaceutically acceptable salt, hydrate or prodrug of the bifunctional molecular compound that induces MMP-7 protein degradation according to claim 1.

3. A pharmaceutical composition, characterized in that The invention comprises the bifunctional molecular compound for inducing MMP-7 protein degradation as claimed in claim 1, or a pharmaceutically acceptable salt, hydrate, or prodrug of the compound; and further comprises a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or a combination thereof.

4. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition is one of injection, tablet and capsule.

5. Use of the bifunctional molecular compound that induces MMP-7 protein degradation according to claim 1, or the pharmaceutically acceptable salt, hydrate or prodrug of the bifunctional molecular compound that induces MMP-7 protein degradation according to claim 2, or the pharmaceutical composition according to any one of claims 3-4 in the preparation of a drug for treating biliary atresia.

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