Preparation method and application of DDB1-E3 ligase ligand of sulfonamide skeleton and derivatives thereof

Abstract

The application discloses a preparation method and application of a DDB1-E3 ligase ligand with a sulfonamide skeleton and a derivative thereof, and belongs to the technical field of medicinal chemistry and targeted protein degradation. The novel E3 ligase ligand with the sulfonamide skeleton is shown as formula I: formula I, wherein X is any one of CH2, O and S; Y is C or N; R is any one of H, F and CF3; and n is an integer of 0-3. The derivative is shown as formula III: formula III, wherein Linker is any chemically feasible connecting structure, and POI ligand is a small molecule with binding affinity to a target protein. The preparation process is simple and easy to implement, the prepared proteolysis targeting chimera or pharmaceutically acceptable salt thereof has the effect of efficiently targeting and degrading EGFR, and can inhibit the proliferation of tumor cells, thereby exerting high-efficiency antitumor activity, and is suitable for development of an antitumor drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and targeted protein degradation technology, and specifically relates to a novel E3 ligase ligand containing a sulfonamide backbone and its derivatives, as well as the application of a protein degrading agent (PROTAC) constructed based on this ligand in disease treatment. Background Technology

[0002] Targeted protein degradation (TPD) technology has developed rapidly in recent years. It utilizes the ubiquitin-proteasome system to selectively degrade pathogenic proteins, and has advantages over traditional inhibitors such as sustained action and the ability to target "undruggable" proteins.

[0003] Proteolytic target chimeras (PROTACs), which induce the degradation of target proteins through bifunctional small molecules, represent one of the most exciting new drug development paradigms in recent years. PROTACs consist of a target protein ligand, an E3 ubiquitin ligase ligand, and a suitable linker, forming a "target protein-PROTAC-E3 ligase" ternary complex that degrades the target protein via the ubiquitin-proteasome system (UPS). Compared to traditional small molecule inhibitors, PROTACs offer unique advantages such as catalytic properties, reduced dosage and frequency of administration, more effective and longer-lasting effects, increased selectivity, reduced potential toxicity, overcoming drug resistance, and expanding the target space. The design of PROTACs requires known target protein ligands and E3 ligase ligands as protein baits, and their development depends on the discovery and optimization of these ligands. The lack of available E3 ligase ligands and the low drugability of existing major E3 ligase ligands limit the development of PROTACs.

[0004] Currently, the most commonly used E3 ligases include CRBN, VHL, IAP, and MDM2. For example, the invention patent with publication number CN109952299A discloses a series of fluorinated VHL ligands and corresponding PROTAC molecules (such as compounds 18d and 18e). The introduction of fluorine atoms can regulate the binding affinity and conformational rigidity between the ligand and VHL, providing a new approach for optimizing the performance of PROTACs. Another example is the invention patent with publication number CN119707950A, which discloses a PROTAC molecule (IPT) targeting the immunosuppressive protein IDO using thalidomide (CRBN ligand) as the E3 ligand ligand. Its degradation activity and anti-proliferative effect on IDO were verified in SKOV3 ovarian cancer cells. However, existing E3 ligands still have the following problems: limited ligand types, restricted tissue expression, easy induction of drug resistance, high toxicity, and insufficient modifiable sites.

[0005] Therefore, developing E3 ligase ligands with novel backbones, easy derivatization, and good pharmacokinetic and cellular activity is of great significance for expanding the field of targeted protein degradation. Summary of the Invention

[0006] This invention addresses the technical problems of existing E3 ligands, such as limited types, restricted tissue expression, easy development of drug resistance, high toxicity, and insufficient modifiable sites. It proposes a method for preparing DDB1-E3 ligands with a sulfonamide backbone and their derivatives, as well as their applications.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a DDB1-E3 ligase ligand with a sulfonamide backbone, the structural formula of which is shown in Formula I:

[0009] Formula I;

[0010] Where X is one of CH2, O, and S; Y is one of C and N; R is one of H, F, and CF3; and n is an integer from 0 to 3.

[0011] The structural formula of the DDB1-E3 ligase ligand is shown in further formula II:

[0012] Formula II.

[0013] This invention provides a protein degrading agent derivative, the structural formula of which is shown in Formula III:

[0014] Formula III;

[0015] Wherein, Linker can be any chemically feasible linking structure, preferably, Linker is a saturated fatty acid chain, an unsaturated fatty acid chain or a fatty acid chain.

[0016] POI ligands are small molecules with binding affinity to target proteins, including but not limited to: EGFR ligand, BRD4 ligand, KRAS ligand, CDK ligand, AR ligand, SHP2 ligand, MEK ligand, and PD-L1 ligand;

[0017] X is one of CH2, O, and S; Y is one of C and N; R is one of H, F, and CF3; and n is an integer from 0 to 3.

[0018] Preferably, the Linker is selected from , , , , , any one of them;

[0019] POI ligands are selected from , , , , Any one of them;

[0020] X is preferably either O or S;

[0021] Y is preferably N;

[0022] R is preferably any one of F and CF3;

[0023] n is preferably an integer between 1 and 2.

[0024] The structural formula of the protein degrading agent derivative is further shown in Formula IV:

[0025] Formula IV;

[0026] Where m is a positive integer from 1 to 6.

[0027] Preferably, the molecular structural formula of the protein degrading agent derivative is as follows:

[0028] .

[0029] Secondly, the present invention provides a method for preparing the protein degrading agent derivatives (compounds 1-6), the synthetic route of which is as follows:

[0030] ;

[0031] Includes the following steps:

[0032] (1) Compound S1, compound S2, and triethylamine were dissolved in dichloromethane and stirred under nitrogen protection to carry out a condensation reaction. After the reaction was completed, the mixture was concentrated and purified to obtain compound A, which is the DDB1-E3 ligase ligand of the sulfonamide skeleton described in this invention. The structural formula of compound S1 is as follows: The structural formula of compound S2 is as follows: ;

[0033] (2) Compound A, compound S3, and potassium carbonate obtained in step (1) were dissolved in DMF and subjected to a substitution reaction under nitrogen protection with stirring. After filtration, concentration, and purification, intermediate Z1 was obtained. The structural formula of compound A is as follows: The structural formula of compound S3 is as follows: ;

[0034] (3) Compound S4, alkynyl bromide, and potassium carbonate were dissolved in DMF, and a substitution reaction was carried out under nitrogen protection with stirring and heating. After cooling, the mixture was filtered, concentrated, and purified to obtain intermediate Z2. The structural formula of compound S4 is: The bromine compound is selected from any one of the following ω-bromo-1-yne: 4-bromo-1-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, 7-bromo-1-heptyne, 8-bromo-1-octyne, and 9-bromo-1-nonyne.

[0035] (4) After dissolving intermediate Z1 obtained in step (2) and intermediate Z2 obtained in step (3) in an organic solvent, copper sulfate and sodium ascorbate are added sequentially, and the reaction is carried out under nitrogen protection with stirring. After the reaction is completed, the protein degradation agent derivative is obtained by concentration and purification. The structural formula of intermediate Z1 is as follows: .

[0036] In step (1), the molar ratio of compound S1, compound S2 and triethylamine is 1:0.8-1.2:2-5, the concentration of dichloromethane is 0.1-0.5M, and the reaction time is 0.5-6h.

[0037] In step (2), the molar ratio of compound A, compound S3 and potassium carbonate is 1:0.95-1.5:2-3, the concentration of DMF is 0.2-0.5M, and the reaction time is 8-24 h.

[0038] In step (3), the molar ratio of compound S4, alkynyl bromide compound and potassium carbonate is 1:1-2:2-3, the concentration of DMF is 0.2-0.5M, the heating temperature is 40-100℃, and the time is 12-24 h.

[0039] In step (4), the molar ratio of intermediate Z1, intermediate Z2, copper sulfate and sodium ascorbate is 1:0.8-1.2:0.05-0.3:0.05-0.5. The organic solvent is a mixture of tert-butanol and water in a volume ratio of 1:1. The concentration of tert-butanol is 0.05-0.2M. The reaction time is 12-24h.

[0040] Thirdly, the present invention also provides the use of the protein degrading agent derivatives thereof or pharmaceutically acceptable salts thereof in the preparation of treatments or preventives of cancer.

[0041] The cancers include non-small cell lung cancer, head and neck squamous cell carcinoma, glioblastoma, colorectal cancer, pancreatic cancer, breast cancer, gastric cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, liver cancer, and melanoma, with non-small cell lung cancer being the preferred option.

[0042] The present invention provides a pharmaceutical composition comprising the aforementioned protein degrading agent derivative and pharmaceutically acceptable excipients.

[0043] The beneficial effects of this invention are:

[0044] Compound A of this invention is a high-affinity ligand for the E3 ligase DDB1. The preparation process of protein degrading agent derivatives based on compound A is simple and easy. The prepared protein degrading agent derivatives or their pharmaceutically acceptable salts exhibit highly efficient targeted degradation of EGFR with high selectivity. Compound 6 can effectively induce EGFR degradation in the non-small cell lung cancer cell line H1975 in a dose-dependent manner, demonstrating considerable bioavailability and excellent in vivo anti-non-small cell lung cancer effects. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 The MST diagram shows the binding affinity of compound A prepared in Example 1 to the E3 ligase DDB1.

[0047] Figure 2 Compounds 1-6 prepared for Example 2 degraded EGFR protein in the non-small cell lung cancer H1975 cell line.

[0048] Figure 3 Compound 6, prepared in Example 2, degrades EGFR protein in a concentration-dependent manner in the non-small cell lung cancer H1975 cell line.

[0049] Figure 4 The results show the antitumor efficacy of compound 6 prepared in Example 2 in mice. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] A method for preparing a DDB1-E3 ligase ligand (compound A) with a sulfonamide backbone, the synthetic route is as follows:

[0053] ;

[0054] The specific steps are as follows: 4-Amino-2-(aminomethyl)thiazole (S1) (1.0 eq), 2-fluoropyridine-5-sulfonyl chloride (S2) (0.9 eq), and triethylamine (TEA, 2.0 eq) were dissolved in dichloromethane (DCM) (concentration approximately 0.25 M), and the mixture was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (eluting with a gradient of petroleum ether / ethyl acetate = 3:1) to obtain a white solid compound A, whose structural formula is [insert structural formula here]. .

[0055] The characterization and detection results of compound A are as follows:

[0056] 1 H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.17 (ddd,J = 8.8, 4.9, 1.8 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.19 (dd, J = 8.9, 8.0Hz, 1H), 7.10 (s, 1H), 6.51 (d, J = 7.1 Hz, 1H), 6.31 (d, J = 7.1 Hz, 1H), 4.21 (d, J = 7.1 Hz, 2H).

[0057] HRMS (ESI) calculated for C9H 10 FN4O2S2 + [M+H] + : 289.0224, found.289.0229.

[0058] Example 2

[0059] A method for preparing a protein degrading agent derivative (compounds 1-6), the synthetic route is as follows:

[0060] ;

[0061] The specific steps are as follows:

[0062] (1) Preparation of compound A: 4-amino-2-(aminomethyl)thiazole (S1) (1.0 eq), 2-fluoropyridine-5-sulfonyl chloride (S2) (0.9 eq) and triethylamine (TEA, 2.0 eq) were dissolved in dichloromethane (DCM) (concentration approximately 0.25 M) and stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (eluting agent: petroleum ether / ethyl acetate = 3:1 gradient elution) to obtain white solid compound A.

[0063] (2) Preparation of intermediate Z1: Compound A (1.0 eq), ethyl azide p-toluenesulfonate (S3) (1.2 eq), and potassium carbonate (K2CO3, 2.5 eq) were added to N,N-dimethylformamide (DMF) (concentration approximately 0.2 M), and the mixture was stirred at room temperature for 12 hours under nitrogen protection. After the reaction solution was cooled to room temperature, the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4:1) to obtain a pale yellow solid intermediate Z1, the structural formula of which is [insert structural formula here]. .

[0064] The compound Z1 was characterized, and the detection results are as follows:

[0065] HRMS (ESI) calculated for C 11 H 13 FN7O2S2 + [M+H] + : 358.0551, found.358.0554.

[0066] (3) Preparation of intermediate Z2: 7-(4-chloro-3-fluoroanilino)-6-hydroxy-4-methoxyquinazoline (S4) (1.0 eq), 4-bromo-1-butyne (1.5 eq) with the corresponding alkyl chain length, and potassium carbonate (K2CO3, 2.5 eq) were weighed and added to DMF (concentration approximately 0.2 M). The mixture was stirred at 80 °C for 24 hours under nitrogen protection. After the reaction solution was cooled to room temperature, it was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2:1) to obtain intermediate Z2-1, whose structural formula is [insert structural formula here]. .

[0067] Intermediate Z2-1 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 19 H 16 ClFN3O2 + [M+H] + : 372.0910, found. 372.0914.

[0068] Replacing 4-bromo-1-butyne with 5-bromo-1-pentyne in step (3) yields intermediate Z2-2, whose structural formula is: .

[0069] Intermediate Z2-2 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 20 H 18 ClFN3O2 + [M+H] + : 386.1066, found. 386.1070.

[0070] Replacing 4-bromo-1-butyne with 6-bromo-1-hexyne in step (3) yields intermediate Z2-3, whose structural formula is: .

[0071] Intermediate Z2-3 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 21 H 20 ClFN3O2 + [M+H] + : 400.1223, found. 400.1225.

[0072] Replacing 4-bromo-1-butyne with 7-bromo-1-heptyne in step (3) yields intermediate Z2-4, whose structural formula is: .

[0073] Intermediate Z2-4 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 22 H 22 ClFN3O2 + [M+H] + : 414.1379, found. 414.1381.

[0074] Replacing 4-bromo-1-butyne with 8-bromo-1-octyne in step (3) yields intermediate Z2-5, whose structural formula is: .

[0075] Intermediate Z2-5 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 23 H 24 ClFN3O2 + [M+H] + : 428.1536, found. 428.1537.

[0076] Replacing 4-bromo-1-butyne with 9-bromo-1-nonyne in step (3) yields intermediate Z2-6, whose structural formula is: .

[0077] Intermediate Z2-6 was characterized, and the detection results are as follows: HRMS (ESI) calculated for C 24 H 26 ClFN3O2 + [M+H] + : 442.1692, found. 442.1694.

[0078] (4) Preparation of compounds 1-6: Intermediate Z1 (1.0 eq) and intermediate Z2-1 (1.0 eq) were dissolved in a tert-butanol / water (volume ratio 1:1) mixed solvent (concentration approximately 0.05 M). Copper sulfate (CuSO4, 0.1 eq) and sodium ascorbate (0.2 eq) were added sequentially, and the mixture was stirred at room temperature for 24 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure to remove the organic solvent, and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1) to obtain target compound 1, whose structural formula is [insert structural formula here]. .

[0079] Compound 1 was characterized, and the detection results are as follows:

[0080] 1 H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.26 – 7.15 (m, 3H), 7.13 (s, 1H), 6.56 (t, J = 3.8 Hz, 1H), 4.53 (t, J = 4.1Hz, 2H), 4.41 (t, J = 5.8 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 3.89 (d, J = 3.7Hz, 5H), 3.04 (t, J = 5.8 Hz, 2H).

[0081] HRMS (ESI) calculated for C 30 H 28 ClF2N 10 O4S2 + [M+H] + : 729.1388, found.729.1391.

[0082] Replacing intermediate Z2-1 with intermediate Z2-2 in step (4) yields compound 2, whose structural formula is: .

[0083] Compound 2 was characterized, and the detection results are as follows:

[0084] 1 H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.57 (s, 1H), 7.52 (ddd,J = 7.4, 4.9, 2.2 Hz, 1H), 7.24 – 7.18 (m, 2H), 7.17 (s, 1H), 7.13 (s, 1H), 6.56 (t, J = 3.8 Hz, 1H), 4.53 (t, J = 4.1 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 4.14 (t, J = 6.6 Hz, 2H), 3.89 (d, J = 3.7 Hz, 5H), 2.83 (t, J = 8.3 Hz, 2H),2.26 – 2.17 (m, 2H).

[0085] HRMS (ESI) calculated for C 31 H 30 ClF2N 10 O4S2 + [M+H] + : 743.1544, found.743.1549.

[0086] Replacing intermediate Z2-1 in step (4) with intermediate Z2-3 yields compound 3, whose structural formula is: .

[0087] Compound 3 was characterized, and the detection results are as follows:

[0088] 1 H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.58 (s, 1H), 7.55 –7.48 (m, 1H), 7.24 – 7.15 (m, 3H), 7.13 (s, 1H), 6.56 (t, J = 3.8 Hz, 1H),4.53 (t, J = 4.1 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 4.04 (t, J = 5.7 Hz, 2H), 3.89 (d, J = 3.7 Hz, 5H), 2.76 (t, J = 7.7 Hz, 2H), 1.97 – 1.80 (m, 4H).

[0089] HRMS (ESI) calculated for C 32 H 32 ClF2N 10 O4S2 + [M+H] + : 757.1701, found.757.1704.

[0090] Replacing intermediate Z2-1 in step (4) with intermediate Z2-4 yields compound 4, whose structural formula is: .

[0091] Compound 4 was characterized, and the detection results are as follows:

[0092] 1H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.58 (s, 1H), 7.52 (ddd,J = 7.4, 4.9, 2.2 Hz, 1H), 7.24 – 7.15 (m, 3H), 7.13 (s, 1H), 6.56 (t, J =3.8 Hz, 1H), 4.53 (t, J = 4.1 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 4.00 (t, J =6.0 Hz, 2H), 3.89 (d, J = 3.7 Hz, 5H), 2.70 (t, J = 8.0 Hz, 2H), 1.83 (p, J =8.0 Hz, 2H), 1.71 (ddd, J = 13.2, 7.2, 6.0 Hz, 2H), 1.53 – 1.43 (m, 2H).

[0093] HRMS (ESI) calculated for C 33 H 34 ClF2N 10 O4S2 + [M+H] + : 771.1857, found.771.1862.

[0094] Replacing intermediate Z2-1 in step (4) with intermediate Z2-5 yields compound 5, whose structural formula is: .

[0095] Compound 5 was characterized, and the detection results are as follows:

[0096] 1H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.57 (s, 1H), 7.52 (ddd,J = 7.4, 4.9, 2.2 Hz, 1H), 7.24 – 7.15 (m, 3H), 7.13 (s, 1H), 6.56 (t, J =3.8 Hz, 1H), 4.53 (t, J = 4.1 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 4.00 (t, J =6.0 Hz, 2H), 3.89 (d, J = 3.7 Hz, 5H), 2.66 (t, J = 8.0 Hz, 2H), 1.84 – 1.73 (m, 4H), 1.48 – 1.34 (m, 4H).

[0097] HRMS (ESI) calculated for C 34 H 36 ClF2N 10 O4S2 + [M+H] + : 785.2014, found.785.2015.

[0098] Replacing intermediate Z2-1 in step (4) with intermediate Z2-6 yields compound 6, whose structural formula is: .

[0099] Compound 6 was characterized, and the detection results are as follows:

[0100] 1H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.0 Hz, 1H), 8.60 (s,1H), 8.29 (s, 1H), 8.17 (ddd, J = 8.8, 4.9, 1.8 Hz, 1H), 7.94 (s, 1H), 7.80(dd, J = 5.0, 2.3 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.57 (s, 1H), 7.52 (ddd,J = 7.4, 4.9, 2.2 Hz, 1H), 7.24 – 7.15 (m, 3H), 7.13 (s, 1H), 6.56 (t, J =3.8 Hz, 1H), 4.53 (t, J = 4.0 Hz, 2H), 4.30 (d, J = 7.1 Hz, 2H), 4.00 (t, J =6.0 Hz, 2H), 3.89 (d, J = 3.7 Hz, 5H), 2.66 (t, J = 8.0 Hz, 2H), 1.84 – 1.73 (m, 4H), 1.48 – 1.29 (m, 6H).

[0101] HRMS (ESI) calculated for C 35 H 38 ClF2N 10 O4S2 + [M+H] + : 799.2170, found.799.2176.

[0102] Example of implementation effect 1

[0103] The DDB1 binding affinity of the DDB1-E3 ligase ligand (compound A) with the sulfonamide backbone prepared in Example 1 was determined as follows:

[0104] Microscale thermophoresis (MST) was used to detect the binding affinity between compound A and the E3 ligase DDB1. First, purified recombinant human DDB1 protein was fluorescently labeled using a Monolith NT Protein Labeling Kit RED-NHS and incubated at room temperature in the dark for 30 min. Free dye was then removed using a desalting column. The labeled DDB1 protein was diluted to a final concentration of approximately 50 nM and mixed with different concentration gradients of compound A (maximum concentration 50 μM, serially diluted 1:1 for 16 concentration points). The mixture was incubated in PBS buffer containing 0.05% Tween-20 at room temperature for 15 min to reach binding equilibrium. The samples were then aspirated into standard capillaries and detected using a NanoTemper Monolith NT.115 instrument with LED power set to 40% and MST power set to Medium. The experimental results were analyzed using MO.Affinity Analysis software, and the equilibrium dissociation constant (Kd) between compound A and DDB1 was found to be approximately 105 nM. Figure 1 This indicates that the compound has a strong binding affinity for DDB1. All experiments were independently repeated at least three times, and results are expressed as mean ± SD.

[0105] Example of implementation effect 2

[0106] The degradation activity of the protein degrading agent derivatives (compounds 1-6) prepared in Example 2 was tested, as follows:

[0107] Western blotting was used to evaluate the target protein degradation activity of compounds 1-6 in H1975 non-small cell lung cancer cells. H1975 cells were seeded in 6-well plates and cultured overnight at 37°C with 5% CO2. Once cell confluence reached approximately 60%-70%, compounds 1-6 were added for treatment. The initial screening concentration for each compound was set at 1 μM, with DMSO as a negative control. Treatment time was 24 h. After treatment, the culture medium was discarded, and cells were washed twice with pre-cooled PBS buffer. Lysis was then performed using RIPA lysis buffer containing protease inhibitors. After incubation on ice for 30 min, cell lysates were collected and centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was used to determine protein concentration. Equal amounts of total protein from each group were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk powder for 1 h, and then incubated overnight at 4°C with the target protein primary antibody. The following day, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing with TBST, the membrane was developed using ECL chemiluminescence. The results are as follows: Figure 2As shown, compounds 1-6 can all reduce the expression level of the target protein to varying degrees, indicating that they have certain protein degradation activity.

[0108] Example of implementation effect 3

[0109] The anti-tumor cell proliferation effects of the protein degradation agent derivatives (compounds 1-6) prepared in Example 2 were screened as follows:

[0110] The above-mentioned protein degrading agent derivatives were screened for anti-tumor cell proliferation experiments. MTT assays were performed in three different non-small cell lung cancer cell lines (H1975, HCC827, and PC-9). The results are shown in the table below. To more intuitively compare the differences in compound activity, IC50 was used. 50 Divided into four categories: IC 50 < 1 μM (A), 1 μM < IC 50 < 3 μM (B), 3 μM < IC 50 (C). The test results are shown in Table 1:

[0111] Table 1. Anti-tumor cell proliferation results of protein degradation agent derivatives (compounds 1-6)

[0112]

[0113] As shown in Table 1, the above compounds exhibited good antiproliferative activity in several different cell lines, and these compounds generally performed better in H1975 cells; among them, compound 6 showed the best performance. Therefore, compound 6 will be selected for further evaluation in the H1975 cell line in subsequent evaluations.

[0114] Example of implementation effect 4

[0115] The protein degrading agent derivative (compound 6) prepared in Example 2 was subjected to dose-dependent degradation of EGFR, as detailed below:

[0116] H1975 cells were cultured according to the method in Example 6, and then treated with different concentrations of compound 6 at concentration gradients of 0.1, 0.3, 1, 3, and 10 μM. After 24 h of treatment, the expression level of the target protein was detected using the same Western blotting method. The experimental results are as follows: Figure 3 As shown, the results indicate that as the concentration of compound 6 increases, the expression of the target protein gradually decreases, exhibiting a clear concentration-dependent degradation trend, suggesting that compound 6 has good protein degradation activity in H1975 cells.

[0117] Example of implementation effect 5

[0118] The bioavailability of the protein degrading agent derivative (compound 6) prepared in Example 2 was evaluated as follows:

[0119] The low bioavailability of PROTAC molecules usually limits their efficacy in vivo. One of the key factors limiting the bioavailability of PROTAC molecules is the poor solubility of conventional E3 ligase ligands. The compound A selected in this invention is a novel E3 ligase ligand, which is expected to achieve higher bioavailability in vivo. The pharmacokinetic evaluation of the preferred compound 6 in rats was performed. After fasting overnight, three rats were administered the drug via gavage (10 mg / kg), and three rats were administered it via tail vein (1 mg / kg). Blood samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, and placed in pre-heparinized tubes. After gently tapping the tubes to thoroughly mix the blood with heparin sodium, the samples were centrifuged (4 ℃, 3000 rpm, 10 min) to obtain plasma. 50 μL of plasma sample was taken, and 50 μL of diluent (50% methanol / water) and 250 μL of methanol precipitant were added. The mixture was vortexed and centrifuged (4 ℃, 12000 rpm, 10 min). The supernatant was sealed and sent for LC-MS / MS analysis. Parameters were analyzed using WinNonlin software, and the details are shown in Table 2.

[0120] Table 2 WinNonlin Software Analysis Parameters

[0121]

[0122] The results in Table 2 show that compound 6 has good oral bioavailability, at 21.6%.

[0123] Example of implementation effect 6

[0124] The in vivo efficacy evaluation of the protein degrading agent derivative (compound 6) prepared in Example 2 is as follows:

[0125] To evaluate the in vivo antitumor efficacy of compound 6, a nude mouse xenograft model using H1975 cells was employed. H1975 cells were subcutaneously inoculated into the axillary region of nude mice, and tumors were allowed to grow to an average volume of 70-10 mm. 3 In this study, tumor-bearing mice were randomly divided into two groups: a control group (solvent control, n=5) and a compound 6 administration group (10 mg / kg, administered by gavage once daily, n=5), for 15 consecutive days. During the experiment, the body weight of the mice in the administration group did not fluctuate significantly compared with the control group, and no abnormalities were observed in their mental state or activity, indicating that compound 6 had good safety under this administration regimen. After the administration was completed, the tumor weight and tumor volume of the mice in the administration group were significantly lower than those in the control group (…). Figure 4The tumor inhibition rate was 76.9% as calculated by the formula (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%. These results indicate that compound 6 has strong in vivo antitumor efficacy in this H1975 xenograft model.

[0126] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DDB1-E3 ligase ligand with a sulfonamide backbone, characterized in that: The structural formula of the DDB1-E3 ligase ligand is shown in Formula I: Equation I; Where X is one of CH2, O, and S; Y is one of C and N; R is one of H, F, and CF3; and n is an integer from 0 to 3.

2. The sulfonamide backbone DDB1-E3 ligase ligand according to claim 1, characterized in that: The structural formula of the DDB1-E3 ligase ligand is further shown in Formula II: Formula II.

3. A protein degrading agent derivative, characterized in that: The structural formula of the protein degrading agent derivative is shown in Formula III: Formula III; Linker can be any chemically feasible linker structure; POI ligands are small molecules with binding affinity to target proteins, including but not limited to: EGFR ligand, BRD4 ligand, PD-L1 ligand, CDK ligand, and AR ligand. The definitions of X, Y, R, and n are as described in claim 1.

4. The protein degrading agent derivative according to claim 3, characterized in that: In structural formula III of the protein degrading agent derivative: Linker selected , , , , , Any one of them; POI ligands are selected from , , , , Any one of them; X is preferably either O or S; Y is preferably N; R is preferably any one of F and CF3; n is preferably an integer between 1 and 2.

5. The protein degrading agent derivative according to claim 4, characterized in that: The structural formula of the protein degrading agent derivative is further shown in Formula IV: Formula IV; Where m is a positive integer from 1 to 6.

6. The method for preparing the protein degrading agent derivative according to claim 5, characterized in that, Includes the following steps: (1) Compound S1, compound S2, and triethylamine were dissolved in dichloromethane and reacted under nitrogen protection with stirring. After the reaction was completed, the mixture was concentrated and purified to obtain compound A. The structural formula of compound S1 is as follows: The structural formula of compound S2 is as follows: ; (2) Compound A, compound S3, and potassium carbonate obtained in step (1) are dissolved in DMF and reacted under nitrogen protection with stirring. After filtration, concentration, and purification, intermediate Z1 is obtained. The structural formula of compound A is: The structural formula of compound S3 is as follows: ; (3) Compound S4, alkynyl bromide, and potassium carbonate were dissolved in DMF, and the mixture was heated under nitrogen protection with stirring. After cooling, the mixture was filtered, concentrated, and purified to obtain intermediate Z2. The structural formula of compound S4 is: The bromine compound is selected from any one of the following ω-bromo-1-yne: 4-bromo-1-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, 7-bromo-1-heptyne, 8-bromo-1-octyne, and 9-bromo-1-nonyne. (4) After dissolving intermediate Z1 obtained in step (2) and intermediate Z2 obtained in step (3) in an organic solvent, copper sulfate and sodium ascorbate are added sequentially, and the reaction is carried out under nitrogen protection with stirring. After the reaction is completed, the protein degradation agent derivative is obtained by concentration and purification; the structural formula of intermediate Z1 is as follows: .

7. The preparation method according to claim 6, characterized in that: In step (1), the molar ratio of compound S1, compound S2 and triethylamine is 1:0.8-1.2:2-5, the concentration of dichloromethane is 0.1-0.5M, and the reaction time is 0.5-6h. In step (2), the molar ratio of compound A, compound S3 and potassium carbonate is 1:0.95-1.5:2-3, the concentration of DMF is 0.2-0.5M, and the reaction time is 8-24 h. In step (3), the molar ratio of compound S4, alkynyl bromide compound and potassium carbonate is 1:1-2:2-3, the concentration of DMF is 0.2-0.5M, the heating temperature is 40-100℃, and the time is 12-24 h. In step (4), the molar ratio of intermediate Z1, intermediate Z2, copper sulfate and sodium ascorbate is 1:0.8-1.2:0.05-0.3:0.05-0.

5. The organic solvent is a mixture of tert-butanol and water in a volume ratio of 1:

1. The concentration of tert-butanol is 0.05-0.2M. The reaction time is 12-24h.

8. The use of the protein degrading agent derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 3-5 in the preparation of a treatment or preventive for cancer.

9. The application according to claim 8, characterized in that: The cancer in question is non-small cell lung cancer.

10. A pharmaceutical composition, characterized in that: It comprises the protein degrading agent derivative as described in any one of claims 3-5 and pharmaceutically acceptable excipients.

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