Bio-orthogonal group covalent inhibitor derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical synthesis, and particularly discloses a covalent inhibitor derivative of a bio-orthogonal group and a preparation method and application of the covalent inhibitor derivative of the bio-orthogonal group, the covalent inhibitor derivative of the bio-orthogonal group takes a KRASG12C inhibitor sotorasib as a modification molecule, polyethylene glycol with different chain lengths is introduced as fusion protein, and different bio-orthogonal groups are connected to obtain a sotorasib derivative; the biological orthogonal groups comprise dibenzocyclooctyne amine, alkynyl and tetrazinyl; the chain length of the polyethylene glycol is 4-16. According to the covalent inhibitor derivative of the biological orthogonal group as well as the preparation method and the application of the covalent inhibitor derivative, the covalent inhibitor modified by the biological orthogonal group is designed and synthesized to induce generation of a tumor specific new antigen, and a new way is provided for solving the problems of target spot selection limitation and heterogeneity in tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, and in particular to covalent inhibitor derivatives of bioorthogonal groups, their preparation methods, and applications. Background Technology

[0002] In clinical practice, cancer treatment methods, including surgery, radiotherapy, cytotoxic chemotherapy, hormone therapy, targeted therapy, and immunotherapy, are used alone or in combination, depending on tumor stage, resectability, biological characteristics, comorbidities, and the patient's overall condition. Among these, targeted therapy (such as small molecule signal transduction inhibitors and receptor tyrosine kinase antibodies) that targets specific mutated oncogenes or their downstream signal transduction cascades, and immunotherapy that activates or enhances the body's immune system to specifically recognize and kill tumor cells, can achieve precise killing of cancer cells, profoundly changing the treatment landscape and survival prognosis for cancer patients.

[0003] However, with the deepening of clinical applications, both targeted therapy and immunotherapy have revealed their respective limitations. Targeted therapy is limited by the scarcity of druggable targets and often develops resistance due to mechanisms such as secondary mutations of targets, bypass activation, and phenotypic transformation. Immunotherapy, on the other hand, is limited by problems such as insufficient overall response rate and frequent immune-related adverse reactions, and its underlying mechanisms involve tumor heterogeneity and immune editing.

[0004] Neoantigens are novel peptides encoded by gene mutations specific to tumor cells, differing from wild-type protein sequences. These are presented to the cell surface via the major histocompatibility complex (MHC), where they can be specifically recognized by T cells and activate an anti-tumor immune response. As a highly promising therapeutic target, neoantigens are expressed only in tumor tissues and not in normal tissues. They possess high specificity and strong immunogenicity, with an extremely low risk of off-target effects, making them ideal targets for precision immunotherapy.

[0005] In existing technologies, neoantigen therapy suffers from limited accuracy in neoantigen prediction, immune escape problems, and insufficient production of neoantigen-specific T cells. These problems severely restrict the development of neoantigen therapy. Summary of the Invention

[0006] The purpose of this invention is to provide covalent inhibitor derivatives of bioorthogonal groups, their preparation methods, and applications. By designing and synthesizing covalent inhibitors modified with bioorthogonal groups, the invention induces the generation of tumor-specific neoantigens, providing a new approach to solving the problems of target selection limitations and heterogeneity in tumor immunotherapy.

[0007] To achieve the above objectives, the present invention provides covalent inhibitor derivatives of bioorthogonal groups. Using the KRASG12C inhibitor sotorasib as a modified molecule, polyethylene glycol of different chain lengths is introduced as a fusion protein and different bioorthogonal groups are linked to obtain sotorasib derivatives. The bioorthogonal groups include dibenzocyclooctylenylamine, alkynyl, and tetraazinyl; The polyethylene glycol chain length is 4 to 16.

[0008] Preferably, the sotorasib derivatives include sotorasib-PEG4-alkynyl, sotorasib-PEG5-alkynyl, sotorasib-PEG4-tetraazine, sotorasib-PEG4-DBCO, sotorasib-PEG10-DBCO, and sotorasib-PEG16-DBCO; The structural formula of the sotorasib-PEG4-alkynyl group is: ; The structural formula of the sotorasib-PEG5-acetylenic group is: ; The structural formula of the sotorasib-PEG4-tetraazine is: ; The structural formula of sotorasib-PEG4-DBCO is: ; The structural formulas of sotorasib-PEG10-DBCO and sotorasib-PEG16-DBCO are as follows: ; Where n is 10 or 16.

[0009] This invention also provides a method for preparing covalent inhibitor derivatives of bioorthogonal groups, comprising the following steps: S1. Dissolve one or more of the following in N,N-dimethylformamide: KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, potassium carbonate, or tert-butyl 2-bromoethyl (methyl)carbamate, and react to obtain an intermediate. S2. Then dichloromethane and trifluoroacetic acid are added sequentially, and the reaction is carried out to obtain the intermediate. S3. Then, the intermediate obtained from compounds a and S2 is added to N,N-dimethylformamide (DMF) solvent or dichloromethane (DCM) solvent and reacted to obtain the target product. The compound a is a small molecule compound with PEG linked to a bioorthogonal group or amino-polyethylene glycol-tert-butyl propionate.

[0010] Preferably, step S3 further includes adding a condensing agent and an organic base to a DMF solvent or a DCM solvent, wherein the condensing agent is HATU (2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) and the organic base is DIPEA (N,N-diisopropylethylamine), specifically including the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol: 0.18 mmol: 0.18 mmol: 1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Dissolve intermediate 2 in dichloromethane and add trifluoroacetic acid. The volume ratio of dichloromethane to trifluoroacetic acid is 3:1. React at room temperature for 3 hours to obtain intermediate 3. S3. Then, compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.07 mmol: 0.07 mmol: 0.14 mmol: 50 µL: 1 mL. The mixture was stirred at room temperature for 1 h to obtain the target product sotorasib-PEG4-DBCO. Among them, compound a is polyethylene glycol linked to dibenzocyclooctylamine with a chain length of 4.

[0011] Preferably, S3 further includes adding the condensing agent and organic base to the DMF solvent or DCM solvent; S3 is followed by the addition of trifluoroacetic acid and dichloromethane, followed by reaction, then the addition of compound b, condensing agent, and organic base, followed by reaction to obtain the target product; Specifically, the following steps are included: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Then, compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.08 mmol: 0.08 mmol: 0.13 mmol: 30 µL: 1 mL. The mixture was stirred overnight at room temperature to obtain intermediate 4. Then, trifluoroacetic acid and dichloromethane were added in a volume ratio of 1:3. The mixture was reacted at room temperature for 3 h to obtain intermediate 5. Then, compound b, HATU, and DIPEA were added in a ratio of 0.05 mmol: 0.06 mmol: 14 µL. The mixture was stirred overnight at room temperature to obtain the target product. Among them, compound a is amino-polyethylene glycol-tert-butyl propionate with a chain length of 10 or 16, and compound b is azirdibenzocyclooctyneamine.

[0012] Preferably, S3 further includes adding the condensing agent and organic base to the DMF solvent or DCM solvent, specifically including the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.1 mmol: 0.08 mmol: 0.16 mmol: 55 µL: 1 mL and reacted at room temperature for 4 h to obtain the target product sotorasib-PEG4-alkynyl. Compound a is a polyethylene glycol chain with a chain length of 4 linked to an alkynyl group.

[0013] Preferably, S3 further includes adding the condensing agent and organic base to the DMF solvent or DCM solvent, specifically including the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.08 mmol: 0.07 mmol: 0.14 mmol: 48 µL: 1 mL and reacted at room temperature for 4 h to obtain the target product sotorasib-PEG5-alkynyl. Compound a is a polyethylene glycol chain with a chain length of 5 linked to an alkynyl group.

[0014] Preferably, it includes the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl 2-bromoethyl (methyl)carbamate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol: 0.18 mmol: 0.18 mmol: 1 mL and reacted at 55 °C for 2 h to obtain intermediate 8. S2. Then, intermediate 8 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 9. S3. Then, compound a and intermediate 9 were added to dichloromethane at a ratio of 0.05 mmol: 0.05 mmol: 1 mL and reacted at room temperature for 5 h to obtain the target product sotorasib-PEG4-tetraazine. Compound a is a polyethylene glycol chain with a chain length of 4 linked to a tetrazine group.

[0015] This invention also provides the application of covalent inhibitor derivatives of bioorthogonal groups in tumor immunotherapy.

[0016] Preferably, the covalent inhibitor derivative of the bioorthogonal group targets and binds to oncoproteins.

[0017] Therefore, the present invention utilizes the above-mentioned covalent inhibitor derivatives of bioorthogonal groups, their preparation methods, and applications, with the following beneficial effects: This invention establishes a "neoantigen induction-T cell recruitment" regulatory system based on bioorthogonal reactions: by designing and synthesizing covalent inhibitors modified with bioorthogonal groups to induce the generation of tumor-specific neoantigens, and simultaneously using anti-CD3 antibodies modified with complementary bioorthogonal groups to achieve T cell-specific recruitment, the two are precisely coupled using bioorthogonal reactions to achieve precise killing of tumor cells.

[0018] This invention combines chemical biology tools with targeted and immunotherapy to obtain highly abundant and stably presented neoantigens. Through a bioorthogonal-mediated targeted recognition mechanism, T cells are activated by antibody bridging. This simultaneously addresses the problems of drug resistance in targeted therapies and low response rates in immunotherapy due to insufficient neoantigen quantity, low presentation efficiency, and immunosuppression in the tumor microenvironment. It provides a new approach to solving the challenges of target selection limitations and heterogeneity in tumor immunotherapy.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is a diagram showing the intracellular binding of KRAS G12C cells to the covalent inhibitor derivatives of the bioorthogonal group of the present invention, their preparation method, and application examples. The compounds corresponding to the numbers are: 3-3 (sotorasib-PEG5-alkynyl), 3-4 (sotorasib-PEG4-alkynyl), 3-16 (sotorasib-PEG4-tetraazine), P4 (sotorasib-PEG4-DBCO), P10 (sotorasib-PEG10-DBCO), and P16 (sotorasib-PEG16-DBCO). Wherein, a is the binding diagram of 3-16, 3-4, and 3-3 in KRAS G12C mutant cells, b is the binding diagram of P4, P10, and P16 in KRAS G12C mutant cells, c is the binding diagram of 3-16, 3-4, and 3-3 in KRAS wild-type cells, and d is the binding diagram of P4, P10, and P16 in wild-type cells. Figure 2 These are the covalent inhibitor derivatives of the bioorthogonal group of the present invention and their preparation method, and the cell membrane surface complex detection diagrams of application examples 2-4, wherein a is the cell membrane surface complex detection diagram of P4, P10, and P16 in MIAPaCa-2 cells, and b is the cell membrane surface complex detection diagram of P4, P10, and P16 in BxPC3 cells. Figure 3 This is a covalent inhibitor derivative of the biological orthogonal group of the present invention, its preparation method, and a crystal violet staining diagram of the co-culture experiment in Example 1. Figure 4 This is a quantitative statistical chart of cells in Example 1 of the present invention, which is a covalent inhibitor derivative of the biological orthogonal group and its preparation method. In the chart, a is a cell viability detection chart after co-culturing with BxPC3, and b is a cell viability detection chart after co-culturing with MIAPaCa-2. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] This invention uses the KRASG12C inhibitor sotorasib as a modified molecule, introduces polyethylene glycol (PEG) of different chain lengths as a fusion protein linker, and connects different bioorthogonal groups, such as dibenzocyclooctylenine (DBCO), alkynyl, and tetrazine, to obtain sotorasib derivatives containing different bioorthogonal groups and PEG chains of different lengths, including sotorasib-PEG4-DBCO (named P4), sotorasib-PEG10-DBCO (named P10), sotorasib-PEG16-DBCO (named P16), sotorasib-PEG4-alkynyl, sotorasib-PEG5-alkynyl, and sotorasib-PEG4-tetrazine, for a total of 6 covalent inhibitor derivatives (sotorasib derivatives).

[0024] Example 1: A covalent inhibitor derivative of a bioorthogonal group, sotorasib-PEG4-DBCO, has the following structural formula: .

[0025] Its synthetic route is as follows: .

[0026] Its preparation method is as follows: S1. KRASG12C inhibitor sotorasib (50 mg, 0.09 mmol), tert-butyl bromoacetate (35 mg, 0.18 mmol), and potassium carbonate (25 mg, 0.18 mmol) were dissolved in 1 mL of N,N-dimethylformamide and stirred at 55 °C for 2 h to obtain intermediate 2.

[0027] S2. Dissolve intermediate 2 in 600µL of dichloromethane and add 200µL of trifluoroacetic acid. React at room temperature for 3 hours to obtain intermediate 3.

[0028] S3. Then, compound a (40 mg, 0.07 mmol), intermediate 3 (43 mg, 0.07 mmol), HATU (53 mg, 0.14 mmol), and DIPEA (50 µL) were added to 1 mL of DMF and stirred at room temperature for 1 h to obtain the target product sotorasib-PEG4-DBCO, denoted as P4, with a yield of 45%.

[0029] Among them, compound a is a PEG with a chain length of 4 linked to a bioorthogonal group DBCO, which was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0030] Example 2: A covalent inhibitor derivative of a bioorthogonal group, sotorasib-PEG10-DBCO, has the following structural formula: ; Where n is 10.

[0031] Its synthetic route is as follows: .

[0032] Its preparation method is as follows: S1. KRASG12C inhibitor sotorasib (50 mg, 0.09 mmol), tert-butyl bromoacetate (35 mg, 0.18 mmol), and potassium carbonate (25 mg, 0.18 mmol) were dissolved in 1 mL of DMF and reacted at 55 °C for 2 h to obtain intermediate 2.

[0033] S2. Then, intermediate 2 is dissolved in 1.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid is added. The mixture is reacted at room temperature for 3 h to obtain intermediate 3.

[0034] S3. Then, compound a (49 mg, 0.08 mmol), intermediate 3 (51 mg, 0.08 mmol), HATU (47 mg, 0.13 mmol), and DIPEA (30 µL) were added to 1 mL of DMF and stirred overnight at room temperature to obtain intermediate 4.

[0035] S4. Then add 200µL of trifluoroacetic acid and 600µL of dichloromethane, and react at room temperature for 3h to obtain intermediate 5. Then add compound b (13mg, 0.05mmol), HATU (23mg, 0.06mmol), and DIPEA (14µL), and stir overnight at room temperature to obtain the target product, sotorasib-PEG10-DBCO, denoted as P10, with a yield of 59%.

[0036] Among them, compound a is amino-polyethylene glycol-tert-butyl propionate with a chain length of 10, which was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0037] Compound b is azirdibenzocyclooctyneamine, purchased from Chongqing Platinum Strontium Titanium Technology Co., Ltd.

[0038] Example 3: A covalent inhibitor derivative of a bioorthogonal group, sotorasib-PEG16-DBCO, differs from the one in Example 2 in that n is 16.

[0039] Its synthesis route is exactly the same as that in Example 2.

[0040] The preparation method differs from that in Example 2 in that: compound a (92 mg, 0.1 mmol) is added to S3 to obtain the target product sotorasib-PEG16-DBCO, denoted as P16, with a yield of 46%.

[0041] Among them, compound a is amino-polyethylene glycol-tert-butyl propionate with a chain length of 16, which was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0042] Example 4: A covalent inhibitor derivative of a bioorthogonal group, sotorasib-PEG4-alkynyl, has the following structural formula: .

[0043] Its synthetic route is as follows: .

[0044] Its preparation method is as follows: S1. KRASG12C inhibitor sotorasib (50 mg, 0.09 mmol), tert-butyl bromoacetate (35 mg, 0.18 mmol), and potassium carbonate (25 mg, 0.18 mmol) were dissolved in 1 mL of N,N-dimethylformamide and reacted at 55 °C for 2 h to obtain intermediate 2.

[0045] S2. Then, intermediate 2 is dissolved in 600µL of dichloromethane and 200µL of trifluoroacetic acid is added. The mixture is reacted at room temperature for 3 hours to obtain intermediate 3.

[0046] S3. Then, compound a (20 mg, 0.1 mmol), intermediate 3 (49 mg, 0.08 mmol), HATU (60 mg, 0.16 mmol), and DIPEA (55 µL) were added to 1 mL of DMF to obtain the target product sotorasib-PEG4-alkynyl with a yield of 64%.

[0047] Compound a is a PEG-linked bioorthogonal alkynyl group with a chain length of 4, purchased from Chongqing Platinum Strontium Titanium Technology Co., Ltd.

[0048] Example 5: A covalent inhibitor derivative of a bioorthogonal group, sotorasib-PEG5-alkynyl, has the following structural formula: Its synthetic route is as follows: .

[0049] Its preparation method is as follows: S1. KRASG12C inhibitor sotorasib (50 mg, 0.09 mmol), tert-butyl bromoacetate (35 mg, 0.18 mmol), and potassium carbonate (25 mg, 0.18 mmol) were dissolved in 1 mL of N,N-dimethylformamide and reacted at 55 °C for 2 h to obtain intermediate 2.

[0050] S2. Then, intermediate 2 is dissolved in 600µL of dichloromethane and 200µL of trifluoroacetic acid is added. The mixture is reacted at room temperature for 3 hours to obtain intermediate 3.

[0051] S3. Then, compound a (23 mg, 0.08 mmol), intermediate 3 (43 mg, 0.07 mmol), HATU (52 mg, 0.14 mmol), and DIPEA (48 µL) were added to 1 mL of DMF and reacted at room temperature for 4 h to obtain the target product sotorasib-PEG5-alkynyl with a yield of 56%.

[0052] Compound a is a PEG chain with a chain length of 5 linked to a bioorthogonal alkynyl group, and was purchased from Chongqing Platinum Strontium Titanium Technology Co., Ltd.

[0053] Example 6: Sotorasib-PEG4-tetraazine, a covalent inhibitor derivative of a bioorthogonal group, has the following structural formula: .

[0054] Its synthetic route is as follows: .

[0055] Its preparation method is as follows: S1. KRASG12C inhibitor sotorasib (50 mg, 0.09 mmol), tert-butyl 2-bromoethyl (methyl)carbamate (40 mg, 0.18 mmol), and potassium carbonate (25 mg, 0.18 mmol) were dissolved in 1 mL of N,N-dimethylformamide and reacted at 55 °C for 2 h to obtain intermediate 8.

[0056] S2. Then, intermediate 2 was dissolved in 600µL of dichloromethane and 200µL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 3 hours to obtain intermediate 9.

[0057] S3. Then, compound a (28 mg, 0.05 mmol) and intermediate 9 (32 mg, 0.05 mmol) were added to 1 mL of DCM and reacted at room temperature for 5 h to obtain the target product sotorasib-PEG4-tetraazine with a yield of 63%.

[0058] Among them, compound a is a PEG-linked bioorthogonal tetraazine group with a chain length of 4, which was purchased from Chongqing Platinum Strontium Titanium Technology Co., Ltd.

[0059] Experimental testing: 1. The structures of the covalent inhibitor derivatives obtained in Examples 1-6 were confirmed, and their proton and carbon spectral data are as follows: Example 1: 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 4.9 Hz, 1H), 7.93 (d, J = 9.9 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.40 – 7.26 (m, 8H), 7.23 (d, J = 7.5 Hz, 1H), 7.06 (s, 1H), 6.77 (t, J = 8.9 Hz, 1H), 6.65 (d, J = 8.6 Hz, 1H), 6.57 (q, J = 8.5 Hz, 3H), 6.38 (dd, J = 16.6, 1.9 Hz, 1H), 5.79 (dd, J = 10.4, 2.0 Hz, 1H), 5.10 (d, J = 14.0 Hz, 1H), 4.86 – 4.67 (m, 1H), 4.38 (p, J = 17.7 Hz, 3H), 3.95 (dd, J = 60.1, 12.8 Hz, 1H), 3.65 (d, J = 13.9 Hz, 2H), 3.59 (dd, J = 9.6, 6.1 Hz, 1H), 3.54 – 3.49 (m, 7H), 3.48 – 3.37 (m, 9H), 3.25 (td, J = 11.9, 6.8 Hz, 2H), 2.74 (d, J = 16.8 Hz, 1H), 2.46 (ddd, J = 16.6, 7.1, 4.9 Hz, 1H), 2.26 (td, J = 6.3, 4.0 Hz, 2H), 2.16 (s, 3H), 2.02 – 1.90 (m, 4H), 1.51 (d, J = 20.6 Hz, 3H), 1.20 (d, J = 6.7 Hz, 3H), 1.09 – 0.94 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 172.07, 170.98, 167.65, 151.14, 149.18, 148.14, 132.17, 130.50, 129.30, 129.15, 128.70, 128.45, 128.30, 127.89, 127.29, 126.72, 125.65, 123.59, 123.11, 122.55, 114.78, 110.07, 109.86, 107.90, 70.54, 70.51, 70.37, 70.24, 70.08, 69.45, 67.17, 55.56,38.82, 36.90, 35.25, 34.80, 17.76. Example 2: 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 4.9 Hz, 1H), 7.98 (s,1H), 7.66 (dd, J = 7.5, 1.3 Hz, 1H), 7.44 – 7.26 (m, 9H), 7.11 (s, 1H), 6.80(t, J = 8.7 Hz, 1H), 6.68 (d, J = 8.5 Hz, 1H), 6.62 (s, 1H), 6.56 – 6.50 (m,1H), 6.40 (dd, J = 16.8, 1.9 Hz, 2H), 5.81 (dd, J = 10.4, 1.9 Hz, 1H), 5.13(d, J = 13.9 Hz, 1H), 4.90 – 4.69 (m, 1H), 4.42 (d, J = 19.0 Hz, 3H), 4.08 –3.83 (m, 1H), 3.81 – 3.64 (m, 4H), 3.61 (d, J = 1.6 Hz, 18H), 3.58 (t, J =3.5 Hz, 13H), 3.57 – 3.53 (m, 4H), 3.49 (dt, J = 7.6, 4.7 Hz, 6H), 3.34 –3.21 (m, 2H), 2.76 (s, 1H), 2.48 (ddd, J = 16.6, 7.2, 4.8 Hz, 1H), 2.37 –2.24 (m, 2H), 2.07 – 1.91 (m, 5H), 1.52 (d, J = 26.9 Hz, 3H), 1.23 (d, J =6.8 Hz, 3H), 1.04 (d, J = 31.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 170.92,170.31, 149.71, 148.11, 146.84, 146.61, 130.80, 130.66, 129.53, 127.69,127.21, 126.87, 126.45, 126.16, 125.57, 125.36, 123.74, 122.19, 121.50,120.79, 112.85, 106.09, 68.73, 68.57, 68.44, 68.30, 67.38, 65.27, 53.79,47.16, 47.14, 37.12, 34.71, 33.84, 32.64, 28.65.17.63. Example 3: 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 4.9 Hz, 1H), 7.94 (d, J = 9.9 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.39 – 7.26 (m, 8H), 7.23 (d, J = 7.4 Hz, 1H), 7.09 – 7.01 (m, 1H), 6.77 (t, J = 8.7 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 6.59 (d, J = 7.0 Hz, 3H), 6.37 (d, J = 16.7 Hz, 1H), 5.82 – 5.75 (m, 1H), 5.10 (d, J = 14.0 Hz, 1H), 4.86 – 4.66 (m, 1H), 4.47 – 4.24 (m, 3H), 4.06 – 3.75 (m, 2H), 3.64 – 3.53 (m, 66H), 3.27 (h, J = 6.7 Hz, 2H), 2.72 (s, 1H), 2.46 (ddd, J = 16.6, 7.1, 5.1 Hz, 4H), 2.28 (q, J = 5.6 Hz, 3H), 1.96 (dd, J = 10.2, 5.1 Hz, 3H), 1.53 (s, 3H), 1.19 (d, J = 6.7 Hz, 3H), 1.00 (d, J = 30.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 172.08, 171.20, 167.67, 151.18, 148.13, 132.21, 130.50, 129.30, 129.17, 128.69, 128.42, 128.32, 127.90, 127.27, 126.72, 125.65, 123.60, 123.10, 122.56, 114.81, 107.88, 70.59, 70.57, 70.54, 70.53, 70.50, 70.39, 70.36, 70.26, 70.06, 69.46, 67.19, 55.55, 38.82, 36.92, 35.24, 34.79, 30.51, 17.72. Example 4: 1H NMR (400 MHz, CDCl3) δ 8.37 (t, J = 4.2 Hz, 1H), 7.96 (s,1H), 7.26 (s, 1H), 7.00 (s, 1H), 6.70 (t, J = 8.8 Hz, 1H), 6.64 – 6.43 (m,3H), 6.29 (d, J = 16.9 Hz, 1H), 5.71 (d, J = 10.4 Hz, 1H), 4.90 (d, J = 63.1Hz, 1H), 4.67 – 4.18 (m, 4H), 4.03 (s, 2H), 4.00 – 3.78 (m, 1H), 3.59 – 3.47(m, 10H), 3.45 – 3.26 (m, 9H), 2.66 (s, 1H), 2.34 (s, 1H), 1.91 (s, 3H), 1.48– 1.38 (m, 3H), 1.11 (dd, J = 7.1, 3.1 Hz, 3H), 0.92 (d, J = 32.8 Hz, 3H);13C NMR (101 MHz, CDCl3) δ 167.59, 154.87, 153.68, 151.16, 150.09, 148.93,132.22, 132.12, 130.60, 129.07, 126.81, 123.55, 109.97, 109.76, 79.51, 74.77,74.76, 70.48, 70.46, 70.34, 70.30, 70.03, 69.34, 69.06, 58.32, 53.59, 52.80,42.13, 38.73, 30.38, 30.14, 29.67, 17.71. Example 5: 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 4.7 Hz, 1H), 7.94 (d,J = 9.5 Hz, 1H), 7.25 (d, J = 7.1 Hz, 1H), 6.98 (s, 1H), 6.69 (t, J = 8.5 Hz,1H), 6.64 – 6.44 (m, 3H), 6.28 (d, J = 16.7 Hz, 1H), 5.70 (d, J = 10.4 Hz,1H), 4.89 (d, J = 59.3 Hz, 1H), 4.66 – 4.15 (m, 4H), 4.04 (d, J = 2.8 Hz,2H), 4.02 – 3.77 (m, 1H), 3.61 – 3.46 (m, 14H), 3.35 (dd, J = 33.1, 17.2 Hz,9H), 2.65 (s, 1H), 2.34 (d, J = 2.6 Hz, 1H), 1.90 (s, 3H), 1.41 (dd, J =15.7, 7.9 Hz, 3H), 1.10 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 32.0 Hz, 3H); 13 C NMR(101 MHz, CDCl3) δ 167.59, 166.28, 154.88, 151.14, 150.08, 148.97, 132.22,132.11, 130.58, 129.05, 126.82, 123.53, 109.95, 109.74, 79.60, 74.73, 70.52,70.50, 70.46, 70.44, 70.31, 70.29, 70.03, 69.33, 69.06, 69.04, 58.34, 58.33,52.77, 38.73, 30.37, 29.66, 17.69. Example 6: ¹H NMR (400 MHz, CDCl₃) δ 8.44 – 8.27 (m, 3H), 7.78 (s, 1H), 7.19 (p, J = 8.3 Hz, 1H), 7.01 – 6.81 (m, 4H), 6.60 (dt, J = 43.3, 8.1 Hz, 3H), 6.32 – 6.20 (m, 1H), 5.68 (t, J = 9.3 Hz, 1H), 4.92 (d, J = 38.3 Hz, 1H), 4.52 (d, J = 81.6 Hz, 1H), 4.09 (p, J = 4.1 Hz, 2H), 3.90 – 3.73 (m, 4H), 3.61 – 3.44 (m, 18H), 3.25 (d, J = 50.1 Hz, 3H), 2.95 – 2.90 (m, 3H), 2.66 (d, J = 34.7 Hz, 1H), 2.30 (s, 2H), 1.87 (d, J = 12.6 Hz, 3H), 1.48 –1.37 (m, 3H), 1.09 (s, 3H), 0.91 (d, J = 23.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 171.63, 166.63, 163.72, 162.47, 148.88, 131.87, 131.77, 130.69, 129.65, 129.64, 128.94, 126.87, 124.31, 123.47, 115.27, 70.83, 70.53, 70.42, 70.23, 70.12, 69.54, 67.66, 67.08, 53.59, 52.90, 36.79, 30.33, 29.65, 21.05. 2. The covalent inhibitor derivatives obtained in Examples 1-6 were subjected to in vivo binding with recombinant protein KRAS G12C, and the results are as follows: Figure 1 As shown.

[0060] Depend on Figure 1 It can be seen that all covalent inhibitor derivatives can bind to KRAS G12C, indicating that the synthetic covalent inhibitors modified with bioorthogonal groups can specifically target and bind to oncoproteins.

[0061] 3. Since the reaction between DBCO and N3 does not require copper ion catalysis, is non-cytotoxic, and has a fast reaction rate, it is stable and efficient in an aqueous environment. The covalent inhibitor derivatives obtained in Examples 1-4 were used to test the presentation of the hapten peptide / MHC complex onto the cell membrane surface of cancer cells. The results are as follows: Figure 2 As shown.

[0062] Depend on Figure 2 It is known that only the hapten peptide formed by P4 and KRAS G12C obtained in Example 1 can be presented to the cell membrane by MHC-I, indicating that after the drug-target protein complex is degraded by the intracellular proteasome, the haptenized peptide containing the conjugated drug can be presented to the surface of tumor cells by MHC-I molecules.

[0063] 4. To evaluate the antitumor effect of P4 from Example 1, another anti-CD3 antibody carrying complementary bioorthogonal groups was used to specifically recognize this neoantigen via a highly efficient click chemistry reaction. The crystal violet staining results are as follows: Figure 3 As shown, the cell quantification results are as follows: Figure 4 As shown.

[0064] Depend on Figure 3 and Figure 4 It was found that P4-treated MIAPaCa-2 (mutant) cells, when combined with Anti-CD3-N3, could enhance the killing effect of T cells, but unlabeled Anti-CD3 antibodies had no significant effect. Furthermore, the killing effect of T cells was detected using the CellTiter-GloLuminescent cell viability assay kit, and results consistent with those observed by crystal violet staining were observed. This indicates that the haptenized peptide / MHC-I complex, as a tumor-specific neoantigen, can be specifically recognized by another anti-CD3 antibody carrying complementary bioorthogonal groups through a click chemistry reaction, thereby recruiting and activating T cells to achieve precise killing of tumor cells, thus verifying the feasibility of the anti-tumor strategy.

[0065] Therefore, this invention utilizes the above-mentioned covalent inhibitor derivatives of bioorthogonal groups and their preparation methods and applications. By designing and synthesizing covalent inhibitors modified with bioorthogonal groups to induce the generation of tumor-specific neoantigens, it provides a new approach to solving the problems of target selection limitations and heterogeneity in tumor immunotherapy.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A covalent inhibitor derivative of a bioorthogonal group, characterized in that: Using the KRASG12C inhibitor sotorasib as a modified molecule, polyethylene glycol of different chain lengths was introduced as a fusion protein and linked with different bioorthogonal groups to obtain sotorasib derivatives. The bioorthogonal groups include dibenzocyclooctylenylamine, alkynyl, and tetraazinyl; The polyethylene glycol chain length is 4 to 16.

2. The covalent inhibitor derivative of the bioorthogonal group according to claim 1, characterized in that: The sotorasib derivatives include sotorasib-PEG4-alkynyl, sotorasib-PEG5-alkynyl, sotorasib-PEG4-tetraazine, sotorasib-PEG4-DBCO, sotorasib-PEG10-DBCO, and sotorasib-PEG16-DBCO; The structural formula of the sotorasib-PEG4-alkynyl group is: ； The structural formula of the sotorasib-PEG5-acetylenic group is: ； The structural formula of the sotorasib-PEG4-tetraazine is: ； The structural formula of sotorasib-PEG4-DBCO is: ； The structural formulas of sotorasib-PEG10-DBCO and sotorasib-PEG16-DBCO are as follows: ； Where n is 10 or 16.

3. A method for preparing a covalent inhibitor derivative of a bioorthogonal group, characterized in that, Includes the following steps: S1. Dissolve one or more of the following in N,N-dimethylformamide: KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, potassium carbonate, or tert-butyl 2-bromoethyl (methyl)carbamate, and react to obtain an intermediate. S2. Then dichloromethane and trifluoroacetic acid are added sequentially, and the reaction is carried out to obtain the intermediate. S3. Then, the intermediate obtained from compounds a and S2 is added to N,N-dimethylformamide solvent or dichloromethane solvent and reacted to obtain the target product. The compound a is a small molecule compound with PEG linked to a bioorthogonal group or amino-polyethylene glycol-tert-butyl propionate.

4. The method for preparing the covalent inhibitor derivative of the bioorthogonal group according to claim 3, characterized in that, Specifically, the following steps are included: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol: 0.18 mmol: 0.18 mmol: 1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Dissolve intermediate 2 in dichloromethane and add trifluoroacetic acid. The volume ratio of dichloromethane to trifluoroacetic acid is 3:

1. React at room temperature for 3 hours to obtain intermediate 3. S3. Then, compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.07 mmol: 0.07 mmol: 0.14 mmol: 50 µL: 1 mL. The mixture was stirred at room temperature for 1 h to obtain the target product sotorasib-PEG4-DBCO. Among them, compound a is polyethylene glycol linked to dibenzocyclooctylamine with a chain length of 4.

5. The method for preparing the covalent inhibitor derivative of the bioorthogonal group according to claim 3, characterized in that, Specifically, the following steps are included: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:

1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Then, compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.08 mmol: 0.08 mmol: 0.13 mmol: 30 µL: 1 mL. The mixture was stirred overnight at room temperature to obtain intermediate 4. Then, trifluoroacetic acid and dichloromethane were added in a volume ratio of 1:

3. The mixture was reacted at room temperature for 3 h to obtain intermediate 5. Then, compound b, HATU, and DIPEA were added in a ratio of 0.05 mmol: 0.06 mmol: 14 µL. The mixture was stirred overnight at room temperature to obtain the target product. Among them, compound a is amino-polyethylene glycol-tert-butyl propionate with a chain length of 10 or 16, and compound b is azirdibenzocyclooctyneamine.

6. The method for preparing the covalent inhibitor derivative of the bioorthogonal group according to claim 3, characterized in that, Includes the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:

1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.1 mmol: 0.08 mmol: 0.16 mmol: 55 µL: 1 mL and reacted at room temperature for 4 h to obtain the target product sotorasib-PEG4-alkynyl. Compound a is a polyethylene glycol chain with a chain length of 4 linked to an alkynyl group.

7. The method for preparing the covalent inhibitor derivative of the bioorthogonal group according to claim 3, characterized in that, Includes the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl bromoacetate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol:0.18 mmol:0.18 mmol:1 mL and reacted at 55 °C for 2 h to obtain intermediate 2. S2. Then, intermediate 2 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:

1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 3. S3. Compound a, intermediate 3, HATU, and DIPEA were added to N,N-dimethylformamide in a ratio of 0.08 mmol: 0.07 mmol: 0.14 mmol: 48 µL: 1 mL and reacted at room temperature for 4 h to obtain the target product sotorasib-PEG5-alkynyl. Compound a is a polyethylene glycol chain with a chain length of 5 linked to an alkynyl group.

8. The method for preparing the covalent inhibitor derivative of the bioorthogonal group according to claim 3, characterized in that, Includes the following steps: S1. KRASG12C inhibitor sotorasib, tert-butyl 2-bromoethyl (methyl)carbamate, and potassium carbonate were dissolved in N,N-dimethylformamide at a ratio of 0.09 mmol: 0.18 mmol: 0.18 mmol: 1 mL and reacted at 55 °C for 2 h to obtain intermediate 8. S2. Then, intermediate 8 is dissolved in dichloromethane and trifluoroacetic acid is added. The volume ratio of dichloromethane to trifluoroacetic acid is 3:

1. The reaction is carried out at room temperature for 3 hours to obtain intermediate 9. S3. Then, compound a and intermediate 9 were added to dichloromethane at a ratio of 0.05 mmol: 0.05 mmol: 1 mL and reacted at room temperature for 5 h to obtain the target product sotorasib-PEG4-tetraazine. Compound a is a polyethylene glycol chain with a chain length of 4 linked to a tetrazine group.

9. The application of a target product prepared by a method for preparing a covalent inhibitor derivative of a bioorthogonal group as described in any one of claims 1-2 or a covalent inhibitor derivative of a bioorthogonal group as described in any one of claims 3-8 in tumor immunotherapy.

10. The application according to claim 9, characterized in that, The covalent inhibitor derivative of the bioorthogonal group targets and binds to oncoproteins.

Images