Small molecule drug conjugate for tumor targeted immunotherapy and application of small molecule drug conjugate

Abstract

The invention relates to a small-molecule drug conjugate for tumor targeted immunotherapy and application thereof, the structure of the small-molecule drug conjugate is as shown in formula I or formula II, and the small-molecule drug conjugate comprises a folate receptor targeted small-molecule ligand, a spacer region, a cleavable sulfur-containing connector and an immunotherapy pharmacophore. Experiments prove that the small molecule drug conjugate provided by the invention can be selectively combined with a ligand part of a folic acid receptor, a payload with an immunosuppressive cell reprogramming drug effect is delivered into folic acid receptor positive immunosuppressive cells in tumor tissues in a targeted manner, and the small molecule drug conjugate is cracked in the cells to release a complete payload; by exerting the immunotherapy function of the effective load, the immunosuppressive tumor microenvironment is improved, T cell immunity is promoted, and the anti-tumor effect is achieved. The invention further discloses a preparation method of the small molecule drug conjugate and application of the small molecule drug conjugate in preparation of anti-tumor targeted immunotherapy drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a small molecule drug conjugate for tumor targeted immunotherapy and an application thereof. Background Art

[0002] As the focus of tumor treatment, immunotherapy research has developed rapidly in recent years. As an emerging tumor treatment method, immunotherapy plays a role in inhibiting tumor growth and spread by regulating or enhancing the function of the body's immune system. The tumor microenvironment (TME) is a key factor leading to tumor growth, metastasis and drug resistance. It is also a complex network composed of immune cells, extracellular matrix, blood vessels and various molecular signals. In the TME, tumor-associated macrophages (TAMs) are gradually revealed as a key component with their plasticity and multifunctionality. Macrophages in the TME show two main phenotypes: M1-TAM and M2-TAM. Among them, M1-TAM secretes pro-inflammatory cytokines, recruits leukocyte infiltration to promote immune response, and promotes the phagocytosis of tumor cells by macrophages, thereby exerting anti-tumor immune effects; M2-TAM secretes anti-inflammatory cytokines, promotes the formation of blood vessels at the tumor site, inhibits the body's immune response, and thus plays a pro-tumor effect. The existence of TAMs not only promotes tumor growth and metastasis, but also contributes significantly to the mechanism of tumor immune escape, hindering the effectiveness of existing immunotherapy strategies. Therefore, targeted regulation of TAMs is crucial to enhancing the effect of immunotherapy and improving the immunosuppressive tumor microenvironment. However, current treatment strategies for TAMs have problems of insufficient selectivity and large side effects, which limit their clinical application. This background has spawned the study of cell-specific drug delivery systems, and the development of targeted immunotherapy drugs that precisely target the TME and reduce toxic side effects is a research focus in this field.

[0003] The classic small molecule drug conjugate SMDC consists of four parts, namely, a targeting ligand, a spacer, a cleavable bond / bridge, and a therapeutic payload. It is composed of small molecules, which makes it easy to control the synthesis process and cost. Compared with antibody drugs, the industrial operation is simple; secondly, SMDC theoretically will not be immunogenic, and safety control is easier to achieve; and SMDC has a small molecular weight, so it can have better cell permeability and better in vitro and in vivo stability in solid tumors. However, the choice of its ligand is limited by factors such as binding affinity, target selectivity, size of the compound, and difficulty in obtaining ligands. The design of the linker also needs to consider its stability and targeting to ensure the effective release and action of the drug in the body, so there are many difficulties in its design and development.

[0004] Based on the above research background, it is necessary to develop new small molecule drug conjugates, especially drugs that improve the therapeutic selectivity of TAMs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a small molecule drug conjugate or its isomers for tumor targeted immunotherapy, a pharmaceutically acceptable salt thereof, and a synthesis method and application thereof.

[0006] To solve the above problems, the technical solution adopted by the present invention is:

[0007] Technical Theme 1

[0008] A small molecule drug conjugate or its isomer, and a pharmaceutically acceptable salt thereof for tumor targeted immunotherapy, wherein the structures thereof are shown in Formula I and Formula II:

[0009]

[0010] W and Q are selected from the following structures:

[0011] (1)W is -(CH2) m C(O)NH-, Q is -(CH2) j -, wherein m is selected from 0, 1, 2, 3, 4, 5, and j is selected from 1, 2, 3, 4 or 5;

[0012] (2)W is -(CH2) n -、-(CH2) n1 CH=CH(CH2) n2 -、-(CH2) n1 C≡C(CH2) n1 -, Q is an aryl group, a 5-6-membered heteroaryl group containing 1-3 selected from N, O, and S, a 5-6-membered heterocyclic group containing 1-3 selected from N, O, and S, or a three-membered to six-membered saturated or unsaturated monocyclic alkyl group, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, or 8; n1 and n2 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0013] X is selected from (i) a sulfur-containing amino acid; (ii) an amide group formed by condensation of a hydrophilic amino acid and a sulfur-containing amino acid;

[0014] L is selected from a sulfur-containing linker that can be cleaved by glutathione GSH reduction;

[0015] Y is selected from a payload having a pharmacodynamic effect on reprogramming immunosuppressive cells.

[0016] As a further improvement of the present invention, the sulfur-containing amino acid is selected from cysteine, methionine, and cystine;

[0017] The hydrophilic amino acid is selected from the group consisting of glycine, tyrosine, cysteine, glutamic acid, aspartic acid, asparagine, glutamine, threonine, serine, lysine, and arginine;

[0018] The 5-6 membered heteroaryl group containing 1-3 selected from N, O, and S is selected from thienyl, pyrrolyl, furanyl, pyridine, and imidazolyl;

[0019] The 5-6 membered heterocyclic group containing 1-3 selected from N, O, and S is selected from piperazinyl, morpholinyl, piperidine, and tetrahydropyrrolyl;

[0020] The Y is selected from a PI3Kγ inhibitor or a TLR agonist.

[0021] As a further improvement of the present invention, the W is -(CH2) m C(O)NH-, Q is -(CH2) j -, wherein m is selected from 0, 1, 2, 3; j is selected from 1, 2, 3, 4 or 5.

[0022] As a further improvement of the present invention, the W is -(CH2) n -、-(CH2) n1 CH=CH-、-(CH2) n1 C≡C-, Q is selected from The n, n1 and n2 are independently selected from 1, 2, 3, 4, 5 or 6.

[0023] As a further improvement of the present invention, the X is selected from cysteine, methionine, cystine or an amide group formed by condensation of any one of them with glutamic acid.

[0024] As a further improvement of the present invention, said L is selected from The k is selected from 0, 1, 2, 3, 4, and 5.

[0025] As a further improvement of the present invention, the Y is selected from PI3Kγ inhibitors AS-252424 or IPI-549, or TLR agonist IMDQ.

[0026] As a further improvement of the present invention, the compound is selected from:

[0027]

[0028]

[0029]

[0030]

[0031] In the present invention, although the compound of formula I or formula II is represented as a 4-oxy form and is so represented throughout the specification, the oxygen exists in a tautomeric equilibrium with the corresponding 4-hydroxyl group. Therefore, the compound of formula I or formula II is considered to include the 4-oxyl group shown in the structural formula and its tautomeric 4-hydroxyl form.

[0032] The "pharmaceutically acceptable salt" of the present invention refers to the salt of the compound of the present invention, which is prepared by the compound of the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of the compound of the present invention with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases, and the salts of the inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. The salts of the organic non-toxic bases include salts of primary amines, secondary amines and tertiary amines, including substituted amines and cyclic amines. For example: N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and the like.

[0033] Technical Theme 2

[0034] A composition comprising the small molecule drug conjugate or its isomers for tumor targeted immunotherapy as described in technical subject one, and a pharmaceutically acceptable salt thereof, which also includes at least one pharmaceutically acceptable carrier or excipient.

[0035] Technical Theme 3

[0036] A use of a small molecule drug conjugate for tumor targeted immunotherapy or its isomers, and a pharmaceutically acceptable salt thereof as described in Technical Topic 1 in the preparation of an anti-tumor targeted immunotherapy drug.

[0037] As a further improvement of the present invention, the tumor is one or more of ovarian cancer, liver cancer, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, and melanoma.

[0038] The beneficial effects of adopting the above technical solution are:

[0039] The present invention provides a class of small molecule drug conjugates. Experiments have shown that the small molecule drug conjugates provided by the present application can selectively bind to the ligand portion of the folate receptor, and deliver the payload having the pharmacodynamic effect of reprogramming immunosuppressive cells to the folate receptor-positive immunosuppressive cells in tumor tissues, and release the complete payload through intracellular cleavage. By exerting the immunotherapeutic function of the payload, the immunosuppressive tumor microenvironment can be improved, T cell immunity can be promoted, and anti-tumor effects can be exerted, which has broad application prospects in tumor targeted immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow cytometry result of M2 macrophages co-incubated with some Ⅰ-P / Ⅱ-P series compounds prepared in the examples and the fluorescent probe GT-NIR-1, CON represents the blank control group;

[0041] Figure 2 The fluorescence intensity analysis results of the Ⅰ-P / Ⅱ-P and Ⅰ-T / Ⅱ-T series compounds prepared in the examples and the fluorescent probe GT-NIR-1 after co-incubation with in vitro macrophages were measured by flow cytometry; wherein: (a) Ⅰ-P / Ⅱ-P series compounds were co-incubated with the fluorescent probe GT-NIR-1; (b) Ⅰ-T / Ⅱ-T series compounds were co-incubated with the fluorescent probe GT-NIR-1; Cell is a macrophage blank control group;

[0042] Figure 3 The in vitro cleavage curves of Ⅰ-P-4a and Ⅰ-T-4a prepared in the example under the action of 10 mM GSH over time; wherein: (a) compound Ⅰ-T-4a; (b) compound Ⅰ-P-4a;

[0043] Figure 4 This is a graph of the in vitro cleavage time of the Ⅰ-P / Ⅱ-P and Ⅰ-T / Ⅱ-T series compounds prepared in the examples under the action of 10 mM GSH; wherein: (a) Ⅰ-T / Ⅱ-T series compounds; (b) Ⅰ-P / Ⅱ-P series compounds; the ordinate is the complete cleavage time;

[0044] Figure 5 The protein expression results of the Ⅰ-P / Ⅱ-P series compounds prepared in the examples after incubation with macrophages were determined by Western blot experiments;

[0045] Figure 6 The expression results of PI3Kγ after incubation with macrophages were determined by Western blot experiment for the I-P / II-P series compounds prepared in the examples;

[0046] Figure 7 The I-P / II-P series compounds prepared in the examples were tested by Western blot for the expression of M1 / M2 markers on the surface of macrophages after incubation with macrophages; wherein: (a) CD206 expression results; (b) iNOS expression results;

[0047] Figure 8 The expression results of IL-10 after incubation with macrophages by Western blot experiment were determined for the Ⅰ-P / Ⅱ-P series compounds prepared in the examples;

[0048] Fig. 9 The expression results of TNF-α (a) and IL-6 (b) of the I-P / II-P series compounds prepared in the examples after incubation with macrophages were determined by Western blot experiments;

[0049] Fig.10 The protein expression results of the Ⅰ-T / Ⅱ-T series compounds and IMDQ prepared in the examples after incubation with macrophages were determined by Western blot experiments;

[0050] Fig.11 The expression results of TLR7 after incubation with macrophages were determined by qRT-PCR experiments for the Ⅰ-T / Ⅱ-T series compounds and IMDQ prepared in the examples;

[0051] Fig.12 The expression results of M1 / M2 markers on the surface of macrophages after incubation with macrophages by qRT-PCR experiments for the Ⅰ-T / Ⅱ-T series compounds prepared in the examples; wherein: (a) CD206 expression results; (b) iNOS expression results;

[0052] Fig.13 The expression results of IL-10 (a) and IL-6 (b) of the Ⅰ-T / Ⅱ-T series compounds prepared in the examples after incubation with macrophages were determined by qRT-PCR experiments;

[0053] Fig.14 The results of in vivo antitumor activity studies of some I-P / II-P series compounds prepared in the examples, (a) is the mouse tumor volume curve; (b) is the mouse tumor weight; (c) is the mouse body weight change curve;

[0054] Fig.15The results of in vivo antitumor activity studies of I-P-4a and I-P-4b prepared in the examples, (a) is the mouse tumor volume curve; (b) is the mouse tumor weight; (c) is the mouse body weight change curve;

[0055] Fig.16 The results of in vivo antitumor activity studies of some Ⅰ-T / Ⅱ-T series compounds prepared in the examples, (a) is the mouse tumor volume curve; (b) is the mouse tumor weight; (c) is the mouse body weight change curve;

[0056] Fig.17 The results of the in vivo selectivity study of Ⅰ-P-4a prepared in the example, (a) is the mouse tumor volume curve; (b) is the mouse tumor weight; (c) is the mouse body weight change curve; FA is folic acid;

[0057] Fig.18 The results of the in vitro cytotoxicity study of the I-P / II-P series compounds prepared in the examples on DMDM ​​cells;

[0058] Fig.19 The results of the in vitro cytotoxicity study of the Ⅰ-T / Ⅱ-T series compounds prepared in the examples on DMDM ​​cells;

[0059] Fig. 20 The H&E staining results of various organs in mice of the Ⅰ-P / Ⅱ-P series compounds prepared in the examples, from left to right are heart, liver, spleen, lung, and kidney;

[0060] Fig.21 H&E staining results of various organs in mice of the Ⅰ-T / Ⅱ-T series compounds prepared in the examples, from left to right are heart, liver, spleen, lung, and kidney;

[0061] Fig. 22 The results of flow cytometry of tumor-associated macrophages (TAMs) in mouse tumors of the I-P / II-P series compounds prepared in the examples; wherein: (a) TAMs in CD45 + (b) The ratio of M1 and M2 macrophages, which is represented by F4 / 80 + CD86 in total macrophages + (M1) and CD206 + (c) is a graph showing the ratio of M1 and M2 macrophages in mouse tumors of the Ⅰ-T / Ⅱ-T series compounds prepared in the examples, which is determined by F4 / 80 + CD86 in total macrophages + (M1) and CD206 + (M2) percentage ratio determination;

[0062] Fig.23 The results of flow cytometry of the compounds prepared in the examples on myeloid-derived suppressor cells (MDSCs) in mouse tumors; wherein: (a) and (b) are the results of flow cytometry of the compounds of the Ⅰ-P / Ⅱ-P series on myeloid-derived suppressor cells (MDSCs) in mouse tumors; (a) PMN-MDSCs in CD45 + The proportion of M-MDSCs in immune cells; (b) M-MDSCs in CD45 + (c) and (d) are the flow cytometry results of the Ⅰ-T / Ⅱ-T series compounds prepared in the examples on myeloid-derived suppressor cells (MDSCs) in mouse tumors; (c) Ly6C + In CD45 + The proportion of Ly6G in immune cells; (d) + In CD45 + The proportion of immune cells;

[0063] Fig.24 The flow cytometry results of the I-P / II-P series compounds prepared in the examples on T cells in mouse tumors; wherein: (a) CD3 + T cells on CD45 + The proportion of immune cells; (b) CD3 + CD4 + T cells on CD45 + The proportion of immune cells; (c) CD3 + CD8 + T cells on CD45 + The proportion of immune cells;

[0064] Fig.25 The flow cytometry results of the I-T / II-T series compounds prepared in the examples on T cells in mouse tumors; wherein: (a) CD3 + CD8 + T cells on CD45 + The proportion of immune cells; (b) CD3 + CD4 + T cells on CD45 + The proportion of immune cells. DETAILED DESCRIPTION

[0065] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0066] Example 1 Substrate III-5a

[0067]

[0068] III-1a (0.98 g, 10 mmol), oxalyl chloride (7.61 g, 60 mmol) and anhydrous CH2Cl2 (20 mL) were added to a 250 mL single-necked flask in sequence. The obtained solution was refluxed for 1 h and then cooled to room temperature. After the solvent was evaporated under reduced pressure, the residue of III-2a was dissolved in 20 mL of ether. The obtained solution was added dropwise to a diazomethane solution (generated by 15 g of Diazald through an Aldrich Mini Diazald device) under ice-water bath conditions, and the addition time was greater than 10 min. After the dropwise addition was completed, it was allowed to stand for 30 min and then stirred for 1 h. 48% aqueous hydrogen bromide solution (20 mL) was added dropwise to the above solution. Refluxed for 1.5 h. After the mixture was cooled to room temperature, the organic layer was separated and the aqueous layer was extracted with ether (200 mL×2). The organic layer and the ether extract were combined, washed twice with 10% Na2CO3 solution, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure to obtain Ⅲ-4a with a yield of 92%. Ⅲ-4a (about 9.2 mmol) was added to anhydrous DMF (25 mL) of 2,4-diamino-6-hydroxypyrimidine (1.26 g, 10 mmol). The resulting mixture was stirred at room temperature under nitrogen protection for 3 days. After the solvent was evaporated under reduced pressure, a sample was prepared and purified by column chromatography (CH2Cl2:CH3OH=100:1.5~100:2.5) to obtain a white powder Ⅲ-5a (1.32 g, yield 71%). 1 H-NMR 1 H-NMR (400MHz, DMSO-d6): δ = 10.82 (s, 1H), 10.17 (s, 1H), 6.02 (s, 2H), 5.94 (s, 1H), 2.79 (s, 1H), 2.67 (t, J = 7.0Hz, 2H), 2.45 (s, 2H). HRMS (APCI): Calcd.for C 10 H 10 N4O[M+H] + :203.0927,found 203.0930.

[0069] Example 2 Substrate III-5b

[0070] A method similar to Example 1 was used to obtain white powdery solid substrate III-5b (1.12 g, yield 52%). 1 H-NMR (400MHz, DMSO-d6)δ=11.35(s,1H),10.99(s,1H),6.89(s,2H),6.00(s,1H),2.81(s,1H) ,2.60(t,J=7.2Hz,2H),2.27–2.12(m,2H),1.93–1.61(m,J=6.5Hz,2H).HRMS(APCI):Calcd.for C11 H 12 N4O[M+H] + :217.1084,found 217.1082.

[0071] Example 3 Substrate III-6a

[0072]

[0073] In a 250 ml round-bottom flask equipped with a magnetic stirrer and a gas inlet, tetrakis(triphenylphosphine)palladium (185 mg, 0.16 mmol), triethylamine (1.01 g, 10 mmol), methyl p-iodobenzoate (393 mg, 1.5 mmol) and anhydrous DMF (20 mL) were added. To the stirred mixture, copper iodide (30 mg, 0.16 mmol) and substrate III-5a (202 mg, 1 mmol) were added under N2, and the reaction mixture was stirred at room temperature overnight. After evaporating the solvent under reduced pressure, column chromatography (CH2Cl2:CH3OH=25:1-7:1) was used to obtain yellow powder III-6a (1.12 g, yield 80%). 1 H NMR (600MHz, DMSO-d6) δ10.89(s,1H),10.23(d,J=13.6Hz,1H),7.90(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),6.08(d,J=14.6Hz ,2H),6.00(d,J=2.1Hz,1H),4.30(q,J=7.1Hz,2H),2.77(dd,J=13.6,5.9Hz,4H),1.30(d,J=7.1Hz,3H).HRMS(APCI):Calcd.for C 19 H 18 N4O3[M+H] + :351.1452,found351.1445.

[0074] Example 4 Substrate III-6b

[0075] A similar method to Example 3 was used to obtain yellow powdery solid substrate III-6b (1.20 g, yield 82%). 1HNMR(600MHz,DMSO-d6)δ10.89(s,1H),10.23(d,J=13.6Hz,1H),7.90(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),6.08(d,J=14.6Hz,2H),6.0 0(d,J=2.1Hz,1H),4.30(q,J=7.1Hz,2H),2.77(dd,J=13.6,5.9Hz,4H),1.67–1.61(m,2H),1.30(d,J=7.1Hz,3H).HRMS(APCI):Calcd.for C 20 H 20 N4O3[M+H] + :365.1608, found 365.1619.

[0076] Example 5 Substrate III-7a

[0077]

[0078] Compound III-6a (0.62 g, 1.77 mmol) was added into a 25 mL single-necked flask equipped with a magnetic stirrer, dissolved with NaOH (6 mL, 1 mol / L) solution at room temperature, and then hydrochloric acid solution (1 mol / L) was slowly added dropwise to adjust the pH of the solution to about 2-3. A large amount of solid precipitated, filtered and washed with a little cold distilled water, and the filter cake was dried to obtain a brown solid substrate III-7a (0.40 g, yield 70%). 1 H NMR (600MHz, DMSO-d6) δ10.89(s,1H),10.23(d,J=13.6Hz,1H),7.90(d,J=8.4Hz,2H),7.49(d,J=8.4Hz, 2H),6.08(d,J=14.6Hz,2H),6.00(d,J=2.1Hz,1H),2.77(dd,J=13.6,5.9Hz,4H).HRMS(APCI):Calcd.for C 17 H 14 N4O3[M+H] + :323.1139,found323.1146.

[0079] Example 6 Substrate III-7b

[0080] A similar method to Example 5 was used to obtain a brown solid substrate III-7b (0.41 g, yield 72%). 1H NMR (600MHz, DMSO-d6) δ10.89(s,1H),10.23(d,J=13.6Hz,1H),7.90(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),6.08 (d,J=14.6Hz,2H),6.00(d,J=2.1Hz,1H),2.77(dd,J=13.6,5.9Hz,4H),1.67–1.61(m,2H).HRMS(APCI):Calcd.for C 18 H 16 N4O3[M+H] + :337.1295,found337.1291.

[0081] Example 7 Substrate III-8a

[0082]

[0083] Substrate III-7a (0.19 g, 0.59 mmol), CDMT (0.19 g, 1.08 mmol), NMM (0.11 g, 1.08 mmol) and 40 mL of anhydrous DMF were added to a 250 mL single-necked flask equipped with a magnetic stirrer and stirred at room temperature for 2 h. Samples were taken, and then S-(triphenylmethyl)-L-cysteine ​​tert-butyl ester hydrochloride (0.31 g, 0.74 mmol) and NMM (0.11 g, 1.08 mmol) dissolved in 32 mL of DMF were added to the reaction solution under stirring at room temperature. The reaction was allowed to proceed for 4 h. The reaction was stopped after TLC detection of the complete reaction. The solvent was evaporated under reduced pressure and the sample was prepared. The compound was purified by column chromatography (CH2Cl2:CH3OH=25:1-7:1) to obtain a yellow solid compound. 24 mL of anhydrous CH2Cl2, 9.2 mL of TFA, and 0.92 mL of triethylsilane were added thereto in sequence. The reaction was allowed to proceed for 3 h, and the color of the solution changed from yellow to light yellow. The solvent was evaporated under reduced pressure to obtain yellow-white solid substrate III-8a (0.22 g, yield 88%). 1 H NMR (600MHz, DMSO-d6) δ10.89(s,1H),10.23(d,J=13.6Hz,1H),7.90(d,J=8.4Hz,2H),7.76(d,J=6.6Hz,1H),7.49(d,J=8.4Hz,2H),6.08(s,2H),6. 00(s,1H),4.07(q,J=5.4Hz,1H),2.95(dd,J=13.1,5.0Hz,1H),2.79(dd,J=13.1,4.5Hz,1H),2.77(dd,J=13.6,5.9Hz,4H).HRMS(APCI):Calcd.forC 20 H19 N5O4S[M+H] + :426.1231, found 426.1239.

[0084] Example 8 Substrate III-8b

[0085] A similar method to Example 7 was used to obtain yellow-white powder solid substrate III-8b (0.23 g, yield 87%). 1 HNMR(600MHz,DMSO-d6)δ10.85(s,1H),10.17(s,1H),8.21(d,J=6.4Hz,1H), 7.82(d,J=8.2Hz,2H),7.47(d,J=8.2Hz,2H),6.07(s,2H),5.92(s,1H),4.47 (s,1H),3.05(dd,J=13.3,5.0Hz,1H),2.85(dd,J=13.0,5.7Hz,1H),2.64(t,J=7.1Hz,2H),2.48–2.45(m,2H),1.88–1.84(m,2H).HRMS(APCI):Calcd.for C 21 H 21 N5O4S[M+H] + :440.1387, found 440.1392.

[0086] Example 9 Substrate III-9a

[0087]

[0088] A similar method to Example 7 was used to obtain yellow-white powder solid substrate III-9a (0.21 g, yield 65%). 1 HNMR(600MHz,DMSO-d6)δ10.86(s,1H),10.18(s,1H),8.69(d,J=6.9Hz,1H),7.99(s, 1H),7.85(d,J=8.1Hz,2H),7.47(d,J=8.1Hz,2H),5.99(s,2H),5.92(s,1H),4.45(s,1 H),4.33(t,J=10.5Hz,1H),3.17(s,1H),2.84–2.78(m,1H),2.66–2.63(m,2H),2.51– 2.48(m,2H),2.12(dd,J=15.4,9.9Hz,2H),2.07–1.95(m,2H).HRMS(APCI):Calcd.for C 25 H 26 N6O7S[M+H] +:555.1656, found 555.1668.

[0089] Example 10 Substrate III-9b

[0090] A similar method to Example 7 was used to obtain yellow-white powder solid substrate III-9b (0.20 g, yield 61%). 1 HNMR(600MHz,DMSO-d6)δ10.86(s,1H),10.18(s,1H),8.69(d,J=6.9Hz,1H),7.99(s,1H), 7.85(d,J=8.1Hz,2H),7.47(d,J=8.1Hz,2H),5.99(s,2H),5.92(s,1H),4.45(s,1H),4.33( t,J=10.5Hz,1H),3.17(s,1H),2.84–2.78(m,1H),2.66–2.63(m,2H),2.48–2.45(m,2H),2 .07(dd,J=15.4,9.9Hz,2H),2.00–1.92(m,2H),1.88–1.84(m,2H).HRMS(APCI):Calcd.for C 26 H 28 N6O7S[M+H] + :569.1813, found 569.1804.

[0091] Example 11 Substrate III-10a

[0092]

[0093] Compound III-6a (0.62 g, 1.77 mmol), palladium carbon (0.08 g, 0.75 mmol) and 80 mL MeOH were added to a 250 mL single-necked flask equipped with a magnetic stirrer. After stirring at 40 ° C for 12 h under hydrogen conditions, the reaction of compound III-6a was complete by TLC detection, and the reaction was stopped. Cooled to room temperature, the solvent was evaporated under reduced pressure to obtain a light yellow powder (0.59 g, 1.68 mmol). The above solid was dissolved with NaOH (6 mL, 1 mol / L) solution at room temperature, and then hydrochloric acid solution (1 mol / L) was slowly added dropwise to adjust the solution pH to about 2-3. A large amount of solid precipitated, filtered and washed with a little cold distilled water, and the filter cake was dried to obtain a yellow-white solid III-10a (0.49 g, yield 90%). 1H NMR(600MHz,DMSO-d6)δ10.79(s,1H),10.30–10.06(m,1H),7.86(d,J=8.0Hz,2H),7.32(d,J=8.0Hz,2H),6 .03(s,2H),5.84(s,1H),2.65(d,J=6.5Hz,2H),2.46(d,J=7.8Hz,2H),1.58(s,4H).HRMS(APCI):Calcd.for C 17 H 18 N4O3[M+H] + :327.1412, found 327.1416.

[0094] Example 12 Substrate III-10b

[0095] A similar method to Example 11 was used to obtain yellow powdery solid substrate III-10b (0.50 g, yield 87%). 1 HNMR(600MHz,DMSO-d6)δ10.80(s,1H),10.33–10.24(m,1H),7.85(d,J=8.1Hz,2H),7.33(d,J=7.9Hz,2H),6.13(d,J=14.7Hz,2 H),5.83(s,1H),2.63(t,J=7.6Hz,2H),2.45(t,J=7.5Hz,2H),1.61–1.56(m,4H),1.29(t,J=7.4Hz,2H).HRMS(APCI):Calcd.for C 18 H 20 N4O3[M+H] + :341.1569, found 341.1561.

[0096] Example 13 Substrate III-11a

[0097]

[0098] A yellow-white powdery solid substrate III-11a (0.14 g, yield 55%) was obtained in a similar manner to Example 7. 1HNMR(600MHz,DMSO-d6)δ10.79(s,1H),10.30–10.06(m,1H),7.86(d,J=8.0Hz,2 H),7.81(d,J=8.1Hz,1H),7.32(d,J=8.0Hz,2H),6.03(s,2H),5.84(s,1H),4.11 (q,J=5.4Hz,1H),2.98(dd,J=13.1,4.9Hz,1H),2.72(dd,J=13.3,5.8Hz,1H),2. 65(d,J=6.5Hz,2H),2.46(d,J=7.8Hz,2H),1.58(s,4H).HRMS(APCI):Calcd.for C 20 H 23 N5O4S[M+H] + :430.1544,found 430.1539.

[0099] Example 14 Substrate III-11b

[0100] A yellow-white powdery solid substrate III-11b (0.12 g, yield 46%) was obtained in a similar manner to Example 7. 1 HNMR(600MHz,DMSO-d6)δ10.80(s,1H),10.33–10.24(m,1H),7.85(d,J=8.1Hz, 2H),7.72(d,J=8.2Hz,1H),7.33(d,J=7.9Hz,2H),6.13(d,J=14.7Hz,2H),5.83( s,1H),4.15(q,J=5.4Hz,1H),2.91-2.85(m,2H),2.63(t,J=7.6Hz,2H),2.45(t, J=7.5Hz,2H),1.61–1.56(m,4H),1.29(t,J=7.4Hz,2H).HRMS(APCI):Calcd.for C 21 H 25 N5O4S[M+H] + :444.1700, found 444.1709.

[0101] Example 15 Substrate III-12a

[0102]

[0103] A yellow-white powder solid substrate III-12a (0.11 g, yield 35%) was obtained in a similar manner to Example 7. 1HNMR(600MHz,DMSO-d6)δ10.67(s,1H),8.09(d,J=5.7Hz,2H),7.82(d,J=6.5Hz,2H), 7.75(d,J=7.6Hz,2H),7.33(d,J=7.6Hz,2H),6.20(s,2H),6.15(s,1H),4.15–4.11(m ,2H),3.16–2.78(m,2H),2.60(d,J=7.7Hz,2H),2.46(t,J=6.5Hz,2H),2.25(s,2H),2 .04(dd,J=22.7,9.4Hz,2H),1.58(s,4H),1.30–1.27(m,2H).HRMS(APCI):Calcd.for C 25 H 30 N6O7S[M+H] + :558.1891, found 558.1899.

[0104] Example 16 Substrate III-12b

[0105] A yellow-white powder solid substrate III-12b (0.14 g, yield 41%) was obtained in a similar manner to Example 7. 1 HNMR(600MHz,DMSO-d6)δ10.78(s,1H),8.02(d,J=5.4Hz,2H),7.96(d,J=6.1Hz,2H),7.73(d, J=7.4Hz,2H),7.23(d,J=7.8Hz,2H),6.26(s,2H),6.17(s,1H),4.15–4.11(m,2H),3.18(d,J=1 2.0Hz,1H),3.06–2.98(m,1H),2.60(d,J=7.7Hz,2H),2.46(t,J=6.2Hz,2H),2.21(s,2H),2.02 (dd,J=22.7,9.4Hz,2H),1.58(s,4H),1.28–1.25(m,2H),1.23(s,2H).HRMS(APCI):Calcd.for C 26 H 32 N6O7S[M+H] + :573.2126, found 573.2138.

[0106] Example 17 Substrate III-13a

[0107]

[0108] A yellow-white powder solid substrate III-13a (0.15 g, yield 34%) was obtained in a similar manner to Example 7. 1 HNMR(600MHz,Methanol-d4)δ8.30(d,J=8.2Hz,1H),7.50(d,J=6.6Hz,1H),7. 02(d,J=6.8Hz,1H),6.11(s,1H),5.88(s,2H),4.53(dt,J=8.1,4.9Hz,1H),2.9 5(ddd,J=12.4,6.6,4.8Hz,1H),2.88(ddd,J=12.4,6.7,4.9Hz,1H),2.85–2.68 (m,4H),2.22(t,J=6.7Hz,1H),1.86(qd,J=6.7,0.7Hz,2H),1.80–1.70(m,2H); 13 C NMR(151MHz,Methanol-d4)δ175.67,162.51,159.32,153.24,150.62,149.38,137.01 ,134.58,130.21,126.78,105.49,105.17,56.67,30.83,29.89,29.42,28.84,28.26.

[0109] HRMS(APCI):Calcd.for C 18 H 20 N5O4S2[MH] - :434.0962,found,434.0965.

[0110] Example 18 Substrate III-13b

[0111]

[0112] A similar method to Example 7 was used to obtain yellow-white powder solid substrate III-13b (0.12 g, yield 33%). 1HNMR (600MHz, Methanol-d4) δ8.32(d,J=8.4Hz,1H),8.26(d,J=8.4Hz,1H),7.49(d,J=6.8Hz,1H),7.02(d,J=6.8Hz,1H),6.11(s,1H),5.88(s,2H),4.45 (dt,J=8.6,5.1Hz,1H),4.38(dt,J=8.6,6.5Hz,1H),2.92–2.68(m,6H),2.4 5–2.29(m,2H),2.11(t,J=6.7Hz,1H),1.93–1.83(m,4H),1.80–1.70(m,2H); 13 CNMR(151MHz,Methanol-d4)δ175.89,175.07,174.90,161.91,159.32,153.24,150.62,149.38,137.01,134.58,130 .21,126.78,105.49,105.17,56.04,53.35,36.48,30.83,29.89,29.45,28.84,28.26,27.24.HRMS(APCI):Calcd.for C 23 H 27 N6O7S2[MH] - :563.1388,found,563.1386.

[0113] Example 19 Substrate III-14a

[0114]

[0115] A similar method to Example 7 was used to obtain yellow-white powder solid substrate III-14a (0.17 g, yield 32%). 1 HNMR (600MHz, DMSO-d6) δ8.63(s,1H),7.91(d,J=7.1Hz,1H),7.63(d,J=8.4Hz,2H),6.94(t,J=6.2Hz,1H),6.66(d, J=8.6Hz,2H),4.48(d,J=5.9Hz,2H),4.38–4.33(m,1H),2.97(dd,J=13.4,4.6Hz,1H),2.87(dd,J=13.3,7.2Hz,1H); 13C NMR(151MHz,DMSO-d6)δ173.23,172.56,166.39,161.81,156.76,154.74,151.25,148.95,148.78,1 29.23,128.42,122.01,111.77,56.04,46.40,40.88,31.20,26.59,21.60; HRMS(APCI):Calcd.forC 17 H 17 N7O4S[MH] - :414.0990,found,414.0981.

[0116] Example 20 Substrate III-14b

[0117]

[0118] A yellow-white powder solid substrate III-14b (0.19 g, yield 33%) was obtained in a similar manner to Example 7. 1 HNMR(600MHz,DMSO-d6)δ9.56(s,1H),8.50(s,1H),8.26(d,J=8.4Hz,1H), 7.86–7.80(m,2H),6.80–6.74(m,2H),6.10(s,2H),5.67(t,J=6.0Hz,1H),5 .07(t,J=6.5Hz,1H),4.68(d,J=6.0Hz,2H),4.44(dt,J=8.6,5.1Hz,1H),2 .87(dd,J=6.6,5.1Hz,2H),2.47(td,J=8.5,1.6Hz,2H),2.31–2.08(m,3H). 13 C NMR (151MHz, DMSO-d6) δ175.89,174.49,174.35,166.16,161.42,155.84,155.19,151.77,149.67,149. 60,132.06,125.94,123.99,113.24,72.03,56.04,41.29,32.83,29.45,27.93.HRMS(APCI):Calcd.for C 22 H 22 N7O8S[M+H] - :544.1256,found,544.1259.

[0119] Example 21 Substrate IV-2

[0120]

[0121] Compound IV-1 (3.00 mL, 33.1 mmol) and 30 mL of anhydrous CH2Cl2 were added to a 250 mL single-necked flask equipped with a magnetic stirrer, and 2-mercaptoethanol (2.33 mL, 33.25 mmol) was added dropwise under nitrogen protection and stirred at 0°C. After reacting at 0°C for 30 min, 2-mercaptopyridine (3.68 g, 33.11 mmol) dissolved in 48 mL of anhydrous CH2Cl2 was added dropwise under stirring at 0°C. The mixture was reacted at 0°C for 1 h, and then heated to room temperature for 1 h. A large amount of solid precipitated from the solution. The solvent was evaporated under reduced pressure until the reaction liquid was about 37.5 mL, filtered, and the filter cake was washed with a little cold CH2Cl2 until the filtrate became colorless and transparent, and the filter cake was dried to obtain a yellow-white solid IV-2 (5.86 g, yield 79%).

[0122] Example 22 Substrate IV-3

[0123]

[0124] Di(trichloromethyl carbonate) (1.08 g, 3.64 mmol) and 8 mL of anhydrous CH2Cl2 were added to a 100 mL single-necked flask equipped with a magnetic stirrer. Under nitrogen protection, compound IV-2 (2.0 g, 8.94 mmol) and triethylamine (0.92 g, 9.10 mmol) dissolved in 32 mL of anhydrous CH2Cl2 were added dropwise at 0°C with stirring. The mixture was heated to room temperature for 3 hours, and the white precipitate in the reaction solution turned into a light yellow clear solution. Then, under nitrogen protection, HOBt (1.24 g, 9.18 mmol) and triethylamine (0.92 g, 9.10 mmol) dissolved in 6 mL of anhydrous CH2Cl2 were added dropwise at 0°C with stirring, and the mixture was heated to room temperature for overnight reaction. The solvent was evaporated under reduced pressure until a solid was clearly precipitated. The mixture was filtered and the filter cake was washed with a little cold CH2Cl2 until the filtrate became colorless and transparent. The filter cake was dried to obtain a light white solid IV-3 (1.52 g, yield 44%).

[0125] Example 23 Substrate IV-4

[0126]

[0127] Substrate IV-3 (0.31 g, 0.81 mmol), PI3Kγ inhibitor (0.16 g, 0.52 mmol), and 8 ml of anhydrous CH2Cl2 were added to a 25 mL single-necked flask equipped with a magnetic stirrer, and DIPEA (0.14 g, 1.08 mmol) was added under stirring at room temperature, and the reaction was stopped after 4 h of reaction detection by TLC. The solvent was evaporated under reduced pressure and purified by preparative thin layer chromatography (CH2Cl2:CH3OH=23:1-22:1) to obtain a yellow solid substrate IV-4 (0.33 g, yield 65%). 1H NMR (600MHz, DMSO-d6) δ8.45(d,J=4.1Hz,1H),7.92(dd,J=8.7,6.8Hz,1H),7.79(t,J=7.6Hz,1H),7.75(d,J=7.9Hz,1H),7.62(s,1H),7.48(d,J= 9.3Hz,1H),7.44(t,J=8.3Hz,1H),7.25–7.23(m,1H),7.20(d,J=2.9Hz,1H),6.96(d,J=2.9Hz,1H),4.49(t,J=5.6Hz,2H),3.22(t,J=5.9Hz,2H); 13 C NMR(151MHz,DMSO-d6)δ169.03,167.50,163.02,161.36,159.06,152.24,151.48,150.16,149.43,147.89,138.26 ,121.88,121.70,121.18,119.98,119.02,118.08,115.36,113.10,112.14,67.18,36.93; HRMS(APCI):Calcd.for C 22 H 15 FN2O6S3[M+H] + :519.0149,found,519.0141.

[0128] Example 24IV-5

[0129]

[0130] A similar method to Example 23 was used to obtain yellow solid compound IV-5 (0.53 g, yield 68%). 1 H NMR (600MHz, DMSO-d6) δ8.44(d,J=4.2Hz,1H),7.97(dd,J=8.7,6.5Hz,1H),7.77(t,J=7.7Hz,1H),7.75(d,J=7.9Hz,1H),7.67(s,1H),7.45(d,J=9.1Hz ,1H),7.45(t,J=8.5Hz,1H),7.23–7.21(m,1H),7.18(d,J=2.9Hz,1H),6.94 (d, J=2.9Hz, 1H), 4.47 (t, J=5.6Hz, 2H), 3.31 (s, 3H), 3.20 (t, J=5.8Hz, 2H); 13CNMR(151MHz,DMSO-d6)δ169.43,166.51,163.42,161.39,159.12,152.21,151.43,150.21,149.36,147.99,138.26,1 21.82,121.76,121.43,119.90,119.38,118.17,116.36,113.19,112.17,67.38,38.77,36.99; HRMS(APCI):Calcd.for C 23 H 19 FN3O5S3[M+H] + :532.0456,found,532.0451.

[0131] Example 25IV-6

[0132]

[0133] A similar method to Example 23 was used to obtain yellow solid compound IV-6 (0.25 g, yield 31%). 1 H NMR(600MHz,DMSO-d6)δ8.92(dd,J=6.8,1.7Hz,1H),8.55(dd,J=4.5,1.7Hz,1H),8.44(d, J=4.2Hz,1H),8.00(d,J=6.8Hz,1H),8.00(s,1H),7.69–7.54(m,6H),7.53–7.43(m,4H),7 .41–7.35(m,2H),7.01(dd,J=6.7,4.5Hz,1H),6.74(s,1H),6.42(s,2H),4.56(quin,J=6. 8Hz,1H),4.49(t,J=5.6Hz,2H),3.82(s,3H),3.22(t,J=5.9Hz,2H),1.35(d,J=6.8Hz,3H); 13C NMR(151MHz,DMSO-d6)δ169.43,166.51,163.42,162.73,161.19,160.93,150.06,149.36 ,147.51,146.74,141.05,138.09,137.81,135.42,133.66,132.56,131.90,129.51,129. 24,129.20,129.17,128.50,126.16,123.41,123.31,118.17,116.36,113.19,107.88,10 2.44,101.15,90.40,87.06,85.94,44.88,38.62,36.99,20.69; HRMS(APCI):Calcd.forC 38 H 32 N9O4S2[M+H] + :742.2013,found,742.2021.

[0134] Example 26I-P-1a

[0135]

[0136] Substrate III-8a (0.10 g, 0.24 mmol), substrate IV-4 (0.08 g, 0.15 mmol) and 6 mL of anhydrous DMSO were added to a 10 mL single-necked flask equipped with a magnetic stirrer, and DIPEA (0.04 g, 0.31 mmol) was added under stirring at room temperature, and the reaction was stopped after TLC detection of the reaction being complete. The yellow solid compound I-1a (0.024 g, yield 12%) was obtained by preparative liquid phase purification. 1 HNMR(600MHz,DMSO-d6)δ10.88(s,1H),10.17(s,1H),8.65(s,1H),7.94–7.90(m,1 H),7.82(d,J=8.0Hz,2H),7.45(d,J=7.8Hz,2H),7.43–7.42(m,1H),7.41–7.38(m,1 H),7.19(s,1H),6.86–6.80(m,2H),6.01(s,3H),4.58(s,1H),4.46(t,J=5.9Hz,2H ),3.17(d,J=10.9Hz,2H),3.11–3.08(m,2H),2.81–2.77(m,2H),2.76–2.72(m,2H). 13C NMR (151MHz, DMSO) δ170.14,163.79,156.63,155.46,150.17,149.30,147.82,136.26,129.27,127.46,125.65,124.27,119.91,117.66,11 6.17,110.36,97.83,96.79,90.68,78.52,64.80,50.36,33.91,29.37,27.11,24.90,18.98,17.35,12.03,-0.76.HRMS(APCI):Calcd.forC 37 H 29 F6O 10 S3[MH] - :831.1019, found 831.1041.

[0137] Example 27I-P-1b

[0138]

[0139] A similar method to Example 26 was used to obtain yellow solid compound I-P-1b (21 mg, yield 23%). 1 H NMR (600MHz, DMSO-d6) δ10.87(s,1H),8.54–8.40(m,2H),7.82(d,J=7.7Hz,2H),7.76(dd,J=8.7,6.9Hz,1H),7.44(d ,J=8.2Hz,2H),7.40(td,J=8.6,2.6Hz,1H),7.27–7.22(m,1H),7.13(d,J=2.8Hz,1H),7.02(d,J=3.4Hz,1H),6.84(dd ,J=5.8,2.6Hz,1H),6.03(s,2H),6.01(s,1H),4.22(d,J=4.5Hz,2H),4.10(dt,J=20.6,6.3Hz,2H),3.20(d,J=4.4Hz, 2H),3.05(d,J=6.2Hz,1H),2.92(s,1H),2.81–2.77(m,2H),2.76–2.73(m,2H),2.09–2.00(m,2H),2.00–1.91(m,2H). 13C NMR (151MHz, DMSO) δ182.20,176.33,174.12,164.88,162.88,161.26,158.82,152.39,151.41,151.03,1 49.59,149.48,137.90,134.06,133.49,131.24,129.76,129.40,127.42,126.49,126.42,125.79,121.36 ,121.23,119.37,106.00,105.85,103.41,103.25,99.78,98.78,92.37,80.63,61.65,59.19,54.17,53.6 2,48.69,43.40,41.34,37.51,32.54,29.12,28.67,26.95,22.59,21.89,19.38.HRMS(APCI):Calcd.forC 42 H 36 F7O 13 S3[MH] - :960.1444, found 960.1406.

[0140] Example 28I-P-2a

[0141]

[0142] A similar method to Example 26 was used to obtain yellow solid compound I-P-2a (22 mg, yield 19%). 1 H NMR (600MHz, DMSO-d6) δ10.85(d,J=9.0Hz,1H),10.18(s,1H),8.56(s,1H),7.92(dd,J=8.8,6.4Hz,1H),7.82(d, J=8.3Hz,2H),7.47(d,J=8.4Hz,2H),7.44(dd,J=9.4,2.6Hz,1H),7.40(td,J=8.4,2.6Hz,1H),7.18(s,1H),6.84 (t,J=4.7Hz,2H),6.00(s,2H),5.92(d,J=1.9Hz,1H),4.54(s,1H),4.45(t,J=6.2Hz,2H),3.16(dd,J=13.3,9.3H z,2H),3.09(dd,J=12.4,6.2Hz,2H),2.64(t,J=7.4Hz,2H),2.46(t,J=7.0Hz,2H),1.85(dd,J=14.5,7.2Hz,2H). 13C NMR (151MHz, DMSO) δ172.14,165.71,158.56,157.44,152.05,151.32,149.82,138.26,131.30,130.23,127.60,126.35,121.91,119.65,11 2.25,112.01,103.23,99.79,98.35,92.85,66.79,35.89,29.11,28.78,27.53,26.57,22.17,21.16,18.64,18.31.HRMS(APCI):Calcd.for C 38 H 31 F6O 10 S3[MH] - :845.1175, found 845.1172.

[0143] Example 29I-P-2b

[0144]

[0145] A similar method to Example 26 was used to obtain yellow solid compound I-P-2b (24 mg, yield 19%). 1 H NMR (600MHz, DMSO-d6) δ10.85(s,1H),8.45(d,J=4.5Hz,2H),7.92(dd,J=8.8,6.4Hz,1H),7.82(d,J=6.4Hz,2H),7.4 5(d,J=8.1Hz,2H),7.42–7.38(m,1H),7.26–7.22(m,1H),7.13(s,1H),6.83(d,J=3.7Hz,1H),6.80(d,J=3.5Hz,1H), 6.03(s,2H),5.92(s,1H),4.46(t,J=6.1Hz,1H),4.21(s,2H),4.09(dd,J=16.8,10.4Hz,1H),3.08–3.05(m,2H),2.9 2(t,J=6.3Hz,2H),2.64(t,J=7.4Hz,2H),2.47–2.44(m,2H),2.00(tdd,J=22.2,16.6,8.7Hz,4H),1.87–1.84(m,2H). 13C NMR (151MHz, DMSO) δ172.43,159.59,152.07,150.94,149.59,137.96,137. 90,131.22,130.21,129.75,127.73,121.23,119.37,118.39,116.40,112.1 9,112.11,106.64,106.41,103.49,99.79,98.37,61.17,59.19,53.19,41.3 2,40.51,29.11,27.53,26.59,21.17,18.31,14.04.HRMS(APCI):Calcd.for C 43 H 38 F7O 13 S3[MH] - :974.1601, found 974.1624.

[0146] Example 30I-P-3a

[0147]

[0148] A similar method to Example 26, except that the substrate III-8a was replaced with the substrate III-15a disclosed in Example 8 of application number CN202310703292.4 Compound I-P-3a (24 mg, yield 21%) was obtained as a yellow solid. 1 H NMR (600MHz, DMSO-d6) δ10.80(s,1H),10.26(s,1H),8.17(d,J=6.3Hz,1H),8.11(t,J=5.2Hz,1H ),7.95–7.89(m,1H),7.48(d,J=7.6Hz,1H),7.42(t,J=8.4Hz,1H),7.32(s,1H),6.95(s,1H),6. 88(d,J=3.3Hz,1H),6.08(s,2H),6.01(s,1H),4.47(t,J=5.8Hz,2H),4.40(d,J=4.4Hz,1H),3.7 7(ddd,J=23.0,16.9,5.7Hz,2H),3.57–3.51(m,2H),3.43(s,2H),3.22(dd,J=13.1,4.0Hz,2H); 13C NMR (151MHz, DMSO) δ169.38,168.82,162.15,160.50,158.59,152.17,152.1 3,152.01,151.23,150.42,149.04,147.00,146.93,127.54,127.48,124.70, 119.10,114.75,114.60,112.22,111.64,111.47,100.23,99.93,66.71,52.9 8,52.24,42.04,41.43,41.23,35.92,34.88,17.63; HRMS(APCI):Calcd.forC 30 H 25 F7O 11 S3[M+H] + :776.0909,found,776.0911.

[0149] Example 31I-P-3b

[0150]

[0151] A similar method to Example 26 was used to obtain yellow solid compound I-P-3b (24 mg, yield 20%). 1 H NMR(600MHz,DMSO-d6)δ10.91(s,1H),10.28(s,1H),8.13(s,1H),8.06(s,2H),7.93–7.88(m,1 H),7.44(d,J=9.1Hz,1H),7.40(t,J=8.3Hz,1H),7.29(s,1H),6.93(s,1H),6.87(d,J=3.3Hz,1H ),6.07(s,2H),6.02(s,1H),4.45(s,2H),4.36(s,1H),4.12(s,1H),3.43(s,2H),3.18(t,J=11. 5Hz,1H),3.06(q,J=7.4Hz,4H),2.96(t,J=11.2Hz,1H),2.18(s,2H),1.94(s,1H),1.82(s,1H); 13C NMR(151MHz,DMSO)δ172.33,172.00,169.80,169.06,162.35,160.71,158.99,1 52.24,151.44,150.63,149.17,147.18,147.10,127.69,125.12,119.29,117.00 ,114.94,112.42,111.81,111.64,100.54,100.07,66.91,53.47,52.59,52.35,4 2.23,41.86,40.05,36.09,35.07,32.01,27.44,17.71; HRMS(APCI):Calcd.forC 35 H 33 F8O 14 S3[M+H] + :905.1335,found,905.1336

[0152] Example 32I-P-4a

[0153]

[0154] A similar method to Example 26 was used to obtain yellow solid compound I-P-4a (23 mg, yield 19%). 1 H NMR (600MHz, DMSO-d6) δ10.78(s,1H),10.12(s,1H),8.66(d,J=8.0Hz,1H),7.92(dd,J=8.9,6.3Hz,1 H),7.77(d,J=7.9Hz,2H),7.53–7.40(m,2H),7.36(s,1H),7.28(d,J=8.0Hz,2H),6.98(d,J=3.6Hz,1 H),6.89(d,J=3.6Hz,1H),5.95(s,2H),5.84(d,J=2.2Hz,1H),4.67(td,J=8.9,4.2Hz,1H),4.46(t,J =6.1Hz,2H),3.18–3.14(m,2H),3.11(q,J=5.9Hz,2H),2.63(d,J=7.1Hz,2H),1.58(t,J=3.9Hz,4H); 13C NMR(151MHz,DMSO)δ166.81,162.69,161.05,158.93,152.39,152.30,15 1.54,150.44,147.55,147.47,146.53,131.88,131.51,128.69,127.86, 119.38,115.23,115.08,112.78,112.07,111.90,100.13,98.31,67.18,52.49,40.51,36.28,35.18,30.75,28.81,27.48; HRMS(APCI):Calcd.for C 37 H 33 F6O 10 S3[M+H] + :837.1477,found,837.1445.

[0155] Example 33I-P-4b

[0156]

[0157] A similar method to Example 26 was used to obtain yellow solid compound I-P-4b (27 mg, yield 14%). 1 H NMR (600MHz, DMSO-d6) δ10.78(s,1H),10.17(s,1H),8.48(d,J=22.0Hz,1H),8.18–8.03(m,1H),7.95–7.90(m,1H),7.79–7.77(m, 2H),7.45(dd,J=9.3,2.6Hz,1H),7.40(td,J=8.5,2.5Hz,1H),7.26(d,J=8.1Hz,2H),7.04(d,J=3.5Hz,1H),6.85(d,J=2.6Hz,2H), 6.83(d,J=2.6Hz,1H),5.97(s,2H),5.82(s,1H),4.47(t,J=6.1Hz,2H),4.39(s,1H),4.31(s,1H),3.08(d,J=5.5Hz,2H),2.98(dd, J=13.2,8.4Hz,2H),2.63(dd,J=12.7,4.1Hz,2H),2.41–2.37(m,2H),2.13–2.02(m,2H),1.96(d,J=7.5Hz,2H),1.60–1.55(m,4H). 13C NMR (151MHz, DMSO) δ182.08,176.27,172.57,172.53,171.81,161.24,158.76,156.64,152.16 ,151.30,150.95,149.52,145.62,131.11,128.33,127.55,127.22,126.53,126.46,115.19,11 5.12,114.07,111.51,110.21,110.13,106.13,105.98,103.40,103.24,99.70,97.91,61.65, 53.59,37.51,34.79,32.46,30.39,28.37,27.13,27.03,22.59,21.73.HRMS(APCI):Calcd.for C 42 H 40 F7O 13 S3[MH] - :964,1757,found 964,1778.

[0158] Example 34I-P-5a

[0159]

[0160] A similar method to Example 26 was used to obtain yellow solid compound I-P-5a (20 mg, yield 18%). 1H NMR (600MHz, DMSO-d6) δ10.77(s,1H),10.39(s,1H),8.46–8.38(m,1H),7.92(dd,J=8.8,6.3Hz,1H),7.76(d,J=8.7Hz,2H),7.48–7 .38(m,1H),7.25(t,J=7.8Hz,2H),7.20–7.16(m,1H),6.85–6.78(m,3H),6.05(s,2H),5.82(d,J=22.8Hz,1H),4.46(t,J=6.1Hz,1H) ,4.33–4.23(m,2H),4.11(dt,J=17.1,6.3Hz,1H),3.07(dd,J=9.7,5.9Hz,1H),2.96–2.85(m,3H),2.61(dd,J=15.6,8.0Hz,2H),2. 45(t,J=7.0Hz,2H),2.31–2.21(m,2H),2.11–2.02(m,1H),2.00–1.93(m,1H),1.60(dd,J=14.7,7.6Hz,4H),1.26(d,J=17.7Hz,2H). 13 CNMR(151MHz,DMSO)δ172.24,172.11,166.46,158.61,158.39,158.19,152.00,151 .20,146.26,131.48,131.28,128.32,127.48,118.33,116.35,114.74,112.50,111. 73,99.73,97.87,66.82,52.13,45.70,40.02,39.88,39.74,39.60,39.46,39.32,39 .18,35.91,34.97,30.59,28.58,28.27,27.26,21.17,8.67.HRMS(APCI):Calcd.for C 38 H 35 F6O 13 S3[MH] - :849.1488, found 849.1484.

[0161] Example 35I-P-5b

[0162]

[0163] A similar method to Example 26 was used to obtain yellow solid compound I-P-5b (17 mg, yield 14%). 11H NMR (600 MHz, DMSO-d6) δ 10.77 (s, 1H), 10.39 (s, 1H), 8.46–8.38 (m, 1H), 7.92 (dd, J = 8.8, 6.3 Hz, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.48–7.38 (m, 1H), 7.25 (t, J = 7.8 Hz, 2H), 7.20–7.16 (m, 1H), 6.85–6.78 (m, 3H), 6.05 (s, 2H), 5.82 (d, J = 22.8 Hz, 1H), 4.46 (t, J = 6.1 Hz, 1H), 4.33–4.23 (m, 2H), 4.11 (dt, J = 17.1, 6.3 Hz, 1H), 3.07 (dd, J = 9.7, 5.9 Hz, 1H), 2.96–2.85 (m, 3H), 2.61 (dd, J = 15.6, 8.0 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.31–2.21 (m, 2H), 2.11–2.02 (m, 1H), 2.00–1.93 (m, 1H), 1.60 (dd, J = 14.7, 7.6 Hz, 4H), 1.26 (d, J = 17.7 Hz, 2H). 13 13C NMR (151 MHz, DMSO) δ 182.00, 176.20, 172.43, 171.81, 165.65, 162.83, 161.22, 158.80, 156.64, 156.01, 155.94, 155.54, 152.14, 151.30, 150.92, 149.53, 145.66, 132.30, 131.17, 128.32, 127.21, 126.55, 126.48, 115.16, 114.06, 111.55, 110.19, 106.22, 106.08, 103.37, 103.22, 99.69, 97.96, 62.81, 61.65, 53.84, 40.14, 37.52, 37.05, 34.84, 32.45, 30.44, 28.44, 28.23, 27.91, 27.18, 22.59, 21.59. HRMS (APCI): Calcd. for C 43 H 42 FN7O 13 S3[M-H] - : 978.1914, found 978.1929。

[0164] Example 36 I-P-6a

[0165]

[0166] A similar method to Example 26 was used to obtain yellow solid compound I-P-6a (22 mg, yield 15%). 1 H NMR (600MHz, DMSO-d6) δ10.79(d,J=2.4Hz,1H),10.11(s,1H),8.65(d,J=8.0Hz,1H),7.93(dd,J=8.8,6.3H z,1H),7.63(d,J=3.7Hz,1H),7.46(dd,J=9.4,2.6Hz,1H),7.42(td,J=8.4,2.7Hz,1H),7.32(s,1H),6.94( s,1H),6.88(d,J=3.6Hz,1H),6.87(d,J=3.7Hz,1H),5.95(s,2H),5.85(s,1H),4.62(dt,J=11.7,6.6Hz,1H ),4.47(t,J=6.1Hz,2H),3.29(dd,J=13.6,4.3Hz,2H),3.16–3.06(m,2H),2.81(s,2H),1.64–1.59(m,4H); 13 C NMR (151MHz, DMSO) δ172.49,161.70,161.00,158.90,152.39,152.32,151.55,151.12,136.84,131.34,129.19,125.87,119.44,115.22, 115.07,112.74,112.06,111.89,100.14,98.36,67.17,52.37,40.53,36.26,31.02,29.74,28.57,27.33,21.54; HRMS(APCI):Calcd.forC 35 H 31 F6O 10 S4[MH] - :841.0896,found,841.0928.

[0167] Example 37I-P-6b

[0168]

[0169] A similar method to Example 26 was used to obtain yellow solid compound Ⅰ-P-6b (17 mg, yield 14%). 1H NMR (600MHz, DMSO-d6) δ10.79(d,J=2.3Hz,1H),10.15(s,1H),8.49(s,1H),8.16(s,1H),7.93(dd,J=8.9,6.3Hz,1H),7.66(d,J= 3.7Hz,1H),7.46(dd,J=9.4,2.7Hz,1H),7.41(td,J=8.5,2.7Hz,1H),7.18(s,1H),6.86(d,J=3.7Hz,2H),6.84(d,J=3.8Hz,1H), 5.96(s,2H),5.85(d,J=2.2Hz,1H),4.47(t,J=6.2Hz,2H),4.40(s,1H),4.29–4.23(m,1H),3.19(dd,J=13.4,4.6Hz,1H),3.08(q ,J=6.0Hz,2H),2.98(dd,J=13.4,8.6Hz,1H),2.80(t,J=6.6Hz,2H),2.27(q,J=8.5Hz,1H),2.10–1.91(m,1H),1.63–1.59(m,5H); 13 CNMR(151MHz,DMSO)δ173.89,172.12,162.57,161.72,160.92,152.43,152.34, 151.57,150.88,149.39,147.34,137.15,131.35,129.09,127.97,125.81,119.5 5,115.19,115.04,112.65,112.05,111.89,100.13,98.36,67.18,52.79,40.90, 36.36,32.40,31.03,29.73,28.54,27.33,27.19,21.55; HRMS(APCI):Calcd.for C 40 H 38 F7O 13 S4[MH] - :970.1322,found,970.1339.

[0170] Example 38I-P-7a

[0171]

[0172] A similar method to Example 26 was used to obtain yellow solid compound I-P-7a (27 mg, yield 19%). 11H NMR (600 MHz, DMSO-d6) δ 10.76 (s, 1H), 10.19 (s, 1H), 8.45 (d, J = 22.0 Hz, 1H), 8.15–8.05 (m, 1H), 7.97–7.93 (m, 1H), 7.79–7.75 (m, 2H), 7.45 (dd, J = 9.3, 2.6 Hz, 1H), 7.41 (td, J = 8.5, 2.5 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 3.5 Hz, 1H), 6.89 (d, J = 2.6 Hz, 2H), 6.81 (d, J = 2.6 Hz, 1H), 5.96 (s, 2H), 5.82 (s, 1H), 4.44 (t, J = 6.1 Hz, 2H), 4.39 (s, 1H), 4.34 (s, 1H), 3.78 (s, 3H), 3.08 (d, J = 5.5 Hz, 2H), 2.99 (dd, J = 13.2, 8.4 Hz, 2H), 2.60 (dd, J = 12.7, 4.1 Hz, 2H), 2.41–2.37 (m, 2H), 2.15–2.09 (m, 2H), 1.96 (d, J = 7.5 Hz, 2H), 1.63–1.58 (m, 4H). 13 13C NMR (151 MHz, DMSO) δ 182.06, 176.27, 172.55, 172.51, 171.81, 161.27, 158.76, 156.66, 152.16, 151.33, 150.95, 149.52, 145.62, 131.31, 128.35, 127.53, 127.22, 126.51, 126.46, 115.15, 115.12, 114.27, 111.51, 110.29, 110.13, 106.17, 105.98, 103.44, 103.24, 99.77, 97.91, 61.66, 53.59, 38.51, 34.79, 32.36, 30.39, 28.37, 27.19, 27.03, 22.61, 21.73. HRMS (APCI): Calcd. for C 43 H 44 FN8O 12 S3[M + H] + : 979.2219, found 979.2210。

[0173] Example 39 I-P-8a

[0174]

[0175] A similar method to Example 26 was used to obtain yellow solid compound I-P-8a (26 mg, yield 12%). 1 H NMR(600MHz,DMSO-d6)δ10.54(s,1H),10.15(s,1H),8.50(dd,J=4.5,1.7Hz,1H) ,8.44(d,J=4.2Hz,1H),8.00(d,J=6.8Hz,1H),8.00(s,1H),7.69–7.54(m,8H),7. 53–7.43(m,4H),7.41(td,J=8.5,2.5Hz,2H),7.41–7.35(m,2H),7.27(d,J=8.1Hz ,2H),7.01(dd,J=6.7,4.5Hz,1H),6.74(s,1H),6.42(s,2H),5.96(s,2H),5.82(s ,1H),4.56(quin,J=6.8Hz,1H),4.49(t,J=5.6Hz,2H),4.44(t,J=6.1Hz,2H),4.3 9(s,1H),4.34(s,1H),3.82(s,3H),3.22(t,J=5.9Hz,2H),3.08(d,J=5.5Hz,2H), 2.99(dd,J=13.2,8.4Hz,2H),2.60(dd,J=12.7,4.1Hz,2H),2.41–2.37(m,2H),2. 15–2.09(m,2H),1.96(d,J=7.5Hz,2H),1.63–1.58(m,4H),1.35(d,J=6.8Hz,3H); 13C NMR(151MHz,DMSO-d6)δ172.51,171.81,169.43,166.51,163.42,162.73,161.19,160.93,158.76,156.66,152.16,151.3 3,150.06,149.36,147.51,146.74,141.05,138.09,137.81,135.42,133.66,132.56,131.90,129.51,129.24,129.20,129 .17,128.50,126.16,123.41,123.31,118.17,116.36,113.19,114.27,111.51,110.13,107.88,102.44,101.15,99.77,9 7.91,90.40,87.06,85.94,44.88,61.66,53.59,38.62,36.99,32.36,30.39,27.19,27.03,20.69; HRMS(APCI):Calcd.for C 58 H 57 N 14 O 11 S2[M+H] + :1189.3767,found,1189.3759.

[0176] Example 40II-P-1a

[0177]

[0178] A similar method to Example 26 was used to obtain yellow solid compound II-P-1a (17 mg, yield 15%). 1H NMR (600MHz, DMSO-d6) δ8.64(s,1H),8.28(s,1H),7.92(dd,J=8.8,6.3Hz,1H),7.64(d,J=8.8Hz,2H),7 .45(dd,J=9.4,2.6Hz,1H),7.41(td,J=8.4,2.6Hz,1H),7.25(s,1H),6.94(t,J=6.1Hz,1H),6.88(J=3. 4Hz,1H),6.86(d,J=3.6Hz,1H),6.65(d,J=8.9Hz,2H),4.60(d,J=11.4Hz,1H),4.48(d,J=6.1Hz,2H),4 .45(t,J=6.2Hz,2H),3.28(dd,J=13.7,4.5Hz,1H),3.13(dd,J=13.5,9.9Hz,1H),3.09(q,J=6.0Hz,2H); 13 C NMR(151MHz,DMSO)δ173.09,172.51,166.67,162.62,160.98,154.42,152.40 ,151.32,149.03,148.88,147.48,147.41,138.26,129.37,128.43,127.96,1 21.73,121.59,119.73,119.46,115.20,115.06,112.71,112.07,111.90,111 .71,67.15,59.57,52.54,46.37,40.90,36.27,21.55; HRMS(APCI):Calcd.for C 34 H 27 F8O 10 S3[MH] - :821.0924,found,821.0939.

[0179] Example 41II-P-1b

[0180]

[0181] A similar method to Example 26 was used to obtain yellow solid compound II-P-1b (16 mg, yield 13%). 11H NMR (600 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.16 (d, J = 6.5 Hz, 2H), 7.93 (dd, J = 8.9, 6.3 Hz, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.46 (dd, J = 9.4, 2.6 Hz, 1H), 7.41 (td, J = 8.5, 2.7 Hz, 1H), 7.21 (s, 1H), 6.91 (t, J = 6.1 Hz, 1H), 6.86 (s, 2H), 6.64 (d, J = 8.9 Hz, 2H), 4.51–4.45 (m, 4H), 4.43 (s, 1H), 4.34–4.23 (m, 1H), 3.18 (dd, J = 13.4, 4.7 Hz, 1H), 3.08 (q, J = 5.8 Hz, 2H), 2.96 (dd, J = 13.4, 8.7 Hz, 1H), 2.32–2.19 (m, 2H), 2.05 (d, J = 7.5 Hz, 1H), 1.94 (dd, J = 15.5, 9.2 Hz, 1H); 13 13C NMR (151 MHz, DMSO) δ 174.52, 172.54, 172.16, 166.68, 162.53, 161.83, 160.88, 156.93, 156.74, 154.76, 152.45, 151.20, 148.95, 148.78, 147.39, 129.39, 128.41, 127.87, 121.97, 119.59, 115.15, 115.01, 112.61, 112.33, 112.06, 111.89, 111.69, 67.19, 61.95, 52.99, 46.40, 40.51, 37.68, 36.37, 32.62, 21.60; HRMS (APCI): Calcd. for C 39 H 34 FN9O 13 S3[M-H] - : 950.1349, found, 950.1375.

[0182] Example 42 Substrate IV-7

[0183]

[0184] Substrate IV-3 (0.50 g, 1.44 mmol), TLR7 / 8 agonist (0.34 g, 0.96 mmol), and 8 ml of anhydrous CH2Cl2 were added to a 25 mL single-necked flask equipped with a magnetic stirrer, and DIPEA (0.19 g, 1.44 mmol) was added under stirring at room temperature. The reaction was allowed to proceed for 4 h. After TLC detection, the reaction was complete and the reaction was stopped. The solvent was evaporated under reduced pressure and purified by preparative thin layer chromatography (CH2Cl2:CH3OH=23:1-22:1) to obtain a white solid substrate IV-7 (0.33 g, yield 57%). 1 H NMR (600MHz, DMSO-d6) δ8.43–8.39(m,1H),7.79(d,J=8.4Hz,1H),7.78–7.76(m,1H),7.74(dd,J=6.1,1.6 Hz,2H),7.59(dt,J=8.4,1.5Hz,1H),7.34(t,J=7.6Hz,1H),7.21–7.18(m,2H),7.05(t,J=7.6Hz,1H),6.9 8(d,J=7.9Hz,2H),6.77–6.66(m,2H),5.84(s,2H),4.16(t,J=6.1Hz,2H),4.12(d,J=6.2Hz,2H),3.06(t, J=6.2Hz,2H),2.89(t,J=7.7Hz,2H),1.71(p,J=7.6Hz,2H),1.37(h,J=7.3Hz,2H),0.86(t,J=7.3Hz,3H); 13 C NMR(151MHz,DMSO)δ159.49,156.51,154.26,151.99,149.99,144.51,139.43,138 .22,135.54,133.51,130.12,128.03,126.99,126.80,126.06,125.94,121.64,120 .63,119.66,114.90,62.09,48.32,43.82,37.88,30.01,29.55,29.50,29.30,29.1 6,29.05,27.03,26.69,25.59,22.56,22.31,14.42,14.15; HRMS(APCI):Calcd.for C 30 H 32 N6O2S2[M+H] + :572.2028,found,572.2017.

[0185] Example 43I-T-1a

[0186]

[0187] Substrate III-8a (0.10 g, 0.24 mmol), substrate IV-7 (0.09 g, 0.15 mmol) and 6 mL of anhydrous DMSO were added to a 10 mL single-necked flask equipped with a magnetic stirrer, and DIPEA (0.04 g, 0.31 mmol) was added under stirring at room temperature. The reaction was allowed to proceed for 2 h. When the reaction was complete by TLC, the reaction was stopped. The preparative liquid phase was purified to obtain a white solid compound I-T-1a (0.024 g, yield 12%). 1 HNMR (600MHz, DMSO-d6) δ10.88(s,1H),10.19(s,1H),8.68(s,1H),7.83(d,J=8.0Hz,2H),7.78(s,1H),7.59(d,J=8.3Hz,1H),7.46(d,J =7.9Hz,2H),7.35(t,J=7.7Hz,1H),7.19(d,J=7.9Hz,2H),7.06(t,J=7.7Hz,1H),6.98(d,J=7.8Hz,2H),6.02(d,J=3.3Hz,3H),5.83(s, 2H),4.62(q,J=8.5,8.0Hz,1H),4.14(t,J=6.4Hz,2H),4.11(d,J=6.2Hz,2H),3.31(d,J=3.9Hz,2H),2.95(td,J=11.8,9.9,4.7Hz,2H), 2.89(t,J=7.7Hz,2H),2.79(d,J=7.0Hz,2H),2.75(d,J=7.1Hz,2H),1.70(p,J=7.6Hz,2H),1.36(h,J=7.3Hz,2H),0.85(t,J=7.4Hz,3H); 13 C NMR (151MHz, DMSO) δ172.52,166.04,158.96,156.60,154.54,152.53,151.87,151.6 6,139.49,135.40,133.69,131.64,129.77,128.04,128.01,127.25,126.65,126.55, 125.94,120.76,114.66,100.21,99.17,93.01,62.37,48.35,43.82,37.49,29.98,29 .49,27.41,27.29,27.02,26.68,22.30,21.55,19.72,14.15; HRMS(APCI):Calcd.for C 45 H 46 N10 O6S2[M+H] + :887.3116, found 887.3111.

[0188] Example 44I-T-1b

[0189]

[0190] A method similar to Example 43 was used to obtain white solid compound Ⅰ-T-1b (21 mg, yield 23%). 1 H NMR(600MHz,DMSO-d6)δ10.88(d,J=2.4Hz,1H),10.21(s,1H),8.66–8.57(m,1H),8. 13(s,1H),7.84(d,J=8.0Hz,2H),7.79(d,J=8.0Hz,1H),7.60(d,J=8.3Hz,1H),7.44( d,J=8.0Hz,2H),7.35(t,J=7.7Hz,1H),7.19(d,J=7.9Hz,2H),7.07(t,J=7.6Hz,1H) ,6.97(d,J=7.9Hz,2H),6.03(s,2H),6.01(d,J=2.2Hz,1H),5.83(s,2H),4.41(td,J= 8.2,4.7Hz,1H),4.32(td,J=8.0,5.1Hz,1H),4.14(d,J=6.3Hz,2H),4.11(s,2H),3. 16(dd,J=13.4,4.6Hz,2H),2.97–2.91(m,2H),2.90(d,J=8.2Hz,2H),2.79(t,J=7.3H z,3H),2.74(t,J=7.2Hz,2H),2.28(q,J=7.5Hz,2H),2.11–2.06(m,1H),1.96(dt,J=1 4.5,7.6Hz,1H),1.70(p,J=7.6Hz,2H),1.37(h,J=7.4Hz,2H),0.86(t,J=7.4Hz,3H); 13C NMR (151MHz, DMSO) δ174.62,172.68,166.26,159.07,153.42,152.43,151.71,139. 37,134.56,131.57,129.74,128.04,127.96,126.34,125.92,100.18,99.18,98.22 ,92.86,81.41,54.70,53.81,42.06,32.93,31.17,31.05,30.91,30.01,29.61,29. 56,28.55,27.73,27.30,26.46,23.18,22.31,19.74,14.64; HRMS(APCI):Calcd.for C 50 H 53 N 11 O9S2[M+H] + :1016.3542,found1016.3542.

[0191] Example 45I-T-2a

[0192]

[0193] A similar method to Example 43 was used to obtain white solid compound Ⅰ-T-2a (22 mg, yield 19%). 11H NMR (600 MHz, DMSO-d6) δ 10.88 (s, 1H), 10.19 (s, 1H), 8.68 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.78 (s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.35 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 7.9 Hz, 2H), 7.06 (t, J = 7.7 Hz, 1H), 6.98 (d, J = 7.8 Hz, 2H), 6.02 (d, J = 3.3 Hz, 3H), 5.83 (s, 2H), 4.62 (q, J = 8.5, 8.0 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H), 4.11 (d, J = 6.2 Hz, 2H), 3.31 (d, J = 3.9 Hz, 2H), 2.95 (td, J = 11.8, 9.9, 4.7 Hz, 2H), 2.89 (t, J = 7.7 Hz, 2H), 2.79 (d, J = 7.0 Hz, 2H), 2.75 (d, J = 7.1 Hz, 2H), 1.87 (tt, J = 7.9, 6.0 Hz, 2H), 1.70 (p, J = 7.6 Hz, 2H), 1.36 (h, J = 7.3 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H); 13 13C NMR (151 MHz, DMSO) δ 172.52, 166.04, 158.96, 156.60, 154.54, 152.53, 151.87, 151.66, 139.49, 135.40, 133.69, 131.64, 129.77, 128.04, 128.01, 127.25, 126.65, 126.55, 125.94, 120.76, 114.66, 100.21, 99.17, 93.01, 62.37, 48.35, 43.82, 37.49, 29.98, 29.49, 27.41, 27.29, 27.02, 26.68, 22.30, 21.55, 19.72, 14.15; HRMS (APCI): Calcd. for C 46 H 48 N 10 O6S2 [M + H] + : 901.3272, found 901.3261.

[0194] Example 46 I-T-2b

[0195]

[0196] A similar method to Example 43 was used to obtain white solid compound Ⅰ-T-2b (24 mg, yield 19%). 1 H NMR(600MHz,DMSO-d6)δ10.88(d,J=2.4Hz,1H),10.21(s,1H),8.66–8.57(m,1H),8.13( s,1H),7.84(d,J=8.0Hz,2H),7.79(d,J=8.0Hz,1H),7.60(d,J=8.3Hz,1H),7.44(d,J=8. 0Hz,2H),7.35(t,J=7.7Hz,1H),7.19(d,J=7.9Hz,2H),7.07(t,J=7.6Hz,1H),6.97(d,J =7.9Hz,2H),6.03(s,2H),6.01(d,J=2.2Hz,1H),5.83(s,2H),4.41(td,J=8.2,4.7Hz,1H ),4.32(td,J=8.0,5.1Hz,1H),4.14(d,J=6.3Hz,2H),4.11(s,2H),3.16(dd,J=13.4,4. 6Hz,2H),2.97–2.91(m,2H),2.90(d,J=8.2Hz,2H),2.79(t,J=7.3Hz,3H),2.74(t,J=7.2 Hz,2H),2.28(q,J=7.5Hz,2H),2.11–2.06(m,1H),1.96(dt,J=14.5,7.6Hz,1H),1.87(tt ,J=7.9,6.0Hz,2H),1.70(p,J=7.6Hz,2H),1.37(h,J=7.4Hz,2H),0.86(t,J=7.4Hz,3H). 13 CNMR(151MHz,DMSO)δ174.62,172.68,166.26,159.07,153.42,152.43,151.71,139 .37,134.56,131.57,129.74,128.04,127.96,126.34,125.92,100.18,99.18,98.22 ,92.86,81.41,54.70,53.81,42.06,32.93,31.17,31.05,30.91,30.01,29.61,29.5 6,28.55,27.73,27.30,26.46,23.18,22.31,19.74,14.64; HRMS(APCI):Calcd.forC 51 H 55 N 11 O9S2[M+H] +:1030.3698,found 1030.3678.

[0197] Example 47I-T-3a

[0198]

[0199] A similar method to Example 43, except that the substrate III-8a was replaced with the substrate III-15a disclosed in Example 8 of application number CN202310703292.4 Compound Ⅰ-T-3a (24 mg, yield 21%) was obtained as a yellow solid. 1 H NMR (600MHz, DMSO-d6) δ10.82(s,1H),8.14(t,J=5.7Hz,1H),7.93(s,1H),7.90(s,1H),7.78(d,J=8.3Hz,1H),7.59(d,J=8.3H z,1H),7.34(t,J=7.7Hz,1H),7.20(d,J=7.9Hz,2H),7.06(t,J=7.6Hz,1H),6.98(d,J=7.9Hz,2H),6.12(s,2H),6.00(s,1H),5. 83(s,2H),4.23(d,J=7.8Hz,1H),4.14(t,J=6.3Hz,2H),4.11(d,J=6.3Hz,2H),3.73(qd,J=16.7,5.7Hz,2H),3.43(s,2H),3.2 4(dd,J=13.0,4.6Hz,2H),3.04–2.92(m,2H),2.89(s,2H),1.70(p,J=7.7Hz,2H),1.37(h,J=7.4Hz,2H),0.86(t,J=7.4Hz,3H). 13 CNMR(151MHz,DMSO)δ172.55,169.81,168.87,159.02,156.69,154.37,152.63 ,152.00,151.69,139.54,135.44,133.56,128.04,127.09,126.74,125.94,12 5.12,121.75,120.69,114.79,100.78,100.36,62.48,53.54,48.33,43.82,42 .71,37.88,35.32,30.01,26.68,22.31,21.60,14.16; HRMS(APCI):Calcd.for C 38 H 43 N 11 O7S2[M+H]+ :830.2861,found,830.2850.

[0200] Example 48I-T-3b

[0201]

[0202] A similar method to Example 43 was used to obtain yellow solid compound I-T-3b (24 mg, yield 20%). 1 H NMR(600MHz,DMSO-d6)δ10.98(s,1H),8.23(t,J=5.8Hz,1H),7.90(s,1H),7 .89(s,1H),7.87(s,1H),7.78(d,J=8.3Hz,1H),7.61(d,J=8.3Hz,1H),7.39– 7.33(m,1H),7.18(d,J=8.0Hz,2H),7.08(t,J=7.6Hz,1H),6.96(d,J=7.9Hz, 2H),6.22(s,2H),6.00(s,1H),5.82(s,2H),4.29(q,J=8.2,4.7Hz,1H),4.12 (t,J=6.1Hz,2H),4.10(d,2H),4.07(q,J=8.2,4.7Hz,1H),3.72(ddd,J=51.2 ,16.7,5.8Hz,2H),3.43(s,2H),3.20(dd,J=13.1,4.5Hz,2H),2.98–2.90(m, 2H),2.88(t,J=7.6Hz,2H),2.24–2.11(m,2H),2.01–1.91(m,1H),1.86–1.78 (m,1H),1.69(p,J=7.7Hz,2H),1.35(h,J=7.3Hz,2H),0.84(t,J=7.3Hz,3H). 13CNMR(151MHz,DMSO)δ174.55,173.57,172.64,172.09,169.92,168.96,159.15,156.67,154. 89,152.72,151.71,151.68,139.59,135.23,133.92,128.05,127.59,126.44,125.94,125.23 ,122.33,120.89,114.36,100.70,100.36,62.42,53.49,53.22,49.07,48.39,43.80,42.69,4 2.45,37.74,35.41,32.48,29.94,28.18,26.67,22.28,21.67,14.14.HRMS(APCI):Calcd.for C 43 H 50 N 12 O 10 S2[M+H] + :959.3287,found,959.3258.

[0203] Example 49I-T-4a

[0204]

[0205] A similar method to Example 43 was used to obtain white solid compound I-T-4a (23 mg, yield 19%). 11H NMR (600 MHz, DMSO-d6) δ 10.79 (s, 1H), 10.14 (s, 1H), 8.58 (d, J = 7.7 Hz, 1H), 7.79 (t, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 7.8 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 7.06 (t, J = 7.7 Hz, 2H), 6.98 (d, J = 7.9 Hz, 2H), 5.96 (s, 2H), 5.85 (s, 1H), 5.83 (s, 2H), 4.64 (td, J = 8.9, 8.3, 4.0 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H), 4.10 (d, J = 6.1 Hz, 2H), 3.28 (dd, J = 13.5, 4.1 Hz, 2H), 3.16–3.09 (m, 2H), 2.99–2.90 (m, 2H), 2.89 (t, J = 7.8 Hz, 2H), 2.63 (t, J = 7.1 Hz, 2H), 1.70 (p, J = 7.7 Hz, 2H), 1.63–1.52 (m, 4H), 1.36 (h, J = 7.5 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H); 13 13C NMR (151 MHz, DMSO) δ 166.72, 158.89, 156.60, 152.31, 151.88, 151.53, 146.46, 139.48, 135.41, 133.67, 131.98, 131.46, 128.69, 128.03, 127.85, 127.21, 126.66, 125.94, 121.90, 120.75, 114.68, 100.14, 98.31, 62.38, 52.70, 48.35, 43.82, 37.45, 35.18, 30.77, 29.99, 28.79, 27.50, 26.68, 22.30, 14.15; HRMS (APCI): Calcd. for C 45 H 50 N 10 O6S2 [M + H] + : 891.3429, found, 891.3414.

[0206] Example 50 I-T-4b

[0207]

[0208] A similar method to Example 43 was used to obtain white solid compound I-T-4b (27 mg, yield 14%). 1 H NMR(600MHz,DMSO-d6)δ10.78(s,1H),8.38(d,J=7.2Hz,1H),7.93(s,1H),7.8 9(d,J=8.2Hz,1H),7.79(d,J=7.8Hz,2H),7.77(d,J=8.3Hz,1H),7.60(d,J=8. 3Hz,1H),7.35(t,J=7.6Hz,1H),7.25(d,J=7.9Hz,2H),7.19(d,J=7.9Hz,2H), 7.06(t,J=7.6Hz,1H),6.97(d,J=7.9Hz,2H),6.09(s,2H),5.83(s,1H),5.82( s,2H),4.31–4.25(m,2H),4.13(t,J=6.4Hz,2H),4.11(d,J=6.1Hz,2H),3.20( dd,J=13.1,4.5Hz,2H),2.98–2.94(m,1H),2.94–2.89(m,2H),2.88(d,J=7.8H z,2H),2.61(t,J=6.9Hz,2H),2.30–2.22(m,2H),2.12–1.94(m,2H),1.70(p,J =7.6Hz,2H),1.63–1.51(m,4H),1.36(h,J=7.3Hz,2H),0.85(t,J=7.3Hz,3H); 13 C NMR (151MHz, DMSO) δ172.34,169.93,164.34,156.90,154.54,152.42,150.32,149.78,149.47,144.03,137.44 ,133.22,131.55,130.21,129.31,126.51,125.91,125.67,125.13,124.52,123.80,119.82,118.63,116.42,11 2.51,97.97,96.16,60.32,51.43,51.27,46.23,41.68,40.67,38.28,38.14,38.00,37.86,37.72,37.58,37.4 4,35.68,33.02,30.61,28.60,27.86,26.58,25.71,25.35,24.55,20.17,19.51,12.02; HRMS(APCI):Calcd.for C 50 H 57 N11 O9S2[M+H] + :1020.3855,found 1020.3845.

[0209] Example 51I-T-5a

[0210]

[0211] A similar method to Example 43 was used to obtain white solid compound I-T-5a (20 mg, yield 18%). 1 H NMR(600MHz,DMSO-d6)δ10.79(s,1H),10.14(s,1H),8.58(d,J=7.7Hz,1H),7 .79(t,1H),7.77(d,J=7.8Hz,2H),7.76(d,J=8.3Hz,1H),7.59(d,J=8.3Hz,1H ),7.35(t,J=7.7Hz,1H),7.28(d,J=7.8Hz,2H),7.18(d,J=7.9Hz,2H),7.06( t,J=7.7Hz,2H),6.98(d,J=7.9Hz,2H),5.96(s,2H),5.85(s,1H),5.83(s,2H) ,4.64(td,J=8.9,8.3,4.0Hz,1H),4.14(t,J=6.4Hz,2H),4.10(d,J=6.1Hz,2 H),3.28(dd,J=13.5,4.1Hz,2H),3.16–3.09(m,2H),2.99–2.90(m,2H),2.89( t,J=7.8Hz,2H),2.63(t,J=7.1Hz,2H),1.87(tt,J=7.9,6.0Hz,2H),1.70(p,J =7.7Hz,2H),1.63–1.52(m,4H),1.36(h,J=7.5Hz,2H),0.85(t,J=7.3Hz,3H). 13C NMR(151MHz,DMSO)δ166.72,158.89,156.60,152.31,151.88,151.53,146.46,13 9.48,135.41,133.67,131.98,131.46,128.69,128.03,127.85,127.21,126.66,1 25.94,121.90,120.75,114.68,100.14,98.31,62.38,52.70,48.35,43.82,37.45 ,35.18,30.77,29.99,28.79,27.50,26.68,22.30,14.15.HRMS(APCI):Calcd.for C 46 H 52 N 10 O6S2[M+H] + :905.3585,found,905.3574.

[0212] Example 52I-T-5b

[0213]

[0214] A similar method to Example 43 was used to obtain white solid compound I-T-5b (17 mg, yield 14%). 1H NMR(600MHz,DMSO-d6)δ10.78(s,1H),8.38(d,J=7.2Hz,1H),7.93(s,1H),7.89(d,J=8.2Hz,1H),7.79(d,J=7.8Hz,2H),7.77(d,J=8.3Hz,1H),7.60(d,J=8.3Hz,1H),7.35(t,J=7.6Hz,1H),7.25(d,J=7.9Hz,2H),7.19(d,J=7.9Hz,2H),7.06(t,J=7.6Hz,1H),6.97(d,J=7.9Hz,2H),6.09(s,2H),5.83(s,1H),5.82(s,2H),4.31–4.25(m,2H),4.13(t,J=6.4Hz,2H),4.11(d,J=6.1Hz,2H),3.20(dd,J=13.1,4.5Hz,2H),2.98–2.94(m,1H),2.94–2.89(m,2H),2.88(d,J=7.8Hz,2H),2.61(t,J=6.9Hz,2H),2.30–2.22(m,2H),2.12–1.94(m,2H),1.87(tt,J=7.9,6.0Hz,2H),1.70(p,J=7.6Hz,2H),1.63–1.51(m,4H),1.36(h,J=7.3Hz,2H),0.85(t,J=7.3Hz,3H). 13 C NMR(151MHz,DMSO)δ172.34,169.93,164.34,156.90,154.54,152.42,150.32,149.78,149.47,144.03,137.44,133.22,131.55,130.21,129.31,126.51,125.91,125.67,125.13,124.52,123.80,119.82,118.63,116.42,112.51,97.97,96.16,60.32,51.43,51.27,46.23,41.68,40.67,38.28,38.14,38.00,37.86,37.72,37.58,37.44,35.68,33.02,30.61,28.60,27.86,26.58,25.71,25.35,24.55,20.17,19.51,12.02.HRMS(APCI):Calcd.for C 51 H 59 N 11 O9S2[M+H] +:1034.4011,found1034.4001.

[0215] Example 53I-T-6a

[0216]

[0217] A similar method to Example 43 was used to obtain white solid compound I-T-6a (22 mg, yield 15%). 1 H NMR (600MHz, DMSO-d6) δ10.80(s,1H),10.22(s,1H),8.36(s,1H),7.88(t,J=6.0Hz,1H),7.78(d,J=8.2Hz,1H),7.60(d,J=3.6Hz,1H),7.58(d,J=8 .2Hz,1H),7.34(t,J=7.6Hz,1H),7.19(d,J=7.8Hz,2H),7.05(t,J=7.6Hz ,1H),6.98(d,J=7.9Hz,2H),6.85(d,J=3.7Hz,1H),6.01(s,2H),5.86(s, 1H),5.83(s,2H),4.49(td,J=9.0,4.5Hz,1H),4.13(t,J=6.7Hz,2H),4.1 1(d,J=6.5Hz,2H),3.32–3.28(m,2H),3.08(dd,J=13.3,9.4Hz,2H),2.99 –2.91(m,2H),2.89(t,J=7.8Hz,2H),2.79(t,J=6.9Hz,2H),1.70(p,J=7. 7Hz,2H),1.65–1.55(m,4H),1.37(h,J=7.3Hz,2H),0.86(t,J=7.4Hz,3H). 13 C NMR (151MHz, DMSO) δ161.42,158.94,156.64,154.39,152.37,151.98,151.57,150. 71,139.52,135.43,133.57,131.33,128.85,128.03,127.09,126.73,125.93,125. 82,121.77,120.69,114.77,100.13,98.37,62.39,48.33,43.82,37.73,31.03,30. 00,29.73,28.56,27.34,26.68,22.30,21.58,21.09,14.16; HRMS(APCI):Calcd.for C 43 H 48 N10 O6S3[M+H] + :897.2993,found,897.2989.

[0218] Example 54I-T-6b

[0219]

[0220] A similar method to Example 43 was used to obtain white solid compound I-T-6b (17 mg, yield 14%). 1 H NMR (600MHz, DMSO-d6) δ10.80(s,1H),10.24(s,1H),8.46(d,J=7.4Hz,1H),8.10(d,J=7. 9Hz,1H),7.83(t,J=6.3Hz,1H),7.79(d,J=8.3Hz,1H),7.66(d,J=3.7Hz,1H),7.61(d,J=8 .3Hz,1H),7.36(t,J=7.7Hz,1H),7.19(d,J=7.8Hz,2H),7.08(t,J=7.6Hz,1H),6.97(d,J= 7.9Hz,2H),6.84(d,J=3.7Hz,1H),6.01(s,2H),5.85(d,J=2.2Hz,1H),5.83(s,2H),4.38( td,J=8.2,4.6Hz,1H),4.27(td,J=8.2,5.0Hz,1H),4.14(t,J=6.4Hz,2H),4.12(d,J=6.1 Hz,2H),3.18(dd,J=13.4,4.6Hz,2H),2.97–2.92(m,2H),2.92–2.90(m,2H),2.89(t,J=8. 1Hz,2H),2.79(d,J=6.9Hz,2H),2.26(t,J=7.7Hz,2H),2.01(ddd,J=84.5,15.8,8.2Hz,2H ),1.70(p,J=7.7Hz,2H),1.63–1.60(m,4H),1.36(h,J=7.4Hz,2H),0.86(t,J=7.3Hz,3H). 13C NMR(151MHz,DMSO)δ174.24,173.03,172.11,161.60,158.95,156.64,154.74,152.37,151.79,1 51.57,150.76,139.55,137.27,135.32,133.81,131.33,129.02,128.04,127.44,126.55,125.94 ,125.81,122.15,120.84,114.48,100.13,98.36,62.40,53.17,52.71,48.37,43.80,37.60,32.5 4,31.04,29.97,29.72,28.52,27.46,27.32,26.68,22.30,21.57,14.15.HRMS(APCI):Calcd.For C 48 H 55 N 11 O9S3[M+H] + :1026.3419,found,1026.3403.

[0221] Example 55II-T-1a

[0222]

[0223] A similar method to Example 43 was used to obtain yellow solid compound II-T-1b (16 mg, yield 13%). 1H NMR (600MHz, DMSO-d6) δ8.64(s,1H),8.21(d,J=7.8Hz,1H),7.78(d,J=8.3Hz,1H),7.75(t,J=6.2Hz,1H),7.63(d,J=8.4Hz,2H),7.59(d, J=8.3Hz,1H),7.34(t,J=7.4Hz,1H),7.19(d,J=7.9Hz,2H),7.06(t,J=7.6Hz,1H),6.98(d,J=7.9Hz,2H),6.94(d,J=6.1Hz,1H),6.65(d,J =8.4Hz,2H),5.83(s,2H),4.59(td,J=8.7,4.2Hz,1H),4.48(d,J=6.0Hz,2H),4.13(t,J=6.4Hz,2H),4.10(d,J=6.1Hz,2H),3.10(dd,J=1 3.4, 9.7Hz, 2H), 2.93 (dd, J = 12.9, 6.7Hz, 2H), 2.89 (t, J = 7.8Hz, 2H), 1.70 (p, J = 7.7Hz, 2H), 1.36 (h, J = 7.7Hz, 2H), 0.85 (t, J = 7.3Hz, 3H). 13 C NMR (151MHz, DMSO) δ166.64,156.60,151.87,151.29,139.48,135.42,133.65,129.37,128.44,128.03,125.94,121. 87,121.78,120.74,111.70,62.36,48.35,46.38,43.81,37.45,29.99,26.68,22.30,14.15.HRMS(APCI):Calcd.forC 42 H 44 N 12 O6S2[M+H] + :877.3021,found,877.2999.

[0224] Example 56II-T-1b

[0225]

[0226] A similar method to Example 43 was used to obtain yellow solid compound II-T-1b (16 mg, yield 13%). 1H NMR (600MHz, DMSO-d6) δ8.63 (s, 1H), 8.09 (d, J = 7.2Hz, 1H), 8.04 (d, J = 8.1Hz, 1H),7.80(dd,J=13.0,7.2Hz,2H),7.65(d,J=8.4Hz,2H),7.61(d,J=8.3Hz,1H) ,7.36(t,J=7.8Hz,1H),7.19(d,J=7.8Hz,2H),7.07(t,J=7.6Hz,1H),6.97(d, J=7.9Hz,2H),6.90(t,J=6.2Hz,1H),6.64(d,J=8.3Hz,2H),5.83(s,2H),4.47( d,J=5.9Hz,2H),4.37(td,J=8.2,4.6Hz,1H),4.28(q,J=7.2,6.7Hz,1H),4.14 (d,J=6.3Hz,2H),4.11(s,2H),3.18(dd,J=13.3,4.5Hz,2H),2.97–2.91(m,2H) ,2.90–2.88(m,2H),2.25(t,J=7.8Hz,2H),2.06(q,J=7.1,6.3Hz,1H),1.96–1. 89(m,1H),1.70(p,J=7.6Hz,2H),1.36(h,J=7.4Hz,2H),0.85(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO)δ172.20,166.61,156.64,154.75,151.76,151.17,148 .97,148.81,139.54,135.31,133.82,129.36,128.42,128.04,126.52,12 5.94,122.01,120.84,114.48,111.68,62.39,53.19,48.38,46.40,43.80 ,37.60,32.65,29.96,29.49,26.67,22.29,14.14.HRMS(APCI):Calcd.for C 47 H 51 N 13 O9S2[M+H] + :1006.3447,found,1006.3479.

[0227] Example 57 In vitro selectivity experiment

[0228] In order to determine whether this series of small molecule drug conjugates can selectively target and enter TAMs, the fluorescent probe GT-NIR-1 disclosed in application number CN202310703292.4 was used.

[0229] A series of in vitro experiments were performed.

[0230] First, M2 macrophages were cultured at 5×10 5 / well were inoculated in a 6-well plate and placed in 2 mL of DMEM medium for 12 h. The culture medium of M2 macrophages was replaced with fresh culture medium containing the probe GT-NIR-1 (100 nM) and cultured for 30 min. Then the culture medium was removed, the cells were washed with PBS (pH = 7.4), centrifuged at 2000 rpm for 3 minutes, and repeated three times to remove unbound probes. Finally, the cells were resuspended in 500 μL of PBS containing 3% BSA and stored in BD Falcon tubes in the dark for flow cytometry analysis. In addition, a culture medium containing the probe GT-NIR-1 (100 nM) and a 100-fold excess of the target compound (10 μM) or the PI3Kγ inhibitor AS-252424 (10 μM) or the TLR agonist IMDQ (10 μM) was prepared. M2 macrophages were incubated in the above culture medium for 30 min under the same conditions. The medium was then removed, and the cells were washed with PBS and centrifuged at 2000 rpm for 3 minutes, which was repeated three times to remove unbound probe. The cells were finally resuspended in 500 μL of PBS containing 3% BSA and stored in BD Falcon tubes in the dark for flow cytometry analysis.

[0231] The experimental results are as follows Figure 1 As shown, after the probe GT-NIR-1 was co-incubated with M2 macrophages, the macrophages showed bright fluorescence, which served as a positive control group. When 100-fold excess of the target compound was added to the macrophages co-incubated with the probe GT-NIR-1, a significant decrease in its relative fluorescence intensity was observed. The I-P series compounds, II-P series compounds, I-T series compounds and II-T series compounds were all subjected to in vitro selectivity experiments under this condition. The results are shown in Figure 2 As shown in the figure, all small molecule drug conjugates can significantly reduce the fluorescence intensity of the positive control probe GT-NIR-1, while the single payload, whether PI3Kγ inhibitor AS-252424 or TLR agonist IMDQ, has no significant effect on the fluorescence intensity of the probe. The experimental results show that these small molecule drug conjugates can selectively bind to the folate receptor on the surface of TAMs and enter the cell, and the targeting mechanism depends on the selective specificity of the ligand part.

[0232] Example 58 In vitro cleavage experiment of small molecule drug conjugate

[0233] The small molecule drug conjugate of this application enters the cell through a targeting ligand. After entering the cell, it partially breaks the disulfide bond under the action of glutathione GSH, releasing the payload PI3Kγ inhibitor / TLR agonist, thereby reprogramming macrophages and achieving an anti-tumor effect. The concentration of GSH in the tumor site is about 1000 times that of plasma, while the concentration of GSH in plasma is about 1-10 μM. By simulating the intracellular situation in vitro, an in vitro cleavage experiment was conducted to explore whether small molecule conjugated drugs can release the payload.

[0234] The target compound was dissolved in PBS containing 10 mM GSH to a final concentration of 0.5 mg / mL and incubated at 37°C. High performance liquid chromatography (HPLC) was used to monitor the cleavage in real time. The selected chromatographic column was a C18 column, and the mobile phase was: water + 10 mM ammonium acetate (A), acetonitrile (B); the settings were as follows: A 95% 0 min → 60% 10 min → 45% 22 min → 0% 30 min. The flow rate was 1 mL / min, and the injection volume was 10 μL. Figure 3 As shown, the target compound Ⅰ-T-4a / Ⅰ-P-4a breaks from the disulfide bond under the action of GSH, releasing the complete PI3Kγ inhibitor AS-252424 or TLR agonist IMDQ. The small molecule drug conjugates prepared in this application were tested under the same conditions. The experimental results showed that when GSH was present, all small molecule drug conjugates could spontaneously cleave and release the payload part, and the complete cleavage time was 10 to 15 hours ( Figure 4 ).

[0235] Example 59 Effect of small molecule drug conjugates on polarization of macrophages in vitro

[0236] Mouse primary macrophages BMDM were isolated from mouse femur and tibia, induced with 20 ng / L M-CSF for 5 days, and then differentiated into M2 macrophages by 10 ng / L IL-4. The effects of the target compounds on the expression of PI3Kγ / TLR7 and phenotypic conversion of M2 macrophages were investigated.

[0237] M2 macrophages 1×10 6 Each well was inoculated in a six-well plate. After incubation for 12 hours, the target compound or a single payload (10 nM) was co-incubated with M2 macrophages for 24 hours. The cells in the well plate were lysed, proteins were extracted, and western blot analysis was performed. Figure 5-6As shown, both the I-P series compounds and the II-P series compounds showed inhibitory effects on PI3Kγ, and were significantly higher than AS-252424 without ligand linkage. Figure 7 The experimental results showed that this series of small molecule drug conjugates can reduce the expression of CD206, a marker of M2 macrophages, and increase the expression of inducible nitric oxide synthase (iNOS), a marker of M1 macrophages, indicating that they have the ability to reprogram M2 macrophages into M1 macrophages. In addition, compared with the control group, all drug-treated groups showed a decrease in the expression level of the anti-inflammatory cytokine IL-10, while the expression levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor α (TNF-α) were increased. Data quantitative analysis of all compounds showed that all I-P series compounds and II-P series small molecule drug conjugates were able to inhibit the PI3Kγ signaling pathway, regulate the polarization state of macrophages, and ultimately exert an anti-tumor immunotherapy effect ( Figure 6-9 ).

[0238] Under the same conditions, the Ⅰ-T series compounds and Ⅱ-T series compounds were treated with macrophages for 24 hours, and then western blot was performed. The Ⅰ-T series compounds and Ⅱ-T series compounds all showed activation effects on TLR, which were significantly higher than IMDQ without ligand connection, and were able to reprogram M2 macrophages into M1 macrophages ( Fig.10 ). Subsequently, the data of the compounds were quantitatively analyzed and it was found that all I-T series compounds and II-T series small molecule drug conjugates can activate the TLR7 / 8 signaling pathway, regulate the polarization state of macrophages, and ultimately play an anti-tumor immunotherapy role ( Figure 11-13 ). In summary, the experimental results confirmed that all small molecule drug conjugates can deliver the payload to the target immune cells and exert the immunotherapeutic effect of the payload.

[0239] Example 60 In vivo antitumor activity study

[0240] Female BALB / c mice aged 6 to 8 weeks, weighing 16 to 20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. The animal operation specifications of this study were strictly in accordance with the guidelines of the Chinese Society of Laboratory Animals and approved by the Animal Experiment Ethics Committee. Experimental animal breeding environment (SPF grade): temperature is 22 to 24 ° C, relative humidity is 50 ± 5%. The light cycle is 12 h, that is, dark after 12 h of light. Maintain an SPF-grade special diet without folic acid for at least 1 week before the animal model is established. 4T1 cells (mouse breast cancer cells) were purchased from the Shanghai Institute of Biological Cells, Chinese Academy of Sciences. RPMI-1640 medium containing 10% FBS was used for routine culture of 4T1 cells. The above culture media contain 100 IU / mL penicillin and streptomycin. All cells were cultured in a 5% carbon dioxide, 37 ° C cell culture incubator.

[0241] Digest 4T1 cells, wash the cells three times with PBS to remove residual serum and trypsin, and dilute 4T1 cells to 1×10 7 / mL, placed on ice. The hair under the armpit of the right hind limb of BALB / c mice was shaved one day before tumor implantation. The next day, 5×10 5 50 μL of 4T1 tumor cells were injected into the second-to-last mammary fat pad under the right hind arm. Tumor growth was measured vertically with a caliper every day, and the tumor volume was calculated as 0.5×L×W 2 (L = longest axis, W = axis perpendicular to L, in millimeters).

[0242] Tumor-bearing mice were randomly divided into 6 groups according to body weight, with 5 mice in each group, namely solvent group, Ⅰ-P-3b, Ⅰ-P-4b, Ⅰ-P-6b, Ⅱ-P-1b, and AS-252424. The dose of all drug-treated groups was 3.87 mg / kg. The corresponding dose of small molecule drug conjugate was dissolved in 100 μL of PBS buffer, and the corresponding dose of AS-252424 was dissolved in 100 μL of solvent (5% DMSO, 30% PEG-400, 65% PBS), and administered by tail vein injection. The solvent group was injected with an equal amount of pure solvent (PBS) carrier as a control. The drug was administered once a day for a total of 14 times. During the drug administration period, the tumor volume and weight changes of mice were recorded at the same time every day, and the tumor volume was measured simultaneously with a vernier caliper, with the formula of 0.5×L×W 2 (L = longest axis, W = axis perpendicular to L, in millimeters) During the treatment, when mice showed a significant weight loss of 20% and tumor volume exceeded 2000mm 3 When the tumor length observed at any angle exceeds 20 mm, the observation is terminated. After the administration is completed, the tumor growth and mouse weight curves are drawn. Fig.14As shown, all dosing groups were able to continuously delay tumor growth, including the single payload. All small molecule drug conjugates did not cause the weight of mice to decrease, but maintained a steady growth. Compared with the conjugate, the single payload had the worst efficacy, and the weight of the mice did not increase significantly. These results demonstrate that the small molecule drug conjugates disclosed in this application have good anti-tumor efficacy and safety.

[0243] In order to explore the specific effect of the spacer on the efficacy of the conjugate, compounds Ⅰ-P-4a and Ⅰ-P-4b with excellent effects in in vitro and in vivo experiments were selected and compared. The experimental conditions were the same as above. The experimental results are shown in Figure 2. Fig.15 As shown, compounds Ⅰ-P-4a and Ⅰ-P-4b showed the ability to delay tumor growth. Although the difference was not significant, Ⅰ-P-4b performed best. In addition, the weight of mice treated with Ⅰ-P-4b maintained a steady growth trend, while the weight of mice in the Ⅰ-P-4a group did not increase significantly compared to before treatment, indicating that the introduction of glutamic acid in the spacer region of the conjugate can not only improve the water solubility of the drug, but also be more beneficial to the efficacy of the drug.

[0244] The in vivo anti-tumor experimental conditions of the Ⅰ-T series compounds and Ⅱ-T series compounds were the same as those of the above experiments except for the dosage. 4T1 tumor-bearing mice were randomly divided into 6 groups according to body weight, with 5 mice in each group, namely solvent group, IT-6a, IT-3a, II-T-1a, IT-4a, and IMDQ. The dose of all dosing groups was 1 mg / kg, and the corresponding dose of small molecule drug conjugate was dissolved in 100 μL of PBS buffer, and the corresponding dose of IMDQ was dissolved in 100 μL of solvent (5% DMSO, 25% PEG-400, 70% PBS), and administered by tail vein injection. The solvent group was injected with an equal amount of pure solvent (PBS) carrier as a control. The drug was administered once a day for a total of 14 times. During the administration period, the tumor volume and weight changes of the mice were recorded at the same time every day, and the tumor volume was measured simultaneously with a vernier caliper, with the formula of 0.5×L×W 2 (L = longest axis, W = axis perpendicular to L, in millimeters) During the treatment, when mice showed a significant weight loss of 20% and tumor volume exceeded 2000mm 3 When the tumor length observed at any angle exceeds 20 mm, the observation is terminated. After the administration, the tumor growth and mouse weight curves are drawn. Fig.16 As shown, the Ⅰ-T series compounds and the Ⅱ-T series compounds can also delay tumor growth in a sustained manner, and their effects are better than those of the single payload.

[0245] The results show that the small molecule drug conjugate provided in the present application has anti-tumor activity.

[0246] Example 61 In vivo selectivity study

[0247] The animal tumor model was constructed by the same method as in the above embodiment, and drug administration began on the 7th day after tumor cell inoculation. The tumor-bearing mice were randomly divided into 3 groups according to body weight, with 5 mice in each group, namely solvent group (100ul PBS buffer), I-P-4b, and FA competition group. The dose of I-P-4b in all drug administration groups was 3.87 mg / kg. The I-P-4b group was a corresponding dose of I-P-4b dissolved in 100μL PBS buffer, and the FA competition group was 3.87 mg / kg of I-P-4b and 100 times the dose of folic acid-polyethylene glycol dissolved in 100μL PBS buffer. The drug was administered by tail vein injection, and the control group was injected with an equal amount of pure solvent carrier. The drug was administered once a day for a total of 14 times. During the drug administration period, the mouse tumor volume and mouse weight changes were recorded at the same time every day, and the tumor volume was measured simultaneously with a vernier caliper, and the formula was 0.5×L×W 2 (L = longest axis, W = axis perpendicular to L, in millimeters) During the treatment, when mice showed a significant weight loss of 20% and tumor volume exceeded 2000mm 3 Or when the tumor length observed at any angle exceeds 20 mm, the observation is terminated. After the administration, the tumor growth and mouse weight curves are drawn. The mice are anesthetized by abdominal injection of sodium pentobarbital, euthanized by cervical dislocation, and the tumors are removed by dissection to record the tumor size.

[0248] In order to confirm the selectivity of this series of compounds for folate receptors in vivo, the above compound Ⅰ-P-4b, which has excellent effect in the in vivo experiment, was subjected to folate competition experiment. The experimental results are as follows Fig.17 As shown, when excess free folate was added to block the folate receptor FR, the antitumor activity of I-P-4b was abolished, confirming that the efficacy of this series of small molecule drug conjugates depends on the uptake of FR.

[0249] Example 62 Toxicity Study

[0250] BMDM cells were seeded into 96-well plates at a density of 10,000 cells / well and cultured for 12 h. -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 The target compound of M) was cultured in BMDM medium for 24 hours, and then the Cell Counting Kit-8 reagent was added to evaluate the cell viability. The optical density was analyzed by a microplate reader at a 450nm filter. The experimental results showed that the series of compounds had no significant inhibitory effect on the proliferation of macrophages ( Figure 18-19), preliminarily verified the safety of this series of small molecule drug conjugates.

[0251] The mice described in Example 60 were anesthetized by intraperitoneal injection of sodium pentobarbital, euthanized by cervical dislocation, and the tumors and major tissues and organs (including heart, liver, spleen, lungs and kidneys) were dissected from the mice. The tissues and organs of the mice were removed and fixed with 4% paraformaldehyde, followed by tissue embedding and H&E staining of relevant sections. Through H&E staining analysis of the heart, liver, spleen, lungs and kidneys, it was found that compared with the control group, there was a large amount of inflammatory cell infiltration in the lung tissues of the mice in the non-specific payload administration group, while no obvious pathological damage was found in the small molecule drug conjugate administration group ( Figure 20-21 ). These experimental results show that this series of conjugates have good safety.

[0252] Example 63 Study on Anti-tumor Immune Mechanism

[0253] Finally, it was verified whether this series of small molecule drug conjugates can play an anti-tumor immune role by regulating the immunosuppressive phenotype of TAMs and MDSCs and improving the tumor microenvironment. 24 hours after the last administration in Example 60, the tumor tissue was digested into single cells, and the cells were first stained with Fixable Viability Dye eFluor780 to distinguish between live and dead cells, and then blocked with CD16 / CD32, and finally TAM was labeled with CD45-APC, CD11b-FITC, F4 / 80-percp, CD86-PE, and CD206-PECy7 antibodies; MDSC cells were labeled with CD45-APC, CD11b-FITC, Ly-6G-PE, and Ly-6C-percp for flow analysis.

[0254] Take 1 mL of single cell suspension in a 2.5 ml centrifuge tube and add Fixable Viability Dye eFluor TM1 μL of 780 live / dead dye, vortex gently, and incubate at 4°C, protected from light, for 30 minutes. Centrifuge the cell suspension at 400g for 5 minutes at 4°C, remove the supernatant, and add 1 mL of PBS. Repeat this step once, and finally add 100 μL of PBS to suspend the cells and vortex to mix. Add 1 μL of Fc blocker, vortex gently, protect from light, and incubate on ice for 10 minutes for blocking. Centrifuge the cell suspension at 400g for 5 minutes at 4°C, remove the supernatant, and add 1 mL of PBS. Repeat this step once, and finally add 100 μL of PBS to suspend the cells and vortex to mix. Add 0.625μL of CD45-APC, 1μL of CD11b-FITC, 2.5μL of F4 / 80-PerCP / Cyanine5.5, and 0.625μL of CD86-PE, vortex gently to mix, and incubate at 4°C in the dark for 30 minutes. Centrifuge the cell suspension at 400g for 5 minutes at 4°C, remove the supernatant, and add 1mL of PBS. Repeat this step once. Finally, add 100μL of PBS to suspend the cells and vortex to mix. Add 100μL of IC fixative to the above 100μL cell suspension, vortex gently to mix, and incubate at room temperature in the dark for 60 minutes. Add 2mL of membrane permeabilization solution, centrifuge the cell suspension at 400g for 5 minutes at room temperature, remove the supernatant, and add 1mL of PBS. Repeat this step once. Resuspend the cells in 100 μL of permeabilization buffer, add 1.25 μL of CD206-PE / Cy7, and incubate for 30 minutes at room temperature in the dark. Add 2 mL of permeabilization buffer, centrifuge the cell suspension at 400 g for 5 minutes at room temperature, remove the supernatant, and add 1 mL of PBS. Repeat this step once, resuspend the cells in 500 μL of PBS containing 3% BSA, pass through a 40 μm cell strainer, and store in a BD Falcon tube in the dark for flow cytometry analysis. The flow cytometry analysis steps for MDSCs and T cells are the same as the above experimental steps.

[0255] like Fig. 22 As shown in Figure 2, all small molecule drug conjugates did not significantly change the total number of TAMs, but significantly increased the ratio of M1 / M2. This indicates that the anti-tumor M1 macrophages (CD11b + F4 / 80 + CD86 + CD206 - ) population, which has a typical immunosuppressive and anti-inflammatory M2 phenotype (CD11b + F4 / 80 + CD86 - CD206 +These experimental results revealed a significant shift of TAMs from M2 to M1 phenotype after treatment with this series of small molecule drug conjugates, indicating an enhancement of anti-tumor immune response. Fig.23 It can be seen that the number of MDSCs in the drug-treated group was significantly reduced. Figure 24-25 It can be found that compared with the control group, the treatment group can significantly increase CD3 + The proportion of T cells. + CD4 + T cells and CD3 + CD8 + The proportion of T cells in immune cells also increased significantly, revealing the reactivation of T cell anti-tumor immunity.

[0256] The above experimental results indicate that this series of small molecule drug conjugates selectively bind to the ligand part of the folate receptor, and deliver the payload PI3Kγ inhibitor / TLR agonist with the pharmacodynamic effect of reprogramming immunosuppressive cells to the folate receptor-positive immunosuppressive cells in tumor tissues, followed by intracellular cleavage to release the complete payload PI3Kγ inhibitor / TLR agonist, thereby exerting the immunotherapeutic function of the PI3Kγ inhibitor / TLR agonist, improving the immunosuppressive tumor microenvironment, and ultimately promoting T cell immunity and exerting an anti-tumor effect.

[0257] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A small molecule drug conjugate or its isomer, or a pharmaceutically acceptable salt thereof for tumor targeted immunotherapy, characterized in that: Its structure is shown in Formula I and Formula II: W and Q are selected from the following structures: (1)W is -(CH2) m C(O)NH-, Q is -(CH2) j -, wherein m is selected from 0, 1, 2, 3, 4, 5, and j is selected from 1, 2, 3, 4 or 5; (2)W is -(CH2) n -、-(CH2) n1 CH=CH(CH2) n2 -、-(CH2) n1 C≡C(CH2) n1 -, Q is an aryl group, a 5-6-membered heteroaryl group containing 1-3 selected from N, O, and S, a 5-6-membered heterocyclic group containing 1-3 selected from N, O, and S, or a three-membered to six-membered saturated or unsaturated monocyclic alkyl group, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, or 8; n1 and n2 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8; X is selected from (i) a sulfur-containing amino acid; (ii) an amide group formed by condensation of a hydrophilic amino acid and a sulfur-containing amino acid; L is selected from a sulfur-containing linker that can be cleaved by glutathione GSH reduction; Y is selected from a payload having a pharmacodynamic effect on reprogramming immunosuppressive cells.

2. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: The sulfur-containing amino acid is selected from cysteine, methionine, and cystine; The hydrophilic amino acid is selected from the group consisting of glycine, tyrosine, cysteine, glutamic acid, aspartic acid, asparagine, glutamine, threonine, serine, lysine, and arginine; The 5-6 membered heteroaryl group containing 1-3 selected from N, O, and S is selected from thienyl, pyrrolyl, furanyl, pyridine, and imidazolyl; The 5-6 membered heterocyclic group containing 1-3 selected from N, O, and S is selected from piperazinyl, morpholinyl, piperidine, and tetrahydropyrrolyl; The Y is selected from a PI3Kγ inhibitor or a TLR agonist.

3. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: W is -(CH2) m C(O)NH-, Q is -(CH2) j -, wherein m is selected from 0, 1, 2, 3; j is selected from 1, 2, 3, 4 or 5.

4. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: W is -(CH2) n -、-(CH2) n1 CH=CH-、-(CH2) n1 C≡C-, Q is selected from The n, n1 and n2 are independently selected from 1, 2, 3, 4, 5 or 6.

5. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: The X is selected from cysteine, methionine, cystine or an amide group formed by condensation of any one of them with glutamic acid; the Y is selected from PI3Kγ inhibitor AS-252424 or IPI-549, or TLR agonist IMDQ.

6. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: The L is selected from The k is selected from 0, 1, 2, 3, 4, and 5.

7. The small molecule drug conjugate or its isomer or pharmaceutically acceptable salt for tumor targeted immunotherapy according to claim 1, characterized in that: The compound is selected from:

8. A composition comprising the small molecule drug conjugate for tumor targeted immunotherapy according to any one of claims 1 to 7 or its isomer, or a pharmaceutically acceptable salt thereof, characterized in that: It also includes at least one pharmaceutically acceptable carrier or excipient.

9. Use of the small molecule drug conjugate for tumor targeted immunotherapy or its isomers, or pharmaceutically acceptable salts thereof, as claimed in any one of claims 1 to 7 in the preparation of anti-tumor targeted immunotherapy drugs.

10. The use according to claim 9, characterized in that: The tumor is one or more of ovarian cancer, liver cancer, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, and melanoma.

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