CN116283953A - Indoline compound containing thiazole structure, and preparation method and application thereof - Google Patents

Abstract

Indoline compounds containing a thiazole structure and their preparation methods and applications belong to the field of medical technology, specifically disclosing indoline compounds containing a thiazole structure represented by general formula I, their stereoisomers or pharmaceutically acceptable salts, their preparation methods and their application in the preparation of drugs for treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT. The compounds disclosed in the present invention have a high level of inhibitory activity on PD-1/PD-L1 protein/protein interaction and NAMPT, and have significant anti-proliferation activity on A2780 cells with high expression of NAMPT. Representative compounds can overexpress NAMPT, PD-L1 inhibits tumor growth in an animal model, and can be used to prepare drugs for treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT, such as cancer and viral infection.

Description

Indoline compounds containing thiazole structure and preparation method and application thereof

Technical Field

The present invention belongs to the field of medical technology, and relates to an indoline compound containing a thiazole structure as shown in general formula I or its stereoisomer or a pharmaceutically acceptable salt thereof, as well as a preparation method thereof and the use of the compound or a pharmaceutical composition containing the compound in the preparation of a drug for treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT.

Background Art

In recent years, with the elucidation of the molecular mechanism of immune regulation, tumor immunotherapy has developed rapidly and achieved good clinical efficacy. Programmed cell death receptor 1 (PD-1) is a T cell surface receptor. When it binds to programmed cell death ligand 1 (PD-L1), it produces negative immune regulatory signals, thereby inhibiting T cell activation, proliferation, and the secretion of cytokines such as interleukin 2 (IL-2) and interferon γ (IFN-γ). A large number of studies have shown that the tumor microenvironment in the body induces upregulation of PD-1 expression in infiltrating T cells, while tumor cells highly express PD-L1, resulting in continuous activation of the PD-1/PD-L1-mediated signaling pathway, and inhibition of tumor-specific CD8 ⁺ T cell function, so that they cannot recognize or kill tumor cells, that is, tumor cells escape immune. Therefore, targeted blocking of PD-1/PD-L1 protein/protein interaction can effectively restore T cell function, allowing it to re-recognize and kill tumor cells.

Immunotherapy based on PD-1/PD-L1 has attracted much attention. Currently, the monoclonal antibody drugs that have been approved for marketing include Pembrolizumab from Merck, Nivolumab from Bristol-Myers Squibb, Avelumab from Merck, Durvalumab from AstraZeneca, Atezolizumab from Roche, Cemiplimab from Regeneron/Sanofi, and Dostarlimab from GlaxoSmithKline. The above drugs have shown significant efficacy in the treatment of various tumors, and the approved indications include melanoma, non-small cell lung cancer, gastric cancer, urothelial carcinoma, etc. With the development of clinical research, monoclonal antibody drugs are expected to achieve breakthroughs in more indications.

Although monoclonal antibody drugs have shown advantages in clinical treatment, they also have obvious defects, such as difficulty in preparation and purification, high production cost, easy to be decomposed by proteases, inability to be administered orally, and serious toxic side effects related to the immunogenicity of monoclonal antibodies. Compared with biological macromolecule drugs, the pharmacokinetic properties of small molecule compounds can be controlled after chemical modification, and there is also greater room for exploration and optimization in production processes, administration methods, etc. At the same time, patients treated with PD-1/PD-L1 monoclonal antibodies have extensive primary or secondary drug resistance, and innovative drug research targeting drug resistance is expected to improve the bottleneck problem in tumor immunotherapy. The occurrence of drug resistance is related to many factors. It has been confirmed that high metabolism of tumor cells leads to the inhibition of T cell proliferation and promotes the formation of tumor immune microenvironment, which greatly reduces the efficacy of monoclonal antibody drugs. Therefore, appropriate intervention in tumor cell metabolism and improvement of tumor immune microenvironment can enhance anti-tumor immune response and improve the clinical efficacy of anti-PD-1/PD-L1 treatment.

Nicotinamide adenine dinucleotide (NAD ⁺ ) is one of the important coenzymes in cellular redox reactions and plays a vital role in a variety of cellular physiological processes, especially energy metabolism. In addition, NAD ⁺ is a substrate for deacetylases sirtuins, poly (ADP-ribose) polymerase 1, and ADP cyclase, and has important effects on Ca ⁺ mobilization, gene stability, cell apoptosis, metabolism, etc. In order to maintain a stable intracellular NAD ⁺ concentration, organisms mainly synthesize NAD ⁺ through the following pathways: ① de novo synthesis from tryptophan. ② pararibosomal synthesis from nicotinic acid and nicotinamide. ③ Recycling NAM (NAD ⁺ metabolite) and then remediating NMN. Among them, remedial recycling synthesis is an important pathway for NAD ⁺ biosynthesis, and nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in this pathway.

Tumor cells have a very active metabolism and consume NAD ⁺ faster than normal cells. Therefore, in tumor metabolism, tumor cells will meet the high demand for NAD ⁺ by highly expressing NAMPT. Therefore, in some tumors with overexpression of NAMPT, the production of NAD ⁺ in tumor cells will be directly inhibited by effectively inhibiting NAMPT, leading to cell death. On the other hand, normal cells are not affected by NAMPT inhibition because they can synthesize NAD ⁺ from nicotinic acid through other biosynthetic pathways, such as catalysis by nicotinic acid phosphoribosyltransferase (NAPRT). However, many cancer cell lines cannot synthesize NAD ⁺ through this pathway due to the lack of NAPRT. Therefore, inhibiting NAMPT has become an effective and selective strategy to inhibit the growth of cancer cells. Representative NAMPT inhibitors include CHS-828 and FK866.

Studies have shown that overexpression of NAMPT in tumor cells can promote the proliferation of immunosuppressive cells MDSCs and promote the formation of an immunosuppressive microenvironment. At the same time, the accumulation of lactic acid, a metabolite of tumor cell glycolysis, can promote the differentiation of monocytes into dendritic cells, which directly inhibit the immune response by secreting immunosuppressive factors and inhibiting T cell immune responses. Therefore, the formation of an immunosuppressive microenvironment caused by high tumor cell metabolism is one of the important reasons for the occurrence of acquired resistance to anti-PD-1/PD-L1 therapy. Studies have shown that compared with single treatment, the combined use of PD-1 monoclonal antibody drugs and NAMPT inhibitors significantly reduced tumor volume and weight, showing better anti-tumor activity (Travelli C et al. Cancer Research, 2019, 79 (8): 1938-1951). Simultaneous inhibition of PD-1/PD-L1 interaction and NAMPT theoretically has a synergistic immune activation function, and is expected to overcome the problems of PD-1/PD-L1 inhibitor resistance and NAMPT inhibitor dose-limiting toxicity to a certain extent. Therefore, the targeted development of dual inhibitors of PD-1/PD-L1 interaction and NAMPT will hopefully yield compounds with outstanding anti-tumor activity and unique mechanisms of action, which is of great research significance.

Summary of the invention

The primary purpose of the present invention is to provide an indoline compound containing a thiazole structure as shown in general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

in,

所述苯基可任选被1–3个R¹取代；Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ;

取代； ^R1 is independently selected from hydrogen, halogen, cyano, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino; the (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino may be optionally substituted by 1-3

replace;

R ² and R ³ are independently selected from hydrogen, (C1-C4)alkyl, hydroxy(C1-C4)alkyl;

or R ² , R ³ and the nitrogen atom to which they are attached together form a 3-6-membered nitrogen-containing heterocyclic ring, preferably a 4-6-membered nitrogen-containing heterocyclic ring; the nitrogen-containing heterocyclic ring contains 1-3 heteroatoms selected from N, O and S; the nitrogen-containing heterocyclic ring may be optionally substituted by 1-3 R ⁴ ;

R ⁴ is independently selected from hydrogen, hydroxy, carboxyl, (C1-C4)alkyl, hydroxy(C1-C4)alkyl;

E is selected from O, N–CN;

X is selected from amino (C1-C4) alkyl, (C2-C4) alkenyl,

R ^a is selected from phenyl and pyridyl; the phenyl and pyridyl may be optionally substituted by 1-3 R ⁵ ;

R ⁵ is independently selected from hydrogen, halogen, hydroxy, amino, (C1-C4) alkyl;

The present invention preferably relates to an indoline compound containing a thiazole structure of general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

in,

所述苯基可任选被1–3个R¹取代；Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ;

取代； ^R1 is independently selected from hydrogen, halogen, hydroxyl, amino, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino; the (C1-C4) alkoxy, (C1-C4) alkylformylamino may be optionally substituted by 1-3

replace;

选自：

Selected from:

E is selected from O, N–CN;

X is selected from amino (C1-C4) alkyl, (C2-C4) alkenyl,

^Ra is independently selected from phenyl and pyridyl; the phenyl and pyridyl may be optionally substituted by 1-3 hydrogen or halogen.

The present invention further preferably relates to an indoline compound containing a thiazole structure represented by general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

in,

所述苯基可任选被1–3个R¹取代；Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ;

取代； ^R1 is independently selected from hydrogen, halogen, hydroxyl, amino, methyl, methoxy, ethoxy, propoxy, propionylamino; the ethoxy, propoxy, propionylamino may be optionally substituted by 1-3

replace;

选自：

Selected from:

当E为N–CN时，X为

When E is O, X is selected from

When E is N-CN, X is

^Ra is pyridyl; the pyridyl may be optionally substituted with 1-3 hydrogen or halogen.

The indoline compounds containing thiazole structure represented by the general formula I of the present invention, their stereoisomers or pharmaceutically acceptable salts thereof are ultimately preferably selected from the following compounds:

In addition, the present invention also includes prodrugs of the compounds of the present invention. Prodrugs of the compounds of the present invention are derivatives of the compounds of Formula I, which may have weak or even no activity themselves, but after administration, are converted into corresponding biologically active forms under physiological conditions (e.g., by metabolism, solvolysis or other means).

Among the indoline compounds containing a thiazole structure of the general formula I described above, their stereoisomers or pharmaceutically acceptable salts thereof, the pharmaceutically acceptable salts include salts formed with inorganic acids, organic acids and alkali metal ions; the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid and phosphoric acid; the organic acid is selected from the group consisting of succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid and p-toluenesulfonic acid; the alkali metal ions are selected from the group consisting of lithium ions, sodium ions and potassium ions.

代表取代基连接处。In the present invention, "halogen" refers to fluorine, chlorine, bromine or iodine; "alkyl" refers to a straight chain or branched chain alkyl;

Represents the place where substituents are attached.

The indoline compounds containing thiazole structure shown in the general formula I of the present invention, their stereoisomers or pharmaceutically acceptable salts thereof are used as active ingredients and mixed with pharmaceutically acceptable carriers or excipients to prepare pharmaceutical compositions, wherein the carriers or excipients include diluents, adhesives, wetting agents, disintegrants, lubricants and glidants known in the art. Diluents include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol and calcium hydrogen phosphate; wetting agents include water, ethanol and isopropanol; adhesives include starch slurry, dextrin, syrup, honey, glucose solution, acacia glue slurry, gelatin slurry, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose and polyethylene glycol; disintegrants include dry starch, microcellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch and sodium dodecyl sulfate; lubricants and glidants include talc, silicon dioxide and polyethylene glycol.

The pharmaceutical composition of the present invention can be formulated into several dosage forms, selected from injections, tablets, and capsules.

The indoline compounds containing thiazole structure, stereoisomers thereof or pharmaceutically acceptable salts thereof described in the present invention can be used in combination with other active ingredients to achieve better therapeutic effects.

The present invention also provides the use of the indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and/or treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT. The diseases related to the PD-1/PD-L1 protein/protein interaction and NAMPT are selected from cancer and infectious diseases. The cancer is selected from lymphoma, non-small cell lung cancer, small cell lung cancer, head and neck cell cancer, glioma, neuroblastoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, gastric cancer, colon cancer, colorectal cancer, kidney cancer, bile duct cancer, gastric cancer, esophageal squamous cell carcinoma, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, brain cancer, melanoma, multiple myeloma, skin cancer, epithelial cell cancer, leukemia and cervical cancer; the infectious disease is selected from bacterial infection and viral infection.

The present invention also provides a method for preparing the compound of the general formula I. All raw materials and intermediates are prepared by methods well known to those skilled in the art of organic chemistry in the manner described in the following flow chart or are commercially available.

Route 1:

(a) Using 4-bromo-1H-indole as the starting material, intermediate 2 is prepared under the action of a reducing agent such as sodium cyanoborohydride;

(b) intermediate 2 and 5-(1,3-dioxolan-2-yl)thiazole-2-carboxylic acid are used as raw materials, and intermediate 3 is prepared by amidation reaction under condensation conditions;

(c) intermediate 3 is used as a raw material, and deprotection is performed under the action of p-toluenesulfonic acid to obtain intermediate 4 containing a formyl group;

(d) intermediate 4 is used as a raw material to prepare intermediate 5 under the action of a reducing agent such as sodium borohydride;

(e) using intermediate 5 and benzene, substituted benzene ring boronic acid or boric ester as raw materials, to obtain intermediate 6 through Suzuki-Miyaura coupling reaction;

(f) intermediate 6 is used as a raw material to prepare intermediate 7 under the action of a chlorinating agent such as dichlorothionyl;

(g) intermediate 7 is used as a raw material to prepare intermediate 8 by Gabriel reaction;

(h) Using intermediate 8 as a starting material, the target compound of formula I is prepared by nucleophilic substitution or amidation reaction.

Route 2:

(i) intermediate 5 and m-hydroxyphenylboronic acid are used as raw materials to prepare intermediate 9 through Suzuki-Miyaura coupling reaction;

(j) intermediate 9 is used as a raw material, and reacted with a dihalide to obtain intermediate 10;

(k) intermediate 10 is used as a raw material, and reacted with a small molecule amine to obtain intermediate 11;

(l) Using intermediate 11 as a raw material, intermediate 12 is prepared under the action of a chlorinating agent such as dichlorothionyl;

(m) using intermediate 12 as a raw material, preparing intermediate 13 by Gabriel reaction;

(n) Using intermediate 13 as a starting material, the target compound of formula I is prepared by nucleophilic substitution or amidation reaction.

Route 3:

(o) Using intermediate 9 as a raw material, intermediate 14 is prepared under the action of a chlorinating agent such as dichlorothionyl;

(p) using intermediate 14 as a raw material, preparing intermediate 15 by Gabriel reaction;

(q) Intermediate 15 is used as a starting material to obtain intermediate 16 via nucleophilic substitution or amidation reaction.

(r) intermediate 16 is used as a raw material, and reacted with a dihalide to obtain intermediate 17;

(s) Intermediate 17 is used as a raw material and reacted with a small molecule amine to obtain the target compound of formula I.

Route 4:

(t) intermediate 18 was prepared by Suzuki-Miyaura coupling reaction using intermediate 5 and m-aminophenylboronic acid as raw materials;

(u) Using intermediate 18 as a raw material, intermediate 19 is prepared under the action of a chlorinating agent such as dichlorothionyl;

(v) using intermediate 19 as a raw material, preparing intermediate 20 by Gabriel reaction;

(w) using intermediate 20 as a starting material, preparing intermediate 21 by nucleophilic substitution or amidation reaction;

(x) using intermediate 21 as a raw material, reacting with a halogenated alkyl halide to obtain intermediate 22;

(y) Intermediate 22 is used as a raw material and reacted with a small molecule amine to obtain the target compound of formula I.

The definitions of Cy, E, X, and ^Ra are as described in the claims; n is 1 to 3, and Y is chlorine or bromine. The indoline compounds containing thiazole structure having the general formula I of the present invention can be prepared according to the method described in the above reaction scheme or a similar method.

Beneficial effects of the present invention:

The compound shown in the general formula I provided by the present invention has a high level of inhibitory activity on PD-1/PD-L1 protein/protein interaction and NAMPT, and has significant anti-proliferative activity on A2780 cells with high expression of NAMPT. Representative compounds can inhibit tumor growth in animal models overexpressing NAMPT and PD-L1. Therefore, the indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound can be used to prepare drugs for treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT, such as cancer and viral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the in vivo pharmacodynamic results of the compound prepared in Example 22 in the A20 endogenous high expression PD-L1 and NAD ⁺ model;

Figure 2 shows the in vivo pharmacodynamic results of the compound prepared in Example 22 in the LLC OE NAMPT/PD-L1 model.

DETAILED DESCRIPTION

In the following examples, methods for preparing some of the compounds are described. It should be understood that the following methods and other methods known to those of ordinary skill in the art are applicable to the preparation of all compounds described in the present invention. The examples are intended to illustrate rather than limit the scope of the present invention.

Example 1: (E)-2-cyano-1-{[2-(4-phenylindolin-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-1)

Step 1: 4-Bromoindoline

Dissolve 4-bromoindole (20.0 g, 0.102 mol) in 120 mL of glacial acetic acid, slowly add NaBH ₃ CN (19.3 g, 0.306 mol) under ice bath, react at room temperature for 1 h, and after the reaction is complete, dilute the reaction solution with 200 mL of water, slowly adjust the pH to 7-8 with 40% NaOH solution under ice bath, extract with ethyl acetate (100 mL×3), combine the organic layers, wash with water (100 mL) and saturated brine (100 mL) in turn. Dry the organic layer with anhydrous sodium sulfate, filter with suction, and evaporate the filtrate to dryness under reduced pressure to obtain 18.7 g of white solid, with a yield of 92.5%; MS (ESI) m/z: 198.1 [M+H] ⁺ .

Step 2: Ethyl 5-formylthiazole-2-carboxylate

Dissolve thiooxamate ethyl ester (40.0 g, 0.3 mol) and 2-bromomalonaldehyde (44.8 g, 0.3 mol) in 200 mL of dioxane and react at room temperature for 2 h. After the reaction is complete, pour the reaction solution into 400 mL of water, and a light yellow solid precipitates. Filter it with suction, and extract the filtrate with toluene (150 mL × 3). The organic phases were combined, washed with saturated aqueous sodium bicarbonate solution (150 mL×2), the aqueous layer was back-extracted once with 150 ml of toluene, the organic phases were combined, washed with saturated brine (150 mL×2), the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 26.6 g of light yellow solid, with a yield of 47.8%; MS (ESI) m/z: 201.1 [M+H] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 10.15 (s, 1H), 8.87 (s, 1H), 4.42 (q, J＝7.1 Hz, 2H), 1.35 (t, J＝7.1 Hz, 3H).

Step 3: Ethyl 5-(1,3-dioxolan-2-yl)thiazole-2-carboxylate

At room temperature, ethyl 5-formylthiazole-2-carboxylate (20.0 g, 0.11 mol) was dissolved in 150 mL of toluene, and ethylene glycol (10.1 g, 0.16 mol) and p-toluenesulfonic acid monohydrate (2.05 g, 11 mmol) were added in sequence, and the temperature was raised to reflux for 4 h. During the reaction, the water produced by the reaction was separated by a Dean-Stark device. After the reaction was completed, the reaction solution was cooled to room temperature, and the reaction solution was washed with a saturated sodium carbonate solution (50 mL × 2) and a saturated saline solution (50 mL) in sequence. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 19.8 g of a brown-red oily liquid with a yield of 87.7%. MS (ESI) m/z: 230.3 [M+H] ⁺ ; ¹ HNMR (600MHz, DMSO-d ₆ ) δ8.17 (s, 1H), 6.24 (s, 1H), 4.38 (q, J = 7.1Hz, 2H), 4.05 (dt, J = 13.0, 8.6Hz, 2H), 4.00 (dt, J = 9.1, 8.6Hz ,2H),1.33(t,J=7.1Hz,4H).

Step 4: 5-(1,3-dioxolan-2-yl)thiazole-2-carboxylic acid

Dissolve 5-(1,3-dioxolan-2-yl)thiazole-2-carboxylic acid ethyl ester (20.0 g, 87.3 mmol) in 80 mL tetrahydrofuran, add 40 mL sodium hydroxide (5.2 g, 131.0 mmol) aqueous solution, and react at room temperature for 1 h. After the reaction is completed, the organic solvent is evaporated and the remaining aqueous solution is cooled in an ice bath. Under stirring, 12 N concentrated hydrochloric acid is slowly added dropwise to adjust the pH to 1-2, and the solid is precipitated, filtered, washed with a small amount of water (5 mL × 3), and dried to obtain 11.4 g of yellow solid, with a yield of 64.9%. MS (ESI) m/z: 313.1 [MH] ^- ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ7.73 (s, 1H), 6.03 (s, 1H), 4.04–4.00 (m, 2H), 3.94–3.90 (m, 2H).

Step 5: [5-(1,3-dioxolan-2-yl)thiazol-2-yl](4-bromoindolin-1-yl)methanone

5-(1,3-dioxolan-2-yl)thiazole-2-carboxylic acid (10.6 g, 0.054 mol) and HATU (23.4 g, 0.061 mol) were dissolved in 50 mL DMF, stirred at room temperature for 10 min, 4-bromoindoline (10.0 g, 0.051 mol) was added, reacted at room temperature for 20 min, DIPEA (25.9 g, 0.20 mol) was added, and reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was poured into 150 mL of water, solid precipitated, filtered, the filter cake was washed with water (25 mL × 2), and dried to obtain 17.2 g of light yellow solid, with a yield of 89.6%; MS (ESI) m/z: 381.1 [M+H] ⁺ .

Step 6: 2-(4-bromoindoline-1-carbonyl)thiazole-5-carbaldehyde

Dissolve [5-(1,3-dioxolan-2-yl)thiazol-2-yl](4-bromoindolyl-1-yl)methanone (15.0 g, 39.4 mmol) in 75 mL of a mixed solvent of acetone/water (V/V, 4:1), add p-toluenesulfonic acid monohydrate (15.0 g, 78.7 mmol), and heat to 70°C for 1 h. After the reaction is complete, cool the reaction solution to room temperature to precipitate a solid, wash the filter cake with water (15 mL × 2), and dry to obtain 12.2 g of a light yellow solid with a yield of 91.6%; MS (ESI) m/z: 337.2 [M+H] ⁺ .

Step 7: (4-bromoindolin-1-yl)[5-(hydroxymethyl)thiazol-2-yl]methanone

2-(4-bromoindoline-1-formyl)thiazole-5-carboxaldehyde (13.5 g, 40 mmol) was dissolved in 120 mL of a mixed solvent of EtOH/DCM (V/V, 1:1), and NaBH ₄ (13.5 g, 60 mmol) was slowly added at 0°C, and the mixture was reacted at 0°C for 1 h. After the reaction was completed, 20 mL of 1 mol/L dilute hydrochloric acid aqueous solution was added to the reaction solution to quench, the reaction solution was concentrated, 100 mL of water was added, and DCM was extracted (50 mL×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and evaporated to obtain 12.2 g of a yellow solid, with a yield of 89.7%; MS (ESI) m/z: 339.0 [M+H] ⁺ .

Step 8: [5-(Hydroxymethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone

Dissolve (4-bromoindolyl-1-yl)[5-(hydroxymethyl)thiazol-2-yl]methanone (30mmol), phenylboronic acid (36mmol), PdCl ₂ (dppf)·DCM (1.5mmol) and potassium carbonate (60mmol) in 100mL of a mixed solvent of dioxane/water (V/V, 4:1), and react at 70°C for 0.5-2h under nitrogen protection. After the reaction is completed, cool to room temperature, filter the reaction solution through diatomaceous earth pad, and wash the filter cake with ethyl acetate (50mL×2). Concentrate the filtrate under reduced pressure, add 200mL of ethyl acetate and 100mL of water to the residue, and separate the organic layer. The organic layer was washed with water (100 mL) and saturated brine (100 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The product was separated and purified by column chromatography to obtain 8.4 g of a white solid with a yield of 83.2%; MS (ESI) m/z: 337.2 [M+H] ⁺ .

Step 9: [5-(Chloromethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone

Dissolve [5-(Hydroxymethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone (20 mmol) in 30 mL of acetonitrile, slowly add thionyl chloride (60 mmol) at 0°C, add dropwise, and react at room temperature for 30 min. After the reaction is completed, pour the reaction solution into 250 mL of ice water, precipitate solid, stir for 15 min, filter, and dry to obtain 6.5 g of yellow solid, with a yield of 91.8%; MS (ESI) m/z: 354.1 [M+H] ⁺ .

Step 10: 2-{[2-(4-phenylindolin-1-carbonyl)thiazol-5-yl]methyl}isoindolinone-1,3-dione

[5-(Chloromethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone (10 mmol), phthalimide (12 mmol) and potassium carbonate (15 mmol) were added to 30 mL of DMF and reacted at 40° C. for 4 h. After the reaction was completed, the reaction solution was poured into 100 mL of water, and the precipitated solid was filtered and dried to obtain 4.0 g of a brown-yellow solid with a yield of 86.8%.

Step 11: [5-(Aminomethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone

2-{[2-(4-phenylindolin-1-formyl)thiazol-5-yl]methyl}isoindolinone-1,3-dione (5mmol) was dissolved in 20mL of ethanol, hydrazine hydrate (10mmol) was added dropwise, and the temperature was raised to 80°C for 1h. After the reaction was completed, the reaction solution was cooled, the solid was precipitated, and the filter cake was washed with ethanol (5mL×2). The filtrate was evaporated to dryness and concentrated, water was added, and dichloromethane (10mL×3) was extracted. The organic phases were combined, washed with saturated brine (10mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated to dryness and concentrated to obtain 0.98g of brown solid with a yield of 58.8%.

Step 12: Methyl (Z)-N'-cyano-N-(pyridin-4-yl)carbamoyl imidothioester

4-Aminopyridine (10mmol) and N-cyano-S,S-dimethyl dithiocarbonate (12mmol) were dissolved in 10mL DMF, and NaH (15mmol) was slowly added at 0℃, and then the temperature was raised to 70℃ for reaction for 5h. After the reaction was completed, the reaction solution was cooled, and 20mL of PE/Et ₂ O (V/V, 1:5) mixed solution was added to the reaction solution. After stirring, the supernatant was removed, and the oil layer was washed twice with 30mL PE/Et ₂ O (V/V, 1:5). The residual oil layer was dissolved in 20mL water, and 1mL of glacial acetic acid was added under stirring to precipitate a white solid, which was washed with water (5mL×2) and a small amount of ether (2.5mL×2) and dried to obtain 1.2g of a white solid with a yield of 63.5%; MS (ESI) m/z: 193.1[M+H] ⁺ .

Step 13: (E)-2-cyano-1-{[2-(4-phenylindolin-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Example 1)

[5-(Aminomethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone (0.5mmol), methyl (Z)-N'-cyano-N-(pyridin-4-yl)carbamoyl imide thioester (6mmol) were dissolved in 2mL pyridine, triethylamine (0.75mmol) and 4-dimethylaminopyridine (0.05mmol) were added, and the temperature was raised to 70°C for reaction for 5h. After the reaction was completed, the reaction solution was cooled, water was added, and ethyl acetate was extracted. The organic phases were combined, and the organic layer was washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. 99mg of white solid was obtained by column chromatography separation and purification, and the yield was 42.3%.

MS (ESI) m/z: 478.2 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ9.78 (s, 1H), 8.47 (d, J = 50.7

Hz,3H),8.23(d,J＝7.5Hz,1H),7.97(s,1H),7.54–7.44(m,4H),7.43–7.33(m,2H),7.29–7.06(m,3H) ,4.72(d,J＝3.4Hz,2H),4.68(t,J＝7.9Hz,2H),3.26(t,J＝8.2Hz,2H); ¹³ C NMR(151MHz,DMSO-d ₆ )δ164.97,157.94,150.29,143.25,142.65,141.49,140.86,139.56,138.14,130.54,128.57,128.13,127.82,127.41,124.95,116.22,115.21,1 06.88,50.34,37.75,28.06.

Example 2: 1-{[2-(4-phenylindolin-1-formyl)thiazol-5-yl]methyl}-3-(pyridine-3-methylene)urea (Compound I-2)

[5-(Aminomethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone (0.5mmol) and phenyl(pyridine-3-methylene)carbamate (0.75mmol) were dissolved in 2mL DMF, and DIPEA (1mmol) was added and the temperature was raised to 80°C for 4h. After the reaction was completed, the reaction solution was cooled, water was added, and a solid was precipitated. The filter cake was washed with water, dried, and purified by column chromatography to obtain 122mg of a white solid with a yield of 52.4%.

MS (ESI) m/z: 468.2 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ8.48 (s, 1H), 8.44 (d, J = 3.8Hz,

1H),8.23(d,J＝6.6Hz,1H),7.87(s,1H),7.66(d,J＝7.8Hz,1H),7.52–7.45(m,4H),7.42–7.31(m,3H ),7.16(d,J＝7.6Hz,1H),6.78(t,J＝5.8Hz,1H),6.72(t,J＝5.9Hz,1H),4.66(t,J＝7.8Hz,2H), 4.48(d,J＝5.9Hz,2H), 4.26(d,J＝5.9Hz,2H), 3.25(t,J＝8.2Hz,2H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ164.58,158.51,158.17,148.98,148.29,145.63,143.71,141.63,139.99,138.53,136.54,135.23,130.91,128.98,128.54,128.21,127.81,1 25.27,123.76,116.61,50.76,41.06,36.36,28.47.

Similar to the synthesis method of Example 2, [5-(aminomethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone was used as a raw material and reacted with phenyl(pyridine-4-methylene)carbamate to prepare the compound of Example 3.

Example 3: 1-{[2-(4-phenylindolin-1-formyl)thiazol-5-yl]methyl}-3-(pyridin-4-methylene)urea (Compound I-3)

MS (ESI) m/z: 468.0 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ8.49 (d, J = 4.9Hz, 2H), 8.23

(d,J＝6.9Hz,1H),7.88(s,1H),7.52–7.46(m,4H),7.42–7.33(m,2H),7.24(d,J＝4.1Hz,2H),7.16( d,J＝7.6Hz,1H),6.86(t,J＝5.9Hz,1H),6.76(t,J＝6.0Hz,1H),4.66(d,J＝7.4Hz,2H),4.49(d, J＝5.9Hz, 2H), 4.27 (d, J＝6.0Hz, 2H), 3.26 (t, J＝8.2Hz, 2H); ¹³ CNMR (151MHz, DMSO-d ₆ )δ164.14,164.59,158.51,158.21,150.29,149.79,145.60,143.71,141.64,139.99,138.53,130.91,129.97,128.97,128.54,128.21,127.81,1 25.27,122.29,116.61,50.76,42.41,36.39,28.47.

Example 4: (E)-N-{[2-(4-phenylindole-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-3-yl)acrylamide (Compound I-5)

Trans-3-(3-pyridyl)allylic acid (0.55 mmol) and HATU (0.6 mol) were dissolved in 2 mL of DMF, stirred at room temperature for 10 min, and then [5-(aminomethyl)thiazol-2-yl](4-phenylindolin-1-yl)methanone (0.5 mmol) was added, and the mixture was reacted at room temperature for 20 min. DIPEA (2 mmol) was added, and the mixture was reacted at room temperature for 1 h. After the reaction was completed, the reaction solution was poured into water, and a solid was precipitated. The solid was filtered, and the filter cake was washed with water, dried, and purified by column chromatography to obtain 114 mg of a white solid with a yield of 48.8%.

MS(ESI)m/z:465.0[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ8.96(s,1H),8.79(s,1H),8.57(s,1H),8.22( s,1H),8.02(s,1H),7.97(s,1H),7.56(d,J＝15.5Hz,1H),7.52–7.44(m,5H),7.42–7.33(m,2H),7.16 (s,1H),6.77(d,J＝16.1Hz,1H),4.68(s,4H),3.26(s,2H); ¹³ CNMR(151MHz,DMSO-d ₆ )δ164.15,165.06,158.40,150.68,149.62,143.66,143.32,142.49,139.97,138.54,136.73,134.46,130.92,130.89,128.97,128.53,128.21,1 27.81,125.31,124.36,123.71,116.61,50.74,35.48,28.46 .

The synthesis method of Example 1 was adopted, and (4-bromoindolyl-1-yl)[5-(hydroxymethyl)thiazol-2-yl]methanone was used as a raw material to undergo a Suzuki-Mi yaura coupling reaction with a substituted phenylboronic acid or a boric acid ester, followed by chlorination and Gabriel reaction to obtain a primary amine intermediate, and the primary amine intermediate was subjected to a substitution reaction with methyl (Z)-N'-cyano-N-(pyridin-4-yl)carbamide imidothioester to prepare the compounds of Examples 5-11.

Example 5: (E)-2-cyano-1-({2-[4-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)indolin-1-carbonyl]thiazol-5-yl}methyl)-3-(pyridin-4-yl)guanidine (Compound I-6)

MS(ESI)m/z:560.3[M+Na] ⁺ ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.75(s,1H),8.51(s,1H),8.44(s,2H), 8.18(d,J＝7.3Hz,1H),7.97(s,1H),7.32(t,J＝7.9Hz,1H),7.21(s,2H),7.11(d,J＝7.6Hz,1H), 6.98(s,1H),6.94(s,2H),4.72(s,2H),4.66(t,J＝7.8Hz,2H),4.28(s,4H),3.24(t,J＝8.1Hz,2H ); ¹³ C NMR (151MHz, DMSO-d ₆ )δ165.43,158.31,157.51,150.78,145.69,143.67,143.60,143.33,143.07,138.04,133.13,130.77,128.12,124.17,121.52,117.53,117.08,1 16.29,115.68,64.52,64.48,50.75,38.15,28.54.

Example 6: (E)-1-({2-[4-(3-chlorophenyl)indoline-1-carbonyl]thiazol-5-yl}methyl)-2-cyano-3-(pyridin-4-yl)guanidine (Compound I-7)

MS(ESI)m/z:514.1[M+H] ⁺ ,535.9[M+Na] ⁺ ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.76(s,1H),8.52(s,1H) ,8.44(s,2H),8.25(d,J＝7.5Hz,1H),7.98(s,1H),7.55(s,1H),7.53–7.49(m,1H),7.49–7.45(m,2H ),7.38(t,J＝7.8Hz,1H),7.28–7.13(m,3H),4.73(s,2H),4.68(t,J＝7.8Hz,2H),3.26(t,J＝8.2Hz ,2H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ165.32,158.39,157.51,150.78,145.65,143.75,143.11,142.08,141.89,137.06,133.70,131.14,130.85,128.35,128.25,127.79,127.3 7,125.33,117.12,116.34,115.67,50.75,38.16,28.31.

Example 7: (E)-1-(2-fluoropyridin-4-yl)-2-cyano-3-{[2-(4-phenylindolin-1-carbonyl)thiazol-5-yl]methyl}guanidine (Compound I-8)

MS (ESI) m/z: 496.0 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ9.55 (s, 1H), 8.69 (s, 1H), 8.23 (d, J = 7.4Hz, 1H),8.10(d,J＝5.7Hz,1H),7.99(s,1H),7.52–7.44(m,4H),7.42–7.33(m,2H),7.19–7.11(m,2H),6.93 (s,1H),4.75(s,2H),4.68(t,J＝7.9Hz,2H),3.26(t,J＝8.2Hz,2H); ¹³ CNMR(151MHz,DMSO-d ₆ )δ165.48,165.06,163.53,158.33,157.34,148.45,143.65,143.20,141.56,139.97,138.56,130.96,128.98,128.54,128.23,127.82,125.36,1 16.63,116.13,113.79,99.89,50.75,38.25,28.47.

Example 8: (E)-2-cyano-1-({2-[4-(3-fluorophenyl)indoline-1-carbonyl]thiazol-5-yl}methyl)-3-(pyridin-4-yl)guanidine (Compound I-9)

MS(ESI)m/z:498.3[M+H] ⁺ ,519.9[M+Na] ⁺ ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.76(s,1H),8.58–8.38(m, 3H),8.25(d,J＝7.2Hz,1H),7.98(s,1H),7.56–7.49(m,1H),7.42–7.32(m,3H),7.26–7.15(m,4H),4.72 (s, 2H), 4.70–4.65 (m, 2H), 3.28 (t, J = 8.1Hz, 2H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ165.31,163.41,161.79,158.39,157.53,150.80,145.67,143.75,143.09,142.36,137.23,137.22,131.10,130.99,129.74,128.32,125.33,1 24.81,124.79,117.06,115.67,115.43,114.71,50.75,38.15 ,28.35.

Example 9: (E)-2-cyano-1-({2-[4-(3-hydroxyphenyl)indolin-1-carbonyl]thiazol-5-yl}methyl)-3-(pyridin-4-yl)guanidine (Compound I-10)

MS(ESI)m/z:494.1[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.76(s,1H),9.54(s,1H),8.52(s,1H),8.44(s,2H),8.21(d,J＝7.7Hz,1H),7.97(s,1H), 7.34(t,J＝7.8Hz,1H),7.26(t,J＝7.8Hz,1H),7.21(s,2H),7.12(d,J＝7.6Hz,1H),6.89(d,J＝7.6 Hz,1H),6.86(s,1H),6.78(dd,J＝8.1,1.7Hz,1H),4.72(s,2H),4.67(t,J＝7.7Hz,2H),3.24(t,J =8.1Hz, 2H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ165.42,158.33,157.82,157.52,150.79,145.67,143.61,143.07,141.79,141.26,138.67,130.82,129.96,128.14,124.17,119.24,116.55,1 16.34,115.66,115.38,114.76,50.75,38.15,28.58.

Example 10: (E)-1-({2-[4-(3-aminophenyl)indoline-1-carbonyl]thiazol-5-yl}methyl)-2-cyano-3-(pyridin-4-yl)guanidine (Compound I-11)

MS(ESI)m/z:493.1[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.76(s,1H),8.51(s,1H),8.44(s,2H),8.19( d,J＝6.9Hz,1H),7.97(s,1H),7.32(t,J＝7.6Hz,1H),7.21(s,2H),7.09(t,J＝7.4Hz,2H),6.66( s,1H),6.58(dd,J＝13.7,7.8Hz,2H),4.16(s,2H),4.79–4.59(m,4H),3.23(t,J＝8.0Hz,2H); ¹³ C NMR (151MHz,DMSO-d ₆ )δ165.48,158.31,157.53,150.79,149.21,145.68,143.51,143.06,141.76,140.61,139.46,130.69,129.38,128.00,124.11,116.28,116.03,1 15.67,113.98,113.38,50.72,38.15,28.63.

Example 11: (E)-2-cyano-1-(pyridin-4-yl)-3-({2-[4-(o-methylphenyl)indoline-1-carbonyl]thiazol-5-yl}methyl)guanidine (Compound I-12)

MS(ESI)m/z:494.1[M+H] ⁺ ,515.8[M+Na] ⁺ ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.77(s,1H),8.59–8.37(m, 3H),8.22(d,J＝7.8Hz,1H),7.96(s,1H),7.35–7.28(m,3H),7.25(t,J＝7.2Hz,1H),7.23–7.14(m,3H ¹³ C NMR (151MHz,DMSO-d ₆ )δ165.48,158.48,157.62,150.87,145.81,143.36,143.16,141.91,139.72,138.87,135.42,131.71,130.60,129.25,128.10,127.77,126.24,1 25.77,119.44,116.56,115.74,50.60,38.24,28.03,20.05 .

Example 12: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-morpholinepropionamide (Compound I-13)

Step 1: (E)-3-Chloro-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carboxyl)indolin-4-yl]phenyl}propanamide

(E)-1-({2-[4-(3-aminophenyl)indoline-1-carbonyl]thiazol-5-yl}methyl)-2-cyano-3-(pyridin-4-yl)guanidine (Compound I-11) (2.47g, 5mmol) was dissolved in 20mL of dichloromethane, 3-chloropropionyl chloride (0.76g, 6moml) was added dropwise, and then triethylamine (0.76g, 7.5mmol) was added, and the reaction was carried out at room temperature for 5h. After the reaction was completed, the reaction solution was washed with water (5mL×2) and saturated brine (5mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated and concentrated to obtain 2.49g of light yellow solid, with a yield of 84.1%; MS (ESI) m/z: 585.2[M+H] ⁺ .

Step 2: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carboxyl)indolin-4-yl]phenyl}-3-morpholinepropionamide

(E)-3-chloro-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carbonyl)indolin-4-yl]phenyl}propanamide (0.5mmol), morpholine (2mmol), sodium iodide (0.5mmol) and potassium carbonate (0.75mmol) were added to 2mL DMF and reacted at 80°C for 5h. After the reaction was completed, water was added, extracted with ethyl acetate, the organic phases were combined, washed with water and saturated brine in turn, evaporated to dryness, and purified by column chromatography to obtain 118mg of a white solid with a yield of 37.2%.

MS(ESI)m/z:634.2[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ10.16(s,1H),9.79(s,1H),8.53(s,1H),8.44(s,2H),8.23(d,J＝7.3Hz,1H),7.98(s,1H), 7.75(s,1H),7.59(d,J＝8.0Hz,1H),7.44–7.34(m,2H),7.21(s,2H),7.18–7.12(m,2H),4.78–4.62(m, 4H), 3.58 (s, 4H), 3.25 (t, J = 8.1Hz, 2H), 2.64 (s, 2H), 2.51 (s, 1H), 2.42 (s, 4H); ¹³ C NMR (151MHz, DMSO -d ₆ )δ170.61,165.40,158.37,157.52,150.77,145.66,143.70,143.09,140.37,139.84,138.47,130.86,130.03,129.36,128.25,124.16,123.22,1 19.14,118.39,116.69,115.65,70.16,66.53,54.49,53.39 ,50.72,38.16,28.53.

A synthetic method similar to that of Example 12 was used, using (E)-3-chloro-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}propanamide as a raw material, and a substitution reaction was carried out with a small molecule amine to prepare the compounds of Examples 13-17.

Example 13: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidinyl]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-(piperidin-1-yl)propanamide (Compound I-14)

MS(ESI)m/z:632.2[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ10.33(s,1H),9.82(s,1H),8.52(s,1H),8.43( s,2H),8.23(d,J＝7.1Hz,1H),7.98(s,1H),7.75(s,1H),7.58(d,J＝7.9Hz,1H),7.40(t,J＝7.9 Hz,1H),7.37(t,J＝7 .9Hz,1H),7.28–7.15(m,3H),7.14(d,J＝7.6Hz,1H),4.74(s,2H),4.68(t,J＝7.7Hz,2H),3.25(t, J＝8.1Hz,2H),2.73(s,2H),2.56(s,6H),1.55(s,4H),1.41(s,2H); ¹³ C NMR (151MHz, DMSO-d ₆ ) δ170.41,165.38,158.38,150.75,143.74,143.70,143.05,140.39,139.80,138.46,130.85,130.03,129.39,128.25,124 .15,123.25,119.12,118.36,116.69,115.62 ,70.16,53.77,53.74,50.72,38.16,28.53,25.48,22.56.

Example 14: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidinyl]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-(pyrrolidin-1-yl)propanamide (Compound I-15)

MS(ESI)m/z:618.2[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ10.25(s,1H),9.86(s,1H),8.51(s,1H),8.41( s,2H),8.23(d,J＝7.4Hz,1H),7.98(s,1H),7.76(s,1H),7.60(d,J＝8.0Hz,1H),7.45–7.31(m,2H ),7.28–7.08(m,4H),4.74(s,2H),4.69(t,J＝7.4Hz,2H),3.25(t,J＝8.1Hz,2H),2.85(s,2H),2.71 –2.54(m,6H),1.73(s,4H); ¹³ C NMR (151MHz, DMSO-d ₆ ) δ170.26,165.40,158.38,150.74,143.70,143.05,141.96,140.37,139.82,138.46,130.86,130.03,129.41,129.36,128 .25,124.16,123.24,119.13,118.38,116.69 ,115.58,70.16,53.76,51.56,50.72,38.16,28.53,23.44.

Example 15: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidino]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-(4-methylpiperazin-1-yl)propanamide (Compound I-16)

MS(ESI)m/z:647.2[MH] ^- ; ¹ H NMR(600MHz,DMSO-d6)δ10.35(s,1H),10.03(s,1H),8.57(s,1H),8.40(s,2H),8.22(s,1H),7.99(s,1H ),7.77(s,1H),7.62(d,J＝7.8Hz,1H),7.38(dt,J＝15.2,7.6Hz,2H),7.23(s,2H),7.15(dd,J＝12.3, 7.8Hz,2H),4.79(s,2H),4.68(s,2H),3.27–3.21(m,2H),2.67–2.60(m,2H),2.51–2.26(m,10H),2.19(s ,3H).

Example 16: (R,E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidinyl]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-(diethylamino)propionamide (Compound I-17)

MS (ESI) m/z: 620.2 [MH] ^- .

Example 17: (E)-N-{3-[1-(5-{[2-cyano-3-(pyridin-4-yl)guanidinyl]methyl}thiazole-2-carboxylic acid)indolin-4-yl]phenyl}-3-(3-hydroxypyrrol-1-yl)propanamide (Compound I-18)

MS (ESI) m/z: 634.2 [MH] ^- .

Example 18: (E)-2-cyano-1-[(2-{4-[3-(2-morpholinethoxy)phenyl]indoline-1-carbonyl}thiazol-5-yl)methyl]-3-(pyridin-4-yl)guanidine (Compound I-19)

Step 1: {4-[3-(2-chloroethoxy)phenyl]indolin-1-yl}[5-(hydroxymethyl)thiazol-2-yl]methanone

[5-(Hydroxymethyl)thiazol-2-yl][4-(3-hydroxyphenyl)indolin-1-yl]methanone (10mmol), dichloroethane (12mmol), and cesium carbonate (13mmol) were added to 30mL DMF, and the reaction solution was heated to 50°C for 3h. After the reaction was completed, the reaction solution was poured into 100mL water, and the solid was precipitated. The filter cake was washed with water (10mL×2) and dried to obtain 3.17g of brown-yellow solid, with a yield of 76.5%; MS (ESI) m/z: 414.1[M+H] ⁺ .

Step 2: [5-(Hydroxymethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indolin-1-yl}methanone

{4-[3-(2-chloroethoxy)phenyl]indoline-1-yl}[5-(hydroxymethyl)thiazol-2-yl]methanone (2.5mmol), morpholine (5mmol), sodium iodide (2.5mmol) and potassium carbonate (3.75mmol) were added to 10mL DMF, and the reaction solution was heated to 80°C for 5h. After the reaction was completed, 30mL of water was added, and ethyl acetate (20mL×3) was used for extraction. The organic phases were combined and washed with water (20mL) and saturated brine (20mL) in turn. The organic phase was evaporated to dryness under reduced pressure to obtain 1.03g of brown-yellow solid, with a yield of 88.2%; MS (ESI) m/z: 466.2[M+H] ⁺ .

Step 3: [5-(Chloromethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indolin-1-yl}methanone

The intermediate [5-(hydroxymethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indoline-1-yl}methanone (2mmol) was dissolved in 5mL acetonitrile, and thionyl chloride (6mmol) was slowly added at 0°C, and the mixture was allowed to react at room temperature for 30min. After the reaction was completed, the reaction solution was poured into 50mL ice water, and the solid was precipitated. The solid was filtered and dried to obtain 1.09g of brown-yellow solid, with a yield of 93.2%; MS (ESI) m/z: 584.1[M+H] ⁺ .

Step 4: 2-[(2-{4-[3-(2-morpholinethoxy)phenyl]indoline-1-carbonyl}thiazol-5-yl)methyl]isoquinoline-1,3-dione

[5-(Chloromethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indoline-1-yl}methanone (1.8mmol), phthalimide (2.16mmol) and potassium carbonate (2.7mmol) were added to 10mL DMF and reacted at 40°C for 4h. After the reaction was completed, the reaction solution was poured into 30mL water to precipitate a solid, which was filtered and the filter cake was washed with water (5mL×2) and dried to obtain 0.89g of a brown-yellow solid with a yield of 83.2%; MS (ESI) m/z: 595.2[M+H] ⁺ .

Step 5: [5-(Aminomethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indolin-1-yl}methanone

2-[(2-{4-[3-(2-morpholinethoxy)phenyl]indoline-1-formyl}thiazol-5-yl)methyl]isoquinoline-1,3-dione (1.5mmol) was dissolved in 10mL of ethanol, hydrazine hydrate (3moml) was added dropwise, and the reaction solution was heated to 80°C for 1h. After the reaction was completed, the reaction solution was cooled to room temperature, solids were precipitated, filtered, and the filter cake was washed with ethanol (2.5mL×2). The filtrate was evaporated to dryness and concentrated, water was added, and dichloromethane (5mL×3) was extracted. The organic phases were combined, washed with saturated brine (5mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 0.44g of brown solid, with a yield of 63.2%.

Step 6: (E)-2-cyano-1-[(2-{4-[3-(2-morpholinethoxy)phenyl]indolin-1-carbonyl}thiazol-5-yl)methyl]-3-(pyridin-4-yl)guanidine

Dissolve [5-(aminomethyl)thiazol-2-yl]{4-[3-(2-morpholinethoxy)phenyl]indoline-1-yl}methanone (0.5mmol) and methyl (Z)-N'-cyano-N-(pyridin-4-yl)carbamoyl imide thioester in 2mL pyridine, add triethylamine (0.75mmol) and 4-dimethylaminopyridine (0.05mmol), and heat to 70°C for 5h. After the reaction is completed, cool the reaction solution, add water, extract with ethyl acetate, combine the organic phases, wash the organic layer with water and saturated brine in turn, dry over anhydrous sodium sulfate, filter, evaporate the filtrate to dryness under reduced pressure, and separate and purify the residue by column chromatography to obtain the target compound.

MS(ESI)m/z:607.1[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.84(s,1H),8.54(s,1H),8.44(s,2H),8.22( d,J＝7.6Hz,1H),7.98(s,1H),7.41–7.32(m,2H),7.22(s,2H),7.17(d,J＝7.5Hz,1H),7.07–7.01(m ,2H),6.97(dd, J＝8.2,2.0Hz,1H),4.75(s,2H),4.67(t,J＝7.8Hz,2H),4.14(t,J＝5.8Hz,2H),3.61–3.54(m,4H), 3.26(t,J＝8.2Hz,2H),2.72(t,J＝5.6Hz,2H),2.50–2.45(m,4H); ¹³ C NMR (151MHz, DMSO-d ₆ ) δ165.38,158.93,158.34,150.75,143.63,143.07,141.88,141.39,138.45,130.98,130.04,128.15,125.34,120.89,116 .68,116.36,115.63,114.60,114.08,66.52 ,65.70,57.39,53.98,50.75,38.16,28.51.

The synthesis method of Example 18 was adopted, and [5-(hydroxymethyl)thiazol-2-yl][4-(3-hydroxyphenyl)indolin-1-yl]methanone was used as a raw material, and a substitution reaction was carried out with a dihaloalkane, and then a reaction was carried out with a small molecular amine, followed by chlorination and Gabriel to obtain a primary amine intermediate, and the primary amine intermediate was reacted with methyl (Z)-N'-cyano-N-(pyridin-4-yl)carbamide thioester to produce the compounds of Examples 19-26.

Example 19: (E)-2-cyano-1-(pyridin-4-yl)-3-{[2-(4-{3-[3-(pyrrolidin-1-yl)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}guanidine (Compound I-21)

MS(ESI)m/z:605.2[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ10.15(s,1H),8.57(s,1H),8.39(s,2H),8.23(d,J＝7.3Hz,1H),8.00(s,1H),7.42–7.32(m,2H ),7.28–7.13(m,3H),7.06(d,J＝7.6Hz,1H),7.02(s,1H),6.97(dd,J＝8.2,1.7Hz,1H),4.79(s,2H) ,4.68(t,J＝7.4Hz,2H),4.11(t,J＝6.1Hz,2H),3.26(t,J＝8.1Hz,2H),2.94(d,J＝25.2Hz,6H),2.11 –7.03(m,2H),1.83(s,4H); ¹³ C NMR (151MHz, DMSO-d ₆ ) δ165.35,158.91,158.35,150.26,143.64,143.05,142.02,141.40,138.42,130.97,130.07,130.02,128.16,125.31,120 .96,116.69,116.43,115.46,114.62,114.00 ,65.74,53.53,52.00,50.75,38.18,28.51,26.93,23.26.

Example 20: (E)-2-cyano-1-{[2-(4-{3-[3-(piperidin-1-yl)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-22)

MS (ESI) m/z: 619.2 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ9.84 (s, 1H), 8.46 (d, J = 62.3

Hz,3H),8.22(d,J＝7.2Hz,1H),7.97(s,1H),7.41–7.32(m,2H),7.26–7.09(m,3H),7.05(d,J＝7.7Hz ,1H),7.00(s,1H),6.96(dd,J＝8.2,2.2Hz,1H),4.73(s,2H),4.67(t,J＝7.9Hz,2H),4.06(t,J＝ 6.3Hz,2H),3.26(t,J=8.2Hz,2H),2.58(s,6H),1.94(s,2H),1.55(s,4H),1.41(s,2H); ¹³ CNMR(151MHz ,DMSO-d ₆ )δ165.36,159.03,158.35,150.65,143.64,143.05,141.95,141.39,138.45,130.96,130.06,130.03,128.17,125.31,120.86,116.69,116.46,1 15.59,114.63,113.97,66.12,55.08,53.98,50.74,38.16 ,28.50,25.97,24.19,23.80.

Example 21: (E)-2-cyano-1-[(2-{4-[3-(3-morpholinopropoxy)phenyl]indoline-1-carbonyl}thiazol-5-yl)methyl]-3-(pyridin-4-yl)guanidine (Compound I-23)

MS (ESI) m/z: 621.2 [MH] ^- ; ¹ H NMR (600MHz, DMSO-d ₆ ) δ9.84 (s, 1H), 8.48 (d, J = 61.1

Hz,3H),8.22(d,J＝7.5Hz,1H),7.98(s,1H),7.42–7.32(m,2H),7.22(s,2H),7.16(d,J＝7.5Hz,1H ),7.04(d,J＝7.7Hz,1H),7.00(s,1H),6.95(dd,J＝8.2,2.0Hz,1H),4.74(s,2H),4.67(t,J＝7.8Hz ,2H),4.06(t,J＝6.4Hz,2H),3.56(t,J＝4.4Hz,4H),3.26(t,J＝8.2Hz,2H),2.43(t,J＝7.1Hz,2H ),2.37(s,4H),1.93–1.85(m,2H); ¹³ C NMR (151MHz, DMSO-d ₆ ) δ165.37,159.11,158.34,157.59,150.77,145.69,143.63,143.05,141.90,141.38,138.47,130.96,130.05,128.16,125 .32,120.79,116.68,116.34,115.62,114.63 ,113.95,66.54,66.20,55.23,53.73,50.74,38.16,28.50,26.24.

Example 22: (E)-2-cyano-1-{[2-(4-{3-[3-(diethylamino)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-24)

MS(ESI)m/z:607.2[MH] ^- ; ¹ H NMR(600MHz,DMSO-d ₆ )δ10.01(s,1H),8.54(s,1H),8.40(s,2H),8.23(d,J＝7.1Hz,1H),7.98(s,1H),7.42–7.32(m,2H ),7.27–7.12(m,3H),7.05(d,J＝7.5Hz,1H),7.01(s,1H),6.96(d,J＝8.2Hz,1H),4.76(s,2H),4.68 (t,J＝7.5Hz,2H),4.09(t,J＝6.0Hz,2H),3.26(t,J＝8.1Hz,2H),2.75(s,6H),1.96(s,2H),1.06 (s,6H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ165.36,158.98,158.35,150.64,143.64,143.05,141.99,141.39,138.44,130.96,130.07,130.03,128.17,125.30,120.90,116.69,116.45,1 15.50,114.65,113.96,65.82,50.74,48.70,46.69,38.17 ,28.50,26.93,25.49.

Example 23: (E)-2-cyano-1-{[2-(4-{3-[3-(4-methylpiperazin-1-yl)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-26)

MS(ESI)m/z:634.2[MH] ^- ; ¹ H NMR(600MHz, DMSO-d ₆ )δ9.87(s,1H),8.71–8.31(m,3H),8.22(d,J＝7.3Hz,1H),7.98(s,1H),7.39–7.32(m,2H),7.27–7.10 (m,3H),7.04(d,J＝7.6Hz,1H),7.00(s,1H),6.95(dd,J＝8.2,1.7Hz,1H),4.75(s,2H),4.67(t, J＝7.6Hz,2H),4.05(t,J＝6.3Hz,2H),3.26(t,J＝8.2Hz,2H),2.48–2.21(m,10H),2.17(s,3H),1.90– 1.84(m,2H); ¹³ CNMR (151MHz, DMSO-d ₆ ) δ165.37,159.11,158.35,150.62,143.64,143.05,141.95,141.38,138.48,130.96,130.05,128.16,125.32,120.78,116.68 ,115.57,114.61,113.98,66.24,54.94,54.69 ,52.87,50.74,45.86,38.16,28.51,26.56.

Example 24: (R,E)-2-cyano-1-{[2-(4-{3-[3-(3-hydroxypyrrol-1-yl)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-28)

MS (ESI) m/z: 621.2 [MH] ^- .

Example 25: (E)-2-cyano-1-{[2-(4-{3-[3-(4-hydroxypiperidin-1-yl)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-31)

MS (ESI) m/z: 635.2 [MH] ^- .

Example 26: (E)-2-cyano-1-{[2-(4-{3-[3-(2-hydroxyethylamino)propoxy]phenyl}indoline-1-carbonyl)thiazol-5-yl]methyl}-3-(pyridin-4-yl)guanidine (Compound I-33)

MS (ESI) m/z: 595.2 [MH] ^- .

Study on the biological activity of the compounds described in the present invention

PD-1/PD-L1 interaction inhibitory activity test method:

Homogenous time-resolved fluorescence (HTRF) assay was used to detect the ability of the compounds of the present invention to inhibit PD-1/PD-L1 interaction. The detection kit was purchased from CisBio, which contains Anti-Tagl-Cyptate, Anti-Tag2-XL665/d2, Tagl-PD-Ll, Tag2-PD-1, DilutionBuffer, Detection Buffer and other reagents required for the experiment.

Experimental steps:

(1) Dilute the 10 mM stock solution of the test compound with DMSO to the desired concentration, and then continue to dilute it 4-fold with DMSO, for a total of 5 concentration gradients. Then dilute each concentration of the compound 20 times (1+19 μL) with Dilution buffer to obtain the compound working solution, and test 8-12 concentrations of each compound.

(2) Use Dilution Buffer to prepare 250 nM Tag2-PD-1 and 25 nM Tag1-PD-L1 working solutions.

(3) Add 2 μL of compound working solution, 4 μL of Tag2-PD-1 and 4 μL of Tag1-PD-L1 working solution to the wells of the 96-well plate in sequence (the positive control did not add compound working solution, but added 2 μL of Dilution buffer; the negative control did not add compound working solution and Tag2-PD-1 working solution, but added 6 μL of Dilution buffer), mix thoroughly, and incubate at room temperature for 15 min.

(4) Prepare 250nM Tag2-PD-1 and 25nM Tag1-PD-L1 working solutions with Dilution Buffer; prepare 1X anti-Tag1-Eu3 ⁺ and 1X anti-Tag2-XL665 working solutions with Dilution Buffer, then mix equal volumes and add 10μL of antibody mixture to each reaction well. Seal the membrane and incubate at room temperature for 4h.

(5) The fluorescence signal was detected using the SpectraMax i3X multi-function microplate reader (excitation at 340 nm, emission at 670 nm and 612 nm).

NAMPT inhibitory activity test method:

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) was used to test the inhibitory activity of the target compounds on NAMPT in vitro.

Experimental steps:

(1) Prepare 50 μL of the mixed system required for the enzymatic reaction: 50 mM Tris-HCl (pH=8.0), 12.5 mM MgCl ₂ , 20 μM NAM, 0.4 mM Phosphoribosyl pyrophosphate, 2 mM ATP, 30 μg/mL alcoholdehydrogenase, 10 μg/mL NMNAT, 1.5% alcohol, 1 mM DTT, 0.02% BSA, 0.01% Tween 20, the test compound. Note that the DMSO content in the system should be less than 1%.

(2) The enzymatic reaction was incubated at 30°C for 90 min, and each compound was tested at 8-12 concentrations.

(3) The fluorescence signal was detected using a Tecan Infinite M1000 microplate reader (excitation at 360 nm and emission at 460 nm).

The results of the compounds inhibiting PD-1/PD-L1 interaction and NAMPT activity are shown in Table 1.

Table 1 The activity range or IC ₅₀ value of the compounds prepared in the examples of the present invention for inhibiting PD-1/PD-L1 interaction and NAMPT. The ranges are as follows: A = 1 nM-100 nM; B = 100.01 nM-1 μM; C = 1.01 μM-20 μM.

Table 1

The HTRF test results showed that the example compounds could significantly inhibit PD-1/PD-L1 interaction and NAMPT activity at the biochemical level.

The indoline compounds containing thiazole structure of the general formula I of the present invention were tested for their anti-proliferation activity against human ovarian cancer cell A2780.

Experimental steps: Cells in the logarithmic growth phase were inoculated into a 96-well plate at 1×10 ⁴ cells/mL, and the volume of the cell suspension was 100 μL/well, and cultured under standard conditions. After 24 hours, 100 μL of 5 different concentrations of drug solutions prepared with culture medium were added to each well, and four replicate wells were set for each concentration, and incubated under standard conditions for 96 hours. After the incubation, the 96-well plate was removed, 50 μL of MTT solution (2 mg/mL) was added to each well, and the plate was removed after incubation for another 4 hours. After the plate was shaken and the supernatant was discarded, 100 μL of DMSO solution was added to each well, and the plate was placed on a micro-oscillator for 5 minutes to completely dissolve the crystals. The absorbance (OD) of each well was measured at 570 nm on an ELISA reader.

The inhibition rate (IR) of cell proliferation in vitro was calculated according to the formula:

IR%=(OD _control -OD _sample )/(OD _control -OD _blank )×100%

In the formula, OD _control : OD value of the well without drug addition; OD _sample : OD value of the well with drug addition; OD _blank : OD value of the blank plate;

Finally, SPSS software was used to input the data and draw the inhibition rate-concentration curve to calculate IC ₅₀ .

The results of the inhibition of A2780 cell proliferation activity of some compounds are shown in Table 2.

Table 2

Example No. IC ₅₀ (μM) Example No. IC ₅₀ (μM) 12 0.18 19 0.024 13 0.017 20 0.011 14 0.019 twenty one 0.12 15 0.084 twenty two 0.0088 16 0.28 twenty three 0.014

As shown in the test results in Table 2, the compound of general formula I in the present invention has significant anti-proliferation activity against A2780 cells with high expression of NAMPT.

沈阳药科大学药物化学实验室自制)为PD-1/PD-L1相互作用抑制剂。The compounds prepared in Example 22 were evaluated for in vivo pharmacodynamics in the A20 tumor animal model with endogenous high expression of PD-L1 and NAD ⁺ and the LLC OE NAMPT/PD-L1 tumor animal model. FK866 is a NAMPT inhibitor, CQ-85 (chemical formula:

(made by the Medicinal Chemistry Laboratory of Shenyang Pharmaceutical University) is a PD-1/PD-L1 interaction inhibitor.

A20 endogenous high expression model:

(1) By detecting the levels of PD-L1 and NAD ⁺ , mouse A20 cells with relatively high expression of both were selected to establish an animal model for pharmacodynamic evaluation of the compound prepared in Example 22.

(2) A20 cells were cultured in RPMI-1640 medium containing 10% FBS at 37°C and 5% CO2 and passaged to the third generation. When the cells reached the _logarithmic growth phase, they were digested with 0.25% trypsin and pipetted to prepare a single-cell suspension. The cell density was adjusted to 2.5× ¹⁰⁷ cells/mL with serum-free culture medium, i.e., 5× ¹⁰⁶ cells were contained in 0.2 mL.

(3) Establishment of a subcutaneous heterotopic tumor transplantation model in Balb/c syngeneic mice

In an SPF laboratory, the axillary subcutaneous of Balb/c mice was disinfected with 75% alcohol, and 0.2 mL of A20 cell suspension was inoculated subcutaneously to establish a mouse subcutaneous heterotopic transplant tumor model. The size of the transplanted tumor was measured. The tumor volume grew to 50-80 mm ^3. According to the principle of consistency between groups, the mice were randomly divided into 5 groups: solvent group, FK866 group, CQ-85 group, low-dose group of the compound prepared in Example 22, and high-dose group of the compound prepared in Example 22. Except for the high-dose group of the compound prepared in Example 22, which was intraperitoneally injected with 40 mg/kg of the inhibitor, the other experimental groups were intraperitoneally injected with the same dose of 20 mg/kg of the inhibitor. The mice grew normally in the first week after grouping, and were treated with intraperitoneal injection of the inhibitor in the second week. The drugs were administered once every 2 days for 12 days.

(4) Evaluation indicators

During the period of drug administration, the survival status of the animals was observed, and the animals were weighed and the tumor diameter was measured every other day, and the experimental data, including the long diameter a (mm) and short diameter b (mm) of the tumor, were recorded. The inhibitory effect of the drug on tumor growth was evaluated based on the tumor tissue volume during the drug administration period, and the drug safety was evaluated based on the weight of the mice during the drug administration period.

a. Tumor volume (TV) = 1/2 × a × b ² ;

b. Relative tumor volume (RTV) = V _t /V ₀ (V _t : tumor volume at each measurement, V ₀ : tumor volume measured before administration);

c. Tumor inhibition rate (TIR%) = (1-average tumor weight of the drug-treated group/average tumor weight of the control group) × 100%. In the pharmacodynamics of anti-tumor drugs in the preclinical research guidelines for new drugs, the average tumor weight of the blank group mice is greater than 1 gram, and the inhibition rate>30% is considered to have a certain therapeutic effect.

The in vivo pharmacodynamic results of the compound prepared in Example 22 in the A20 endogenous high expression PD-L1 and NAD ⁺ model are shown in Figure 1.

As shown in the test results of Figure 1, the compound prepared in Example 22 can significantly inhibit the growth of mouse tumors. The tumor inhibition rates at dosages of 20 mg/kg and 40 mg/kg are 73.76% and 83.11%, respectively, which are better than the PD-L1 inhibitor CQ-85 and the NAMPT inhibitor FK866; and there is no significant effect on the body weight of the mice, indicating that the compound prepared in Example 22 has no obvious toxicity to mice.

LLC OE NAMPT/PD-L1 Model:

(1) LLC OE NAMPT/PD-L1 cell construction

By lentiviral transfection, a lentiviral plasmid vector overexpressing NAMPT and PD-L1 was introduced to construct LLCOE NAMPT/PD-L1 cells, and stable overexpression of endogenous NAMPT and PD-L1 was induced by adding puromycin and neomycin.

(2) Cell culture

LLC OE NAMPT/PD-L1 cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO2 and passaged to the third generation. When the cells reached the _logarithmic growth phase, they were digested with 0.25% trypsin, pipetted, and prepared into a single-cell suspension. The cell density was adjusted to 1× ¹⁰⁷ /mL with serum-free culture medium, that is, 2× ¹⁰⁶ cells were contained in 0.2mL.

(3) Establishment of a subcutaneous heterotopic transplant tumor model in C57BLc syngeneic mice

In the SPF laboratory, C57BLc mice were subcutaneously disinfected with 75% alcohol, and 0.2 mL LLC OENAMPT/PD-L1 cell suspension was inoculated subcutaneously to establish a mouse subcutaneous heterotopic transplant tumor model. The size of the transplanted tumor was measured, and when the tumor volume grew to 50-80 mm ³ , it was randomly divided into 4 groups according to the principle of consistency between groups: solvent group, FK866 group, CQ-85 group, and compound group prepared in Example 22. After grouping, the mice grew normally in the first week. Starting from the second week, the experimental groups were all intraperitoneally injected with the same dose of 20 mg/kg inhibitor, and the drug was continuously administered for 14 days in a five-day on and two-day off manner.

(4) Evaluation indicators:

During the period of drug administration, the survival status of the animals was observed, and the animals were weighed and the tumor diameter was measured every other day, and the experimental data, including the long diameter a (mm) and short diameter b (mm) of the tumor, were recorded. The inhibitory effect of the drug on tumor growth was evaluated based on the tumor tissue volume during the drug administration period, and the drug safety was evaluated based on the weight of the mice during the drug administration period.

a. Tumor volume (TV) = 1/2 × a × b ² ;

b. Relative tumor volume (RTV) = V _t /V ₀ (V _t : tumor volume at each measurement, V ₀ : tumor volume measured before administration);

c. Tumor inhibition rate (TIR%) = (1-average tumor weight of the drug-treated group/average tumor weight of the control group) × 100%. In the pharmacodynamics of anti-tumor drugs in the preclinical research guidelines for new drugs, the average tumor weight of the blank group mice is greater than 1 gram, and the inhibition rate>30% is considered to have a certain therapeutic effect.

The in vivo pharmacodynamic results of the compound prepared in Example 22 in the LLC OE NAMPT/PD-L1 model are shown in Figure 2.

As shown in the test results of Figure 2, the compound prepared in Example 22 can significantly inhibit the growth of mouse tumors. The tumor inhibition rate is 76.01% at a dosage of 20 mg/kg, which is better than the PD-L1 inhibitor CQ-85 and the NAMPT inhibitor FK866; and has no obvious effect on the body weight of the administered mice, indicating that the compound prepared in Example 22 has no obvious toxicity to mice.

Claims (10)

1. An indoline compound containing a thiazole structure represented by general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof;

in, Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ; ^R1 is independently selected from hydrogen, halogen, cyano, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino; the (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino may be optionally substituted by 1-3

replace; R ² and R ³ are independently selected from hydrogen, (C1-C4)alkyl, hydroxy(C1-C4)alkyl; or R ² , R ³ and the nitrogen atom to which they are attached together form a 3-6 membered nitrogen-containing heterocyclic ring; the nitrogen-containing heterocyclic ring contains 1-3 heteroatoms selected from N, O and S; the nitrogen-containing heterocyclic ring may be optionally substituted by 1-3 R ⁴ ; R ⁴ is independently selected from hydrogen, hydroxy, carboxyl, (C1-C4)alkyl, hydroxy(C1-C4)alkyl; E is selected from O, N–CN; X is selected from amino (C1-C4) alkyl, (C2-C4) alkenyl,

R ^a is selected from phenyl and pyridyl; the phenyl and pyridyl may be optionally substituted by 1-3 R ⁵ ; R ⁵ is independently selected from hydrogen, halogen, hydroxy, amino, (C1-C4) alkyl. 2. The indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that in the general formula I: Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ; ^R1 is independently selected from hydrogen, halogen, hydroxyl, amino, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylformylamino; the (C1-C4) alkoxy, (C1-C4) alkylformylamino may be optionally substituted by 1-3

replace;

Selected from:

E is selected from O, N–CN; X is selected from amino (C1-C4) alkyl, (C2-C4) alkenyl,

^Ra is independently selected from phenyl and pyridyl; the phenyl and pyridyl may be optionally substituted by 1-3 hydrogen or halogen. 3. The indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that in the general formula I: Cy is selected from phenyl,

The phenyl group may be optionally substituted with 1-3 R ¹ ; ^R1 is independently selected from hydrogen, halogen, hydroxyl, amino, methyl, methoxy, ethoxy, propoxy, propionylamino; the ethoxy, propoxy, propionylamino may be optionally substituted by 1-3

replace;

Selected from:

When E is O, X is selected from

When E is N-CN, X is

^Ra is pyridyl; the pyridyl may be optionally substituted with 1-3 hydrogen or halogen. 4. The indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that the indoline compound containing a thiazole structure is any one of the following compounds:

5. The indoline compound containing a thiazole structure, its stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the pharmaceutically acceptable salt refers to a salt formed by the compound with an inorganic acid, an organic acid or an alkali metal ion; the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid and phosphoric acid; the organic acid is selected from the group consisting of succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid and p-toluenesulfonic acid; the alkali metal ion is selected from the group consisting of lithium ion, sodium ion and potassium ion. 6. A pharmaceutical composition, characterized in that it is prepared by mixing the indoline compound containing a thiazole structure according to any one of claims 1 to 4, its stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient with a pharmaceutically acceptable carrier or excipient. 7. Use of the indoline compound containing a thiazole structure according to any one of claims 1 to 4, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and/or treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT. 8. Use of the pharmaceutical composition according to claim 6 in the preparation of a drug for preventing and/or treating diseases related to PD-1/PD-L1 protein/protein interaction and NAMPT. 9. The use according to claim 7 or 8, characterized in that the disease related to PD-1/PD-L1 protein/protein interaction and NAMPT is selected from cancer and infectious diseases. 10. The use according to claim 9, characterized in that the cancer is selected from lymphoma, non-small cell lung cancer, small cell lung cancer, head and neck cell carcinoma, glioma, neuroblastoma, squamous cell carcinoma of the lung, lung adenocarcinoma, bladder cancer, gastric cancer, colon cancer, large intestine cancer, kidney cancer, bile duct cancer, gastric cancer, esophageal squamous cell carcinoma, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, brain cancer, melanoma, multiple myeloma, skin cancer, epithelial cell carcinoma, leukemia and cervical cancer; and the infectious disease is selected from bacterial infection and viral infection.

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