Indoline-2-ketone compound as well as preparation method and application thereof

Abstract

The invention discloses an indoline-2-ketone compound as shown in a general formula I, or an enantiomer, a diastereoisomer, a raceme and pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the indoline-2-ketone compound, the indoline-2-ketone compound is used as a cGAS-STING pathway targeted inhibitor, and can be used for targeted inhibition of cGAMP excitation of the cGAS-STING pathway, so that the cGAS-STING pathway can be used for inhibiting cGAMP activation of the cGAS-STING pathway. The invention has the application of preparing medicines for treating and / or preventing inflammatory diseases and autoimmune diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an indolin-2-one compound and a preparation method and use thereof. The compound has strong STING inhibitory activity in THP1 cells stably transfected with STING. Background Art

[0002] Stimulator of interferon gene (STING), also known as MITA, ERIS or MPYS, is a protein encoded by the human TMEM173 gene and plays an important regulatory role in the innate immune system. The innate immune system recognizes pathogens and upregulates the immune function of immune cells through pattern recognition receptors (PRRs), while inducing the secretion of a large number of inflammatory factors and chemokines, thereby activating adaptive immunity. Among them, pattern recognition receptors include the upstream protein guanylate-adenylate synthase (Cyclic GMP-AMP synthase, cGAS) of STING. As an intracellular DNA sensor, cGAS can recognize exogenous or endogenous cytoplasmic double-stranded DNA (dsDNA), synthesize and release cyclic dinucleotides (CDNs) such as c-di-GMP, c-di-AMP, 3',3'-cGAMP and 2',3'-cGAMP, etc., which activates STING and transfers it from the endoplasmic reticulum to the Golgi apparatus, and then recruits TANK binding kinase-1 (TBK-1) in the cytoplasm, mediates TBK-1 to phosphorylate interferon regulatory factor-3 (IRF-3), induces the secretion of type I interferon, and simultaneously induces the secretion of multiple inflammatory factors such as TNF, IL-6 and CXCL10. In addition, STING also induces the production of pro-inflammatory factors through the NF-κB pathway.

[0003] Excessive activation of STING can lead to a series of inflammatory diseases and autoimmune diseases. These diseases include STING-associated vasculopathy with onset in infancy (SAVI) syndrome caused by gain-of-function mutations in TMEM173, Aicardi-Goutières syndrome (AGS) and hereditary lupus. In addition to these genetic diseases, STING has a wider pathogenic role in a series of inflammatory and autoimmune-related diseases, such as systemic lupus erythematosus, rheumatoid arthritis, nerve damage, organ fibrosis, psoriasis, systemic sclerosis and some cancer metastasis. STING mutants (V147L, N154S, V155M, V155R or R284S) can cause STING autoactivation and lead to the onset of SAVI. The functional loss of three prime repair exonuclease 1 (Trex1) leads to abnormal self-DNA hydrolysis. Abnormal DNA is recognized by cGAS and activates STING, resulting in aggravation of AGS syndrome. In addition to these two diseases, in other related diseases, abnormal DNA accumulation caused by factors such as Trex1 loss of function, organ damage, STING gain-of-function mutations, or chromosomal DNA instability will activate STING, leading to the expression of a large number of inflammatory factors or aggravating the process of organ fibrosis. Therefore, the above diseases are strongly correlated with excessive activation of STING protein, and inhibiting excessive activation of STING protein will have therapeutic value for the occurrence and development of the above diseases (Nat Rev Genet. 2019, 20(11): 657-674).

[0004] Therefore, small molecule-based drug intervention in the STING signaling pathway has important potential for treating diseases caused by excessive STING activation. Summary of the invention

[0005] Objective of the invention: One of the objectives of the present invention is to provide an indolin-2-one compound of general formula I or its enantiomers, diastereomers, racemates and pharmaceutically acceptable salts thereof:

[0006]

[0007] in,

[0008] Ring A is selected from alkyl-substituted or unsubstituted 5-10-membered aliphatic heterocyclic ring;

[0009] Ring B is selected from 1-2 R 3 Substituted or unsubstituted aromatic ring, aromatic heterocycle, aliphatic heterocycle or condensed ring;

[0010] R 1 is selected from H, substituted or unsubstituted aromatic ring, aromatic heterocycle or condensed ring;

[0011] R 2 is selected from H, an alkyl chain of 1 to 6 carbon atoms;

[0012] n is selected from 0 or 1.

[0013] In certain preferred embodiments,

[0014] Ring A is selected from a tetrahydro-1H,3H-pyrrolo[1,2-c]thiazole ring or a 1-methylpyrrolidine ring.

[0015] In certain preferred embodiments,

[0016] The aromatic ring, aromatic heterocycle, aliphatic heterocycle or condensed ring in ring B is selected from N-methylpyrrole ring, pyrrole ring, pyridine ring, quinoline ring, pyrazole ring, tetrahydropyrrole ring, 1,4-benzodioxetane ring or benzene ring; R 3 Selected from methyl, benzoyl, acetyl, methoxy, piperidine-1-carbonyl, cyclohexanecarbonyl, cyclopentanecarbonyl, 4-methylpiperazine-1-carbonyl, cyclohexylmethyl or hydroxyl.

[0017] In certain preferred embodiments,

[0018] R 1 When selected from substituted or unsubstituted aromatic rings, aromatic heterocycles or condensed rings, the substituted or unsubstituted aromatic rings, aromatic heterocycles or condensed rings are selected from benzo[b]thiophene rings, pyridine rings, thiophene rings, benzene rings, naphthalene rings, indole rings, N-ethylcarbamoylthiophene rings, benzo[b]furan rings or 4,5,6,7-tetrahydrobenzo[b]thiophene rings.

[0019] In certain preferred embodiments,

[0020] The alkyl chain of 1 to 6 carbon atoms is selected from N-(2-methoxyethyl)carbamoyl.

[0021] In certain preferred embodiments,

[0022] R 2 Selected from H, N-(2-methoxyethyl)carbamoyl.

[0023] In certain preferred embodiments,

[0024] When R 1 When selected from H, n is selected from 0, R 2 Selected from H;

[0025] When R 1 is selected from a benzo[b]thiophene ring, when n is selected from 0, R 2Selected from H;

[0026] When R 1 is selected from a benzo[b]thiophene ring, and when n is selected from 1, R 2 is selected from N-(2-methoxyethyl)carbamoyl; when R 1 Selected from pyridine

[0027] Pyridine ring, thiophene ring, benzene ring, naphthalene ring, indole ring, N-ethylcarbamoylthiophene ring, benzene

[0028] When the ring is 4,5,6,7-tetrahydrobenzo[b]thiophene, n is selected from 0, R 2 Selected from H.

[0029] The compounds of the general formula I of the present invention are preferably the following compounds:

[0030]

[0031] The above-mentioned compounds of general formula I of the present invention may also exist in the form of their salts, which are converted in vivo into compounds of general formula I. For example, within the scope of the present invention, the compounds of the present invention are converted into pharmaceutically acceptable salt forms according to processes known in the art, and they are used in the form of salts.

[0032] All tautomeric forms of the compounds of the general formula I of the present invention are included within the scope of the present invention. The compounds of the present invention may exist in specific geometric or stereoisomer forms. Additional asymmetric carbon atoms may exist in substituents such as alkyl, and all these isomers and their mixtures are included within the scope of the present invention.

[0033] In some preferred embodiments, the pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed by the compound of formula I and the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, acetic acid, trifluoroacetic acid, pyruvic acid, citric acid, tartaric acid, lactic acid, maleic acid, benzenesulfonic acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, fumaric acid, salicylic acid or phenylacetic acid; and also include acid salts formed by the compound of formula I and inorganic bases or organic salts made from basic amines.

[0034] The present invention also provides a method for preparing a compound having the general formula I, and the synthetic route thereof is as follows:

[0035]

[0036] Reagents and conditions: (a) methanol, 65-70°C, reflux for 5 h; (b) potassium hydroxide, tetrahydrofuran / methanol / water = 1:1:1 (v / v / v), 60°C, 1-3 h; (c) O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, 25°C, 8 h; (d) reduced iron powder, ammonium chloride, ethanol / water = 3:1 (v / v), 80°C, 4 h; (e) sodium borohydride, anhydrous dichloromethane, anhydrous magnesium sulfate, 25°C, 8-10 h;

[0037] Among them, R 1 , R 2 , A, B, and n are as defined in Formula I.

[0038] The compounds of the general formula I of the present invention can be prepared by the above or similar preparation methods, and the corresponding starting materials can be selected according to the different substituents and the different positions of the substituents. It should be appreciated by those skilled in the art that the above routes are helpful for understanding the present invention, but do not limit the content of the present invention. Unless otherwise specified, the variables are defined as mentioned in the general formula I.

[0039] Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or its enantiomers, diastereomers, racemates, pharmaceutically acceptable salts and a pharmaceutically acceptable carrier.

[0040] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, adhesives, colorants, fillers, emulsifiers, etc.

[0041] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[0042] Topical compositions may be formulated as oils, lotions, creams, etc. Carriers for the compositions include vegetable or mineral oils, animal fats, and high molecular weight alcohols, etc. Pharmaceutically acceptable carriers are carriers in which the active ingredient is soluble.

[0043] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0044] Another object of the present invention is to provide the use of the compound of formula I or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate thereof in the preparation of STING protein inhibitors.

[0045] Another object of the present invention is to provide the use of the compound of formula I or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate thereof in the preparation of drugs for preventing and / or treating inflammatory diseases and autoimmune diseases related to the function of STING protein.

[0046] The disease related to the function of STING protein is a disease caused by excessive activation of the STING pathway.

[0047] The inflammatory diseases and autoimmune diseases related to the function of STING protein include STING-related infantile vasculopathy SAVI, Aicardi-Goutières syndrome AGS, COPA syndrome caused by gene mutation of the α subunit of the symbiotic protein complex, systemic lupus erythematosus SLE, familial pernio lupus erythematosus FCL, Parkinson's disease PD, amyotrophic lateral sclerosis ALS, Huntington's disease, non-alcoholic steatohepatitis NASH, alcoholic liver, nerve damage, rheumatoid arthritis, renal fibrosis, systemic sclerosis, intervertebral disc degeneration, pulmonary fibrosis, aging, scleroderma, psoriasis, inflammatory bowel disease, autoimmune colitis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, uveitis, mucositis, diabetes, cardiovascular disease or neurodegenerative disease.

[0048] Beneficial effects:

[0049] The present invention discovered lead compound 1 based on screening of a DNA-encoded compound library, designed and synthesized a series of indolin-2-one compounds, which exhibited good STING inhibitory activity, had strong STING inhibitory activity in stably transfected THP1 cells, and had strong STING SAVI mutant inhibitory activity against HEK293T cells transfected with SAVI mutants. The compounds can be used to prepare drugs for treating diseases related to STING protein function, have the prospect of preparing drugs for treating and / or preventing inflammatory diseases and autoimmune diseases, and provide a reliable approach for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a data diagram of the evaluation of the binding ability of compound 1 to STING by isothermal titration calorimetry (ITC).

[0051] Figure 2 Compound 1 inhibits the inflammatory factors induced by SAVI mutant plasmid ( Figure 2 a:IFN-β1, Figure 2 b: Data graph of CXCL10) expression. DETAILED DESCRIPTION

[0052] The preparation method of the compound of general formula I of the present invention is described below in conjunction with specific examples, but these specific methods do not constitute any limitation to the present invention. The compound of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such a combination can be easily carried out by a technician in the field to which the present invention belongs.

[0053] The starting materials, reaction reagents, etc. used in the specific embodiments of the present invention are all commercially available. The present invention can be prepared in the form of salt by using a salt-forming method commonly used in the art, for example, dissolving the compound in hydrochloric acid ethanol at room temperature to react to generate a hydrochloride; or adding benzenesulfonic acid to react to generate a benzenesulfonate.

[0054] The experimental methods in the examples of the present invention where specific conditions are not specified are generally carried out under conventional conditions or under conditions recommended by raw material or product manufacturers.

[0055] The structures of all compounds in the examples were determined by H NMR spectroscopy ( 1 HNMR) was measured by Bruker Avance 300 MHz spectrometer, using TMS as internal standard, and the recording temperature was 300 K. The purity of the compound was evaluated by reverse phase HPLC (Agilent 1260, Hanbang C18 4.6×150 mm, 5 μm), and the elution system was methanol: water = 80:20 (v / v).

[0056] Unless otherwise specified in the examples, all reactions were carried out under air atmosphere.

[0057] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0058] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20-30°C.

[0059] Preparation of (S)-2-amino-3-(benzothiophen-3-yl)-N-(2-methoxyethyl)propionamide (Intermediate M-3)

[0060]

[0061] Step 1: Preparation of compound M-2

[0062] Compound M-1 (500 mg, 1.13 mmol) was dissolved in N, N-dimethylformamide (10 mL), O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (520 mg, 1.36 mmol) and N, N-diisopropylethylamine (240 μL, 1.36 mmol) were added under ice bath, and stirred for 0.5 h under ice bath. 2-Methoxyethylamine (110 μL, 1.24 mmol) was added to the reaction solution, and stirred at room temperature for 8 h. After the reaction was completed, 100 mL of water was added to the reaction solution, and ethyl acetate was extracted three times with 50 mL × 3, the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (dichloromethane: ethyl acetate = 20: 1-5: 1, v / v) to obtain 400 mg of light yellow solid with a yield of 71.04%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 8.20 (t, J = 4.8 Hz, 1H), 7.92 (dd, J = 24.3, 7.6 Hz, 4H), 7.80-7.54 (m, 3H), 7. 51-7.22(m,7H),4.49-4.29(m,1H),4.27-3.92(m,3H),3.35-3.14(m,8H),3.08(dd,J=14.4,9.6Hz,1H).

[0063] Step 2: Preparation of compound M-3

[0064] Compound M-2 (400 mg, 0.799 mmol) was dissolved in N, N-dimethylformamide (9 mL), piperidine (1 mL) was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed, 100 mL of water was added to the reaction solution, and ethyl acetate was extracted three times with 50 mL × 3, and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (dichloromethane: ethyl acetate = 20: 1-5: 1, v / v) to obtain 131 mg of a yellow oil with a yield of 59.00%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 8.02 (t, J = 5.5 Hz, 1H), 7.97 (dd, J = 7.1, 1.8 Hz, 1H), 7.87 (dd, J = 7.2, 1.8 Hz, 1H), 7.48 (s, 1H), 7.39 (pd, J=7.1,1.5Hz,2H),3.52(dd,J=8.2,5.0Hz,1H),3.41-3.22(m,6H),3.21(s,3H),3.18(d,J=5.2Hz,1H),2.87(dd,J=14.3,8.2Hz,1H).

[0065] Preparation of (3R,6'S)-5-nitro-2-oxo-1',6',7',7a'-tetrahydro-3'H-pyrroloindole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxylic acid (Intermediate M-5)

[0066]

[0067] Step 1: Preparation of compound M-4

[0068] Dissolve dilute propionylphenyl ester (200 mg, 1.35 mmol), 5-nitroisatin (260 mg, 1.35 mmol) and (R)-2-carboxythiazolidine (180 mg, 1.35 mmol) in methanol (5 mL), heat to reflux at 70 ° C, and stir for 5 h. After the reaction is completed, remove the methanol by vacuum rotary evaporation, add 100 mL of water to the reaction bottle, extract three times with ethyl acetate 50 mL × 3, combine the organic phases, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and remove the organic solvent by vacuum rotary evaporation. The residue is subjected to silica gel column chromatography (dichloromethane: methanol = 100: 1, v / v) to obtain 259 mg of light yellow solid, with a yield of 46.72%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.41 (s, 1H, NH), 8.33 (dd, J = 8.6, 2.3Hz, 1H, Ar-H), 8.29 (d, J = 2.1Hz, 1H, Ar -H),7.32-7.20(m,2H,Ar-H),7.21-7.09(m,2H,Ar-H),6.33(d,J=7.5Hz,2H,Ar-H),4.17(dq,J=9.2,5.7Hz, 1H,NCH),3.87(dd,J=12.8,6.7Hz,1H,COCH),3.74(d,J=11.2Hz,1H,SCH2CH),3.41(d,J=11.2Hz,1H,SCH2CH ),3.22-3.07(m,2H,SCH2N),2.57(dd,J=12.9,6.5Hz,1H,CHCH2CH),2.18(td,J=12.8,9.8Hz,1H,CHCH2CH).

[0069] Step 2: Preparation of compound M-5

[0070] Compound M-4 (200 mg, 0.486 mmol) was dissolved in a mixed solution of tetrahydrofuran / methanol / water (tetrahydrofuran: methanol: water = 1:1:1, v / v / v) (6 mL), potassium hydroxide (82 mg, 1.46 mmol) was added, heated to 60 ° C, and stirred for 3 h. After the reaction was completed, the organic solvent was removed by vacuum rotary evaporation. The reaction solution was diluted with 50 mL of water and the pH was adjusted to 3 with dilute hydrochloric acid (a large amount of solid precipitated at this time). The precipitated solid was filtered and the filter cake was dried to obtain 108 mg of yellow solid with a yield of 66.25%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 12.32 (brs, 1H), 11.27 (s, 1H), 8.24 (dd, J=8.6, 2.1Hz, 2H), 8.21-8.18 (m, 1H), 7.05 (d, J=8.6Hz, 1H), 4.16-3.99 (m,1H),3.70(d,J=11.0Hz,1H),3.48(dd,J=12.8,6.7Hz,1H),3.31(d,J=11.1Hz,1H),3.14-3.00(m,2H),2.47-2.34(m,1H),2.15-1.95(m,1H).

[0071] Preparation of 4-benzoyl-1-methyl-1H-pyrrole-2-carbaldehyde (Intermediate M-6)

[0072]

[0073] Dilute N-methylpyrrole (567 mg, 7 mmol), N,N-dimethylformamide (511 mg, 7 mmol) and oxalyl chloride (888 mg, 7 mmol) with 1,2-dichloroethane (5 mL), respectively. Add the above oxalyl chloride solution in 1,2-dichloroethane dropwise to the N,N-dimethylformamide solution in 1,2-dichloroethane under ice bath, and stir at room temperature for 0.5 h. Add the 1,2-dichloroethane solution of N-methylpyrrole to the reaction solution under ice bath, stir at room temperature for 0.5 h, and set aside. Add aluminum chloride (2.05 g, 15.4 mmol) and benzoyl chloride (984 mg, 7 mmol) to the above reaction solution, and react at room temperature for 3 h. After the reaction is completed, pour the reaction solution onto crushed ice containing 50% sodium hydroxide (w%) and stir for 10 min. Adjust the pH to 4 with dilute hydrochloric acid (2M). The product was extracted with ethyl acetate for three times (50 mL × 3), and the organic phases were combined and washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation under reduced pressure. The residue was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v) to obtain 1013 mg of a light yellow solid with a yield of 67.94%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.67 (s, 1H), 7.92 (s, 1H), 7.86-7.77 (m, 2H), 7.66 (t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.3 Hz, 2H), 7.49 (d, J = 1.7 Hz, 1H), 3.96 (s, 3H).

[0074] Example 1 Preparation of (3R, 6'S)-N-((S)-3-(Benzo[b]thiophen-3-yl)-1-((2-methoxyethyl)amino)-1-oxopropan-2-yl)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 1)

[0075]

[0076] Step 1: Preparation of compound 1-1

[0077] Compound M-5 (108 mg, 0.322 mmol) was dissolved in N, N-dimethylformamide (4 mL), O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (147 mg, 0.386 mmol) and N, N-diisopropylethylamine (68 μL, 0.386 mmol) were added under ice bath, and stirred for 0.5 h under ice bath. M-3 (98 mg, 0.354 mmol) was added to the reaction solution and stirred at room temperature for 8 h. After the reaction was completed, 100 mL of water was added to the reaction solution, and ethyl acetate was extracted three times with 50 mL × 3, the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried with anhydrous sodium sulfate, and the organic solvent was removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v). 126 mg of yellow solid was obtained, and the yield was 65.5%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.10 (d, J = 6.3Hz, 2H), 8.36-7.86 (m, 4H), 7.64 (s, 1H) ),7.51-7.25(m,2H),7.24-7.03(m,1H),6.91(t,J=9.3Hz,1H),4.38-4.15(m,1H),4 .13-3.99(m,1H),3.67(d,J=10.4Hz,1H),3.54-3.40(m,1H),3.32-2.87(m,10H),2. 85-2.68(m,1H),2.43-2.30(m,1H),2.29-2.12(m,1H),2.04(dd,J=19.7,8.7Hz,1H).

[0078] Step 2: Preparation of compound 1-2

[0079] Dissolve compound 1-1 (126 mg, 0.211 mmol) in 4 mL of a mixed solution of ethanol / water = 3:1 (v / v), add reduced iron powder (47 mg, 0.844 mmol) and ammonium chloride (5.5 mg, 0.105 mmol). Heat to

[0080] Reflux at 80℃ for 4h. After the reaction is completed, remove the iron powder by diatomaceous earth filtration and remove the ethanol by vacuum distillation. Add 100mL of water to the reaction solution, extract three times with 50mL×3 of ethyl acetate, combine the organic phases, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and remove the organic solvent by vacuum rotary evaporation. Obtain 90mg of crude yellow oil with a yield of 75.49%, directly for the next step.

[0081] Step 3: Preparation of compound 1

[0082] Compound 1-2 (59 mg, 0.104 mmol) was dissolved in anhydrous dichloromethane (2 mL), and compound M-6 (27 mg, 0.125 mmol) and anhydrous magnesium sulfate (100 mg) were added respectively. Stir at room temperature for 8 h under nitrogen protection. The reaction solution was evaporated to dryness, and the remaining solid was redissolved in methanol (2 mL). Sodium borohydride (8 mg, 0.21 mmol) was added under ice bath and stirred at room temperature for 2 h. After the reaction was complete, 100 μL of water was added to quench the reaction, and methanol was removed by vacuum rotary evaporation. 100 mL of water was added to the reaction bottle, and ethyl acetate was extracted three times with 50 mL × 3, and the organic phases were combined. The organic phases were washed with saturated sodium chloride aqueous solution, and the organic phases were dried over anhydrous sodium sulfate. The organic solvent was removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) to obtain 19 mg of yellow solid with a yield of 23.94%. 1 H NMR(300MHz,DMSO-d6)δ(ppm)10.01(s,1H,CONH),8.13-7.03(m,13H,Ar-H,CONH),6.83(d,J=22.6Hz,1H,Ar-H),6.67-6.36(m,3H,Ar-H,CONH), 5.50(t,J=5.3Hz,1H,NH),4.59-3.94(m,4H,),3.92-3.43(m,4H),3.34- 2.91(m,11H),2.90-2.76(m,1H),2.75-2.54(m,1H),2.38-1.88(m,2H).

[0083] Example 2 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 2)

[0084]

[0085] Step 1: Preparation of compound 2-2

[0086] The preparation method is the same as step 1 in Example 1, M-5 (257 mg, 0.766 mmol), M-3 is replaced by compound 2-1 (150 mg, 0.919 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 170 mg of a yellow solid, with a yield of 35.41%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.07 (s, 1H), 8.33 (t, J = 5.9Hz, 1H), 8.13 (d, J = 2.3Hz, 1H), 8.05 ( dd,J=8.7,2.3Hz,1H),7.90(d,J=7.9Hz,1H),7.52(d,J=7.8Hz,1H),7.39-7.18(m,2H),6.96(s,1 H),6.83(d,J=8.7Hz,1H),4.29-4.15(m,2H),4.09(s,1H),3.67(d,J=10.5Hz,1H),3.47(dd,J=12 .1,6.8Hz,1H),3.26(d,J=10.5Hz,1H),3.14-2.98(m,2H),2.47-2.39(m,1H),2.26-2.08(m,1H).

[0087] Step 2: Preparation of compound 2-3

[0088] The preparation method is the same as step 2 in Example 1, except that compound 1-1 is replaced by compound 2-2 (170 mg, 0.354 mmol). 30 mg of crude yellow oil is obtained with a yield of 18.81%, which is directly used for the next step.

[0089] Step 3: Preparation of compound 2

[0090] The preparation method is the same as step 3 in Example 1, except that compound 1-2 is replaced by compound 2-3 (44 mg, 0.098 mmol). Silica gel column chromatography (dichloromethane:methanol=50:1, v / v) is performed to obtain 21 mg of a yellow solid with a yield of 33.11%. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 9.98 (s, 1H), 8.03 (s, 1H), 7.97-7.83 (m, 1H), 7.70 (d, J = 7.4Hz, 2 H),7.55(d,J=6.5Hz,2H),7.51-7.41(m,2H),7.41-7.13(m,3H),6.85(s,1H),6.78-6.55(m,3H ),6.54(s,1H),5.61(brs,1H),4.22(s,2H),4.13(s,2H),4.03(s,1H),3.79-3.51(m,4H),3.51 -3.39(m,2H),3.03(q,J=6.9Hz,1H),2.92-2.78(m,1H),2.41-2.30(m,1H),2.30-2.14(m,1H).

[0091] Example 3 Preparation of (3R, 6'S)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-N-methyl-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-pyrroloindole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 3)

[0092]

[0093] Step 1: Preparation of compound 3-2

[0094] The preparation method is the same as step 1 in Example 1, M-5 (200 mg, 0.596 mmol), M-3 is replaced by compound 3-1 (49 mg, 0.715 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 107 mg of a yellow solid, with a yield of 51.7%. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.09 (s, 1H), 8.27-8.08 (m, 2H), 7.66 (q, J = 4.8 Hz,1H),7.06-6.91(m,1H),4.16-4.01(m,1H),3.69(d,J=10.5Hz,1H),3.50-3. 38(m,1H),3.33-3.26(m,1H),3.13(ddd,J=13.1,10.6,6.8Hz,1H),3.00(dd,J= 11.2, 3.5Hz, 1H), 2.48-2.35 (m, 1H), 2.23 (d, J = 4.4Hz, 3H), 2.19-2.05 (m, 1H).

[0095] Step 2: Preparation of compound 3-3

[0096] The preparation method is the same as step 2 in Example 1, except that compound 1-1 is replaced by compound 3-2 (250 mg, 0.718 mmol). 200 mg of crude yellow oil is obtained with a yield of 87.59%, which is directly used for the next step.

[0097] Step 3: Preparation of compound 3

[0098] The preparation method is the same as step 3 in Example 1, except that compound 1-2 is replaced by compound 3-3 (200 mg, 0.628 mmol). Silica gel column chromatography (dichloromethane:methanol=50:1, v / v) is performed to obtain 109 mg of a yellow solid with a yield of 29.86%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 9.91 (s, 1H), 7.72 (dd, J = 6.9, 1.7Hz, 2H), 7.61-7.43 (m, 3H), 7.39 (d ,J=2.0Hz,1H),7.35-7.25(m,1H),6.75(d,J=11.1Hz,1H),6.53(d,J=8.7Hz,3H),5.56(d,J=17.3Hz ,1H),4.26-4.08(m,2H),3.99(t,J=6.9Hz,1H),3.91-3.74(m,1H),3.67(s,3H),3.63-3.47(m,1H), 3.31-3.23(m,1H),3.14-2.95(m,1H),2.87-2.67(m,1H),2.45(d,J=4.4Hz,1H),2.36-2.01(m,4H).

[0099] Example 4 Preparation of (3R, 6'S)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-N-(pyridin-3-ylmethyl)-1', 6', 7', 7a'-tetrahydro-3'-pyrroloindole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 4)

[0100]

[0101] Step 1: Preparation of compound 4-2

[0102] The preparation method is the same as step 1 in Example 1, M-5 (335 mg, 1 mmol), M-3 is replaced by compound 4-1 (130 mg, 1.2 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v). 177 mg of yellow-brown solid is obtained, with a yield of 41.64%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 8.44-8.34 (m, 1H), 8.29 (d, J = 4.3 Hz, 1H), 8.20-8.01 (m, 3H), 7.03 (s, 2H), 6.92 (d, J = 9.1 Hz, 1H), 4.15-3.84 (m, 3H), 3.68 ( d,J=10.6Hz,1H),3.43(dd,J=12.0,6.4Hz,1H),3.27(d,J=10.6Hz,1H),3.10(dd,J= 11.1, 6.7Hz, 1H), 3.03 (d, J = 3.4Hz, 1H), 2.46-2.36 (m, 1H), 2.14 (q, J = 11.1Hz, 1H).

[0103] Step 2: Preparation of compound 4-3

[0104] The preparation method was the same as step 2 in Example 1, except that compound 1-1 was replaced by 4-2 (170 mg, 0.4 mmol). 133 mg of crude yellow oil was obtained with a yield of 84.17%, which was directly used for the next step.

[0105] Step 3: Preparation of compound 4

[0106] The preparation method is the same as step 3 in Example 1, except that compound 1-2 is replaced by 4-3 (133 mg, 0.336 mmol). Silica gel column chromatography (dichloromethane: methanol = 30: 1, v / v). 87 mg of yellow solid was obtained, with a yield of 43.88%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.01 (s, 1H), 8.44-8.26 (m, 1H), 8.19 (d, J = 2.3 Hz, 1H), 8.08 (t, J = 5.9 Hz, 1H), 7.78-7.63 (m, 2H), 7.62-7.43 (m, 3H), 7.35 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 7.9, 4.6 Hz, 1H), 6.97-6.69 (m, 2H ),6.68-6.41(m,3H),5.61(s,1H),4.25-3.91(m,5H),3.77-3.53(m,4H),3.42(d,J=7.5Hz,1H),3.28(d,J=9 .1Hz,1H),3.02(dd,J=10.8,6.6Hz,1H),2.83(dd,J=10.8,5.2Hz,1H),2.42-2.30(m,1H),2.30-2.15(m,1H).

[0107] Example 5 Preparation of (3R, 3'S)-N-(Benzothiophen-3-ylmethyl)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-1'-methyl-2-oxospiro[indoline-3,2'-pyrrolidine]-3'-carboxamide (Compound 5)

[0108] 1. Preparation of (3R, 3'S)-1'-methyl-5-nitro-2-oxospiro[indoline-3,2'-pyrrolidine]-3'-carboxylic acid (Intermediate 5-2)

[0109]

[0110] Step 1: Preparation of compound 5-1

[0111] The preparation method is the same as that of intermediate M-5, step 1, except that (R)-2-carboxythiazolidine is replaced with sarcosine (231 mg, 2.6 mmol). Silica gel column chromatography (dichloromethane:methanol=100:1, v / v) was performed to obtain 531 mg of a light yellow solid, with a yield of 55.6%. 1HNMR (300MHz, DMSO-d6) δ (ppm) 11.35 (s, 1H), 8.30 (dd, J = 8.7, 2.4Hz, 1H), 7.90 (d, J = 2.4Hz, 1H), 7.32-7.21 (m, 2H), 7.20-7.07 ( m,2H),6.35(dd,J=7.7,1.7Hz,2H),3.78(t,J=9.3Hz,1H),3.29-3.04(m,2H),2.65-2.53(m,1H),2.44-2.29(m,1H),2.04(s,3H).

[0112] Step 2: Preparation of compound 5-2

[0113] The preparation method is the same as that of intermediate M-5 preparation step 2, except that compound M-4 is replaced by 5-1 (520 mg, 1.42 mmol). 220 mg of yellow solid is obtained, and the yield is 53.6%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 12.31 (s, 1H), 11.24 (s, 1H), 8.22 (dd, J = 8.6, 2.4 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 3.25-3.12 (m, 2H), 3.11-2.99 (m, 1H), 2.48-2.36 (m, 1H), 2.31-2.14 (m, 1H), 2.00 (s, 3H).

[0114] 2. Preparation of Compound 5

[0115]

[0116] Step 1: Preparation of compound 5-3

[0117] The preparation method is the same as step 1 in Example 1, except that M-5 is replaced by 5-2 (207 mg, 0.71 mmol), and M-3 is replaced by 2-1 (140 mg, 0.85 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v) is performed to obtain 128 mg of a light yellow solid, with a yield of 29%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 10.99 (s, 1H), 8.35 (t, J = 5.5Hz, 1H), 8.05 (dd, J = 8. 6,2.5Hz,1H),7.96-7.84(m,2H),7.42(d,J=7.8Hz,1H),7.36-7.21(m,2H),6.94( s,1H),6.83(d,J=8.6Hz,1H),4.23(dd,J=15.2,5.6Hz,1H),4.14(dd,J=15.2,5.1 Hz, 1H), 3.30 (d, J = 8.5Hz, 2H), 3.20-3.04 (m, 2H), 2.19-2.04 (m, 1H), 1.97 (s, 3H).

[0118] Step 2: Preparation of compound 5-4

[0119] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 5-3 (128 mg, 0.29 mmol). 113 mg of crude white solid was obtained with a yield of 95%, which was directly used for the next step.

[0120] Step 3: Preparation of compound 5

[0121] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 5-4 (113 mg, 0.278 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). A white solid of 160 mg is obtained with a yield of 95.8%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.96 (s, 1H), 8.11 (t, J = 5.2 Hz, 1H), 7.93-7.85 (m, 1H), 7.73-7.62 (m, 2H), 7.56 (t, J = 7.3 Hz, 1H), 7.51-7.41 (m, 3H), 7.36-7.23 (m, 3H), 6.68 (d, J = 6.8 Hz, 2H), 6.60 (s, 2H), 6.49 (d, J = 2.0Hz,1H),5.61(t,J=4.9Hz,1H),4.35-4.14(m,2H),4.10(t,J=5.3Hz,2H),3.57(s,3H),3.22(t,J=8.3Hz ,1H),3.11(t,J=8.1Hz,1H),2.99(t,J=7.9Hz,1H),2.47-2.37(m,1H),2.06(q,J=8.9Hz,1H),1.94(s,3H).

[0122] Example 6 Preparation of (3R, 6'S)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-N-(thiophen-3-ylmethyl)-1', 6', 7', 7a'-tetrahydro-3'-pyrroloindole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 6)

[0123]

[0124] Step 1: Preparation of compound 6-2

[0125] The preparation method is the same as step 1 in Example 1, M-5 (335 mg, 1 mmol), M-3 is replaced by 6-1 (136 mg, 1.2 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v). 224 mg of yellow solid is obtained, and the yield is 52.09%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 11.08 (s, 1H), 8.41 (t, J = 5.4 Hz, 1H), 8.18-8.07 (m, 2H), 7.15 (dd, J = 5.1, 1.3 Hz, 1H), 6.92 (d, J = 9.3 Hz, 1H), 6.73 (dd, J = 5.1, 3.4 Hz, 1H), 6. 53(d,J=3.5Hz,1H),4.21-3.98(m,3H),3.68(d,J=10.6Hz,1H),3.48-3.39(m,1H),3 .27(d,J=10.5Hz,1H),3.15-2.96(m,2H),2.46-2.34(m,1H),2.11(q,J=11.6Hz,1H).

[0126] Step 2: Preparation of compound 6-3

[0127] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 6-2 (224 mg, 0.52 mmol). 182 mg of crude light yellow oil was obtained with a yield of 87.5%, which was directly used for the next step.

[0128] Step 3: Preparation of compound 6

[0129] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 6-3 (182 mg, 0.455 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). A yellow solid of 79 mg is obtained with a yield of 29.25%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.93 (s, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.76-7.64 (m, 2H), 7.62-7.53 (m, 1H), 7.49 (dd, J = 8.1, 6.5 Hz, 2H), 7.37 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 5.1, 1.3 Hz, 1H), 6.86-6.71 (m, 2H), 6.61-6.45 (m, 4H), 5.52 (s ,1H),4.11(d,J=5.2Hz,4H),4.01(t,J=6.7Hz,1H),3.66(s,3H),3.60(d,J=9.0Hz,1H),3.44-3.36(m,1H),3.28(d, J=9.0Hz,1H),3.01(dd,J=10.7,6.5Hz,1H),2.81(dd,J=10.7,5.4Hz,1H),2.34(p,J=6.7Hz,1H),2.25-2.05(m,1H).

[0130] Example 7 Preparation of (3R, 6'S)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-N-benzyl-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-pyrroloindole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 7)

[0131]

[0132] Step 1: Preparation of compound 7-2

[0133] The preparation method is the same as step 1 in Example 1, M-5 (335 mg, 1 mmol), M-3 is replaced by 7-1 (129 mg, 1.2 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v) is performed to obtain 374 mg of a yellow solid, with a yield of 88%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 8.26-8.18 (m, 2H), 8.02-7.96 (m, 1H), 7.56 (d, J = 7.5Hz, 1H), 7.35-7.29 (m, 4H), 7.29-7.21 (m, 1H), 4.39-4.33 (m, 2H),4.09-3.99(m,2H),3.95-3.86(m,1H),3.25(t,J=7.0Hz,1H),2.84( dd,J=12.2,6.9Hz,1H),2.73(dd,J=12.3,7.0Hz,1H),2.06-1.94(m,2H).

[0134] Step 2: Preparation of compound 7-3

[0135] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 7-2 (374 mg, 0.88 mmol). 120 mg of a crude light yellow solid was obtained with a yield of 34.59%, which was directly used for the next step.

[0136] Step 3: Preparation of compound 7

[0137] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 7-3 (105 mg, 0.266 mmol). Silica gel column chromatography (dichloromethane: methanol = 50:1, v / v) is performed to obtain 30 mg of a yellow solid, with a yield of 19.1%. NMR (300MHz, DMSO-d6) δ (ppm) 9.98 (s, 1H), 7.94 (t, J = 5.9Hz, 1H), 7.77-7.66 (m, 2H), 7.62-7.54 (m, 1H), 7.49 (dd, J = 8.1, 6.5Hz,2H),7.37(d,J=1.9Hz,1H),7.11(dd,J=5.2,1.9Hz,3H),6.82(s,1H),6.79-6.67(m,2H),6.66-6.55(m,2H),6.53(d ,J=1.9Hz,1H),5.61(t,J=4.6Hz,1H),4.11(d,J=5.2Hz,2H),4.08-3.91(m,3H),3.62(d,J=5.5Hz,4H),3.42(dd,J=11.5, 7.2Hz, 1H), 3.29 (d, J = 9.0Hz, 1H), 3.13-2.96 (m, 1H), 2.83 (dd, J = 10.7, 5.3Hz, 1H), 2.41-2.30 (m, 1H), 2.29-2.14 (m, 1H). Example 8 Preparation of (3R, 6'S)-5-(((4-acetyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-N-(benzothiophen-3-ylmethyl)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiroindole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 8)

[0138]

[0139] Step 1: Preparation of compound 8-1

[0140] The preparation method is the same as that of intermediate M-6, except that benzoyl chloride is replaced by acetyl chloride (610 mg, 7.77 mmol). Silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v) is performed. 876 mg of a white solid is obtained, with a yield of 74.66%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 9.62 (s, 1H), 7.45 (s, 1H), 7.31 (d, J = 1.8 Hz, 1H), 3.99 (s, 3H), 2.44 (s, 3H).

[0141] Step 2: Preparation of compound 8

[0142] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (86 mg, 0.191 mmol), and M-6 is replaced by 8-1 (35 mg, 0.229 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 37 mg of yellow solid is obtained, with a yield of 33.11%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.09 (s, 1H), 9.65 (s, 1H), 8.18-8.00 (m, 1H), 8.00-7.87 (m, 1H), 7.72 (t, J = 5.1 Hz, 1H), 7.51 (s, 1H), 7.35 (d, J = 9.1 Hz, 2H), 6.85-6.72 (m, 1H), 6. 70-6.36(m,3H),5.86-5.15(m,1H),4.38(d,J=6.0Hz,2H),4.25-3.96(m,3H),3.96-3.81 (m,1H),3.83-3.43(m,5H),2.96-2.77(m,1H),2.63(d,J=9.3Hz,2H),2.37-2.09(m,4H).

[0143] Example 9 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 9)

[0144]

[0145] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (85 mg, 0.188 mmol), and M-6 is replaced by 9-1 (25 mg, 0.227 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 28 mg of yellow solid is obtained, with a yield of 27.42%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.96 (s, 1H), 8.03 (t, J = 5.9 Hz, 1H), 7.96-7.83 (m, 1H), 7.58 (dd, J = 6.4, 2.9 Hz, 1H), 7.41-7.26 (m, 2H), 6.85 (s, 1H), 6.74-6.45 (m, 4H), 5.98 (dd, J = 3.6, 1.8 Hz, 1H), 5.87 (t, J =3.0Hz,1H),5.36(t,J=4.8Hz,1H),4.33-4.15(m,2H),4.13-3.95(m,3H),3.64(d,J=8.9Hz,1H),3.50 (s,3H),3.45(d,J=7.7Hz,1H),3.31(s,1H),3.12-2.98(m,1H),2.94-2.81(m,1H),2.42-2.17(m,2H).

[0146] Example 10 Preparation of (3R, 6'S)-5-(((1H-pyrrol-2-yl)methyl)amino)-N-(benzo[b]thiophen-3-ylmethyl)-2-oxo-1',6',7',7a'-tetrahydro-3'-H-spiroindole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 10)

[0147]

[0148] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (220 mg, 0.489 mmol), and M-6 is replaced by 10-1 (51 mg, 0.538 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 127 mg of yellow solid is obtained, with a yield of 49.09%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.68 (s, 1H), 9.96 (s, 1H), 8.01 (s, 1H), 7.97-7.82 (m, 1H), 7.69-7.49 (m, 1H), 7.43-7.21 (m, 3H), 6.83 (s, 1H), 6.69 (s, 1H), 6.58 (d, J = 19.3 Hz, 3 H),6.08-5.81(m,2H),5.32(s,1H),4.23(t,J=6.6Hz,2H),4.05(d,J=7.2Hz,4H),3.64( d,J=8.9Hz,1H),3.46(s,0H),3.15-2.97(m,1H),2.93-2.82(m,1H),2.39-2.16(m,3H).

[0149] Example 11 Preparation of (3R, 6'S)-5-(((1H-pyrrol-2-yl)methyl)amino)-N-(benzo[b]thiophen-3-ylmethyl)-2-oxo-1',6',7',7a'-tetrahydro-3'-H-spiroindole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 11)

[0150]

[0151] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (85 mg, 0.188 mmol), and M-6 is replaced by 11-1 (24 mg, 0.227 mmol). Silica gel column chromatography (dichloromethane: methanol = 50:1, v / v) is performed to obtain 60 mg of a yellow solid, with a yield of 58.99%. NMR (300MHz, DMSO-d6) δ (ppm) 9.96 (s, 1H), 8.49 (d, J = 4.8Hz, 1H), 8.01 (t, J = 5.2Hz, 1H), 7.96-7.81 (m, 1H) ,7.68-7.49(m,2H),7.38-7.26(m,3H),7.20(t,J=6.3Hz,1H),6.75(d,J=2.2Hz,1H),6.61(s,1H),6.55(d, J=8.3Hz,1H),6.51-6.43(m,1H),5.94(s,1H),4.27(s,2H),4.21(s,2H),4.10-3.92(m,1H),3.61(d,J=9.0 Hz, 1H), 3.53-3.39 (m, 1H), 3.28 (d, J = 9.0Hz, 1H), 3.06-2.98 (m, 1H), 2.86-2.65 (m, 1H), 2.43-2.11 (m, 2H).

[0152] Example 12 Preparation of (3R, 6'S)-5-(((1H-pyrrol-2-yl)methyl)amino)-N-(benzo[b]thiophen-3-ylmethyl)-2-oxo-1',6',7',7a'-tetrahydro-3'-H-spiroindole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 12)

[0153]

[0154] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (94 mg, 0.208 mmol), and M-6 is replaced by 12-1 (36 mg, 0.229 mmol). Silica gel column chromatography (dichloromethane: methanol = 50:1, v / v) is performed to obtain 47 mg of a yellow solid, with a yield of 38.21%. NMR (300MHz, DMSO-d6) δ (ppm) 9.99 (s, 1H), 8.83 (d, J = 4.4Hz, 1H), 8.37-8.22 (m, 1H), 8.05 (s, 1H), 7.99 (s, 1H) ,7.94-7.82(m,2H),7.58(t,J=8.0Hz,2H),7.45(dd,J=8.4,4.4Hz,1H),7.38-7.24(m,2H),6.81(s,1H),6.67(s ,1H),6.53(q,J=8.5,8.1Hz,2H),6.07(s,1H),4.53-4.30(m,2H),4.20(d,J=5.6Hz,2H),4.12-3.83(m,2H),3.6 1-3.52(m,1H),3.31-3.19(m,1H),2.98-2.86(m,1H),2.77-2.62(m,1H),2.40-2.29(m,1H),2.25-2.10(m,1H). Example 13 Preparation of (3R, 6'S)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-N-(naphthalen-1-ylmethyl)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-pyrroloindole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 13)

[0155]

[0156] Step 1: Preparation of compound 13-2

[0157] The preparation method is the same as step 1 in Example 1, M-5 (258 mg, 0.77 mmol), M-3 is replaced by 13-1 (133 mg, 0.85 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 126 mg of a yellow solid, with a yield of 34.61%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.07 (s, 1H), 8.33 (t, J = 5.7Hz, 1H), 8.16 (d, J = 2.3Hz, 1H), 8.12-8.00 (m, 1H ),7.87(d,J=8.1Hz,1H),7.75(t,J=9.5Hz,2H),7.47(t,J=7.4Hz,1H),7.42-7.30(m,1H),7.19(t,J=7.6Hz ,1H),6.86(t,J=8.1Hz,2H),4.59-4.27(m,2H),4.17-4.04(m,1H),3.68(d,J=10.5Hz,1H),3.58-3.42(m,1 H), 3.27 (d, J = 10.4Hz, 1H), 3.16-3.06 (m, 1H), 3.04-2.92 (m, 1H), 2.46 (t, J = 6.2Hz, 1H), 2.30-2.10 (m, 1H).

[0158] Step 2: Preparation of compound 13-3

[0159] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 13-2 (126 mg, 0.26 mmol). The crude product was 100 mg of pale yellow oil with a yield of 86.62%, which was directly used for the next step.

[0160] Step 3: Preparation of compound 13

[0161] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 13-3 (120 mg, 0.27 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). A yellow solid of 78 mg is obtained with a yield of 45.08%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.97 (s, 1H), 8.00 (d, J = 5.5 Hz, 1H), 7.93-7.82 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.70 (dd, J = 7.8, 5.3 Hz, 3H), 7.56 (t, J = 7.1 Hz, 1H), 7.46 (t, J = 7.6 Hz, 4H), 7.33 (d, J = 1.9 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 6.87 (s, 1H),6.77(d,J=7.1Hz,1H),6.68-6.49(m,3H),5.62(s,1H),4.46(d,J=4.5Hz,2H),4.12(d,J=3.9Hz,2H),4.04(t,J=6. 8Hz,1H),3.71-3.56(m,4H),3.54-3.44(m,1H),3.30(s,1H),3.11-2.96(m,1H),2.94-2.79(m,1H),2.42-2.18(m,2H).

[0162] Example 14 Preparation of (3R, 6'S)-N-((1H-indol-3-yl)methyl)-5-((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methylamino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 14)

[0163]

[0164] Step 1: Preparation of compound 14-2

[0165] The preparation method is the same as step 1 in Example 1, M-5 (521 mg, 1.56 mmol), M-3 is replaced by 14-1 (250 mg, 1.71 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 512 mg of a yellow solid, with a yield of 70.91%. 1H NMR(300MHz,DMSO-d6)δ(ppm)9.14(d,J=8.1Hz,1H),8.26-8.14(m,3H),7.69(dd,J=7.5,1.4Hz,1H),7.5 8(d,J=7.5Hz,1H),7.35(dd,J=7.6,1.5Hz,1H),7.27(d,J=8.2Hz,1H),7.17(td,J=7.5,1.5Hz,1H),7.07 (td,J=7.4,1.6Hz,1H),4.36(qd,J=12.5,8.2Hz,2H),4.09-3.99(m,2H),3.91(p,J=7.0Hz,1H),3.25(t, J=7.0Hz, 1H), 2.84 (dd, J=12.3, 7.1Hz, 1H), 2.73 (dd, J=12.5, 6.9Hz, 1H), 1.97 (qt, J=12.5, 7.0Hz, 2H).

[0166] Step 2: Preparation of compound 14-3

[0167] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 14-2 (500 mg, 1.08 mmol). 108 mg of a crude light yellow oil was obtained with a yield of 23.1%, which was directly used for the next step.

[0168] Step 3: Preparation of compound 14

[0169] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 14-3 (54 mg, 0.126 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 31 mg of yellow solid was obtained, with a yield of 39.7%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.84-10.72 (m, 1H), 9.98-9.87 (m, 1H), 7.63 (t, J = 5.4 Hz, 1H), 7.42-7.17 (m, 4H), 7.17-6.95 (m, 3H), 6.92 (s, 1H), 6.88-6.80 (m, 1H), 6.69 (s, 1H), 6.60 (t, J = 9.4 Hz, 2H), 6. 55-6.34(m,3H),5.53(s,1H),4.37-4.22(m,1H),4.22-4.07(m,3H),4.07-3.92(m,3H),3.90-3.72(m ,1H),3.70-3.52(m,3H),3.29-3.16(m,1H),2.88-2.78(m,1H),2.37-2.25(m,1H),2.22-2.10(m,1H).

[0170] Example 15 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((4-methoxybenzyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 15)

[0171]

[0172] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (90 mg, 0.2 mmol), and M-6 is replaced by 15-1 (30 mg, 0.22 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) is performed to obtain 56 mg of a yellow solid with a yield of 49.12%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 9.97 (s, 1H), 8.04 (s, 1H), 7.91 (d, J = 6.3Hz, 1H), 7.58 (s, 1H), 7.40-7.17 (m, 4H), 6.96-6.39 (m, 6H), 5.68 (s ,1H),4.22(d,J=4.9Hz,2H),4.12-3.99(m,3H),3.78-3.57(m,5H),3.22-3.12(m,1H),3.02(s,1H),2.79(s,1H),2.35(s,1H),2.21(s,1H).

[0173] Example 16 Preparation of (3R, 6'-)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((1-methyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 16)

[0174] 1. Preparation of 1-methyl-4-(piperidine-1-carbonyl)-1H-pyrrole-2-carbaldehyde (Intermediate 16-4)

[0175]

[0176] Step 1: Preparation of compound 16-1

[0177] 1H-pyrrole-3-carboxylic acid methyl ester (730 mg, 5.84 mmol), N,N-dimethylformamide (427 mg, 5.84 mmol) and oxalyl chloride (741 mg, 5.84 mmol) were diluted with 1,2-dichloroethane (5 mL) respectively. The 1,2-dichloroethane solution of the above oxalyl chloride was added dropwise to the 1,2-dichloroethane solution of N,N-dimethylformamide under ice bath, and stirred at room temperature for 0.5 h. The 1,2-dichloroethane solution of 1H-pyrrole-3-carboxylic acid methyl ester was added to the reaction solution under ice bath, stirred at room temperature for 3 h, and set aside. After the reaction was completed, the reaction solution was poured onto crushed ice containing 50% sodium hydroxide (w%) and stirred for 10 min. The pH was adjusted to 7 with dilute hydrochloric acid (2 M), extracted with ethyl acetate three times 50 mL × 3, the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried with anhydrous sodium sulfate, and the organic solvents were removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v) to obtain 638 mg of a white solid with a yield of 76.1%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 12.73 (s, 1H), 9.55 (d, J = 1.0 Hz, 1H), 7.75 (s, 1H), 7.38 (d, J = 1.5 Hz, 1H), 3.75 (s, 3H).

[0178] Step 2: Preparation of compound 16-2

[0179] Compound 16-1 (190 mg, 1.24 mmol) was dissolved in tetrahydrofuran (8 mL) under ice bath, 60% sodium hydride (148 mg, 3.72 mmol) was slowly added, and the mixture was heated to 50 °C for activation for 0.5 h. Iodomethane (175 mg, 1.24 mmol) was added to the above reaction solution under ice bath, and the mixture was reacted at 25 °C for 3 h. 2 mL of methanol was added to quench the reaction, and the pH was adjusted to 7 with glacial acetic acid. After removing the organic solvent by vacuum rotary evaporation, 50 mL of water was added, and the mixture was extracted with ethyl acetate three times (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and removed by vacuum rotary evaporation. The residue was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v) to obtain 173 mg of white solid with a yield of 87.09%. 1H NMR (300MHz, Chloroform-d) δ (ppm) 9.59 (d, J = 1.0 Hz, 1H), 7.46 (s, 1H), 7.34 (d, J = 1.8 Hz, 1H), 3.98 (s, 3H), 3.84 (s, 3H).

[0180] Step 3: Preparation of compound 16-3

[0181] The preparation method is the same as that of the preparation step 2 of intermediate M-5, except that compound M-4 is replaced by 16-2 (173 mg, 1.08 mmol). 53 mg of yellow solid is obtained, with a yield of 32.1%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 9.62 (s, 1H), 7.53 (s, 1H), 7.39 (s, 1H), 4.01 (s, 3H).

[0182] Step 4: Preparation of compound 16-4

[0183] The preparation method is the same as the preparation step 1 of intermediate M-3, except that M-1 is replaced by 16-3 (50 mg, 0.327 mmol), and 2-methoxyethylamine is replaced by piperidine (33 mg, 0.392 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v) gave 20 mg of a yellow oil with a yield of 27.82%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 9.57 (s, 1H), 7.28-7.18 (m, 1H), 7.00 (d, J = 1.9 Hz, 1H), 3.96 (s, 3H), 3.65 (t, J = 5.4 Hz, 4H), 1.82-1.54 (m, 6H).

[0184] 2. Preparation of Compound 16

[0185]

[0186] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (41 mg, 0.091 mmol), and M-6 is replaced by 16-4 (20 mg, 0.091 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 21 mg of yellow solid is obtained, with a yield of 35.5%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.00 (s, 1H), 8.10 (s, 1H), 8.02-7.86 (m, 2H), 7.59 (dd, J = 6.2, 3.0 Hz, 1H), 7.33 (d, J = 7.0 Hz, 2H), 7.02-6.84 (m, 1H), 6.58 (s, 2H), 6.51 (s, 1H), 6.20 (s, 1H), 5.58 ( t,J=5.6Hz,1H),4.70(d,J=7.1Hz,1H),4.31–4.15(m,2H),4.15-3.97(m,2H),3.70-3.52(m,5H) ,3.34-3.18(m,5H),3.14-3.01(m,1H),2.95-2.81(m,1H),2.42-2.17(m,2H),1.67-1.22(m,6H).

[0187] Example 17 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((1-methyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 17)

[0188] 1. Preparation of 4-(aminomethyl)-N-ethylthiophene-2-carboxamide (Intermediate 17-2)

[0189]

[0190] Step 1: Preparation of compound 17-1

[0191] The preparation method is the same as the preparation step 1 of intermediate M-3, except that M-1 is replaced by 4-cyanothiophene-2-carboxylic acid (250 mg, 1.63 mmol), and 2-methoxyethylamine is replaced by ethylamine hydrochloride (146 mg, 1.79 mmol). Silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1-1: 1, v / v) gives 171 mg of a yellow solid with a yield of 57.96%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 8.03 (t, J = 1.1 Hz, 1H), 7.60 (s, 1H), 6.17 (s, 1H), 3.64-3.32 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H).

[0192] Step 2: Preparation of compound 17-2

[0193] Compound 17-1 (165 mg, 0.92 mmol) was dissolved in 3 mL of lithium aluminum hydride tetrahydrofuran solution (1 M) under ice bath and reacted at room temperature for 3 h. After the reaction was completed, 0.12 mL of water was added and stirred for 10 min under ice bath, 0.24 mL of 10% sodium hydroxide aqueous solution was added and stirred for 10 min, and 0.36 mL of water was added and stirred for 10 min to quench. Celite was filtered, the solvent was evaporated under reduced pressure, 50 mL of water was added, and ethyl acetate was extracted three times with 20 mL × 3, the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure, and the residue was chromatographed on a silica gel column (petroleum ether: ethyl acetate = 100: 1, v / v) to obtain 160 mg of a yellow oil with a yield of 95%. 1H NMR(300MHz,Chloroform-d)δ(ppm)7.87(t,J=6.2Hz,1H),7.59(d,J=1.6Hz,1H),7.35(d,J=1.4Hz,1H),3.79(t,J=6. 3Hz, 2H), 3.32 (qd, J = 8.0, 6.2Hz, 2H), 2.71 (dt, J = 7.1, 6.3Hz, 1H), 2.51 (dt, J = 7.0, 6.2Hz, 1H), 1.21 (t, J = 8.0Hz, 3H).

[0194] 2. Preparation of Compound 17

[0195]

[0196] Step 1: Preparation of compound 17-3

[0197] The preparation method is the same as step 1 in Example 1, M-5 (266 mg, 0.794 mmol), M-3 is replaced by 17-2 (160 mg, 0.874 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v). A yellow solid of 160 mg is obtained, with a yield of 36.6%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 8.23 ​​(dd, J = 7.4, 1.5 Hz, 1H), 8.19 (d, J = 1.4 Hz, 1H), 8.14 (t, J = 8.5 Hz, 1H), 7.96 (t, J = 6.2 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.23 (d, J = 1.6 Hz, 1H),4.49-4.38(m,2H),4.10-4.00(m,2H),3.91(p,J=7.0Hz,1H),3.42-3.23(m,3H),2.84(dd,J=1 2.3, 7.1Hz, 1H), 2.73 (dd, J=12.5, 6.9Hz, 1H), 1.97 (qt, J=12.5, 7.0Hz, 2H), 1.21 (t, J=8.0Hz, 3H).

[0198] Step 2: Preparation of compound 17-4

[0199] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 17-3 (150 mg, 0.3 mmol). 90 mg of crude yellow oil was obtained with a yield of 63.3%, which was directly used for the next step.

[0200] Step 3: Preparation of compound 17

[0201] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 17-4 (90 mg, 0.19 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 44 mg of yellow solid was obtained, with a yield of 34.92%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.68 (s, 1H), 8.36 (t, J = 5.6 Hz, 1H), 8.06 (d, J = 5.8 Hz, 1H), 7.76-7.68 (m, 2H), 7.58-7.45 (m, 4H), 7.39 (d, J = 1.9 Hz, 1H), 7.27 (s, 1H), 6.82-6.77 (m, 1H), 6.62-6.48 (m, 3H), 5.62 (s, 1H), 4.30-4. 14(m,2H),4.14-4.03(m,2H),3.92-3.78(m,2H),3.68(s,3H),3.56-3.50(m,1H),3.21(q,J=6.9Hz,2H),3.14-3 .02(m,1H),2.85-2.73(m,1H),2.66-2.57(m,1H),2.36-2.21(m,1H),2.06-1.94(m,1H),0.85(t,J=6.1Hz,3H).

[0202] Example 18 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((4-(cyclohexanecarbonyl)-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 18)

[0203]

[0204] Step 1: Preparation of compound 18-1

[0205] The preparation method is the same as that of intermediate M-6, except that benzoyl chloride is replaced by cyclohexylcarbonyl chloride (730 mg, 5 mmol). Silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v). 887 mg of white solid is obtained, with a yield of 81%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 9.62 (d, J = 1.0 Hz, 1H), 7.47 (s, 1H), 7.32 (dd, J = 1.8, 0.9 Hz, 1H), 3.99 (s, 3H), 2.90 (tt, J = 11.7, 3.2 Hz, 1H), 1.87 (dt, J = 7.0, 3.8 Hz, 4H), 1.79-1.67 (m, 1H), 1.54-1.25 (m, 5H).

[0206] Step 2: Preparation of compound 18

[0207] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (187 mg, 0.415 mmol), and M-6 is replaced by 18-1 (100 mg, 0.457 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 103 mg of yellow solid is obtained, with a yield of 38%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.97 (s, 1H), 8.01 (d, J = 5.6 Hz, 1H), 7.94-7.83 (m, 1H), 7.59-7.49 (m, 2H), 7.36-7.26 (m, 2H), 6.83 (s, 1H), 6.58 (d, J = 10.6 Hz, 3H), 6.41 (d, J = 1.9 Hz, 1H), 5.54 (d, J = 5.8 Hz, 1 H),4.22(s,2H),4.12-3.96(m,3H),3.69-3.59(m,2H),3.57(s,3H),3.52-3.41(m,1H),3.09-3.00(m ,1H),2.90-2.81(m,2H),2.44-2.30(m,1H),2.31-2.15(m,1H),1.84-1.49(m,8H),1.44-1.21(m,2H).

[0208] Example 19 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((4-(cyclopentanecarbonyl)-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 19)

[0209]

[0210] Step 1: Preparation of compound 19-1

[0211] The preparation method is the same as that of intermediate M-6, except that benzoyl chloride is replaced by cyclopentylcarbonyl chloride (660 mg, 5 mmol). Silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1, v / v) is performed. 759 mg of a white solid is obtained, with a yield of 74.04%. 1H NMR (300 MHz, Chloroform-d) δ (ppm) 9.61 (s, 1H), 7.53-7.42 (m, 1H), 7.35-7.28 (m, 1H), 3.99 (s, 3H), 3.39 (p, J = 7.9 Hz, 1H), 1.95-1.81 (m, 4H), 1.79-1.59 (m, 4H).

[0212] Step 2: Preparation of compound 19

[0213] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (199 mg, 0.443 mmol), and M-6 is replaced by 19-1 (100 mg, 0.488 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 124 mg of yellow solid is obtained, with a yield of 43.8%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 8.12 (s, 1H), 8.04 (d, J = 4.6 Hz, 1H), 7.99-7.87 (m, 1H), 7.58-7.50 (m, 1H), 7.43-7.26 (m, 3H), 6.73-6.36 (m, 5H), 5.63-5.52 (m, 1H), 4.38 (d, J = 5.7 Hz ,1H),4.22(s,1H),4.09(s,2H),3.93-3.83(m,1H),3.63(s,3H),3.60-3.48(m,2H),3.20- 2.98(m,2H),2.89-2.81(m,1H),2.68-2.58(m,1H),2.47-2.08(m,2H),1.79-1.53(m,8H).

[0214] Example 20 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((1-methyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 20)

[0215]

[0216] Step 1: Preparation of compound 20-1

[0217] The preparation method is the same as that of intermediate M-3 preparation step 1, except that M-1 is replaced by 16-3 (117 mg, 0.764 mmol), and 2-methoxyethylamine is replaced by N-methylpiperazine (92 mg, 0.916 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-10: 1, v / v) is performed to obtain 76 mg of a yellow solid with a yield of 42.3%. 1H NMR(300MHz,Chloroform-d)δ(ppm)9.69(s,1H),7.78(d,J=1.6Hz,1H),6.91(d,J=1.6Hz,1H),3.93(s,3H),3.48(dt,J= 12.3,7.0Hz,2H),3.39(dt,J=12.5,7.1Hz,2H),3.08(dt,J=12.3,7.1Hz,2H),2.64(dt,J=12.5,7.1Hz,2H),2.31(s,3H).

[0218] Step 2: Preparation of compound 20

[0219] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (57 mg, 0.127 mmol), and M-6 is replaced by 19-1 (30 mg, 0.127 mmol). Silica gel column chromatography (dichloromethane: methanol = 20:1, v / v) is performed to obtain 27 mg of a yellow solid, with a yield of 31.76%. NMR (300MHz, DMSO-d6) δ (ppm) 9.98 (s, 1H), 8.06 (t, J = 6.2 Hz, 1H), 7.92-7.89 (m, 1H), 7.60-7.55 (m, 2H), 7.34 (d, J =3.0Hz,1H),6.98(d,J=1.9Hz,1H),6.86(s,1H),6.68(d,J=9.6Hz,1H),6.56(s,1H),6.50(d,J=4.2Hz,1H),6.21( d,J=1.9Hz,1H),5.57(t,J=6.2Hz,1H),4.68-4.64(m,1H),4.23-4.15(m,2H),4.07(d,J=4.6Hz,2H),3.67-3.61(m ,1H),3.59-3.51(m,8H),3.12-3.01(m,2H),2.92-2.85(m,1H),2.38-2.32(m,1H),2.26-2.21(m,5H),2.15(s,3H).

[0220] Example 21 Preparation of (3R, 6'S)-N-(Benzofuran-3-ylmethyl)-5-(((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 21)

[0221]

[0222] Step 1: Preparation of compound 21-2

[0223] The preparation method is the same as step 1 in Example 1, M-5 (335 mg, 1 mmol), M-3 is replaced by 21-1 (147 mg, 1 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 180 mg of a yellow solid, with a yield of 38.79%. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.05 (s, 1H), 8.35-8.14 (m, 2H), 8.05 (d, J = 2.4Hz ,1H),7.95(dd,J=8.6,2.3Hz,1H),7.45(d,J=8.3Hz,2H),7.30-7.19(m,2H),6.75 (d,J=8.6Hz,1H),4.14-4.00(m,3H),3.65(d,J=10.5Hz,1H),3.50-3.40(m,1H), 3.23(d,J=10.4Hz,1H),3.12-2.98(m,2H),2.45-2.34(m,1H),2.27-1.95(m,1H).

[0224] Step 2: Preparation of compound 21-3

[0225] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 21-2 (170 mg, 0.366 mmol). 85 mg of crude yellow oil was obtained with a yield of 53.51%, which was directly used for the next step.

[0226] Step 3: Preparation of compound 21

[0227] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 21-3 (85 mg, 0.19 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 36 mg of yellow solid is obtained, with a yield of 30%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 10.00 (s, 1H), 8.00 (t, J = 5.8 Hz, 1H), 7.78-7.67 (m, 2H), 7.64-7.46 (m, 4H), 7.44-7.12 (m, 5H), 6.82 (s, 1H), 6.64-6.49 (m, 3H), 5.60 (t, J = 5.5 Hz, 1H), 4 .25-3.96(m,5H),3.62(d,J=11.4Hz,4H),3.48-3.40(m,1H),3.30(d,J=8.9Hz,1H),3.03(d d, J=10.6, 6.5Hz, 1H), 2.85 (dd, J=10.6, 5.5Hz, 1H), 2.43-2.28 (m, 1H), 2.28-2.12 (m, 1H).

[0228] Example 22 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((3,4-dihydroxybenzyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'-H-spiro[indole-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 22)

[0229]

[0230] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (55 mg, 0.344 mmol), and M-6 is replaced by 22-1 (57 mg, 0.413 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) is performed to obtain 67 mg of a yellow solid with a yield of 34.05%. 1H NMR(300MHz,)δ(ppm)9.65(s,1H),8.13(s,1H),8.02-7.86(m,1H),7.81-7.56(m,1H),7.53-7.10(m,4H),6.88-6.58(m,2H),6.40(t,J=7. 8Hz,2H),5.77-5.24(m,1H),4.68-4.14(m,4H),4.06-3.82(m,3H),3.19-3.03(m,2H),2.94-2.74(m,1H),2.75-2.60(m,1H),2.27(s,1H).

[0231] Example 23 Preparation of (3R, 6'S)-5-((4-benzoyl-1-methyl-1H-pyrrol-2-yl)methyl)-2-oxo-N-(4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)methyl)-1',6',7',7'a-tetrahydro-3'H-spiro[indole-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 23)

[0232]

[0233] Step 1: Preparation of compound 23-2

[0234] The preparation method is the same as step 1 in Example 1, M-5 (335 mg, 1 mmol), M-3 is replaced by 23-1 (167 mg, 1 mmol). Silica gel column chromatography (dichloromethane: methanol = 100: 1-20: 1, v / v) is performed to obtain 194 mg of a yellow solid, with a yield of 40%. 1HNMR (300MHz, DMSO-d6) δ (ppm) 11.10 (s, 1H), 8.18-8.09 (m, 3H), 6.94 (d, J = 8.4Hz, 1H), 6.40 (s, 1H), 3.88-3.74 (m, 2H), 3.68 (d, J = 10.5Hz, 1H) ,3.48-3.40(m,1H),3.27(d,J=10.4Hz,1H),3.12-2.99(m,2H),2.63-2. 57(m,2H),2.43(q,J=6.4Hz,1H),2.20-2.09(m,3H),1.79-1.50(m,5H).

[0235] Step 2: Preparation of compound 23-3

[0236] The preparation method was the same as step 2 in Example 1, except that 1-1 was replaced by 23-2 (194 mg, 0.4 mmol). 153 mg of crude yellow oil was obtained with a yield of 84.25%, which was directly used for the next step.

[0237] Step 3: Preparation of compound 23

[0238] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 23-3 (153 mg, 0.337 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v). 86 mg of yellow solid was obtained, with a yield of 39.2%. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 9.97 (s, 1H), 7.87-7.62 (m, 3H), 7.57 (t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.4 Hz, 2H), 7.37 (d, J = 2.0 Hz, 1H), 6.79 (s, 1H), 6.68-6.40 (m, 3H), 6.24 (s, 1H), 5.57 (s, 1H), 4.24-4.04 (m, 2H), 4 .04-3.96(m,1H),3.80(d,J=6.7Hz,2H),3.72-3.60(m,3H),3.61-3.52(m,1H),3.49-3.38(m,1H),3.10-2 .94(m,1H),2.91-2.76(m,1H),2.61(d,J=6.0Hz,2H),2.43-2.08(m,4H),1.99(s,1H),1.78-1.52(m,4H).

[0239] Example 24 Preparation of (3R, 6'S)-N-(Benzo[b]thiophene-3-methyl)-5-((4-(cyclohexylmethyl)-1-methyl-1H-pyrrole-2-methyl)amino)-2-oxo-1', 6', 7', 7'-tetrahydro-3'H-spiro[indole-3, 5'-pyrrole[1, 2-c]thiazole]-6'-carboxamide (Compound 24)

[0240] 1. Preparation of 4-(cyclohexylmethyl)-1-methyl-1H-pyrrole-2-carbaldehyde (Intermediate 24-4)

[0241]

[0242] Step 1: Preparation of compound 24-1

[0243] Compound 18-1 (170 mg, 0.77 mmol) was dissolved in dioxane (4 mL), water (1 mL) was added, potassium hydroxide (52 mg, 0.93 mmol) and potassium permanganate (146 mg, 0.93 mmol) were added under ice bath, and the mixture was transferred to room temperature and stirred for 3 h. After the reaction was completed, 30% hydrogen peroxide (2 mL) was slowly added dropwise under ice bath. The precipitate was filtered, the filtrate was adjusted to pH 2 with dilute hydrochloric acid, and extracted with ethyl acetate three times (50 mL × 3), the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried with anhydrous sodium sulfate, and the organic phases were evaporated to dryness under reduced pressure. The solid was slurried with an organic solvent (petroleum ether: ethyl acetate = 30: 1, v / v), filtered, and the filter cake was dried to obtain 140 mg of a white solid with a yield of 77.4%. 1H NMR (300MHz, Chloroform-d) δ (ppm) 7.48 (d, J = 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 3.97 (s, 3H), 3.04-2.77 (m, 1H), 1.94-1.66 (m, 5H), 1.59-1.17 (m, 5H).

[0244] Step 2: Preparation of compound 24-2

[0245] Compound 24-1 (140 mg, 0.596 mmol) was dissolved in methanol (5 mL), and thionyl chloride (0.423 mL, 5.96 mmol) was slowly added dropwise under ice bath. After addition, nitrogen protection was applied, and the mixture was heated to 60 ° C and refluxed for 3 h. After the reaction was completed, the solvent was evaporated under reduced pressure and saturated sodium bicarbonate was added under ice bath to neutralize until no carbon dioxide gas was generated. Ethyl acetate was extracted three times with 50 mL × 3, and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure and dried, and the residue was chromatographed on a silica gel column (petroleum ether: ethyl acetate = 20:1, v / v). 90 mg of white solid was obtained, and the yield was 60.64%. 1HNMR (300MHz, Chloroform-d) δ (ppm) 7.39 (d, J = 2.0Hz, 1H), 7.32 (d, J = 2.0Hz, 1H), 3.96 (s, 3H), 3.85 (s, 3H), 2.93-2.85 (m, 1H), 1.84-1.26 (m, 10H).

[0246] Step 3: Preparation of compound 24-3

[0247] Aluminum chloride (360 mg, 2.7 mmol) was placed in a reaction bottle, tetrahydrofuran (5 mL) was added, lithium aluminum hydride (52 mg, 1.35 mmol) was added to the reaction solution in batches under an ice bath, and stirred at room temperature for 5 min. Compound 24-2 (90 mg, 0.361 mmol) was added to the reaction solution, heated to 50 ° C and refluxed for 2 h, cooled to room temperature after the reaction, and the reactant was dripped onto ice, extracted with ethyl acetate three times 50 mL × 3, the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure and dried, and the residue was chromatographed on a silica gel column (petroleum ether: ethyl acetate = 20: 1, v / v). 64 mg of yellow oil was obtained with a yield of 85.64%. 1H NMR (300MHz, Chloroform-d) δ (ppm) 6.53 (d, J = 2.1 Hz, 1H), 6.02 (d, J = 2.0 Hz, 1H), 4.53 (s, 2H), 3.64-3.61 (m, 5H), 2.06-1.92 (m, 1H), 1.78-1.52 (m, 10H).

[0248] Step 4: Preparation of compound 24-4

[0249] Compound 24-3 (64 mg, 0.309 mmol) was dissolved in anhydrous dichloromethane (2 mL), and then added dropwise to an anhydrous dichloromethane solution (2 mL) of pyridinium chlorochromate (133 mg, 0.618 mmol), and stirred at room temperature for 2 h under nitrogen protection. After the reaction was completed, the mixture was filtered, and 50 mL of water was added to the filtrate. The mixture was extracted with ethyl acetate three times, 50 mL × 3. The organic phases were combined and washed with 1 M sodium hydroxide solution and 1 M dilute hydrochloric acid, respectively. The organic phase was evaporated under reduced pressure and dried, and then chromatographed on a silica gel column (petroleum ether: ethyl acetate = 20:1, v / v). 28 mg of a white oily product was obtained with a yield of 44.01%. 1H NMR(300MHz,Chloroform-d)δ(ppm)6.57(s,1H),4.76(d,J=1.6Hz,1H),4.68(d,J=1.6Hz,1H),3. 09(s,3H),2.22(dd,J=7.0,1.3Hz,2H),1.75-1.64(m,5H),1.29-1.11(m,4H),0.97-0.87(m,2H).

[0250] 2. Preparation of Compound 24

[0251]

[0252] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (74 mg, 0.163 mmol), and M-6 is replaced by 24-4 (28 mg, 0.136 mmol). Silica gel column chromatography (dichloromethane: methanol = 50:1, v / v) is performed to obtain 21 mg of a yellow solid, with a yield of 24.13%. NMR (300MHz, DMSO-d6) δ (ppm) 9.96 (s, 1H), 8.04 (t, J = 5.7Hz, 1H), 7.90 (dt, J = 7.0, 3.4Hz, 1H), 7.57 (dt, J = 6.7,3.5Hz,1H),7.33(dt,J=6.0,3.5Hz,3H),6.72-6.65(m,2H),6.55-6.42(m,3H),4.70(s,2H),4.40(t,J =5.1Hz,1H),4.29-4.19(m,2H),4.07-3.98(m,1H),3.63(d,J=8.8Hz,1H),3.51-3.42(m,2H),3.33(d,J=8. 8Hz,1H),3.08-2.99(m,6H),2.88-2.82(m,1H),2.41-2.29(m,1H),2.28-2.15(m,1H),1.76-1.71(m,10H).

[0253] Example 25 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-(((1-(cyclohexanecarbonyl)-3-methyl-1H-pyrazol-4-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiro[indoline-3, 5'-pyrrolo[1, 2-c]thiazole]-6'-carboxamide (Compound 25)

[0254]

[0255] Step 1: Preparation of compound 25-2

[0256] Disperse 60% sodium hydride (80 mg, 2 mmol) in 10 mL toluene under ice bath. Add compound 25-1 (200 mg, 1.82 mmol) to the reaction system, heat to 70 °C and heat to activate for 0.5 h. After activation, cool the reaction system to room temperature, transfer to ice bath and drop cyclohexanecarbonyl chloride (265 μL, 2 mmol) into the reaction solution. After 4 h of reaction at room temperature, add water (100 μL) to quench the reaction. Evaporate the reaction solution to dryness under reduced pressure rotary evaporation, redissolve the remaining oil in 100 mL ethyl acetate, and extract with water (100 mL). Wash the organic phase with 10% sodium hydroxide aqueous solution and saturated brine respectively. Collect the organic phase, remove the organic solvent by reduced pressure rotary evaporation, and chromatograph on a silica gel column (petroleum ether: ethyl acetate = 20:1, v / v). Obtain 347 mg of white solid, yield: 87.01%. 1 H NMR (300MHz, Chloroform-d) δ (ppm) 10.02 (s, 1H), 8.65 (s, 1H), 3.66-3.50 (m, 1H), 2.53 (s, 3H), 2.03-1.74 (m, 6H), 1.67-1.27 (m, 4H).

[0257] Step 2: Preparation of compound 25

[0258] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (100 mg, 0.22 mmol), and M-6 is replaced by 25-2 (59 mg, 0.264 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) is performed to obtain 41 mg of a yellow solid with a yield of 28.7%. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm)9.98(s,1H),8.16(s,1H),8.02(s,1H),7.89(d,J=7.4Hz,1H),7.53(d,J=7.2Hz,1H) ,7.37-7.26(m,2H),6.78(s,1H),6.67(s,1H),6.58(s,2H),4.19(s,2H),4.01(s,2H),3.62(d ,J=9.1Hz,1H),3.48-3.42(m,2H),3.27(d,J=9.1Hz,2H),3.03(t,J=8.4Hz,1H),2.81(dd,J=1 0.3,5.3Hz,1H),2.37-2.19(m,5H),1.84-1.76(m,2H),1.74-1.67(m,2H),1.41-1.21(m,6H).

[0259] Example 26 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((((1-(cyclohexanecarbonyl)pyrrolidin-3-yl)methyl)amino)-2-oxo-1', 6', 7', 7a'-tetrahydro-3'H-spiro[indoline-3, 5'-pyrro[1, 2-c]thiazole]-6'-carboxamide (Compound 26)

[0260]

[0261] Step 1: Preparation of compound 26-2

[0262] The preparation method is the same as step 1 in Example 25, except that compound 25-1 is replaced by compound 26-1 (200 mg, 2 mmol), the amount of cyclohexanecarbonyl chloride used is 291 μL, 2.2 mmol. The amount of 60% sodium hydride used is 88 mg, 2.2 mmol. Silica gel column chromatography is not required for post-treatment. 80 mg of yellow oil is obtained, and the yield is 19.1%. 1 H NMR (300MHz, Chloroform-d) δ (ppm) 11.15 (s, 1H), 3.86-3.51 (m, 2H), 3.23-2.97 (m, 2H), 2.06 (s, 1H), 1.58-1.50 (m, 7H), 1.45 (d, J = 6.4Hz, 6H).

[0263] Step 2: Preparation of compound 26

[0264] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (100 mg, 0.22 mmol), and M-6 is replaced by 26-2 (55 mg, 0.264 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) is performed to obtain 38 mg of a brown solid, with a yield of 26.9%. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm)9.95(s,1H),8.06(s,1H),7.89(s,1H),7.55(s,1H),7.32(s,2H),6.72(s,1H),6.61-6.43(m,3H),4.22(s,2H), 3.65(d,J=8.7Hz,2H),3.47(s,2H),3.19-2.90(m,7H),2.41-2.17(m,4H),1.98(s,1H),1.63(s,6H),1.36-1.15(m,6H).

[0265] Example 27 Preparation of (3R, 6'S)-N-(Benzo[b]thiophen-3-ylmethyl)-5-((((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-2-oxo-1',6',7',7a'-tetrahydro-3'H-spiro[indoline-3,5'-pyrrolo[1,2-c]thiazole]-6'-carboxamide (Compound 27)

[0266]

[0267] The preparation method is the same as step 3 in Example 1, except that 1-2 is replaced by 2-3 (100 mg, 0.22 mmol), and M-6 is replaced by 27-1 (44 mg, 0.264 mmol). Silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) is performed to obtain 43 mg of a yellow solid with a yield of 32.4%. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm)9.94(s,1H),8.02(d,J=5.3Hz,1H),7.96-7.87(m,1H),7.60-7.52(m,1H),7.41-7.25(m,2H),6 .87-6.70(m,4H),6.66(s,1H),6.52(d,J=8.3Hz,1H),6.43(dd,J=8.3,2.2Hz,1H),5.72(t,J=5.7Hz,1H ),4.29-4.12(m,6H),4.03(d,J=6.5Hz,3H),3.60(d,J=8.9Hz,1H),3.45(dd,J=11.4,7.5Hz,1H),3.29( d,J=8.9Hz,1H),3.08-2.96(m,1H),2.76(dd,J=10.7,5.6Hz,1H),2.41-2.27(m,1H),2.28-2.11(m,1H).

[0268] Example 28 Biological Activity

[0269] The inhibitory effect of the compound on the cGAMP-stimulated STING pathway in THP1-Dual cells was detected by the luciferase method as follows:

[0270] (1) 4*10 THP1-Dual cells in the logarithmic growth phase were cultured. 4 Each well was seeded in a 96-well plate, and different concentrations of compounds were added (three-fold dilution from 50 μM, a total of five concentration gradients), and the DMSO group was used as a blank control.

[0271] (2) Cells were transfected with 10 μg / mL cGAMP and lipofectamine 2000 (Invitrogen) and cultured for 24 h.

[0272] (3) Using QUANTI-Luc TM 10 μL of cell culture supernatant was added to a 96-well plate, and then 50 μL of QUANTI-Luc TM The reagents were read using a multifunctional microplate reader (Thermo).

[0273] Relative luciferase activity calculation method: cells treated with lipofectamine 2000 were used as blank control, and cells treated with lipofectamine 2000:cGAMP complex were used as negative control.

[0274] Relative luciferase activity = (RLU - RLU blackcontrol) / (RLU negativecontrol - RLU blackcontrol), RLU represents the original luciferase value.

[0275] Measured compound IC 50 , and compared with the positive control drug H151 STING antagonist as shown in Table 1:

[0276] Table 1 Compound IC 50

[0277]

[0278]

[0279] It can be seen from the above table that the above compounds of the present invention have a good inhibitory effect on the cGAS-STING pathway, can provide a basis for the preparation of drugs for treating diseases related to the function of the STING protein, have the prospect of preparing drugs for treating and / or preventing inflammatory diseases and autoimmune diseases, and provide a reliable approach for clinical application.

[0280] Example 29 Evaluation of the binding ability of compound 1 to STING by isothermal titration calorimetry (ITC)

[0281] The ITC experiment uses the MicroCal ITC 200 instrument to measure the binding affinity between proteins and ligands. The ITC experiment can fit and quantitatively calculate the binding thermodynamic parameters, including the number of binding sites (n), binding constant (Ka), reaction enthalpy change (ΔH), entropy change (ΔS), etc., by detecting the heat change (endothermic or exothermic) when the protein solution and the small molecule (ligand) solution interact.

[0282] The high-concentration, high-purity STING-CTD protein was obtained through extraction, dialysis, and ultrafiltration, and the buffer consistent with the protein component system was retained as much as possible during the dialysis process. WT CTD (aa 149-379) was cloned into the pET28a expression vector and transformed into the Escherichia coli BL21 (DE3) strain. Then, after induction with isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM) at 16°C for 20 hours, the transformed bacteria were cultured at 37°C for 12 hours. The bacteria were lysed with an ultrasonic lysator in lysis buffer (20 mM tris-HCl (pH 7.4), 200 mM NaCl, 0.1% Tween 20, and 0.1% mercaptoethanol), and then collected, centrifuged, and the supernatant was filtered and lysed with Ni. 2+ -NTA column purification. The obtained protein was dialyzed overnight with 1× phosphate buffered saline (PBS) buffer, and its purity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). First, 100 μL of STING-CTD protein (214 μM) and 400 μL of compound (10 μM) were prepared, in which the DMSO content in the protein and compound 1 was consistent. Then an isothermal titration calorimeter (MicroCal iTC200) was used, and the operating parameters were set: temperature 25°C; speed 750 rpm. Clean the sample pool and titration needle, aspirate about 50 μL of STING-CTD protein into the titration needle, add about 280 μL of compound to the sample pool, and drip into the sample pool at a rate of 2 μL per drop, with an interval of 2 minutes between each drop, for a total of 19 drops. After the experiment, MicroCal.Origin version 7.0 software was used for data processing and analysis to obtain the entropy change ΔS, enthalpy change ΔH and free energy ΔG change of the compound binding to the STING-CTD protein, and then the KA value and KD value (KD = 1 / KA) were obtained. The smaller the KD value, the better the binding strength and selectivity of the compound to the protein.

[0283] The compound 1 tested showed binding activity to STING. Figure 1 As shown, the binding affinity constant KD of compound 1 is 1.92 μM. This indicates that compound 1 has a good binding affinity to the STING-CTD protein.

[0284] Example 30 Real-time fluorescence quantitative polymerase chain reaction detection (RT-qPCR)

[0285] STING SAVI mutant (V155M, R284G, R281Q, G166E and C206Y) plasmid (pcDNA3.1-3×Flagvector) was purchased from youbio Biotechnology Co. The mutant plasmid was transfected into HEK293T cells using jetPRIME (polyplus, Cat#101000046) to construct a cell model of SAVI self-activation.

[0286] HEK293T cells (1×106 cells / mL) were added to a 12-well plate, 1 mL per well, adhered overnight, and then transfected with the corresponding plasmid 1 μg / well using jetPRIME transfection reagent (Polyplus). After 6 hours, the medium was changed and different concentrations of compound 1 were administered. The cells were incubated at 37°C for 18 hours, and then the medium was discarded and RZ lysis solution was added. RNA was extracted using an RNA extraction kit (Tiangen Bio) as required. Finally, 50 μL ddH2O was used to dissolve the RNA, and 1 μL of RNA extraction solution was taken to determine the concentration using a NanoDrop UV spectrophotometer. Then, 4×gDNA wiper Mi and 5×HiScript III RT SuperMix reverse transcription reagent (R341-02, Vazyme) were used to reverse transcribe the RNA into cDNA at the corresponding temperature. Subsequently, RT-qPCR experiments were performed using 2×ChamQ SYBR qPCR Master Mix dye and corresponding DNA primers (GAPDH as an internal reference), and the mRNA expression levels of IFN-β and CXCL10 were calculated and analyzed using the 2-ΔΔCt method.

[0287] Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of inflammatory factors in different groups. Figure 2 As shown. Compared with the blank group (DMSO group), the mRNA expression levels of IFN-β1 and CXCL10 in HEK293T cells transfected with STING SAVI mutant plasmid were increased, indicating that the STING SAVI mutant plasmid caused an inflammatory response in HEK293T cells. The drug-treated group (ie, IS-1 4μM and 8μM) can significantly reduce the mRNA expression levels of IFN-β1 and CXCL10 in HEK293T cells transfected with STING SAVI mutant plasmid, indicating that compound 1 has a strong STINGSAVI mutant inhibitory activity against HEK293T cells transfected with SAVI mutants. It reflects that compound 1 has a certain therapeutic effect on diseases caused by excessive activation of STING, which can provide a basis for the preparation of drugs for treating diseases related to the function of STING protein, and has the prospect of preparing drugs for the treatment and / or prevention of inflammatory diseases and autoimmune diseases, providing a reliable way for clinical application.

[0288] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An indolin-2-one compound of general formula I or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt: in, Ring A is selected from alkyl-substituted or unsubstituted 5-10-membered aliphatic heterocyclic ring; Ring B is selected from an aromatic ring, an aromatic heterocycle, an aliphatic heterocycle or a condensed ring substituted or unsubstituted by 1-2 R3; R1 is selected from H, substituted or unsubstituted aromatic ring, aromatic heterocycle or condensed ring; R2 is selected from H, an alkyl chain of 1 to 6 carbon atoms; n is selected from 0 or 1.

2. The compound according to claim 1, characterized in that Ring A is selected from a tetrahydro-1H,3H-pyrrolo[1,2-c]thiazole ring or a 1-methylpyrrolidine ring; The aromatic ring, aromatic heterocycle, aliphatic heterocycle or condensed ring in ring B is selected from N-methylpyrrole ring, pyrrole ring, pyridine ring, quinoline ring, pyrazole ring, tetrahydropyrrole ring, 1,4-benzodioxetane ring or benzene ring; R3 is selected from methyl, benzoyl, acetyl, methoxy, piperidine-1-carbonyl, cyclohexanecarbonyl, cyclopentanecarbonyl, 4-methylpiperazine-1-carbonyl, cyclohexylmethyl or hydroxyl; When R1 is selected from a substituted or unsubstituted aromatic ring, aromatic heterocyclic ring or condensed ring, the substituted or unsubstituted aromatic ring, aromatic heterocyclic ring or condensed ring is selected from a benzo[b]thiophene ring, a pyridine ring, a thiophene ring, a benzene ring, a naphthalene ring, an indole ring, an N-ethylcarbamoylthiophene ring, a benzo[b]furan ring or a 4,5,6,7-tetrahydrobenzo[b]thiophene ring; The alkyl chain of 1 to 6 carbon atoms is selected from N-(2-methoxyethyl)carbamoyl.

3. The compound according to claim 1, characterized in that When R1 is selected from H, n is selected from 0, and R2 is selected from H; When R1 is selected from a benzo[b]thiophene ring, n is selected from 0, and R2 is selected from H; When R1 is selected from a benzo[b]thiophene ring and n is selected from 1, R2 is selected from N-(2-methoxyethyl)carbamoyl; When R1 is selected from a pyridine ring, a thiophene ring, a benzene ring, a naphthalene ring, an indole ring, an N-ethylcarbamoylthiophene ring, a benzo[b]furan ring or a 4,5,6,7-tetrahydrobenzo[b]thiophene ring, n is selected from 0 and R2 is selected from H.

4. The compound according to claim 1, characterized in that Selected from:

5. A method for preparing the compound according to any one of claims 1 to 4, characterized in that: The synthetic route is as follows: Reagents and conditions: (a) methanol, 65-70°C, reflux for 5 h; (b) potassium hydroxide, tetrahydrofuran / methanol / water = 1:1:1 (v / v / v), 60°C, 1-3 h; (c) O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, 25°C, 8 h; (d) reduced iron powder, ammonium chloride, ethanol / water = 3:1 (v / v), 80°C, 4 h; (e) sodium borohydride, anhydrous dichloromethane, anhydrous magnesium sulfate, 25°C, 8-10 h; Wherein, R1, R2, A, B, and n are as defined in Formula I.

6. The compound according to claim 1, characterized in that The pharmaceutically acceptable salts include acid addition salts formed between the compound of formula I and the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, acetic acid, trifluoroacetic acid, pyruvic acid, citric acid, tartaric acid, lactic acid, maleic acid, benzenesulfonic acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, fumaric acid, salicylic acid or phenylacetic acid; and also include acid salts formed between the compound of formula I and inorganic bases or organic salts made from basic amines.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of one or more of the compounds according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 4 in the preparation of a STING protein inhibitor.

9. Use of the compound according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating inflammatory diseases and autoimmune diseases associated with STING protein function.

10. The use according to claim 9, characterized in that The inflammatory diseases and autoimmune diseases related to the function of STING protein include STING-related infantile vasculopathy SAVI, Aicardi-Goutières syndrome AGS, COPA syndrome caused by gene mutation of the α subunit of the symbiotic protein complex, systemic lupus erythematosus SLE, familial pernio lupus erythematosus FCL, Parkinson's disease PD, amyotrophic lateral sclerosis ALS, Huntington's disease, non-alcoholic steatohepatitis NASH, alcoholic liver, nerve damage, rheumatoid arthritis, renal fibrosis, systemic sclerosis, intervertebral disc degeneration, pulmonary fibrosis, aging, systemic sclerosis, psoriasis, inflammatory bowel disease, autoimmune colitis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, uveitis, mucositis, diabetes, cardiovascular disease or neurodegenerative disease.

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