CN115919872B - Use of tricyclic compounds and pharmaceutical compositions containing the same - Google Patents

Abstract

The invention provides an application of tricyclic compounds and a pharmaceutical composition containing the tricyclic compounds, the application of tricyclic compounds as STING agonists and the preparation of drugs for treating diseases related to STING protein functions, wherein the tricyclic compounds are compounds shown in a formula I, tautomers, racemates, meso, enantiomers, diastereoisomers and pharmaceutically acceptable salts thereof, and the formula I is as follows: The tricyclic compound or the pharmaceutical composition containing the tricyclic compound can be used for preparing interferon gene stimulation factor agonists or immunoadjuvants or preparing medicines for treating diseases related to STING protein functions.

Description

Application of tricyclic compound and pharmaceutical composition containing tricyclic compound

Technical Field

The invention relates to the application field of organic compounds, in particular to application of a tricyclic compound and a pharmaceutical composition containing the tricyclic compound.

Background

The interferon gene stimulators (Stimulator of interferon gene, STING) are a key node protein for specific regulation of the innate immune system. The innate immune system serves as a first line of defense in humans, and is capable of rapidly recognizing invasion of foreign pathogenic microorganisms through pattern recognition receptors (Pattern recognition receptors, PRRs), inducing Macrophages (MC) and natural killer cells (NK) to exert phagocytic and degradeable functions, while secreting a large number of inflammatory or chemotactic factors, promoting presentation of antigens by Dendritic Cells (DCs), and activating adaptive immunity. Among them, pattern recognition receptors include nucleotide binding domain-like receptors (NLRs), toll-like receptors (TLRs), retinoic acid-induced gene I-like receptors (RIG-I like receptors, RLRs), C-type lectin receptors (C-TYPE LECTIN receptors, CLRs), guanylate-adenylate synthetase (CYCLIC GMP-AMP SYNTHASE, CGAS), and the like. Studies have shown that PRRs recognize pathogenic molecular related patterns (Pathogen associated molecular patterns, PAMPs, such as single/double-stranded ribonucleotides and lipopolysaccharides) or lesion-related molecular patterns (Damage associated molecular patterns, DAMPs) and further initiate downstream signaling pathways to release cytokines, regulating the immune response in the body. As a key linker protein in the innate immune response, the activation of STING can enhance the immune effect of immunotherapy, and more widely activate and enhance the innate immune system and adaptive immune function of human body, so that the preparation is expected to be applicable to various cancers, even for treating other diseases besides cancers, and is one of possible effective targets for small molecule tumor immunotherapy. Likewise, aberrant activation of the SITNG signaling pathway may trigger autoimmune and inflammatory diseases.

Therefore, research on compounds capable of activating STING signal pathway has important significance, chinese patent application 2022107988350 discloses benzimidazole compounds, pharmaceutical compositions containing the benzimidazole compounds and application of the benzimidazole compounds, the benzimidazole compounds have the effect of activating STING signal pathway, and in view of wide application prospect of STING activation, more compounds for activating STING signal pathway are to be discovered.

Disclosure of Invention

The invention aims to provide an application of tricyclic compound as STING agonist or immune adjuvant and a pharmaceutical composition containing the tricyclic compound.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

An application of tricyclic compounds as STING agonists, immunoadjuvants or in preparing medicines for treating diseases related to STING protein functions, the tricyclic compound is a compound shown in a formula I, a tautomer, a racemate, a meso, an enantiomer, a diastereomer and a pharmaceutically acceptable salt thereof, wherein the formula I is as follows:

Wherein,

R ¹、R² is independently selected from one of hydrogen, halogen, substituted or unsubstituted C _1-4 alkyl, aryl and heteroaryl, the substituent of C _1-4 alkyl is alkoxy, thio, cyano, amino, amido or carboxyl, and the heteroaryl is preferably triazole.

X is selected from N or C, Y is selected from S, O, C, N and one of sulfoxide groups, and N is selected from 0, 1 or 2.

L is a substituted or unsubstituted main chain containing 1-4 atoms, the main chain is C _1-4 alkylene, or the main chain contains carbonyl or amido, the rest is alkylene, and the substituent of the main chain is methyl or hydroxyl.

The ring A, B is independently selected from one of an aromatic ring and a C _5-6 aromatic heterocycle.

The ring Q is selected from 5-7 membered aliphatic heterocyclic ring or aromatic ring, the hetero atom is selected from one or more of N, O and S, and the hetero atom number is 1-3.

Z is a substituted or unsubstituted C _1-4 alkyl group, and the substituent of the C _1-4 alkyl group is a hydroxyl group, an amino group or an amide group. As a further improvement of the technical scheme, the chemical structural formula of the tricyclic compound is shown as a formula II,

Wherein n is 0 or 1;

R ³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰ is independently selected from one of hydrogen, halogen, substituted or unsubstituted C _1-4 alkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, the substituent of C _1-4 alkyl is alkoxy, thio, cyano, amino, amido or carboxyl, and the aryl or heteroaryl is triazole.

As a further improvement of the technical scheme, R ³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰ are hydrogen atoms;

Or R ³、R⁶、R⁷、R⁹ is a hydrogen atom, R ⁴ and R ⁹ are independently selected from one of hydrogen, halogen, cyano, formamide, methoxy, methylthio, triazole and formyl;

Or R ³、R⁴、R⁶、R⁷、R⁹、R¹⁰ is hydrogen atom, R ⁵ and R ⁸ are simultaneously selected from cyano or formamide;

Or R ³、R⁴、R⁵、R⁶、R⁸、R¹⁰ is hydrogen atom, R ⁷ and R ⁹ are simultaneously selected from cyano or formamide.

As a further improvement of the technical scheme, L is a substituted or unsubstituted main chain containing 1-4 atoms, the main chain is C _1-4 alkylene, or the main chain contains carbonyl or amido, the rest is alkylene, and the substituent of L is methyl or hydroxyl.

As a further improvement of the technical scheme, Q is selected from 5-7 membered aliphatic heterocyclic ring selected from piperidine or piperazine or aromatic ring, and the aromatic ring is benzene ring.

As a further improvement of the technical scheme, Z is selected from hydroxyethyl, N-ethyl, aminoethyl or aminoacetyl.

As a further improvement of the technical scheme, the tricyclic compound is any one of the following compounds:

another aspect of the technical scheme of the invention is to provide a preparation method of the compound with the general structural formulas I and I, wherein the synthetic route is as follows:

Wherein the reaction conditions are (a) sodium hydride, anhydrous N, N-dimethylformamide, room temperature, 2-3 hours, and (b) potassium carbonate, sodium iodide, N, N-dimethylformamide or acetonitrile. When A, B rings are benzene rings, the structure of the general formula I is synthesized. R ₁、R₂ and n, A, B, X, Y, L, Q, Z are as defined above.

Because the tricyclic compound has the application of activating the STING signal pathway and has the application prospect of treating diseases related to the STING protein function, the invention also provides a pharmaceutical composition based on the application of the compound for activating the STING signal pathway, and the pharmaceutical composition comprises a therapeutically effective amount of one or a mixture of the tricyclic compound, a pharmaceutically acceptable crystal form, a hydrate, a solvate, a prodrug and a metabolite thereof, and also comprises a pharmaceutical auxiliary material and/or a pharmaceutically acceptable carrier.

The pharmaceutical excipients refer to excipients and additives used in the production of medicines and formulation prescriptions, including solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, inclusion agents, moisturizers, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders, etc., preferably oral pharmaceutical excipients such as disintegrants, co-solvents, lubricants, etc.

Pharmaceutically acceptable carriers refer to systems, including microcapsules and microspheres, nanoparticles, liposomes, and the like, that alter the manner and distribution of the drug into the body, control the release rate of the drug, and deliver the drug to targeted organs. When the pharmaceutical composition is used in solid tumor diseases, it is preferable that the composition is in the form of an active ingredient+carrier.

The pharmaceutical composition has the application in medicines for treating diseases related to the function of STING proteins, wherein the diseases related to the function of STING proteins are one or more of inflammation, autoimmune diseases, infectious diseases and tumor-related diseases. The inflammatory disease is selected from acne vulgaris, rheumatoid arthritis, pelvic inflammatory disease, asthma, celiac disease, inflammatory bowel disease, reperfusion injury, chronic prostatitis, glomerulonephritis, sarcoidosis, vasculitis, airway inflammation caused by house dust mites, and interstitial cystitis. The infectious disease is selected from bacterial and viral infections, in particular Hepatitis B Virus (HBV), hepatitis C Virus (HCV), nasopharyngeal carcinoma-associated EBV (EBV), human Papilloma Virus (HPV) and Human Immunodeficiency Virus (HIV) infections.

The tumor-associated disease is selected from cancers such as colon cancer, gastric cancer, breast cancer, fibrosarcoma and squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, brain cancer and spinal cancer, liver cancer, cervical cancer, head and neck cancer, leukemia and blood cancer, skin cancer, cancer of the reproductive system, lung cancer, malignant mesothelioma, sarcoma, lymphoma, adenocarcinoma, thyroid cancer, heart tumor, germ cell tumor, gastrointestinal cancer, liver and bile duct cancer, kidney cancer and bladder cancer, bone cancer, etc.

Compared with the prior art, the tricyclic compound has outstanding substantive characteristics and remarkable progress, and particularly has the effect of exciting interferon gene Stimulation (STING) proteins, and the pharmaceutical composition containing the tricyclic compound can be used for treating cancers, inflammations, autoimmune diseases, infectious diseases and the like.

Drawings

FIG. 1 is a graph showing the ITC titration of Compound 1 with STING protein.

FIG. 2 is a STD map of the effect of Compound 1 on STING proteins.

FIG. 3 is a graph showing the results of qPCR experiments examining the effect of Compound 1 on mRNA downstream of STING.

FIG. 4 is a graph showing the result of examining the effect of Compound 1 on protein downstream of STING by Western-Blot.

Detailed Description

The experimental methods in the examples of the present invention, for which specific conditions are not noted, were selected according to conventional methods and conditions, or according to the commodity specifications.

The structure of the compound is determined by Nuclear Magnetic Resonance (NMR) or/and High Resolution Mass Spectrometry (HRMS), ¹ H NMR spectrum is determined by Bruker AV-300 NMR, the solvent is deuterated dimethyl sulfoxide (DMSO-d ₆), deuterated chloroform (CDCl ₃), and the internal standard is Tetramethylsilane (TMS). High resolution mass spectrometry was determined using a Water Q-Tof micro mass spectrometer from Volter, america.

The chemical reaction was detected using a 0.25mm GF254 thin layer chromatography silica gel plate and observed by a ZF7 three-purpose uv analyzer.

In the examples, the reaction was carried out under an air atmosphere unless otherwise specified.

In the examples, the solution refers to an aqueous solution unless otherwise specified.

In the embodiment, if no special description exists, the reaction temperature is 20-30 ℃ at room temperature.

The purity of the compounds in the examples was measured by HPLC using an Agilent C18 (4.6mm.times.150mm, 3.5 μm) reverse phase column under conditions of a methanol/water (80 to 20) mixed solvent as a mobile phase at a flow rate of 1mL/min by UV absorption at 254nm and 365 nm.

EXAMPLE 1 preparation of Compound 2

Step 1 Synthesis of Compound 1-1

Hydroxyethyl piperazine (2 g,15.4 mmol), 1-bromo-3-chloropropane (3.63 g,23.1 mmol) was dissolved in acetonitrile, et ₃ N (3.12 g,30.8 mmol) was added and stirred overnight at room temperature. After the reaction was completed, it was distilled off by spin-drying, followed by purification by column chromatography to give 1-1 (2.56 g, yield) as a pale yellow or white oil 80.62％).¹H NMR(300MHz,DMSO-d₆)δ4.21(t,J＝8.3Hz,1H),3.68(t,J＝6.5Hz,2H),3.58(t,J＝6.0Hz,2H),2.63(t,J＝6.0Hz,2H),2.54–2.41(m,2H),1.93(s,8H),1.89(d,J＝6.8Hz,2H).

Step 2 Synthesis of Compound 2

NaH (60%, 4476 mg,11.15 mmol) was placed in a three-necked flask, filled with N ₂ under ice bath, anhydrous DMF was added to mix the system, phenothiazine (1 g,4.45 mmol) was added, and after stirring for 1h, the prepared compound 1-1 was added and reacted at room temperature for 3-4h. After confirming the completion of the reaction, water was slowly added in an ice bath to quench. Extracting with water, ethyl acetate, washing with saturated NaCl, drying with anhydrous sodium sulfate, concentrating by rotary evaporation, making sand, and purifying by column chromatography to obtain white solid 2 (300 mg, yield) 15％).¹H NMR(300MHz,Chloroform-d)δ7.23–7.12(m,4H),7.02–6.83(m,4H),3.97(t,J＝6.8Hz,2H),3.66(t,J＝5.4Hz,2H),2.57(dt,J＝18.1,6.4Hz,12H),2.09–1.92(m,2H).HRMS(ESI):calcd for C₂₁H₂₈N₃OS⁺[M+H]⁺370.1947,found 370.1942.Purity:99.94％by HPLC(t_R＝2.86min,MeOH/H₂O＝80:20).

EXAMPLE 2 preparation of Compound 3

The synthesis was as for compound 2 in example 1, except that 2-carboxyphenothiazine was used instead of phenothiazine.

The synthesis gave 53mg (yield) 21％).¹H NMR(300MHz,Chloroform-d)δ7.53–7.45(m,2H),7.25–7.12(m,3H),7.00–6.90(m,2H),4.02(t,J＝6.8Hz,2H),3.71(t,2H),2.81–2.64(m,6H),2.59(d,J＝11.8Hz,9H),2.02(m,2H).HRMS(ESI):calcd for C₂₃H₃₀N₃O₂S⁺[M+H]⁺412.2053,found 412.2046.Purity:95.08％by HPLC(t_R＝4.10min,MeOH/H₂O＝80:20).

EXAMPLE 3 preparation of Compound 4

The synthesis was as for compound 2 in example 1, except that 2-methoxyphenothiazine was used instead of phenothiazine.

Synthesis of 34mg (yield) 16％).¹H NMR(300MHz,Chloroform-d)δ7.22–7.14(m,2H),7.09–7.03(m,1H),6.99–6.88(m,2H),6.56–6.49(m,2H),3.94(t,J＝6.7Hz,2H),3.82(s,3H),3.76–3.70(m,2H),2.80–2.66(m,6H),2.60(m,J＝13.0,5.9Hz,6H),2.03(m,J＝2.3Hz,2H).HRMS(ESI):calcd for C₂₂H₂₉N₃O₂S⁺[M+H]⁺400.2053,found 400.2050.Purity:99.55％by HPLC(t_R＝3.29min,MeOH/H₂O＝80:20).

EXAMPLE 4 preparation of Compound 5

Step 1 Synthesis of Compound 5-1

NaH (60%, 7g,27 mmol) was placed in a three-necked flask, filled with N ₂ under ice bath, anhydrous DMF was added to mix the system, 2-cyanophenothiazine (5 g,22 mmol) was added, and after stirring for 1h, 1-bromo-3-chloropropane was added and reacted at room temperature for 3-4h. After confirming the completion of the reaction, water was slowly added in an ice bath to quench. Extraction with water, ethyl acetate, washing with saturated NaCl, drying over anhydrous sodium sulfate, concentration by rotary evaporation, sand making, column chromatography purification gave a colorless oil (4.3 g, 65% yield).

Step 2 Synthesis of Compound 5

Compound 5-1 (1 g,3.32 mmol) was dissolved in DMF and K ₂CO₃ (0.918 g,6.65 mmol), hydroxyethylpiperazine (0.399 g,3.99 mmol) and sodium iodide (0.498 g,3.32 mmol) were added and reacted for 8h at 90 ℃. After the completion of the reaction, water, ethyl acetate extraction, saturated NaCl washing, anhydrous sodium sulfate drying, rotary evaporation concentration, sand preparation, column chromatography purification, obtaining a pale yellow solid 350mg (yield) 26.68％).¹H NMR(300MHz,Chloroform-d)δ7.26–7.17(m,3H),7.16–7.07(m,2H),7.05–6.89(m,2H),3.95(t,J＝6.7Hz,2H),3.79–3.73(t,2H),2.84–2.71(m,6H),2.68–2.51(m,6H),2.01–1.92(m,2H).HRMS(ESI):calcd for C₂₀H₂₈N₄OS⁺[M+H]⁺395.1876,found 395.1895.Purity:98.77％by HPLC(t_R＝6.09min,MeOH/H₂O＝80:20).

EXAMPLE 5 preparation of Compound 6

The synthesis was as for compound 5 in example 4, except that 2-methylthiophenothiazine was used instead of 2-cyanophenothiazine.

Synthesis yield 62mg (yield) 53.24％).¹H NMR(300MHz,Chloroform-d)δ7.15(t,J＝7.6Hz,2H),7.05(d,J＝7.8Hz,1H),6.96–6.85(m,2H),6.85–6.78(m,2H),3.92(t,J＝6.7Hz,2H),3.67(t,J＝5.2Hz,2H),2.76–2.58(m,6H),2.58–2.49(m,6H),2.47(s,3H),1.99–1.92(m,2H).HRMS(ESI):calcd for C₂₂H₃₀N₃OS₂ ⁺[M+H]⁺416.1824,found 416.1823.Purity:95.67％by HPLC(t_R＝3.29min,MeOH/H₂O＝80:20).

EXAMPLE 6 preparation of Compound 7

Step 1, synthesis of Compound 5-1 (see preparation step 1 of Compound 5).

Step 2 Synthesis of Compound 7-1

Compound 5-1 (1.0 g,3.33 mmol) was dissolved in EtOH/DMSO, aqueous sodium hydroxide solution (1.33 g) was added under ice-bath conditions, and after stirring for 5 minutes, 30% hydrogen peroxide (4 ml) was added and the reaction was resumed at room temperature for 3h. After completion of the reaction by TLC, the reaction system was poured into ice water, and the solid was separated out and dried to give 0.56g of a solid (yield 52.84％).¹H NMR(300MHz,DMSO-d₆)δ8.04(s,1H),7.51(d,J＝7.8Hz,2H),7.43(s,1H),7.31–7.17(m,3H),7.12(dd,J＝8.3,1.2Hz,1H),7.00(td,J＝7.4,1.2Hz,1H),4.12(t,J＝6.6Hz,2H),3.75(t,J＝6.3Hz,2H),2.15(p,J＝6.4Hz,2H).

Step 3 Synthesis of Compound 7

The synthesis was as for compound 5 in example 4, except that 2-carboxamido phenothiazine was used instead of 2-cyanophenothiazine.

The synthesis gave 43mg (yield) 23.6％).¹H NMR(300MHz,Chloroform-d)δ7.44(s,1H),7.25–7.06(m,4H),6.94(dd,J＝11.1,7.7Hz,2H),4.00(t,J＝6.9Hz,2H),3.61(t,J＝5.4Hz,2H),2.54(dd,J＝11.9,6.7Hz,12H),2.04–1.89(m,2H).HRMS(ESI):calcd for C₂₂H₂₉N₄O₂S⁺[M+H]⁺413.2005,found 413.1999.Purity:95.09％by HPLC(t_R＝2.34min,MeOH/H₂O＝80:20).

EXAMPLE 7 preparation of Compound 8

Step 1, step 2, synthesis of the same compounds 5-1 and 7-1.

Step 3 Synthesis of Compound 8-1

Compound 7-1 (600 mg,1.89 mmol) was dissolved in DNF-DMA solution, warmed to 100℃and stirred for 1h. Cooled to room temperature and concentrated in vacuo. Ethanol, hydrazine acetate and 50 ℃ are added in sequence and reacted for 30 minutes. Concentrated in vacuo, diluted with water, filtered, the filter residue washed with ethanol and dried to give 213mg of an off-white solid (yield 33.01%) which is ready for further use.

Step 4 Synthesis of Compound 8

The synthesis was as for compound 5 in example 4, except that 2-cyanophenothiazine was replaced with 2-triazolothiazine.

Synthesis yield 26mg (yield 10.21％).¹H NMR(300MHz,Chloroform-d)δ8.19(s,1H),7.61(d,J＝8.9Hz,2H),7.24–7.13(m,3H),7.01–6.88(m,2H),4.00(t,J＝6.9Hz,2H),3.67(t,J＝6.1,5.5Hz,2H),2.62–2.46(m,12H),2.06(p,J＝8.1,7.2Hz,2H).HRMS(ESI):calcd for C₂₃H₂₉N₆OS⁺[M+H]⁺437.2118,found437.2110.Purity:96.24％by HPLC(t_R＝3.56min,MeOH/H₂O＝80:20).

EXAMPLE 8 preparation of Compound 9

Step 1 Synthesis of Compound 9-1

2-Cl phenothiazine (1 g,4.27 mmol) was placed in a round bottom flask under ice bath, 20ml of glacial acetic acid was added, 30% hydrogen peroxide (0.75 ml,6.5 mmol) was added, and the reaction was allowed to return to room temperature overnight with stirring. After completion of TLC monitoring, vacuum concentration, EA, water extraction, drying of the organic phase over anhydrous sodium sulfate, concentration gives an off-white solid, 0.96g (yield 89.85％).¹H NMR(300MHz,DMSO-d₆)δ11.09(s,1H),8.02-7.87(m,2H),7.64(s,1H),7.43-7.34(m,2H),7.30-7.18(m,2H).

Step 2 Synthesis of Compound 9-2

The synthesis was as for compound 5-1 in example 4, except that 2-chlorophenothiazine-5-oxide was used instead of 2-cyanophenothiazine.

Synthesized to yield 0.75g of oil (yield 71.76％).¹H NMR(300MHz,Chloroform-d)δ7.97(dd,J＝7.7,1.7Hz,1H),7.90(d,J＝8.3Hz,1H),7.69(ddd,J＝8.7,7.1,1.6Hz,1H),7.58–7.50(m,2H),7.35(d,J＝7.1Hz,1H),7.27(dd,J＝8.3,1.8Hz,1H),4.52(t,J＝8.3,6.4Hz,2H),3.75(t,J＝5.8Hz,2H),2.42(p,J＝6.0,2.1Hz,2H).

Step 3 Synthesis of Compound 9

The synthesis was as for compound 5 in example 4, except that 2-chlorophenothiazine-5-oxide was used instead of 2-cyanophenothiazine.

Synthesis gave 54mg of a white solid (yield 13.98％).¹H NMR(300MHz,Chloroform-d)δ8.09(d,J＝7.7Hz,1H),8.02(d,J＝8.3Hz,1H),7.83–7.74(m,1H),7.73–7.64(m,2H),7.47–7.42(m,1H),7.39–7.31(m,1H),4.50(t,J＝7.6Hz,2H),3.78(t,J＝4.6,3.9Hz,2H),2.83–2.56(m,12H),2.22(p,J＝7.3Hz,2H).HRMS(ESI):calcd for C₂₁H₂₇ClN₃O₂S⁺[M+H]⁺420.1507,found420.1504.Purity:96.35％by HPLC(t_R＝4.68min,MeOH/H₂O＝80:20).

EXAMPLE 9 preparation of Compound 10

Step 1 Synthesis of Compound 10-2

Compound 10-1 (1 g,5.46 mmol), naH (262 mg,6.55 mol) was placed in a three-necked flask, put three times under nitrogen, anhydrous DMF was added, and after half an hour of stirring, 1-bromo-3-chloropropane was added and reacted at room temperature for 3-4h. After confirming the completion of the reaction, water was slowly added in an ice bath to quench. Extraction with water, ethyl acetate, washing with saturated NaCl, drying over anhydrous sodium sulfate, and concentration by rotary evaporation gives a crude colorless oil which is carried out in the next step without purification.

Step 2 Synthesis of Compound 10

The synthesis was as for compound 5 in example 4, except that the 2-cyanophenothiazine was replaced with phenoxazine.

Synthesis gave 79mg (yield) 36.78％).¹H NMR(300MHz,Chloroform-d)δ6.87–6.78(m,2H),6.70–6.65(m,4H),6.62(dd,2H),3.70(t,J＝5.4Hz,2H),3.63(t,2H),3.10(s,1H),2.72–2.58(m,8H),2.50(t,J＝6.8Hz,4H),1.88(p,2H).HRMS(ESI):calcd for C₂₁H₂₇N₃O₂ ⁺[M+H]⁺354.2176,found 354.2169.Purity:97.35％by HPLC(t_R＝2.68min,MeOH/H₂O＝80:20).

EXAMPLE 10 preparation of Compound 11

Step 1 Synthesis of Compound 11-2

The synthesis was as for compound 10-2 in example 9, except that 9, 10-dihydroacridine was used instead of phenoxazine.

Step 2 Synthesis of Compound 11

The synthesis was as for compound 5 in example 4, except that 9, 10-dihydroacridine was used instead of 2-cyanophenothiazine.

Synthesis gave 54mg (yield) 46.23％).¹H NMR(300MHz,Chloroform-d)δ7.23–7.10(m,4H),6.94(dd,J＝12.1,7.7Hz,4H),3.97(s,2H),3.95(t,2H),3.66(t,J＝5.4Hz,2H),2.73–2.56(m,8H),2.51(t,J＝7.1Hz,4H),2.01(q,J＝7.4Hz,2H).HRMS(ESI):calcd for C₂₂H₃₀N₃O⁺[M+H]⁺352.2383,found352.2383.Purity:98.65％by HPLC(t_R＝2.96min,MeOH/H₂O＝80:20).

EXAMPLE 11 preparation of Compound 12

Step 1 Synthesis of Compound 12-2

The synthesis was as for compound 10-2 in example 9, except that 2-chloro-9H-carbazole was used instead of phenoxazine.

Step 2 Synthesis of Compound 12

The synthesis was as for compound 5 in example 4, except that 2-chloro-9H-carbazole was used instead of 2-cyanophenothiazine.

Synthesis yield 46mg (yield) 33.35％).¹H NMR(300MHz,Chloroform-d)δ8.09(dt,J＝7.8,1.1Hz,1H),8.01(d,J＝8.3Hz,1H),7.59(d,J＝1.8Hz,1H),7.52–7.46(m,2H),7.29–7.25(m,1H),7.21(dd,J＝8.3,1.8Hz,1H),4.41(t,J＝6.3Hz,2H),3.70(t,J＝5.3Hz,2H),3.02(s,1H),2.67(t,J＝5.4Hz,6H),2.50(s,4H),2.31(d,J＝6.6Hz,2H),2.06(p,2H).HRMS(ESI):calcd for C₂₁H₂₇ClN₃O⁺[M+H]⁺372.1837,found 372.1833.Purity:98.85％by HPLC(t_R＝3.66min,MeOH/H₂O＝80:20).

EXAMPLE 12 preparation of Compound 13

Step 1 Synthesis of Compound 13-2

The synthesis was as for compound 10-2 in example 9, except that carbazole was used in place of phenoxazine.

Step 2 Synthesis of Compound 14

The synthesis was as for compound 5 in example 4, except that carbazole was used in place of 2-cyanophenothiazine.

The synthesis gave 95mg (yield) 43.69％).¹H NMR(300MHz,Chloroform-d)δ8.16(d,J＝7.8Hz,2H),7.58–7.47(m,4H),7.34–7.25(m,2H),4.47(t,J＝6.6Hz,2H),3.69(t,J＝5.4Hz,2H),2.64(t,J＝5.4Hz,10H),2.38(t,J＝6.8Hz,2H),2.11(p,J＝6.7Hz,2H).HRMS(ESI):calcd for C₂₁H₂₈N₃O⁺[M+H]⁺338.2226,found 338.2223.Purity:95.34％by HPLC(t_R＝4.68min,MeOH/H₂O＝80:20).

EXAMPLE 13 preparation of Compound 14

Step 1 Synthesis of Compound 14-2

The synthesis was as for compound 10-2 in example 9, except that 2-bromo-9H-carbazole was used instead of phenoxazine.

Step 2 Synthesis of Compound 14

The synthesis was as for compound 5 in example 4, except that 2-bromo-9H-carbazole was used in place of 2-cyanophenothiazine.

The synthesis gave 57mg (yield) 26.78％).¹H NMR(300MHz,Chloroform-d)δ8.07(d,J＝7.8Hz,1H),7.94(d,J＝8.2Hz,1H),7.74(d,J＝1.8Hz,1H),7.49(d,J＝6.3Hz,2H),7.34(dd,J＝8.3,1.7Hz,1H),7.29–7.21(m,1H),4.39(t,J＝6.3Hz,2H),3.67(t,J＝5.4Hz,2H),2.64(q,J＝5.3Hz,6H),2.46(s,4H),2.28(t,J＝6.5Hz,2H),2.10–1.97(m,2H).HRMS(ESI):calcd for C₂₁H₂₇BrN₃O⁺[M+H]⁺416.1332,found416.1326.Purity:99.06％by HPLC(t_R＝2.98min,MeOH/H₂O＝80:20).

EXAMPLE 14 preparation of Compound 15

Step 1 Synthesis of Compound 15-3

15-1 (5 G,27 mmol), 15-2 (6.01 g,27 mmol) were dissolved in a suitable amount of DMSO solution, anhydrous K ₂CO₃ (11.2 g,81 mmol) was added, warmed to 100℃and reacted for 8h. TLC plate monitoring reaction completion, dilution with water, EA extraction, drying of the organic phase with anhydrous sodium sulfate, vacuum concentration, column chromatography separation and purification gave 8.36g of compound 15-3 (yield) 83.37％).¹H NMR(300MHz,DMSO-d₆)δ8.73(d,J＝2.1Hz,1H),8.09(dd,J＝8.8,2.1Hz,1H),8.02(dd,J＝7.9,1.6Hz,1H),7.56(td,1H),7.37(dd,J＝8.1,1.5Hz,1H),7.18(td,J＝7.6,1.5Hz,1H),6.95(d,J＝8.8Hz,1H).

Step 2 Synthesis of Compound 15-4

Compound 15-3 (3 g,8.20 mmol) was placed in a eggplant-shaped reaction flask, 20ml of water/ethanol (1:3) was added, NH ₄ Cl (219.309 mg,4.1 mmol) was added, stirring was turned on, reduced iron powder (1.813 g,32.8 mmol) was added, and the temperature was raised to 80℃for 1h of reaction. TLC monitoring reaction completion, filtering while it is still hot, vacuum concentrating the filtrate, extracting with water and ethyl acetate, drying the extracted organic phase with anhydrous sodium sulfate, concentrating, and separating and purifying by column chromatography to obtain 2.13 Compound 16-4 (yield) 77.33％).¹H NMR(300MHz,DMSO-d₆)δ8.00–7.87(m,1H),7.50–7.35(m,1H),7.13(d,J＝2.1Hz,1H),7.04(t,J＝6.9Hz,2H),6.91(dd,J＝8.3,2.1Hz,1H),6.52(d,J＝8.3Hz,1H),5.59(s,2H).

Step 3 Synthesis of Compound 15-5

Compound 15-4 (1 g,3.0 mmol) was dissolved in toluene and Pd ₂(dba)₃ (140 mg,0.15 mmol), t-Buona (404 mg,4.2 mmol) and DPPF (166.32 mg,0.3 mmol) were added. The reaction was carried out for 20h at 130℃under nitrogen. TLC monitored completion of the reaction, cooled to room temperature, filtered through celite, and the residue washed with DCM and EA, and the filtrate was collected and concentrated in vacuo. Diluting with water, extracting with ethyl acetate, washing with saturated NaCl, vacuum concentrating, separating and purifying by column chromatography to obtain 0.4g (yield) 64.57％).¹H NMR(300MHz,DMSO-d₆)δ8.55(s,1H),7.02(dd,J＝8.2,1.8Hz,1H),6.77(d,J＝1.9Hz,1H),6.72(d,J＝8.3Hz,1H),6.68(d,J＝2.0Hz,1H),6.58–6.65(m,2H),6.50(dd,J＝8.3,1.6Hz,1H).

Step 4 Synthesis of Compound 15-6

The synthesis was as for compound 10-2 in example 9, except that 2-cyano-phenoxazine was used instead of phenoxazine.

Step 2 Synthesis of Compound 15

The synthesis was as for compound 5 in example 4, except that 2-cyano-phenoxazine was used instead of 2-cyano phenothiazine.

Synthesis of pale yellow solid 46mg (yield 13.98％).¹H NMR(300MHz,Chloroform-d)δ7.06–6.96(m,3H),6.80(d,J＝3.1Hz,1H),6.72(d,J＝3.7Hz,3H),3.71(q,J＝4.2,2.9Hz,4H),2.86–2.58(m,9H),2.53(d,J＝6.3Hz,3H),1.91(t,J＝6.7Hz,2H).HRMS(ESI):calcd for C₂₂H₂₇N₄O₂ ⁺[M+H]⁺379.2128,found 379.2128.Purity:93.86％by HPLC(t_R＝6.38min,MeOH/H₂O＝80:20).

EXAMPLE 15 preparation of Compound 16

Step 1 Synthesis of Compound 16-3

Compound 16-1 (2 g,18.32 mmol), 16-2 (2.54 g,18.32 mmol) was dissolved in DMF and anhydrous K ₂CO₃ (7.6 g,54.96 mmol) was added and reacted at 130℃for 3h. After completion of TLC monitoring, water was added for dilution, extraction with ethyl acetate, washing with saturated NaCl, vacuum concentration, column chromatography separation and purification, 1.6g (yield) 41.93％).¹H NMR(300MHz,DMSO-d₆)δ8.94(s,1H),7.18(dd,J＝8.2,1.8Hz,1H),6.99(d,J＝1.9Hz,1H),6.78(ddd,J＝7.7,6.2,2.8Hz,1H),6.70–6.61(m,2H),6.50(dd,J＝8.3,1.6Hz,2H).

Step 2 Synthesis of Compound 16-4

The synthesis was as for compound 10-2 in example 9, except that 3-cyano-phenoxazine was used instead of phenoxazine.

Step 3 preparation of Compound 16

The synthesis was as for compound 5 in example 4, except that 3-cyano-phenoxazine was used instead of 2-cyano phenothiazine.

Synthesis yielded 32mg (yield) of pale yellow solid 36.54％).¹H NMR(300MHz,Chloroform-d)δ7.11(dd,J＝8.4,2.0Hz,1H),6.90–6.73(m,3H),6.72–6.58(m,3H),3.70(t,J＝5.4Hz,2H),3.65(t,2H),2.82(s,1H),2.69–2.62(m,5H),2.58(s,4H),2.48(t,J＝6.5Hz,3H),1.85(p,J＝6.8Hz,2H).HRMS(ESI):calcd for C₂₂H₂₇N₄O₂ ⁺[M+H]⁺379.2128,found 379.2126.Purity:93.86％by HPLC(t_R＝5.35min,MeOH/H₂O＝80:20).

EXAMPLE 16 preparation of Compound 17

Step 1 Synthesis of Compound 17-2

The synthesis was as for the synthesis of compound 7-1 in example 6, except that 3-cyano-phenoxazine was used instead of 2-cyano-phenothiazine.

Synthesis gave a yellow solid (300 mg, yield 63.96％).¹H NMR(300MHz,DMSO-d₆)δ7.74(s,1H),7.42(d,J＝8.5Hz,1H),7.17(d,J＝2.0Hz,2H),6.89(t,J＝6.9Hz,1H),6.84–6.76(m,2H),6.75–6.66(m,2H),3.84(t,J＝6.3Hz,2H),3.75(t,J＝7.9Hz,2H),2.03(p,J＝7.4Hz,2H).

Step 2 Synthesis of Compound 17

The synthesis was as for compound 5 in example 4, except that 3-cyano-phenoxazine was used instead of 2-cyano phenothiazine.

Synthesis yield 23mg (yield) 16.35％).¹H NMR(300MHz,DMSO-d₆)δ7.75(s,1H),7.41(dd,J＝8.4,2.1Hz,1H),7.16(d,J＝2.1Hz,2H),6.91–6.76(m,3H),4.67(s,1H),3.65(t,J＝7.4Hz,2H),3.59(s,2H),2.62(s,7H),2.51(d,J＝2.3Hz,5H),1.73(p,J＝7.2Hz,2H).HRMS(ESI):calcd for C₂₂H₂₉N₄O₃ ⁺[M+H]⁺397.2234,found 397.2230.Purity:97.86％by HPLC(t_R＝4.56min,MeOH/H₂O＝80:20).

EXAMPLE 17 preparation of Compound 18

Step 1 Synthesis of Compound 18-3

Compound 18-1 (500 mg,3.73 mmol), 18-2 (520 mg,3.73 mmol) and KOH (420 mg,7.46 mmol) were placed in a three-necked flask, and DMSO was added under nitrogen with stirring and heated to 100℃for 3h. After completion of TLC monitoring, water was added for dilution, and the solid was precipitated, filtered and dried to give 560mg (yield 64.41％).1H NMR(300MHz,DMSO-d₆)δ9.25(s,1H),7.23(dd,J＝8.2,1.8Hz,1H),7.13(dd,J＝8.3,2.0Hz,1H),7.08(d,J＝1.8Hz,1H),6.84–6.73(m,2H),6.54(d,J＝8.1Hz,1H).

Step 2 Synthesis of Compound 18-4

The synthesis was as for the synthesis of compound 7-1 in example 6, except that 3, 7-dicyano-phenoxazine was used instead of 2-cyanophenothiazine.

Step 3 preparation of compound 18,

The synthesis was as for compound 5 in example 4, except that 3, 7-dicyano-phenoxazine was used instead of 2-cyanophenothiazine.

The synthesis gave 216mg (yield) 41.46％).¹H NMR(300MHz,Deuterium Oxide)δ7.12(dd,J＝8.4,1.9Hz,1H),7.02(dd,J＝8.2,1.7Hz,1H),6.95(d,J＝1.8Hz,1H),6.81(d,J＝1.9Hz,1H),6.64(d,J＝8.3Hz,2H),3.66(t,J＝5.3Hz,2H),3.60(t,2H),2.72–2.60(m,7H),2.54(s,3H),2.43(t,J＝6.2Hz,2H),1.78(p,J＝6.7Hz,2H).HRMS(ESI):calcd for C₂₃H₂₆N₅O₂ ⁺[M+H]⁺404.2081,found 404.2076.Purity:96.38％by HPLC(t_R＝2.35min,MeOH/H₂O＝80:20).

EXAMPLE 18 preparation of Compound 19

Step1 Synthesis of Compound 19

The synthesis was as for the synthesis of compound 7-1 in example 6, except that 3, 7-dicyano-phenoxazine was used instead of 2-cyanophenothiazine.

Synthesis yield 36mg (yield) 33.05％).¹H NMR(300MHz,DMSO-d₆)δ7.87(s,1H),7.75(s,1H),7.41(dd,J＝8.4,2.1Hz,1H),7.34–7.22(m,2H),7.17(t,J＝2.4Hz,3H),6.83(d,J＝8.5Hz,1H),6.71(d,J＝8.2Hz,1H),4.37(t,J＝5.1Hz,1H),3.69(t,J＝7.3Hz,2H),3.50(t,J＝5.9Hz,2H),2.51–2.21(m,12H),1.72(t,J＝7.1Hz,2H).HRMS(ESI):calcd for C₂₃H₃₀N₅O₄ ⁺[M+H]⁺440.2292,found 440.2285.Purity:93.36％by HPLC(t_R＝4.38min,MeOH/H₂O＝80:20).

EXAMPLE 19 preparation of Compound 20

The synthesis was as for compound 5 in example 4, except that N-ethylpiperazine was used instead of hydroxyethylpiperazine.

Synthesis yield 268mg of a white solid (yield 76.36％).¹H NMR(300MHz,Chloroform-d)δ7.16(ddd,J＝15.2,7.5,1.5Hz,2H),7.03(d,J＝8.0Hz,1H),6.99–6.85(m,4H),3.91(t,J＝6.8Hz,2H),2.83–2.48(m,8H),2.48–2.23(m,4H),1.96(p,J＝7.0Hz,2H),1.11(t,J＝7.2Hz,3H).HRMS(ESI):calcd for C₂₁H₂₇ClN₃S⁺[M+H]⁺388.1608,found 388.1600.Purity:96.46％by HPLC(t_R＝3.19min,MeOH/H₂O＝80:20).

EXAMPLE 20 preparation of Compound 21

Step 1 Synthesis of Compound 21-2

21-1 (2.0 G,8.56 mmol) was dissolved in toluene and chloroacetyl chloride (1.5 g,12.84 mmol) was slowly added dropwise under ice bath, warmed to 80℃and reacted for 6-8h. Diluting with water, extracting with EA, washing with saturated NaCl, concentrating the organic phase, and separating and purifying by column chromatography to obtain white solid 1.89g (yield) 71.20％).¹H NMR(300MHz,Chloroform-d)δ7.61(dd,J＝6.9,1.4Hz,1H),7.55(d,J＝2.2Hz,1H),7.43(dd,J＝7.1,1.3Hz,1H),7.40–7.34(m,2H),7.28–7.24(m,1H),7.20(dd,J＝8.1,2.3Hz,1H),4.21(s,2H).

Step 2 Synthesis of Compound 21-3

To 21-2 (800 mg,2.58 mmol) was added 3ml BH ₃ -THF under ice-bath, BF ₃-Et₂ O (3.66 g,25.8 mmol) was added and the reaction was stirred overnight. After the completion of the TLC monitoring reaction, water was slowly added dropwise under ice bath to quench, EA was extracted, and the organic phase was washed with saturated NaCl, and then concentrated, followed by separation and purification by column chromatography to obtain 0.43g (yield 56.29%) of a white solid.

Step 3 Synthesis of Compound 21

The synthesis was as for compound 5 in example 4, except that 2-chloro-10- (2-chloroethyl) -10H-phenothiazine was used instead of 2-chloro-10- (3-chloropropyl) -10H-phenothiazine.

The synthesis gave 95mg (yield) 36.08％).¹H NMR(300MHz,Chloroform-d)δ7.26–7.14(m,2H),7.07(d,J＝8.1Hz,1H),7.02–6.89(m,4H),4.03(t,2H),3.87(t,J＝5.1Hz,2H),3.15–2.99(m,6H),2.91–2.78(m,6H).HRMS(ESI):calcd for C₂₀H₂₅ClN₃OS⁺[M+H]⁺390.1401,found390.1397.Purity:95.66％by HPLC(t_R＝4.82min,MeOH/H₂O＝80:20).

EXAMPLE 21 preparation of Compound 22

Step 1 Synthesis of Compound 22-2, synthesis of Compound 21-2

Step 2 Synthesis of Compound 22

The synthesis was as for compound 5 in example 4, except that 2-chloro-10- (3-chloropropyl) -10H-phenothiazine was replaced with 2-chloro-10-chloroacetyl-10H-phenothiazine.

22-1 (500 Mg,1.62 mmol) was dissolved in acetonitrile, and anhydrous K ₂CO₃ (4478 mg,3.24 mmol) was added to synthesize a white solid 365mg (yield) 56.06％).¹H NMR(300MHz,Chloroform-d)δ7.70(d,J＝2.2Hz,1H),7.57(dd,J＝7.9,1.4Hz,1H),7.48(dd,J＝7.6,1.6Hz,1H),7.37(td,J＝8.2,2.6Hz,2H),7.29–7.22(m,2H),3.62(t,J＝5.4Hz,2H),3.33(d,J＝4.8Hz,2H),2.64–2.39(m,10H).HRMS(ESI):calcd for C₂₀H₂₃ClN₃O₂S⁺[M+H]⁺404.1194,found404.1187.Purity:96.29％by HPLC(t_R＝3.33min,MeOH/H₂O＝80:20).

EXAMPLE 22 preparation of Compound 23

23-1 (2.0 G,8.56 mmol) and triphosgene (1.52 g,5.136 mmol) were placed in a three-necked flask, nitrogen blanket, 1, 2-dichloroethane was added, 1ml pyridine was added, and the temperature was raised to 75℃for 3h of reaction. After cooling to room temperature and completion of the reaction by TLC, hydroxyethylpiperazine (1.12 g,8.56 mmol) was added, and the temperature was raised to 50℃and the reaction was continued for 2-3 hours. After completion of the TLC monitoring reaction, water was slowly added dropwise under ice bath to quench, dichloromethane was extracted, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 56mg of a white solid (two-step yield 1.5％).¹H NMR(300MHz,Chloroform-d)δ7.89(d,J＝2.1Hz,1H),7.47(dd,J＝8.1,1.3Hz,1H),7.35–7.20(m,3H),7.14(m,J＝7.9,1.7Hz,2H),3.65(t,J＝5.3Hz,2H),3.48(t,J＝5.0Hz,4H),2.58(t,J＝5.3Hz,2H),2.49(t,J＝5.0Hz,4H).HRMS(ESI):calcd for C₁₉H₂₁ClN₃O₂S⁺[M+H]⁺390.1037,found390.1038.Purity:96.44％by HPLC(t_R＝3.21min,MeOH/H₂O＝80:20).

EXAMPLE 23 preparation of Compound 24

The synthesis was as for compound 5 in example 4, except that 2-chloro-10- (4-chlorobutyl) -10H-phenothiazine was used instead of 2-chloro-10- (3-chloropropyl) -10H-phenothiazine.

Synthesis yield 106mg (yield 41.12％).¹H NMR(300MHz,Chloroform-d)δ7.44–7.33(m,2H),7.27(d,J＝8.1Hz,1H),7.22–7.04(m,4H),4.10(t,J＝6.9Hz,2H),3.86(t,J＝5.4Hz,2H),2.96(s,1H),2.85–2.55(m,12H),2.08(p,J＝7.2Hz,2H),1.87(p,J＝7.3Hz,2H).HRMS(ESI):calcd for C₂₂H₂₉ClN₃OS⁺[M+H]⁺418.1714,found 418.1708.Purity:95.38％by HPLC(t_R＝3.34min,MeOH/H₂O＝80:20).

EXAMPLE 24 preparation of Compound 25

Step 1 preparation of Compound 25-2

The synthesis was as for compound 21-2 in example 21, except that 2-cyanophenothiazine was used instead of 2-chlorophenothiazine.

Step 2 preparation of Compound 25-3

The synthesis was the same as that of the compound 21-3 in example 20 except that 2-cyanophenothiazine was used instead of 2-chlorophenothiazine and the reaction time was adjusted to 10 min.

Step 3 preparation of Compound 25-4

The synthesis was as for the synthesis of compound 7-1 in example 6, except that 2-cyano-10- (2-chloroethyl) -10H-phenothiazine was used instead of 2-cyano-10- (3-chloropropyl) -10H-phenothiazine.

Step 4 preparation of Compound 25

The synthesis was as for compound 5 in example 4, except that 2-carboxamide-10- (2-chloroethyl) -10H-phenothiazine was used instead of 2-carboxamide-10- (3-chloropropyl) -10H-phenothiazine.

Synthesis yield 16mg of pale yellow solid (yield 36.68％).¹H NMR(300MHz,Chloroform-d)δ7.76(dd,J＝7.5,1.5Hz,1H),7.54(s,2H),7.54(s,1H),7.21–7.17(m,2H),7.14–7.11(m,1H),7.09–7.03(m,2H),4.03(t,J＝7.1Hz,2H),3.99(t,J＝7.5Hz,1H),3.54(q,J＝7.2Hz,2H),2.95(t,J＝7.1Hz,2H),2.65–2.49(m,10H).HRMS(ESI):calcd for C₂₁H₂₇N₄O₂S⁺[M+H]⁺399.1849,found 399.1846.Purity:98.35％by HPLC(t_R＝2.68min,MeOH/H₂O＝80:20).

Examples 25 and 26 preparation of Compounds 26 and 27

Step 1,2 Synthesis of Compounds 26-2, 26-3 the same as Synthesis of Compound 5 except that hydroxyethylpiperazine was replaced with anhydrous piperazine.

Step 3 Synthesis of Compound 26-4

Compound 26-3 (500 mg,1.39 mmol), bromoacetonitrile (331.2 mg,2.78 mmol) was dissolved in acetonitrile, anhydrous K ₂CO₃ was added, the reaction was carried out at room temperature for 16h, after TLC monitoring the reaction was complete, the inorganic salts were removed by suction filtration, and vacuum concentration sand column chromatography separation purification gave 536mg of white solid (yield 96.71%).

Step 4 Synthesis of Compound 26

The synthesis was as for compound 7-1 in example 6, except that 26-4 was used instead of 2-cyanophenothiazine.

Synthesis gave 32mg of a white solid (yield 30.62％)¹H NMR(300MHz,Chloroform-d)δ7.22(d,J＝8.0Hz,1H),7.23–7.16(m,2H),7.16–7.07(m,3H),7.05(d,J＝2.2Hz,1H),6.78(d,J＝7.7Hz,1H),6.64(d,J＝7.5Hz,1H),3.93(t,J＝6.5Hz,2H),3.13(s,2H),2.67(t,J＝4.6Hz,2H),2.61(dd,J＝5.0,4.3Hz,2H),2.54(t,J＝4.6Hz,4H),2.45(t,J＝6.2Hz,2H),1.82(q,J＝6.4Hz,2H).HRMS(ESI):calcd for C₂₁H₂₆ClN₄OS⁺[M+H]⁺417.1510,found 417.1511.Purity:96.36％by HPLC(t_R＝3.69min,MeOH/H₂O＝80:20).

Step 5 preparation of Compound 27

Dropwise adding an ether solution of LiAlH4 into an ether solution of 27-4 in ice bath, reacting for 2h at room temperature after the dropwise adding, monitoring the reaction completely by TLC, slowly dropwise adding a saturated NaHCO ₃ solution into the reaction solution in ice bath for quenching, extracting by EA, and separating and purifying by column chromatography. Yield 68mg of white solid (yield 33.66％).¹H NMR(300MHz,Chloroform-d)δ7.18(m,2H),7.05(d,J＝8.1Hz,1H),7.01–6.84(m,4H),3.93(t,J＝6.9Hz,2H),3.55(t,J＝29.1Hz,2H),2.66–2.20(m,12H),1.97(p,J＝7.0Hz,2H),1.31(d,J＝8.8Hz,2H).HRMS(ESI):calcd for C₂₁H₂₈ClN₄S⁺[M+H]⁺403.1717,found403.1719.Purity:95.86％by HPLC(t_R＝2.36min,MeOH/H₂O＝80:20).

EXAMPLE 27 preparation of Compound 28

Preparation scheme of compound 28 the synthesis was as for compound 5 in example 4, except 1-bromo-3-chloro-2-methylpropane was used instead of 1-bromo-3-chloropropane.

The synthesis gave 68mg of a white solid (yield 68.56％).¹H NMR(300MHz,Chloroform-d)δ7.03(dd,J＝7.9,5.7Hz,2H),6.92(d,J＝8.7Hz,1H),6.85–6.73(m,4H),3.95(dd,J＝13.3,3.9Hz,1H),3.52(t,J＝5.3Hz,2H),3.37(dd,J＝13.4,8.2Hz,1H),3.05(s,2H),2.52–2.44(m,8H),2.35–2.25(m,2H),2.21–2.15(m,1H),2.12(s,2H),0.83(d,J＝6.2Hz,3H).HRMS(ESI):calcd for C₂₂H₂₉ClN₃OS⁺[M+H]⁺418.1714,found 418.1714.Purity:98.62％by HPLC(t_R＝4.33min,MeOH/H₂O＝80:20).

EXAMPLE 28 preparation of Compound 29

Step 1 preparation of Compound 29-2

29-1 (1.5 G,6.42 mmol) was dissolved in DMF and epibromohydrin (2.64 g,19.25 mmol) and Cs ₂CO₃ (8.37 g,25.68 mmol) were added and reacted overnight at room temperature. TLC monitoring reaction completion, dilution with water, EA extraction, drying of the organic phase with anhydrous sodium sulfate, concentration followed by column chromatography separation and purification gave 1.68g of oil (yield 90.33％).¹H NMR(300MHz,Chloroform-d)δ7.25–7.06(m,6H),7.04(d,J＝2.2Hz,1H),4.16(dd,J＝11.9,3.1Hz,2H),3.91–3.85(m,1H),2.82(dd,J＝12.5,2.6Hz,2H).

Step 2 preparation of Compound 29

29-2 (200 Mg,0.69 mmol) was added to DMF, hydroxyethylpiperazine (108 mg,0.83 mmol) was added, and anhydrous K ₂CO₃ (284 mg,2.07 mmol) was added and reacted at 80℃for 10h. After TLC monitoring the completion of the reaction, water was added for dilution, EA extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 89mg of a white solid (yield 30.79％).¹H NMR(300MHz,Chloroform-d)δ7.23(dd,J＝8.7,7.1Hz,2H),7.11(d,J＝8.0Hz,1H),7.07–6.94(m,4H),4.21(ddt,J＝9.4,7.0,3.4Hz,1H),4.15–3.87(m,2H),3.72(t,J＝5.2Hz,2H),2.69(ddt,J＝29.2,21.6,12.0Hz,12H).HRMS(ESI):calcd for C₂₁H₂₇ClN₃O₂S⁺[M+H]⁺420.1507,found 420.1506.Purity:98.11％by HPLC(t_R＝4.27min,MeOH/H₂O＝80:20).

EXAMPLE 29 preparation of Compound 30

Preparation scheme of Compound 30 in addition to the substitution of 2-chlorophenothiazine with phenoxazine, preparation scheme of assimilated Compound 29.

The synthesis gave 132mg of a white solid (yield 42.74％).¹H NMR(300MHz,Chloroform-d)δ6.83–6.73(m,2H),6.70–6.57(m,6H),4.12–4.01(m,1H),3.71–3.50(m,4H),3.30–2.86(m,2H),2.70(d,J＝9.7Hz,2H),2.65–2.40(m,10H).HRMS(ESI):calcd for C₂₁H₂₈N₃O₃ ⁺[M+H]⁺370.2125,found 370.2120.Purity:97.72％by HPLC(t_R＝2.27min,MeOH/H₂O＝80:20).

EXAMPLE 30 preparation of Compound 31

Preparation of Compound 31 assimilation Compound 30 was prepared except for the substitution of hydroxyethylpiperazine with 4-piperidineethanol.

Synthesis of Compound 31 gave 57mg (yield) 37.01％).¹H NMR(300MHz,Chloroform-d)δ6.80(td,J＝7.2,6.7,2.1Hz,2H),6.73–6.56(m,6H),4.11(m,1H),3.71(t,J＝6.3Hz,2H),3.59(td,J＝15.4,5.6Hz,2H),2.96(dd,J＝32.8,11.4Hz,2H),2.63–2.43(m,2H),2.36(td,J＝11.5,2.5Hz,1H),2.05(td,J＝11.8,2.6Hz,1H),1.84–1.66(m,2H),1.62–1.29(m,5H).HRMS(ESI):calcd for C₂₂H₂₉N₂O₃ ⁺[M+H]⁺369.2172,found 369.2172.Purity:99.03％by HPLC(t_R＝2.35min,MeOH/H₂O＝80:20).

EXAMPLE 31 preparation of Compound 32

Preparation scheme of compound 32 assimilation compound 29 was prepared except that 2-cyanophenoxazine was substituted for phenoxazine.

Synthesis yield 26mg (yield) 21.77％).¹H NMR(300MHz,Chloroform-d)δ7.51(dd,J＝8.6,2.2Hz,1H),7.42(d,J＝2.2Hz,1H),7.18–7.07(m,3H),6.90(d,J＝8.6Hz,1H),6.88–6.81(m,1H),4.10–3.98(m,1H),3.63(td,J＝13.9,12.4,6.8Hz,4H),2.80–2.39(m,12H).HRMS(ESI):calcd for C₂₂H₂₇N₄O₃ ⁺[M+H]⁺395.2078,found395.2081.Purity:98.11％by HPLC(t_R＝4.17min,MeOH/H₂O＝80:20).

Examples 32 and 33 preparation of Compounds 33 and 34

Preparation scheme of compound 34 preparation of compound 29 was followed by replacement of the phenoxazine with 3-cyanophenoxazine.

The synthesis gave 136mg (yield) 45.56％).¹H NMR(300MHz,Chloroform-d)δ7.08(d,J＝8.4Hz,1H),6.86–6.71(m,4H),6.65(s,2H),4.10–3.98(m,1H),3.63(td,J＝13.9,12.4,6.8Hz,4H),2.77(d,J＝7.5Hz,2H),2.70–2.44(m,10H).HRMS(ESI):calcd for C₂₂H₂₇N₄O₃ ⁺[M+H]⁺395.2078,found395.2075.Purity:97.55％by HPLC(t_R＝3.88min,MeOH/H₂O＝80:20).

Preparation of Compound 33 preparation of Compound 7-1 was carried out in place of 2-cyanophenothiazine with Compound 33.

Synthesis yield 26mg (yield) 31.08％).¹H NMR(300MHz,Chloroform-d)δ7.74(dd,J＝8.1,1.9Hz,1H),7.34–7.29(m,2H),7.18–7.09(m,3H),6.86–6.79(m,1H),4.10–3.98(m,1H),3.63(td,J＝13.9,12.4,6.8Hz,4H),2.77(d,J＝7.5Hz,2H),2.70–2.44(m,10H).HRMS(ESI):calcd for C₂₂H₂₈N₄O₄ ⁺[M+H]⁺413.2183,found 413.2180.Purity:98.11％by HPLC(t_R＝4.27min,MeOH/H₂O＝80:20).

EXAMPLE 34 preparation of Compound 35

Preparation scheme of compound 35 preparation of assimilation compound 229 except for the replacement of the phenoxazine with 2-chlorocarbazole.

The synthesis gave 165mg of a white solid (yield 62.23％).¹H NMR(300MHz,Chloroform-d)δ8.06(d,J＝7.8Hz,1H),8.00(d,J＝8.3Hz,1H),7.53(d,J＝1.8Hz,1H),7.49(d,J＝4.0Hz,2H),7.28(d,J＝2.3Hz,1H),7.26–7.18(m,1H),4.35(dd,J＝5.1,2.7Hz,2H),4.22(dt,J＝8.4,5.1Hz,1H),3.63(t,J＝5.3Hz,2H),2.67(s,2H),2.56(t,J＝5.4Hz,5H),2.51–2.37(m,5H).HRMS(ESI):calcd for C₂₁H₂₆ClN₃O₂ ⁺[M+H]⁺388.1786,found388.1788.Purity:98.11％by HPLC(t_R＝4.27min,MeOH/H₂O＝80:20).

Example 35 evaluation of the binding Capacity of Compound 1 to STING by Isothermal Titration Calorimetry (ITC)

Compound 1 is commercially available under CAS number 58-39-9 at a purity of 99.72wt%.

ITC experiments were performed using a MicroCal ITC 200 instrument to determine the binding affinity of proteins to ligands. ITC experiments enable the fit calculation of quantitative binding thermodynamic parameters, including binding site number (n), binding constant (Ka), reaction enthalpy change (Δh), entropy change (Δs), etc., by detecting thermal changes (endothermic or exothermic) when a protein solution and a small molecule (ligand) solution interact.

Extraction and purification of STING protein the STING (residues 137-375 at the C-terminus) gene encoded by DNA was cloned into pET28a vector and transfected into competent BL21 (DE 3) cells were cultured overnight on solid medium. After picking up the monoclonal and amplifying in liquid medium, IPTG (1 mM) was added and the culture was continued at low temperature (16 ℃) for 16 hours to induce protein expression. Cells were sonicated with lysis buffer [20mM Tris-HCl (pH 7.4), 200mM NaCl,and 0.02mM β -mercaptoethanol ] for 30min, centrifuged at 12000rpm for 20min at 4℃and the supernatant was purified in AKTA (equilibration: 300mM NaCl,20mM Tris-HCl,5mM imidazole, pH=7.4; elution: 300mM NaCl,20mM Tris-HCl,400mM imidazole, pH=7.4). The resulting protein was dialyzed overnight against PBS buffer (0.2 mM KH ₂PO₄,1mM Na₂HPO₄, 20mM NaCl,0.3mM,pH =7.4).

The protein used in this experiment was self-extracting purified STING ^WT protein with buffer of 20mM Tris and 200mM NaCl (pH 8.2). 100. Mu.M of compound (100. Mu.M) was prepared using Tris buffer, and the DMSO content was 1% (molar ratio) since the compound stock solution was 10mM, and 500. Mu.L of 5. Mu.M of STING ^WT protein solution was prepared, taking care to ensure consistent DMSO content (1%) between the protein and the compound. Then the protein is slowly pumped into a sample cell (cell) after the cleaning, the compound is sucked into a titration needle (syringe) according to the instruction of an instrument, the instrument parameters are set, the total drop number is 19 drops, the titration volume is 2 mu l each time, the balance is 600s, the temperature of the sample cell is 25 ℃, the first drop is delayed for 60s, the first drop volume is 0.5 mu l, the stirring speed is 750rpm, and the interval time is 120s. After titration, the analysis was fitted using Origin software to obtain enthalpy change (Δh), entropy change (Δs), number of binding sites (n), binding constant (Ka).

Compound 1 was assayed to exhibit binding to STING and as shown in figure 1, the binding affinity constant K _d for compound 1 was 1 μm.

Example 36 spin echo sequence pulse experiments (CPMG) and Nuclear magnetic resonance saturation transfer Spectrometry (STD-NMR) experiments verify the binding of Compound 1 to STING.

STING ^WT protein was first displaced with heavy water (D ₂ O) and then separately assayed using a 600MHz nuclear magnetic resonance spectrometer (Bruker) on 100 μm compound (2 wt% dmso), 100 μm compound and 2 μm protein in mixed solution (2 wt% dmso), and the data analyzed using the MestReNova software.

As shown in fig. 2, the CPMG results showed that the blue color is the nuclear magnetic pattern of compound 1, the red color is the nuclear magnetic pattern of STING ^WT type protein after incubation with compound, and after the two patterns are superimposed, the characteristic peak of compound 1 is significantly reduced, which indicates that compound 1 is bound to STING ^WT type protein. STD-NMR analysis showed characteristic peaks of Compound 1 in the difference spectrum, so that the binding with STING protein could also be demonstrated.

EXAMPLE 37 RT-qPCR experiments to detect the Effect of Compound 1 on STING downstream cytokines

THP1 cells (Human acute monocytic leukemia, invitogen) were cultured in RPMI 1640 containing 10% foetal calf serum to a density of 80% -90% based on 37 ℃ in incubator with 5% co ₂, cells were collected by centrifugation at 1000rpm, diluted and inoculated into 6 well clear cell culture plates at 1×10 ⁶ cells per well and treated with test compounds at different concentrations (DMSO group, 10 μm,20 μm,30 μm) for 24h. After the incubation, the cells were collected, the medium was removed as much as possible, and RNA was extracted using an RNA extraction kit (Tiangen organism, cat# DP 419) according to the instructions, and then reverse transcribed into cDNA using HISCRIPT III RT SuperMix (gDNA wiper) kit (Vazyme, cat# R323-01), and RT-qPCR analysis was performed using ChamQ SYBR QPCR MASTER Mix (Vazyme, Q341-02/03) and primers (GAPDH as an internal reference), and the expression levels of IFN-. Beta.and CXCL10 mRNA were analyzed using the 2 ^-ΔΔCt method.

As shown in FIG. 3, compound 1 was administered at concentrations of 10. Mu.M, 20. Mu.M, and 30. Mu.M for 24 hours in the RT-qPCR assay, and the results showed that compound 1 was able to induce the concentration-dependent expression of mRNA of the downstream cytokines CXCL10 and IFN-. Beta.at 24 hours.

The primer sequences are as follows ：GAPDH-F：AAGGCTGTGGGCAAGGTCATC,GAPDH-R：AGGTGGAGGAGTGGGTGTCG;CXCL10-F：ATTTGCTGCCTTATCTTTCTG,CXCL10-R：CTTGATGGCCTTCGATTCTG;IFNβ-F：CATTACCTGAAGGCCAAGGA,IFNβ-R：AGCAATTGTCCAGTCCCAGA.

EXAMPLE 38 Western-Blot experiments investigating the influence of Compound 1 on STING and downstream proteins

Firstly, performing an aging Western-Blot experiment, namely, treating THP-1 cells with a compound 1 (10 mu M) for 1h,3h,6h and 12h, centrifuging at 1000rpm to collect cells, washing with PBS for three times, adding RIPA lysate, protease inhibitor and phosphatase inhibitor, and performing ice lysis for 15min. After completion of lysis, the supernatant was centrifuged at 12000rpm for 10min, and the protein concentration was measured using a BCA (Thermo, waltham, mass.). Adding 5× Loading Buffer (volume ratio (4:1)) into supernatant, mixing, decocting at 100deg.C for 10min, and storing in-20 refrigerator. 40ug samples were each loaded onto a 15-well SDS-PAGE gel, switched to 120V after running at 60V for 30min, and transferred to PVDF membrane (Perkinelmer, northwalk, CT, USA) after running, and run on a transfer membrane apparatus with an equicurrent of 0.3A. The PVDF membrane was then blocked with 1% BSA for 1h, and the corresponding antibody was incubated overnight at 4℃as required, after which the PVDF membrane was washed three times with TBST, and then with Dylight 800-labeled secondary antibody for 1h at room temperature, after which the PVDF membrane was washed three times with TBST again, and finally developed using ECL luminescence solution, and the strips were scanned using an Odyssey INFRARED IMAGING SYSTEM (LI-COR; lincoln, NE) instrument, stored and analyzed.

The Western Blot experiments showed that THP-1 cells were lysed on ice by treatment with Compound 1 (10. Mu.M, 20. Mu.M, 30. Mu.M) for 3h, washing with PBS. The rest of the operation steps are the same as those of the aging WB experiment.

As shown in FIG. 4, in the measurement of the aging Western-Blot experiment, the administration time was 1h,3h,6h,12h, and the administration concentration was 20. Mu.M, and the result showed that compound 1 induced phosphorylation of STING, IRF3 and TBK1 more significantly at the administration time of 3h, so the administration time was 3h.

In the measurement of the dose-response Western-Blot experiment, the administration concentration of the compound 1 is 10 mu M, 20 mu M and 30 mu M, and the administration time is 3 hours. The results show that compound 1 is able to induce the concentration-dependent phosphorylation of downstream STING, IRF3 and TBK 1. Compound 1 was therefore identified as a STING agonist, and was able to induce expression of downstream proteins by activating STING-TBK1-IRF signaling pathway.

The information for the antibodies used in the experiments is as follows:

GAPDH(ProteinTech,1E6D9),STING(Cell Signaling Technology,13647S),P-STING(Cell Signaling Technology,19781S),IRF3(ProteinTech,11312-1-AP),p-IRF3(ser386)(Abcam,ab76493),TBK1(ProteinTech,2D7B1),p-TBK1(Abcam,ab109272).

example 39 agonist activity of compounds on STING signaling pathway was determined based on 293-Dual ^TM hssting R232 cells.

Principle of the experiment the activation of STING signaling pathway by compounds was evaluated by detecting their activation of IRF, their binding to interferon stimulation response element (interferon stimulated response element, ISRE) and their expression. 293T-Dual ^TM hSTING-R232 cells (Invivogen: 293 d-R232) were obtained by stably transfecting the R232 subtype of hSTING in 293-Dual ^TM Null (ISG/KI-IFN. Beta.) cells. It is capable of stably expressing two inducible reporter genes, a Secreted Embryo Alkaline Phosphatase (SEAP) reporter gene and a secreted luciferases reporter gene, wherein the level of the luciferases can be measured by reading an optical density value (OD value) at 620nm using a QUANTI-Blue ^TM reagent (Invivogen: rep-qbs, rep-qbs 2) to evaluate the induced activation of interferon regulatory factor (IRF 3), and further by measuring the level of luciferases by reading Relative Light Units (RLU) using a QUANTI-Luc ^TM reagent (Invivogen: rep-qlc1, rep-qlc) to evaluate the induced expression of IFN- β.

Experimental procedure 293-Dual ^TM hSTING-R232 cells were incubated with DMEM medium containing penicillin, streptomycin, normocin ^TM, blasticidin, hygromycin and Zeocin ^TM antibiotics (10% fetal bovine serum FBS) in a incubator of 37℃and 5% CO ₂ to 80% -90% density, collected by digestion centrifugation (1000 rpm), diluted with DMEM medium containing 10% fetal bovine serum, plated at 100. Mu.L/well with 1X 10 ⁵ cells per well in 96 well clear cell plates, incubated at 37℃for 18-24h, after 60-70% adherence of the cells, residual medium was aspirated, a gradient dilution of the test compound (10 concentrations were diluted as required by the experiment, 3 duplicate wells were set) was added, and incubation at 37℃for 24h was performed. Then 10. Mu.L of cell supernatant was aspirated into a new 96-well cell culture plate per well, then 90. Mu. L QUANTI-Blue ^TM solution was added in the dark, incubated at 37℃for 3h, the optical density value (OD value) at 620nm was read using a microplate reader to measure SEAP levels, and further induction activation of interferon regulatory factor (IRF 3) was assessed, in addition 20. Mu.L of cell supernatant per well was aspirated into a white 96-well cell culture plate, and then 50. Mu. L QUANTI-Luc ^TM solution was added in the dark, and immediately the Relative Light Unit (RLU) was read using a luminometer to determine the levels of Lucia luciferase, and further induction expression of interferon-beta (IFN-. Beta.) was assessed. Half maximal effect concentration (EC ₅₀) was calculated by GRAPHPAD PRISM software log (agonist) vs response-variable slope fitting.

The experimental results are shown in table 1:

TABLE 1 agonistic Activity of partial Compounds of the invention on STING signalling pathway

As can be seen from table 1, some of the compounds of the present invention were able to significantly activate STING signaling pathway.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the specific embodiments of the present invention may be modified or some technical features may be equivalently replaced, and they are all included in the scope of the technical solution of the present invention claimed herein without departing from the spirit of the technical solution of the present invention.

Claims (2)

1. A tricyclic compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

。

2. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or pharmaceutical excipient.