Carbazole methyl phenyl ether derivatives, preparation method and application thereof

Abstract

The present invention discloses carbazole methyl phenyl ether derivatives, preparation methods and applications thereof. The structure of the carbazole methyl phenyl ether derivatives is shown in general formula I or II: Currently, most of the small molecule compounds targeting PD-L1 protein use a biphenyl structure as a parent nucleus. The carbazole methyl phenyl ether derivatives in the present invention patent have made a breakthrough in the structural skeleton of the compound, with a more novel structure and stronger innovation. Compounds Z8 and Z9 also show good biological activity. Therefore, this series of carbazole methyl phenyl ether derivatives have higher development potential and research value.

Description

Technical Field

[0001] The present invention discloses a class of carbazole methyl phenyl ether derivatives, and a preparation method and use thereof. Specifically, it relates to carbazole methyl phenyl ether derivatives shown in general formula I and general formula II, a preparation method thereof, and the use of such compounds in treating diseases related to PD-1 / PD-L1 signaling pathways such as cancer, infectious diseases, and autoimmune diseases. Background Art

[0002] Today, cancer is one of the most harmful diseases to humans in the world and the main cause of death. Every year, a large number of people suffer from various cancers, and a large amount of resources are used for cancer treatment. In recent years, tumor immunotherapy has become one of the important means in the field of malignant tumor treatment, and it is bringing revolutionary changes to the treatment of various types of tumors.

[0003] The immune system can not only play a role in regulating cancer fate by affecting tumor suppressor genes that destroy the ability of tumors to invade and metastasize; it can also promote tumor cell transformation and promote tumor growth, thereby shaping the immunogenicity of tumor cells. The immune system is regulated by co-stimulatory signals (T cell receptors) and co-inhibitory signals (immune checkpoints). Co-stimulatory signaling pathways stimulate the immune system to release cascades of immunomodulatory proteins to eliminate pathogens by preventing their growth. Immune system checkpoints not only maintain self-tolerance, but also protect normal tissues from unnecessary damage when responding to pathogenic infections. Unfortunately, tumor cells have learned to exploit mechanisms that stimulate immune checkpoints and evade the cytotoxic immune system, especially those targeting tumor antigen-specific T cells. This explains to some extent the metastatic nature of malignant tumors, including solid and blood types, and clarifies the nature of tumor immune escape. Immune checkpoint inhibitors block immune checkpoints through ligand-receptor interactions, thereby restoring the anti-tumor immune response of co-stimulatory T cells to achieve the purpose of tumor treatment. Inhibitory immune checkpoint molecules have attracted widespread attention as targets for cancer immunotherapy, and they have been widely used in the treatment of various cancers. For discovering the immune checkpoint molecules CTLA-4 and PD-1, American immunologist James P Alison and Japanese immunologist Tasuku Honjo jointly won the 2018 Nobel Prize in Physiology or Medicine. As we all know, one of the most important advances in the field of tumor immunotherapy is the discovery, development and clinical application of PD-1 / PD-L1 pathway inhibitors, making the PD-1 / PD-L1 pathway one of the most promising targets in the field of tumor immunology.

[0004] Programmed cell death protein-1 (PD-1) (also known as CD279) is an immune co-inhibitory molecule. It is a type I transmembrane glycoprotein composed of 288 amino acids. It was discovered by Japanese scholar Honjo Tasuku from apoptotic mouse T cell hybridoma 2B4.11. PD-1 protein is expressed by activated T cells, B cells, natural killer cells, macrophages and dendritic cells. PD-1 protein has two ligands: programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), which belong to the B7 family and have 37% sequence homology. PD-L1 protein (also known as B7H1 or CD274) is present on the surface of many different immune cells (antigen presenting cells, B cells, T cells), tissue cells (including heart, lung, liver, pancreatic islets, vascular endothelium, epithelial cells, muscle cells, etc.) and tumor cells. The expression of PD-L2 protein (CD273) is limited and is only found in dendritic cells (DCs) and a few tumor cell lines. PD-1 / PD-L1 pathway inhibitors can block the pathway by interfering with the interaction between PD-1 protein and PD-L1 protein, thereby enhancing the activity of T cells, promoting the proliferation of cytotoxic T lymphocytes, and enhancing the body's anti-tumor immune response. Studies have shown that PD-1 / PD-L1 pathway inhibitors have shown good therapeutic effects on various types of tumors, such as melanoma, non-small cell lung cancer, urothelial cell carcinoma, renal cell carcinoma, head and neck squamous cell carcinoma, classical Hodgkin's lymphoma, etc. In recent years, monoclonal antibody drugs (MAbs) targeting the PD-1 / PD-L1 signaling pathway have shown significant anti-tumor effects in clinical studies with low toxicity, so they have developed rapidly and are increasingly widely used. To date, the U.S. Food and Drug Administration (FDA) has approved seven monoclonal antibody drugs targeting the PD-1 / PD-L1 pathway for clinical treatment, including four PD-1 monoclonal antibodies (Pembrolizumab, Nivolumab, Cemiplimab, and Dostarlimab) and three PD-L1 monoclonal antibodies (Atezolizumab, Durvalumab, and Avelumab). Summary of the invention

[0005] The technical problem solved by the present invention is to provide a carbazole methyl phenyl ether derivative having the ability to inhibit PD-1 / PD-L1 interaction and a preparation method thereof, and use thereof in preparing a drug for preventing or treating diseases related to the PD-1 / PD-L1 signaling pathway.

[0006] Carbazole methyl phenyl ether derivatives and stereoisomers thereof and pharmaceutically acceptable salts thereof as shown in general formula I or II,

[0007]

[0008] R1 can be one of the following: hydrogen, C1-8 saturated alkyl, C1-5 alkoxy;

[0009] R2 can be one of the following: R3 can be one of the following: substituted C1-8 saturated alkylamino, substituted C2-6 unsaturated alkylamino, substituted C2-6 azacyclo-1-yl, the substituent can be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-5 alkyl, C1-5 alkoxy, amino, C1-6 alkylamino, acetamido, cyano, urea, guanidine, ureaamino, guanidineamino, sulfonylamino, sulfamoyl, methylsulfonylamino, hydroxyformyl, C1-8 alkoxyformyl, mercapto, imidazolyl, thiazolyl, oxazolyl or tetrazolyl;

[0010] R4 can be one of the following: R5 can be one of the following: substituted C1-8 saturated alkylamino, substituted C2-6 unsaturated alkylamino, substituted C2-6 azacyclo-1-yl, and the substituent can be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-5 alkyl, C1-5 alkoxy, amino, C1-6 alkylamino, acetylamino, cyano, urea, guanidine, ureaamino, guanidineamino, sulfonylamino, sulfamoyl, methylsulfonylamino, hydroxyformyl, C1-8 alkoxyformyl, mercapto, imidazolyl, thiazolyl, oxazolyl or tetrazolyl.

[0011] In addition, the starting materials and intermediates in the above reactions are relatively easy to obtain. The pharmaceutically acceptable salts described in Formula I or Formula II include salts formed by different acids, such as salts formed with the following inorganic acids or organic acids: hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid. The pharmaceutically acceptable salts described in Formula I or Formula II also include different alkali metal salts (lithium, sodium, potassium salts), alkaline earth metal salts (calcium, magnesium salts) and ammonium salts. All of these salts within the scope of the present invention can be prepared by conventional methods. In the preparation process of the compounds of Formula I, Formula II, their solvates and their salts, different crystallization conditions may result in polymorphism or eutectic.

[0012] The second aspect of the technical solution of the present invention is to provide a method for preparing the compound described in the first aspect: in order to prepare the compound described in general formula I of the present invention, according to the structure of general formula I, the preparation of the compound of general formula I of the present invention will be divided into eight steps (route one).

[0013] Route 1:

[0014]

[0015] (a) using p-nitrobenzyl alcohol as shown in formula 1 as a raw material and triisopropylsilyl chloride as a protecting group to carry out a reaction to protect the hydroxyl group on the nitrobenzyl alcohol to obtain an intermediate 2;

[0016] (b) using iron powder and ammonium chloride as reducing agents to reduce the nitro group on intermediate 2 to an amino group to obtain intermediate 3;

[0017] (c) using intermediate 3 and a 4-bromo-3-iodobenzene derivative shown in formula 4 as raw materials, and obtaining intermediate 5 by Buchward reaction;

[0018] (d) Intermediate 5 undergoes intramolecular N-aromatization reaction at high temperature catalyzed by palladium acetate to directly generate intermediate 6;

[0019] (e) deprotecting the hydroxyl protecting group on intermediate 6 under the action of tetrabutylammonium fluoride (TBAF) to obtain intermediate 7;

[0020] (f) intermediate 7 is used as a raw material, and undergoes Mitsunobu reaction with 2,5-dihydroxybenzaldehyde shown in formula 8 to obtain intermediate 9;

[0021] (g) Using intermediate 9 as a starting material, reacting with various substituted aryl halides R2-Cl under alkaline conditions to obtain intermediate 10;

[0022] (h) using the aldehyde-containing intermediate 10 as a raw material, and subjecting it to a reductive amination reaction with substituted N-acetylethylenediamine to obtain target compounds Z1-Z4;

[0023] The substituent R1 in compound 4, the substituent R2 in the substituted aryl halide R2-Cl and the substituent R3 in the substituted N-acetylethylenediamine are defined the same as in the target compound.

[0024] In order to prepare the compound of general formula II of the present invention, according to the structure of general formula II, the preparation of the compound of general formula II of the present invention is divided into six steps, and intermediate 3 is the same as intermediate 3 in route 1 (route 2).

[0025] Route 2:

[0026]

[0027] (a) Intermediate 3 and intermediate 10 were subjected to Buckward coupling reaction to obtain intermediate 11;

[0028] (b) Intermediate 11 undergoes intramolecular N-aromatization reaction at high temperature catalyzed by palladium acetate to directly generate intermediate 12;

[0029] (c) intermediate 12 was desilylated in the presence of tetrabutylammonium fluoride to obtain key intermediate 13;

[0030] (d) Intermediate 13 is used as a raw material, and undergoes Mitsunobu reaction with 2,4-dihydroxybenzaldehyde to obtain intermediate 15; (e) Intermediate 15 reacts with various substituted aryl halides R4-Cl under alkaline conditions to obtain intermediate 16;

[0031] (f) Using the aldehyde-containing intermediate 16 as a raw material, a reductive amination reaction is carried out with a substituted N-acetylethylenediamine or a substituted aminoethanol to obtain the target compound Z5-Z9; the substituent R1 in the intermediate 10, the substituent R4 in the substituted aryl halide R4-Cl and the substituent R5 in the substituted N-acetylethylenediamine or the substituted aminoethanol are defined as the same as in the target compound.

[0032] The beneficial effects achieved by the present invention are: currently, most of the small molecule compounds targeting PD-L1 protein are based on biphenyl structure as the parent core, and the carbazole methyl phenyl ether derivatives in the present invention patent have made breakthroughs in the structural skeleton of the compound, with a more novel structure and stronger innovation, while compounds Z8 and Z9 also show good biological activity. Therefore, this series of carbazole methyl phenyl ether derivatives have higher development potential and research value. DETAILED DESCRIPTION

[0033] The invention will be further described below in conjunction with the examples, but the scope of the invention is not limited thereto. Measuring instruments: Bruker AV-400 Bruker AV-600 NMR spectrometers were used for nuclear magnetic resonance spectroscopy. ZAD-2F and VG300 mass spectrometers were used for mass spectrometry. HPLC purity determination method: The purity of the compound was determined by HPLC (high performance liquid chromatography) analysis, and its purity was confirmed to be above 96%. C18 column (InertSustain C18, 4.6×250 mm, 5 μm) and Shimadzu LC-20A were used for HPLC analysis, and UV detection was performed at 254 nm.

[0034] Embodiment 1:

[0035] N-{2-[2-(4-fluorophenyloxy)-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z1)

[0036]

[0037] (1) Triisopropyl-(4-nitrobenzyloxy)silane

[0038] In a 250mL single-mouth round-bottom flask, add 4-nitrobenzyl alcohol (9.2g, 60.1mmol, 1.0eq) and dichloromethane (130mL). After stirring and dissolving, add imidazole (8.5g, 126.2mmol, 2.1eq), and then slowly add triisopropylsilyl chloride (13.8g, 72.2mmol, 1.2eq). After the addition is completed, a white precipitate appears in the system, and the reaction solution changes from clear to turbid. Stir the reaction at room temperature overnight. After TLC detects that the raw material has reacted completely, the reaction system is washed twice with water, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain a crude product, namely intermediate 2: 20.3 grams, which does not need to be purified and is directly used in the next step.

[0039] (2) 4-Triisopropylsilylmethylaniline

[0040] At room temperature, triisopropyl-(4-nitrobenzyloxy)silane (Intermediate 2) (17.2 g, 55.3 mmol, 1.0 eq), methanol (100 mL) and water (33 mL) were added to a 250 mL single-necked round-bottom flask and stirred. Iron powder (9.2 g, 165.4 mmol, 3.0 eq) and ammonium chloride (8.8 mg, 165.1 mmol, 3.0 eq) were added to the reaction mixture. Then, the reaction mixture was heated to 50 ° C and the reaction was continued for 8 hours. After the reaction was completed by TLC detection, the reaction mixture was cooled to room temperature and filtered through a pressed diatomaceous earth pad. The filtrate was concentrated to dryness under reduced pressure, and the residue was poured into water and extracted with DCM (twice). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain the residue, which was purified by column (eluent: PE / EA=10:1, v / v) to obtain the product, namely Intermediate 3: 14.5 g, off-white solid, yield: 93.5%. 1H NMR (600MHz, Chloroform-d): δ7.19–7.15(m,2H),6.71–6.67(m,2H),4.75(s,2H),3.62(s,2H),1.24–1.15(m,3H),1.11(d,J=7.1Hz,18H).

[0041] (3) (2-bromo-5-methylphenyl)-(4-triisopropylsilyloxymethylphenyl)amine

[0042] In a 50mL single-mouth round-bottom flask, palladium acetate (22.4mg, 0.1mmol, 0.05eq), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (Xantphos) (86.8mg, 0.15mmol, 0.075eq), DMF (15mL), nitrogen purged twice, stirred at room temperature for 20 minutes. Then, 4-triisopropylsilyloxymethylaniline (intermediate 3) (614.9mg, 2.2mmol, 1.1eq), 1-bromo-2-iodo-4-methylbenzene (compound 4) (593.8mg, 2.0mmol, 1.0eq), cesium carbonate (912.2mg, 2.8mmol, 1.4eq) were added. After the addition was completed, nitrogen purged three times, heated to 120℃ and reacted for 4 hours. After the reaction was completed by TLC detection, the heating was stopped, the reaction system was cooled to room temperature, and filtered with diatomaceous earth. The filtrate was concentrated under reduced pressure, and then extracted three times with water and ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by column to obtain the product, intermediate 5: 731.9 mg, yield: 81.6%.

[0043] (4) 2-Methyl-6-triisopropylsilyloxymethyl-9H-carbazole

[0044] In a 50mL single-mouth round-bottom flask, (2-bromo-5-methylphenyl)-(4-triisopropylsilyloxymethylphenyl)amine (intermediate 5) (448.5mg, 1.0mmol, 1.0eq) and N,N-dimethylacetamide (12mL) were added, and after stirring and dissolving, potassium carbonate (276.4mg, 2.0mmol, 2.0eq), palladium acetate (11.2mg, 0.05mmol, 0.05eq) and 2-(dicyclohexylphosphino)biphenyl (CyJohnPhos) (35.0mg, 0.1mmol, 0.1eq) were added. After the addition was completed, the reaction system was replaced with nitrogen three times, stirred at room temperature for 15 minutes, and then heated to 130℃ for 4 hours. After the reaction was completed by TLC detection, the heating was stopped, the reaction system was cooled to room temperature, water and ethyl acetate were added, stirred and allowed to stand for stratification, and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain a residue, which was purified by column to obtain the product, intermediate 6: 205.1 mg, yield: 55.8%. 1H NMR (600MHz, Chloroform-d): δ8.04(d,J=1.6Hz,1H),7.96(d,J=7.9Hz,1H),7.89(d,J=22.7Hz,1H),7.44–7.35(m, 2H),7.22(s,1H),7.08(dd,J=8.0,1.4Hz,1H),5.02(s,2H),2.55(s,3H),1.31–1.21(m,3H),1.16(d,J=7.3Hz,18H).

[0045] (5) (7-methyl-9H-carbazol-3-yl)methanol

[0046] At room temperature, 1M TBAF aqueous solution (1.0 ml, 1.0 mmol, 2.0 eq) was added to a tetrahydrofuran (8 mL) solution of 2-methyl-6-triisopropylsilyloxymethyl-9H-carbazole (i.e., intermediate 6) (183.8 mg, 0.5 mmol, 1.0 eq). After the addition was completed, the mixture was stirred at room temperature overnight. After TLC detection showed that the raw material had reacted completely, the solvent was evaporated under reduced pressure, ethyl acetate and water were added, and after stirring for 5 minutes, the mixture was allowed to stand for stratification. The organic phase was washed with water once more, dried over anhydrous sodium sulfate, and concentrated to a small amount of solvent remaining. 10 mL of petroleum ether was added, stirred for 20 minutes, and filtered. After drying the filter cake, a white solid, i.e., intermediate 7, was obtained: 97.4 mg, yield: 92.2%. 1H NMR (400MHz, DMSO-d6): δ11.05(s,1H),7.99–7.91(m,2H),7.38(d,J=8.2Hz,1H),7.30(dd,J=8.3,1.6Hz, 1H),7.25(s,1H),6.96(dd,J=8.0,1.5Hz,1H),5.11(t,J=5.7Hz,1H),4.61(d,J=5.7Hz,2H),2.46(s,3H).

[0047] (6) 2-Hydroxy-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzaldehyde

[0048] In a 25mL three-necked round-bottom flask with a thermometer and a dropping device, (7-methyl-9H-carbazole-3-yl)methanol (i.e., intermediate 7) (422.6mg, 2.0mmol, 1.0eq), 2,5-dihydroxybenzaldehyde (303.8mg, 2.2mmol, 1.1eq) and triphenylphosphine (577.0mg, 2.2mmol, 1.1eq) were added, and tetrahydrofuran (30mL) was added. After stirring and dissolving, the mixture was cooled to 0°C in an ice bath, and DIAD (444.8mg, 2.2mmol, 1.1eq) was slowly added dropwise. After the addition was completed, the mixture was slowly heated to room temperature and reacted overnight. After the reaction reached equilibrium by TLC detection, the reaction solution was directly purified by column without treatment to obtain an off-white solid, i.e., intermediate 9: 64.9 mg, yield: 9.8%.

[0049] (7) At room temperature, DMF (5.0 mL) was added to a mixture of the intermediate 2-hydroxy-5-(7-methyl-9H-carbazole-3-ylmethoxy)benzaldehyde (i.e., intermediate 9) (214 mg, 0.65 mmol 1.0 eq), cesium carbonate (274 mg, 0.84 mmol, 1.3 eq), sodium iodide (50 mg, 0.33 mmol 0.5 eq) and different substituted arylmethyl chloride derivatives (4-fluorobenzyl chloride) (103 mg, 0.71 mmol, 1.1 eq). After the addition was completed, stirring was started and the reaction mixture was heated to 60°C for two hours. After TLC detection showed that the reaction of the raw materials was complete, saturated brine was added to the reaction mixture. A large amount of solid precipitated in the system, which was filtered and the filter cake was dried to obtain intermediate 10, which was directly used for the next step without purification.

[0050] (8) N-{2-[2-(4-fluorophenyloxy)-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z1)

[0051]

[0052] At room temperature, the intermediate 2-(4-fluorobenzyloxy)-5-(7-methyl-9H-carbazole-3-ylmethoxy)-benzaldehyde (Intermediate 10) (43.9 mg, 0.1 mmol, 1.0 eq), N-(2-aminoethyl)-acetamide (20.4 mg, 0.2 mmol, 2.0 eq) and DMF (2 mL) were added to a 25 mL single-mouth round-bottom flask. After stirring and dissolving, 1 drop of glacial acetic acid was added dropwise. After stirring for 20 minutes, sodium cyanoborohydride (12.6 mg, 0.2 mmol, 2.0 eq) was added and stirred at room temperature overnight. After the reaction was completed by thin layer chromatography (TLC), the solvent was evaporated under reduced pressure to obtain a residue, which was purified by preparative HPLC to obtain the target compound Z1: 15.2 mg, yield: 28.1%, off-white solid. HPLC purity: 96.56%. 1H NMR (400MHz, Chloroform-d): δ8.12(s,1H),8.07(d,J=1.3Hz,1H),7.95(d,J =8.0Hz,1H),7.45–7.36(m,4H),7.25(s,1H),7.13–7.04(m,3H),7.02–6.91( m,2H),6.87(d,J=8.9Hz,1H),6.50(s,1H),5.18(s,2H),5.03(s,2H),3.87(s ,2H),3.30(q,J=5.5Hz,2H),2.74(t,J=5.4Hz,2H),2.54(s,3H),1.91(s,3H).

[0053] Embodiment 2:

[0054] N-{2-[2-(3,5-difluorobenzyloxy)-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z2).

[0055]

[0056] 3,5-difluorobenzyl chloride was used to replace the same molar amount of 4-fluorobenzyl chloride, and the other operations were the same as in Example 1 to obtain an off-white solid: 10.2 mg, yield: 21.2%. HPLC purity: 96.32%. 1H NMR (600MHz, Chloroform-d): δ8.09(s,2H),7.95(d,J=7.9Hz,1H),7.47–7.40(m,2 H),7.26(s,1H),7.09(d,J=8.1Hz,1H),6.96(ddd,J=12.7,9.5,3.9Hz,3H),6.90(d d,J=8.8,3.1Hz,1H),6.83–6.75(m,2H),6.04(s,1H),5.19(s,2H),5.05(s,2H),3. 83(s,2H),3.30(q,J=5.6Hz,2H),2.71(t,J=5.8Hz,2H),2.55(s,3H),1.94(s,3H).

[0057] Embodiment 3:

[0058] N-{2-[2-(3-Fluoro-benzyloxy)-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z3).

[0059]

[0060] Substituting m-fluorobenzyl chloride for an equal molar amount of 4-fluorobenzyl chloride, the other operations were the same as in Example 1 to obtain an off-white solid: 14.4 mg, yield: 24.3%, HPLC purity: 95.78%. 1 H NMR (600MHz, Chloroform-d): δ8.24(s,1H),8.08–8.05(m,1H),7.94(d,J=7.9Hz,1H),7.50–7.29(m,3H),7.2 3(s,1H),7.18(dt,J=7.9,1.2Hz,1H),7.13(dt,J=9.5,2.1Hz,1H),7.07(dd,J=7.9,1.2Hz,1H),7.03(td,J=7. 9,2.2Hz,1H),6.99(d,J=3.0Hz,1H),6.92(dd,J=8.8,3.0Hz,1H),6.83(d,J=8.9Hz,1H),6.50(s,1H),5.17(s ,2H),5.06(s,2H),3.86(s,2H),3.30(q,J=5.5Hz,2H),2.72(dd,J=7.2,3.9Hz,2H),2.53(s,3H),1.91(s,3H).

[0061] Embodiment 4:

[0062] N-{2-[2-Benzyloxy-5-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z4).

[0063]

[0064] Benzyl chloride was used to replace the same molar amount of 4-fluorobenzyl chloride, and the other operations were the same as in Example 1 to obtain an off-white solid: 9.3 mg, yield: 18.2%. HPLC purity: 96.22%. 1 H NMR (400MHz, Chloroform-d): δ8.19(s,1H),8.08(d,J=1.5Hz,1H),7.95(d,J=7.9Hz,1H),7.49–7.31(m,7H),7.24(s,1H),7.08(dd,J=8.1,1.4Hz,1 H),6.98–6.86(m,3H),6.18(s,1H),5.18(s,2H),5.07(s,2H),3.81(s,2H ),3.26(q,J=5.5Hz,2H),2.67(t,J=5.6Hz,2H),2.54(s,3H),1.90(s,3H).

[0065] The preparation method of target compounds Z5-Z9 is similar to that of Z1, except that 2,4-dihydroxybenzaldehyde (compound 14) is used instead of 2,5-dihydroxybenzaldehyde (compound 8) to carry out Mitsunobu reaction with intermediate 13 to obtain intermediate 15, which is then subjected to nucleophilic substitution reaction with different substituted arylmethyl chloride derivatives to obtain intermediate 16, and finally, a reductive amination reaction is carried out with appropriate amines to obtain target compounds Z5-Z9.

[0066] Embodiment 5:

[0067] N-{2-[2-(4-Fluorobenzyloxy)-4-(7-methyl9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z5).

[0068]

[0069] The substituent R1 is methyl, and the preparation method of compound 15 repeats the preparation process of intermediate 9 in Example 1, the only difference is that "2,5-dihydroxybenzaldehyde (compound 8) is replaced by an equal molar amount of 2,4-dihydroxybenzaldehyde (compound 14)".

[0070] Secondly, in the preparation process of intermediate 10 in Example 1, "intermediate 9 raw material is replaced by an equal molar amount of compound 15" to obtain compound 16.

[0071] The target compound of this example was obtained by replacing the intermediate 10 raw material in the preparation process of the target compound of Example 1 with an equal molar amount of compound 16. An off-white solid was obtained: 9.2 mg, yield: 16.1%. HPLC purity: 97.24%. 1 H NMR (500MHz, Chloroform-d): δ8.10(s,1H),8.03(s,1H),7.94(d,J=7.9Hz,1H),7.39–7.37(m,1H),7.37–7.32(m,2H),7.22–7.12(m,4H),7.12–7.0 6(m,1H),7.02(dd,J=11.3,6.7Hz,2H),6.66–6.54(m,2H),5.11(d,J=2.9 Hz,4H),4.05(s,2H),3.43(s,2H),3.00(s,2H),2.54(s,3H),1.91(s,3H).

[0072] Embodiment 6:

[0073] N-{2-[2-(3,5-difluorobenzyloxy)-4-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z6).

[0074]

[0075] The substituent R1 is methyl, and the preparation method of compound 15 repeats the preparation process of intermediate 9 in Example 2, the only difference is that "2,5-dihydroxybenzaldehyde (compound 8) is replaced by an equal molar amount of 2,4-dihydroxybenzaldehyde (compound 14)".

[0076] Secondly, in the preparation process of intermediate 10 in Example 2, "intermediate 9 raw material is replaced by an equal molar amount of compound 15" to obtain compound 16.

[0077] The target compound of this example was obtained by replacing the intermediate 10 raw material with the compound 16 of the same molar amount in the preparation process of the target compound of Example 2. An off-white solid was obtained: 8.1 mg, yield: 12.8%. HPLC purity: 98.22%. 1H NMR (500MHz, Chloroform-d): δ8.03 (d, J = 1.5 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.43-7.34 (m, 2H), 7.30-7.23 (m, 2H), 7.07 (dt, J = 8.1, 2.1 Hz, 2H), 6.99 (dd, J = 8.0, 1.5 Hz, 1H), 6 .88(ddd,J=9.2,6.8,2.4Hz,1H),6.71(d,J=7.3Hz,2H),5.22(s,2H),5.15(s,2H),4.0 0(s,2H),3.35(dd,J=6.9,5.2Hz,2H),2.92(t,J=6.0Hz,2H),2.49(s,3H),1.89(s,3H).

[0078] Embodiment 7:

[0079] N-{2-[2-(3-fluorobenzyloxy)-4-(7-methyl-9H-carbazol-3-ylmethoxy)benzylamino]ethyl}acetamide (Z7).

[0080]

[0081] The substituent R1 is methyl, and the preparation method of compound 15 repeats the preparation process of intermediate 9 in Example 3, the only difference is that "2,5-dihydroxybenzaldehyde (compound 8) is replaced by an equal molar amount of 2,4-dihydroxybenzaldehyde (compound 14)".

[0082] Secondly, in the preparation process of intermediate 10 in Example 3, "intermediate 9 raw material is replaced by an equal molar amount of compound 15" to obtain compound 16.

[0083] The target compound of this example was obtained by replacing the intermediate 10 raw material with the compound 16 of the same molar amount in the preparation process of the target compound of Example 3. A light yellow solid was obtained: 11.3 mg, yield: 21.2%. HPLC purity: 97.65%. 1H NMR (500MHz, Chloroform-d): δ8.12 (d, J = 6.0 Hz, 1H), 8.07 (s, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.47-7.34 (m, 4H), 7.25 (dt, J = 1.5, 0.8 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.11-7. 04(m,3H),6.66–6.56(m,2H),5.16(s,2H),5.04(d,J=4.7Hz,2H),3.86(d,J=5.0Hz,2 H), 3.35 (d, J = 5.4Hz, 2H), 2.80 (t, J = 5.5Hz, 2H), 2.54 (s, 3H), 1.93 (d, J = 2.8Hz, 3H).

[0084] Embodiment 8:

[0085] 3-{5-(9H-carbazol-3-ylmethoxy)-2-[(2-hydroxy-ethylamino)methyl]phenoxymethyl}benzonitrile (Z8).

[0086]

[0087] 3-[5-(9H-carbazol-3-ylmethoxy)-2-formylphenoxymethyl]benzonitrile was reacted with 2-aminoethanol. The preparation conditions were as described in Example 7 (the corresponding intermediates with different substituents were replaced with equal molar amounts) to obtain an off-white solid: 8.2 mg, yield: 16.1%. HPLC purity: 98.12%. 1H NMR (500MHz, Methanol-d4): δ8.11 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.67 (dt, J = 7.8, 1.4 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.49 –7.43(m,3H),7.42–7.33(m,2H),7.21–7.14(m,1H),6.84–6.76(m,2H),5.29(s,2H) ,5.24(s,2H),4.22(d,J=5.8Hz,2H),3.77(dd,J=6.1,4.4Hz,2H),3.12–3.05(m,2H).

[0088] Embodiment 9:

[0089] 2-{5-(9H-carbazol-3-ylmethoxy)-2-[2-hydroxyethylamino)methyl]phenoxymethyl}isonicotinonitrile (Z9).

[0090]

[0091] 2-[5-(9H-carbazol-3-ylmethoxy)-2-formylphenoxymethyl]isonicotinonitrile reacts with 2-aminoethanol. The preparation conditions are as in Example 7 (the corresponding intermediates with different substituents are replaced with equal molar amounts) to obtain an off-white solid: 5.3 mg, yield: 10.6%. HPLC purity: 98.36%. 1H NMR (500MHz, Chloroform-d): δ8.79 (d, J = 2.1 Hz, 1H), 8.73 (d, J = 1.9 Hz, 1H), 8.15 (d, J = 2.3 Hz, 1H), 8.01 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.34 (dt, J = 32.0, 7.9 Hz, 4H), 7.24 (d, J = 8.4 Hz, 1H), 7.20–7. 05(m,2H),6.64(d,J=2.3Hz,2H),5.16(d,J=8.4Hz,4H),4.05(s,2H),3.67(t,J=5.2Hz,2H),2.98– 2.89(m,2H).HRMS(ESI):exactmasscalculatedforC29H27N4O3[M+H]+479.2078,found479.2081.

[0092] Pharmacological activity of the compound:

[0093] 1. In vitro activity evaluation of the inhibitory activity of the compound on the interaction between PD-1 and PD-L1: The in vitro protein level detection method adopts the PD-1 / PD-L1 binding assay kit of Cisbio.

[0094] Screening principles and methods for PD-1 / PD-L1 small molecule inhibitors

[0095] (1) Principle: PD-1 protein carries a HIS tag, and the ligand of PD-1, PD-L1, carries an hFc tag. Eu-labeled anti-hFc antibody and XL665-labeled anti-HIS antibody are respectively bound to the two tagged proteins. After laser excitation, energy can be transferred from the donor Eu to the acceptor XL665, causing XL665 to emit light. When an inhibitor (compound or antibody) is added, the binding of PD-1 and PD-L1 is blocked, so that Eu and XL665 are far apart, energy cannot be transferred, and XL665 does not emit light.

[0096] (2) Experimental method: The specific method can be referred to Cisbio's PD-1 / PD-L1 kit (Cat. No. 64ICP01PEG) and is briefly described as follows: 384-well white ELISA plate, add 4μl PD-1 protein and 4μl PD-L1 protein to each well, then add 2μl diluent or target compound diluted with diluent to each well, incubate at room temperature for 15 minutes, then add 10μL of anti-Tag1-Eu3+ and anti-Tag2-XL665 mixture to each well, incubate at room temperature for 1h-4h, and then use a multifunctional ELISA reader to detect the fluorescence signals at 665nm and 620nm. HTRF rate = (665nm / 620nm)*10 4 .

[0097] Each compound was tested at 8-10 concentrations and IC was calculated using Graphpad software 50 .

[0098] (3) The screening results are shown in Table 1:

[0099] Table 1. Screening results of the inhibitory activity evaluation of the example compounds on the interaction between PD-1 and PD-L1 at the molecular level

[0100]

[0101]

Claims

1. Carbazole methyl phenyl ether derivatives and pharmaceutically acceptable salts thereof as shown in general formula II, R1 is one of the following: hydrogen, C1-8 saturated alkyl, C1-5 alkoxy; R4 is one of the following: R5 is a substituted C1-8 saturated alkylamino group, and the substituent is a hydroxyl group.

2. The carbazole methyl phenyl ether derivative and the pharmaceutically acceptable salt thereof according to claim 1, wherein the carbazole methyl phenyl ether derivative is one of the following: 3-{5-(9H-carbazol-3-ylmethoxy)-2-[(2-hydroxy-ethylamino)methyl]phenoxymethyl}benzonitrile, 2-{5-(9H-carbazol-3-ylmethoxy)-2-[2-hydroxyethylamino)methyl]phenoxymethyl}isonicotinonitrile, 3. The carbazole methyl phenyl ether derivative and the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pharmaceutically acceptable salts are salts formed by combining the carbazole methyl phenyl ether derivatives with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions or organic bases that can provide physiologically acceptable cations, as well as ammonium salts.

4. The carbazole methyl phenyl ether derivative and the pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The inorganic acid is hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is lithium ion, sodium ion or potassium ion; the alkaline earth metal ion is calcium ion or magnesium ion; the organic base that can provide physiologically acceptable cations is methylamine, dimethylamine, trimethylamine, piperidine or morpholine.

5. The method for preparing the carbazole methyl phenyl ether derivative according to claim 1, characterized in that: The preparation route is as follows: (a) Intermediate 3 and intermediate 10 were subjected to Buckward coupling reaction to obtain intermediate 11; (b) Intermediate 11 undergoes intramolecular N-aromatization reaction at high temperature catalyzed by palladium acetate to directly generate intermediate 12; (c) intermediate 12 was desilylated in the presence of tetrabutylammonium fluoride to obtain key intermediate 13; (d) intermediate 13 is used as a raw material, and intermediate 15 is obtained by Mitsunobu reaction with 2,4-dihydroxybenzaldehyde; (e) Intermediate 15 reacts with various substituted aryl halides R4-Cl under basic conditions to give intermediate 16; (f) using the aldehyde-containing intermediate 16 as a raw material, and reacting it with substituted N-acetylethylenediamine or substituted aminoethanol to undergo a reductive amination reaction to obtain the target compound of formula II; wherein the substituents R1, R4 and R5 are as defined in claim 1.

6. Use of the carbazole methyl phenyl ether derivative and the pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating diseases related to the PD-1 / PD-L1 signaling pathway.

7. The use according to claim 6, characterized in that The disease related to the PD-1 / PD-L1 signaling pathway is selected from cancer, infectious disease or autoimmune disease.

8. Use according to claim 7, characterized in that The cancer is skin cancer, lung cancer, urinary system tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor or head and neck cancer; the infectious disease is bacterial infection or viral infection; the autoimmune disease is organ-specific autoimmune disease or systemic autoimmune disease, wherein the organ-specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis, and the systemic autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma or autoimmune hemolytic anemia.

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