A macrocyclic compound, a preparation method and application thereof, and a pharmaceutical composition

Abstract

The present application relates to the field of biological medicine, and discloses a kind of macrocyclic compound and preparation method and application, a kind of pharmaceutical composition, and the compound has the structure shown in formula (I). The compound provided in the present application or its pharmaceutically acceptable salt, or its stereoisomer shows excellent inhibitory activity to HPK1 kinase, and can be used for preventing and / or treating HPK1-mediated diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a macrocyclic compound, a preparation method and application thereof, and a pharmaceutical composition. BACKGROUND

[0002] T cell receptor (TCR) mediated T cell activation plays a crucial role in thymic T cell development, T cell subset differentiation and effector T cell function. TCR can specifically recognize the antigen peptide presented by MHC (major histocompatibility complex) on the surface of antigen presenting cells, and convert the extracellular antigen peptide into a signal that can be transmitted to the cell interior through MHC recognition. Among them, the MHC molecules on the surface of antigen presenting cells include MHC class II and MHC class I molecules, which can be specifically recognized by CD4+ and CD8+ T cell surface corresponding helper receptors CD4 and CD8 molecules, respectively, and then cause the activation of downstream signaling pathways. The typical intracellular signals of TCR activation include mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and calcium ion signaling pathways. The activation of these signals finally activates the specific gene expression of T cells, causes the proliferation of cells, and makes T cells differentiate into effector T cells.

[0003] HPK1, also known as MAP4K1, is a serine / threonine kinase and is one of the members of the MAP4K family, which also includes five members, MAP3K2, MAP4K3, MAP4K4, MAP4K5 and MAP4K6. HPK1 can interact with many linker proteins, such as SLP-76 family, CARD11, HIS, HIP-55, GRB2 family, LAT, CRK family, etc., activate the JNK / SAPK signaling pathway of hematopoietic stem cells, and thus negatively regulate the TCR pathway. MAP4K3, also known as GLK kinase, has a biological effect opposite to that of HPK1. GLK can promote the activation of the TCR pathway by binding to downstream linker proteins. Therefore, we need to screen HPK1 inhibitors that are selective for GLK.

[0004] The main processes in which HPK1 is involved in regulating TCR are: (1) TCR binds with extracellular antigen through MHC to activate TCR pathway to transmit signals to downstream linker protein molecules; (2) the activated SLP76 of linker protein tyrosine kinases Lck and Zap70, and then phosphorylates HPK1; (3) the activated HPK1 phosphorylates the receptor protein SLP-76 in turn; (4) the phosphorylation of SLP-76 provides various protein binding sites for the 14-3-3 (TCR pathway inhibitor) receptor protein to form a complex; (5) the SLP-76 phosphorylated complex is involved in the down-regulation of the Erk signal pathway and the ubiquitination degradation process of SLP76, thereby causing the decrease of TCR signal pathway and T cell proliferation. In summary, HPK1 can negatively regulate the TCR signal pathway, and therefore, HPK1 can serve as a new regulatory mechanism of T cell-mediated immune response and become a new research hotspot of immune anti-tumor.

[0005] HPK1 inhibitors play an important role in malignant solid tumors or hematological cancers (such as acute myeloid leukemia, urothelial carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, melanoma), autoimmune diseases (such as systemic lupus erythematosus, psoriatic arthritis) and inflammatory reactions.

[0006] At present, there is no drug on the market targeting HPK1, in order to better meet the clinical needs, we aim to develop a highly selective and active HPK1 inhibitor, and the present application will provide a novel structure of selective HPK1 inhibitor with good physicochemical properties and drug properties. SUMMARY

[0007] The purpose of the present application is to provide a macrocyclic compound containing a pyrazole-substituted pyrimidine structure.

[0008] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a macrocyclic compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the compound has a structure represented by formula (I):

[0009]

[0010] In formula (I),

[0011] R 11 , R 12 , R 13 , R 14 are each independently selected from at least one of H, halogen;

[0012] R2 is selected from H, substituted or unsubstituted C 1-8 alkyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted C 2-8oxaalkyl;

[0013] at least one of fluorine, chlorine, bromine, hydroxyl, cyano, (C 1-3 alkyl)2N-, morpholinyl, pyrrolidinyl, methyl, ethyl, n-propyl, iso-propyl, C 2-6 alkyl, C 2-4 acyl, C 3-6 cycloalkyl.

[0014] The second aspect of the present application provides a method for preparing a macrocyclic compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, having a structure represented by formula (I), the method comprising: contacting a compound represented by formula (A) with a compound represented by formula (B),

[0015]

[0016] wherein, in formula (A), R 11 , R 12 , R 13 , R 14 are the same as the definitions described in the first aspect;

[0017] in formula (B), R2 is the same as the definition described in the first aspect; B is boron.

[0018] The third aspect of the present application provides use of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof having a structure represented by formula (I) described in the first aspect in the preparation of a medicament for preventing and / or treating a disease mediated by HPK1.

[0019] The fourth aspect of the present application provides a pharmaceutical composition containing an active ingredient and an excipient, wherein the active ingredient comprises at least one of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof having a structure represented by formula (I) described in the first aspect.

[0020] The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof having a structure represented by formula (I) provided by the present application exhibits excellent inhibitory activity on HPK1 kinase, and can be used for preventing and / or treating a disease mediated by HPK1, exhibits good selectivity on GLK kinase, and exhibits good pharmacokinetic properties and in vivo efficacy. DETAILED DESCRIPTION

[0021] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0022] The following terms are explained for the present invention:

[0023] In this article, the wavy lines in the groups Indicates the bonding position.

[0024] "Halogen" includes fluorine, chlorine, bromine and iodine.

[0025] "Morpholinyl" refers to The group shown in FIG. 4 , wherein the wavy line indicates the bonding position with the group R2; that is, the morpholinyl group is a substituent of the group R2.

[0026] "Pyrrolidyl" refers to The group shown in FIG. 4 , wherein the wavy line indicates the bonding position with the group R2; that is, the morpholinyl group is a substituent of the group R2.

[0027] “C 1-6 "alkyl" means an alkyl group having a total carbon number of 1 to 6, including C 1-6 The straight chain alkyl and C 1-6 The branched alkyl group may be, for example, a straight-chain alkyl group or a branched-chain alkyl group having a total carbon atom count of 1, 2, 3, 4, 5 or 6, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, etc. 1-3 The "alkyl group" has a similar explanation, except that the number of carbon atoms is different.

[0028] "C substituted by 1-3 halogen atoms 1-6 "alkyl" means an alkyl group having a total carbon number of 1 to 6, including straight-chain alkyl and branched-chain alkyl groups, and the C 1-6 1-3 H atoms in the alkyl group are substituted by halogen atoms selected from halogen, for example, 1-6 1, 2 or 3 H in the alkyl group is substituted by any one or more halogen atoms selected from fluorine, chlorine, bromine and iodine, for example, monochloromethyl, fluoromethyl, difluoromethyl, trifluoroethyl and the like. 1-3 The difference is that the number of carbon atoms is different.

[0029] “(C 1-6alkyl)2N-" means that the two H groups on the amino group are substituted by branched or straight chain alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms, and the two C 1-6 The alkyl groups may be the same or different, for example, (CH3)2N-, (CH3CH2)2N-, etc. “(C 1-3 The same explanation applies to "alkyl)2N-" except that the number of carbon atoms is different.

[0030] "C containing 1-3 O atoms 2-6 "Alkyl" means a branched chain alkyl group with a total carbon atom count of 2 to 6 or a straight chain alkyl group in which the carbon atoms may be interrupted by 1 to 3 oxygen atoms, for example, CH3OCH2CH2-, CH3OCH2-, wait.

[0031] “C 2-6 "Acyl" means an acyl group having a total carbon number of 2 to 6, such as acetyl, CH3CH2CO-, etc. 2-4 The "acyl group" has a similar explanation, except that the number of carbon atoms is different.

[0032] “C 3-8 "Cycloalkyl" means a cycloalkyl group having 3 to 8 carbon atoms, for example, a cycloalkyl group having 3, 4, 5, 6, 7 or 8 carbon atoms, for example, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. 3-6 Cycloalkyl", "C 3-5 The "cycloalkyl" has a similar interpretation, except that the number of carbon atoms is different.

[0033] "Substituted or unsubstituted C 1-12 "alkyl" means an alkyl group having a total number of carbon atoms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, including C 1-12 The straight chain alkyl and C 1-12 branched alkyl, and C 1-12 The H on the alkyl group may be arbitrarily substituted or unsubstituted by the corresponding group defined herein, and the carbon atoms in the substituent are not counted in the C 1-12 The total number of carbon atoms in the alkyl group. 1-10 Alkyl", "substituted or unsubstituted C 1-8 Alkyl", "substituted or unsubstituted C 1-6 The "alkyl group" has a similar explanation, except that the number of carbon atoms in the alkyl group is different.

[0034] "Substituted or unsubstituted C 3-12 "substituted or unsubstituted cycloalkyl" 6-12"substituted or unsubstituted aryl" has a similar definition, C 3-12 "substituted or unsubstituted cycloalkyl" has a similar definition, C 6-12 "substituted or unsubstituted aryl" has a similar definition, C

[0035] "substituted or unsubstituted C 2-12 "substituted or unsubstituted oxa-cycloalkyl" means a cycloalkyl group having a total number of carbon atoms of 2 to 12, and at least one O atom among the atoms forming the ring, and H on the oxa-cycloalkyl group can be optionally substituted with or without the corresponding group defined herein, the number of carbon atoms contained in the substituent not being included in the aforementioned total number of carbon atoms. For example, it can be and the like, wherein the wavy line indicates the bonding position to the structure shown in formula (I). "substituted or unsubstituted C 2-10 "substituted or unsubstituted oxa-cycloalkyl" means a cycloalkyl group having a total number of carbon atoms of 2 to 12, and at least one O atom among the atoms forming the ring, and H on the oxa-cycloalkyl group can be optionally substituted with or without the corresponding group defined herein, the number of carbon atoms contained in the substituent not being included in the aforementioned total number of carbon atoms. For example, it can be 2-8 "substituted or unsubstituted oxa-cycloalkyl" means a cycloalkyl group having a total number of carbon atoms of 2 to 12, and at least one O atom among the atoms forming the ring, and H on the oxa-cycloalkyl group can be optionally substituted with or without the corresponding group defined herein, the number of carbon atoms contained in the substituent not being included in the aforementioned total number of carbon atoms. For example, it can be

[0036] In the case of "substituted or unsubstituted" related groups of the present application, if it is a substituted group, the substituent can be at any position of the corresponding group that can be substituted, unless otherwise specified.

[0037] As described above, the first aspect of the present application provides a compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, the compound having a structure shown in formula (I):

[0038]

[0039] wherein, in formula (I),

[0040] R 11 , R 12 , R 13 , R 14 each is independently selected from at least one of H, halogen;

[0041] R2 is selected from H, substituted or unsubstituted C 1-8 alkyl, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted C 2-8 oxa-cycloalkyl;

[0042] The substituent optionally contained in R2 is at least one of fluorine, chlorine, bromine, hydroxyl, cyano, (C 1-3 alkyl)2N-, morpholinyl, pyrrolidinyl, methyl, ethyl, n-propyl, isopropyl, C 2-6 alkyl having 1 to 3 O atoms, C 2-4 acyl, C 3-6 cycloalkyl.

[0043] Several preferred embodiments are provided below for the compounds described in the present application.

[0044] Preferred Embodiment 1:

[0045] In formula (I),

[0046] R 11 , R 12 , R 13 , R 14 are each independently selected from at least one of H, fluorine;

[0047] R2is selected from H, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-6 cycloalkyl, substituted or unsubstituted C 2-6 oxacycloalkyl;

[0048] The substituent optionally contained in R2is at least one of fluorine, hydroxyl, cyano, (CH3)2N-, (CH3CH2)2N-, morpholinyl, pyrrolidinyl, methyl, ethyl, isopropyl, CH3OCH2CH2-, CH3OCH2-, acetyl, cyclopropyl, cyclobutyl.

[0049] Preferred Embodiment 2:

[0050] The compound described in the present application is selected from any one of the following compounds:

[0051]

[0052]

[0053] As described above, the second aspect of the present application provides a method for preparing a macrocyclic compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, represented by formula (I), the method comprising: contacting a compound represented by formula (A) with a compound represented by formula (B),

[0054]

[0055] wherein, in formula (A), R 11 , R 12 , R 13 , R 14 are each independently selected from at least one of H, fluorine;

[0056] In formula (B), R2is selected from H, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-6 cycloalkyl, substituted or unsubstituted C 2-6 oxacycloalkyl;

[0057] Preferably, the molar ratio of the compound represented by formula (A) to the compound represented by formula (B) is 1:1-2.

[0058] Preferably, the conditions of the contact reaction include: temperature of 110-120℃, time of 1-2h.

[0059] It should be noted that the catalyst and solvent used in the contact reaction are not particularly limited in the present application, and can be selected by those skilled in the art as needed. Exemplarily, the compound of formula (A) is contacted with the compound of formula (B), anhydrous sodium carbonate, 1,4-dioxane and water in the presence of tetrakis-triphenylphosphine palladium.

[0060] The aforementioned preparation method of the present application can also involve various post-treatment operations known in the art, such as extraction, washing, filtration, drying, concentration under reduced pressure, column chromatography, etc., and the present application is not particularly limited thereto, and those skilled in the art should not be construed as a limitation on the present application.

[0061] It should be noted that the raw materials involved in the preparation method of the present application can be synthesized according to the structure of the raw materials, combined with the organic synthesis method in the art, or can be obtained by commercial purchase. The preparation methods of several raw materials are exemplarily given in the following of the present application, and those skilled in the art should not be construed as a limitation on the present application.

[0062] The third aspect of the present application provides the use of the compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof of the first aspect in the preparation of a medicament for preventing and / or treating a HPK1-mediated disease.

[0063] Preferably, the HPK1-mediated disease is at least one of cancer or non-cancer proliferative disease, inflammation, and immune disease.

[0064] Preferably, the immune disease is at least one of lupus erythematosus, glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes.

[0065] Preferably, the cancer or non-cancer proliferative disease is at least one of lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, breast cancer, breast ductal carcinoma, head and neck cancer, endometrial cancer, corpus uteri cancer, rectal cancer, liver cancer, kidney cancer, pancreatic cancer, renal pelvis cancer, esophageal cancer, esophageal adenocarcinoma, glioma, prostate cancer, thyroid cancer, female reproductive system cancer, carcinoma in situ, lymphoma, neurofibromatosis, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, oral cancer, pharyngeal cancer, multiple myeloma, leukemia, non-Hodgkin's lymphoma, large intestinal villous adenoma, melanoma, cytoma, sarcoma, and myelodysplastic syndrome.

[0066] The fourth aspect of the present application provides a pharmaceutical composition containing an active ingredient and an excipient, wherein the active ingredient comprises at least one of the compound having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to the first aspect.

[0067] Preferably, the content of the active ingredient is 1-99.9% by weight.

[0068] More preferably, in the antitumor drug, the content of the active ingredient is 5-99.9% by weight, and the content of the active ingredient is 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, 40% by weight, 45% by weight, 50% by weight, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, etc.

[0069] In the present application, the excipient is various excipients conventionally used in the art, such as solvents, antioxidants, fillers, etc.

[0070] The present application will be described in detail below by way of examples. In the following examples, the raw materials used are commercially available analytical pure products unless otherwise specified.

[0071] Example 1: Preparation of Compound 1

[0072] The synthetic route of Compound 1 is as follows:

[0073]

[0074] In this synthetic route, -OTBS represents Boc represents tert-butyloxycarbonyl.

[0075] Step 1: Preparation of tert-butyl (3-bromo-4-cyanophenyl)carbamate

[0076] Into a 500 mL single-necked round-bottom flask, 4-amino-2-bromobenzonitrile (152.3 mmol) and N,N'-dimethylformamide (DMF, 250 mL) were added, followed by N,N'-carbonyldiimidazole (CDI, 456.9 mmol), and the reaction was carried out at 80°C for 5 h, then tert-butanol (761.5 mmol) was added, and the reaction was continued at 80°C for 2 h. TLC monitoring showed that the raw material was completely reacted, and after cooling to room temperature, it was diluted with ethyl acetate (1000 mL) and washed with saturated brine (500 mL) for 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain a white solid (yield 85%).

[0077] Step 2: Preparation of (E)-tert-butyldimethyl(4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)but-3-en-l-yloxy)silane

[0078] Into a 100 mL two-necked round bottom flask was added bis(cyclopentadienyl)zirconium chloride (Schwartz reagent, 54.3 mmol) and argon was bubbled for 3 times. Then 4-(tert-butyldimethylsilyloxy)-l-butyne (54.3 mmol), triethylamine (5.43 mmol) were added successively, and finally pinacolborane (59.8 mmol) was slowly added into the mixture. The resulting solution was heated to 60 °C and stirred for 8 hours under argon protection. TLC was used to monitor the completion of the reaction. The mixture was cooled to room temperature and loaded onto a short silica gel column. Column chromatography with 10% ethyl acetate in hexane gave a colorless oil (yield 80.5%).

[0079] Step 3: Preparation of (E)-(3-(4-((tert-butyldimethylsilyl)oxy)but-l-en-l-yl)-4- cyano phenyl)carbamic acid tert-butyl ester

[0080] Into a 100 mL Schlenk tube was added tert-butyl (3-bromo-4-cyanophenyl)carbamate (16.8 mmol), palladium acetate (1.68 mmol), 2-dicyclohexylphosphino-2',6'- dimethoxybiphenyl (s-Phos, 3.36 mmol), cesium carbonate (50.4 mmol), and argon was bubbled for 3 times after the addition was completed. Then tetrahydrofuran (THF, 20 mL), water (4.0 mL), (E)-tert-butyldimethyl(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)but-3-en-l- yloxy)silane (16.8 mmol) were added successively, and the resulting solution was heated to 80 °C and stirred for 2 hours under argon. TLC was used to monitor the completion of the reaction. The reaction system was transferred to a separatory funnel with ethyl acetate (500 mL), washed with saturated brine (200 mL) for 3 times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to give a light yellow oil (yield 79.4%).

[0081] Step 4: Preparation of (E)-(4-cyano-3-(4-hydroxybut-l-en-l-yl)phenyl)carbamic acid tert-butyl ester

[0082] Into a 250 mL single necked round bottom flask was added (E)-(3-(4-((tert- butyldimethylsilyl)oxy)but-1-en-1-yl)-4-cyanophenyl)carbamic acid tert-butyl ester (13.4 mmol), tetrahydrofuran (THF, 100 mL) and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 mol / L, 20.1 mL), the reaction was stirred at room temperature for 8 h, TLC monitoring showed the starting material was consumed, the reaction was transferred to a separatory funnel with ethyl acetate (500 mL), washed with saturated brine (200 mL) 3 times, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was purified by column chromatography to give a colorless oil (80.2% yield).

[0083] Step 5: Preparation of (4-cyano-3-(4-hydroxybutyl)phenyl)carbamic acid tert-butyl ester

[0084] Into a 250 mL single necked round bottom flask was added (E)-(3-(4-((tert- butyldimethylsilyl)oxy)but-1-en-1-yl)-4-cyanophenyl)carbamic acid tert-butyl ester (13.4 mmol), tetrahydrofuran (THF, 100 mL) and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 mol / L, 20.1 mL), the reaction was stirred at room temperature for 8 h, TLC monitoring showed the starting material was consumed, the reaction was transferred to a separatory funnel with ethyl acetate (500 mL), washed with saturated brine (200 mL) 3 times, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was purified by column chromatography to give a colorless oil (80.2% yield).

[0085] Step 6: Preparation of (S)-(2-hydroxy-1-(2-hydroxyphenyl)ethyl)carbamic acid tert-butyl ester

[0086] Into a 500 mL single necked round bottom flask was added (S)-2-(1-amino-2- hydroxyethyl)phenol (150.0 mmol), anhydrous ethanol (250 mL), di-tert-butyl dicarbonate (150.0 mmol), the reaction was stirred at room temperature for 1 h, the reaction was directly concentrated under reduced pressure to give a white solid (100% yield).

[0087] Step 7: Preparation of (S)-(2-((tert-butyldimethylsilyl)oxy)-1-(2- hydroxyphenyl)ethyl)carbamic acid tert-butyl ester

[0088] Into a 500 mL single necked round bottom flask was added (S)-(2-hydroxy-1-(2- hydroxyphenyl)ethyl)carbamic acid tert-butyl ester (150.0 mmol), dichloromethane (DCM, 250 mL), imidazole (225.0 mmol), tert-butyldimethylsilyl chloride (165.0 mmol). The reaction was stirred at room temperature for 30 min, TLC monitoring showed the starting material was consumed, the reaction was filtered under reduced pressure, the filter cake was rinsed with dichloromethane, the filter cake was discarded, the filtrate was washed with saturated brine (200 mL) 3 times, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, to give a colorless oil (93% yield).

[0089] Step 8: Preparation of (S)-(l-(2-(4-(5-((tert-butoxycarbonyl)amino)-2- isocy anophenyl) butoxy) phenyl) -2-((tert-butyldimethylsilyl) oxy) ethyl) carbamic acid tert-butyl ester

[0090] Into a 100 mL single necked round bottom flask was added tert-butyl (4-cyano-3- (4-hydroxybutyl)phenyl)carbamate (10.3 mmol), triphenylphosphine (20.6 mmol), (S)-(2-((tert-butyldimethylsilyl)oxy)-l-(2-hydroxyphenyl)ethyl)carbamic acid tert-butyl ester (10.3 mmol), toluene 10 mL, heated to 80 °C stirred to dissolve, then added dropwise diisopropyl azodicarboxylate (20.6 mmol), stirred for 30 min after dropwise addition was complete, TLC monitored for complete reaction of starting material. The system was directly concentrated under reduced pressure and loaded onto a short silica gel column. Column chromatography with 10% ethyl acetate in hexane gave a colorless oil (50.1% yield).

[0091] Step 9: Preparation of (S)-4-amino-2-(4-(2-(l-amino-2-hydroxyethyl)phenoxy)butyl)benzonitrile hydrochloride

[0092] Into a 100 mL single necked round bottom flask was added (S)-(l-(2-(4-(5-((tert- butoxycarbonyl)amino)-2-isocy anophenyl) butoxy) phenyl) -2-((tert-butyldimethylsilyl) oxy) ethyl) carbamic acid tert-butyl ester (5.2 mmol), ethyl acetate (5.0 mL), hydrogen chloride in ethyl acetate (4 mol / L, 10.0 mL), stirred at room temperature for 20 h after which TLC monitored for complete reaction of starting material, the reaction was filtered under reduced pressure, the filter cake was rinsed with dry ethyl ether and dried to give a white solid (88.4% yield).

[0093] Step 10: Preparation of (S)-4-amino-2-(4-(2-(l-((5-bromo-2-chloropyrimidin-4- yl)amino)-2-hydroxyethyl)phenoxy)butyl)benzonitrile

[0094] Into a 50 mL single necked round bottom flask was added (S)-4-amino-2-(4-(2-(l- amino-2-hydroxyethyl)phenoxy)butyl)benzonitrile hydrochloride (4.6 mmol), acetonitrile (5.0 mL), N,N'-diisopropylethylamine (DIPEA, 14.8 mmol), 5-bromo-2,4- dichloropyrimidine (4.6 mmol), stirred at room temperature for 5 h after which TLC monitored for complete reaction of starting material, the reaction was transferred to a separatory funnel with ethyl acetate (200 mL), washed 3 times with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was purified by column chromatography to give a light yellow oil (48.4% yield).

[0095] Step 11 : Preparation of (S)-3 5 - bromo-5-hydroxymethyl-7-oxa-2,4-diaza-3(2,4)- pyrimidin-1(1,3),6(1,2)-dibenzenacycloundecyl-1 4 - Preparation of cyanide

[0096] Into a 50 mL single necked round bottom flask was added acetonitrile (10 mL), water (1.0 mL), hydrogen chloride in dioxane (4 mol / L, 0.1 mL), and the reaction flask was warmed to reflux. Then (S)-4-amino-2-(4-(2-(1-((5-bromo-2-chloropyrimidin-4-yl)amino)-2-hydroxyethyl)phenoxy)butyl)benzyl cyanide (1.0 mmol) was dissolved in 3 mL of acetonitrile and added dropwise to the reaction flask. After the addition was complete, the reaction was refluxed for 5 h. TLC was used to monitor the completion of the reaction. The reaction mixture was directly concentrated under reduced pressure to give a yellow solid. The crude product was purified by trituration with anhydrous methanol to give a white solid (30.4% yield).

[0097] Step 12: Preparation of compound 1

[0098] Into a 10 mL microwave tube was added (S)-3 5 - bromo-5-hydroxymethyl-7-oxa-2,4-diaza-3(2,4)- pyrimidin-1(1,3),6(1,2)-dibenzenacycloundecyl-1 4 - cyanide (2.3 mmol), 1-tert-butoxycarbonyl-pyrazole-4- boronic acid pinacol ester (3.49 mmol), tetrakis triphenylphosphine palladium (0.233 mmol), anhydrous sodium carbonate (6.96 mmol), 1,4-dioxane (7.0 mL), water (1.5 mL), and the reaction was heated at 115 °C for 1.5 h. TLC was used to monitor the completion of the reaction. The reaction mixture was transferred to a separatory funnel with ethyl acetate (150 mL), washed with saturated brine (100 mL) three times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to give a light yellow solid (25.8% yield).

[0099] 1H NMR (600 MHz, DMSO-d6) δ 13.17 (s, 1H), 9.73 (s, 1H), 8.54 (s, 1H), 7.97 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 7.9 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.47 (d, J = 9.7 Hz, 1H), 5.93 - 5.84 (m, 1H), 4.97 (t, J = 5.4 Hz, 1H), 4.28 - 4.15 (m, 2H), 4.14 - 4.09 (m, 1H), 3.78 - 3.63 (m, 2H), 3.19 - 3.13 (m, 1H), 2.74 - 2.57 (m, 2H), 2.09 - 1.95 (m, 2H), 1.91 - 1.71 (m, 2H).

[0100] Example 2: Preparation of compound 2

[0101]

[0102] The preparation method is similar to Example 1, except that 1-tert-butoxycarbonyl- pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of 1- methylpyrazole-4-boronic acid pinacol ester to obtain compound 2 as a white solid with a yield of 60.5%.

[0103] 1 H NMR (600 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.53 (d, J = 2.4 Hz, 1H), 8.05 (s, 1H), 7.93 (s, 1H), 7.75 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.27 - 7.17 (m, 2H), 7.10 - 7.00 (m, 2H), 6.93 (t, J = 7.8 Hz, 1H), 6.46 (d, J = 9.6 Hz, 1H), 5.88 (dd, J = 9.6, 4.8 Hz, 1H), 4.96 (t, J = 5.4 Hz, 1H), 4.28 - 4.15 (m, 2H), 3.95 (s, 3H), 3.79 - 3.72 (m, 1H), 3.68 - 3.61 (m, 1H), 2.76 - 2.58 (m, 2H), 2.11 - 1.98 (m, 2H), 1.89 - 1.74 (m, 2H).

[0104] Example 3: Preparation of compound 3

[0105]

[0106] The preparation method is similar to that in Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 1-ethylpyrazole-4-boronic acid pinacol ester, to obtain compound 3 in white solid, with a yield of 55.9%.

[0107] 1 H NMR (600 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.50 (s, 1H), 8.07 (s, 1H), 7.92 (s, 1H), 7.74 (s, 1H), 7.56-7.43 (m, 1H), 7.25-7.10 (m, 2H), 7.10-6.95 (m, 2H), 6.90 (s, 1H), 6.50-6.35 (m, 1H), 5.91-5.76 (m, 1H), 4.95 (s, 1H), 4.30-4.07 (m, 3H), 3.92 (s, 1H), 3.78-3.68 (m, 1H), 3.69-3.54 (m, 1H), 2.74-2.55 (m, 2H), 2.10-1.92 (m, 2H), 1.87-1.68 (m, 2H), 1.43 (t, J = 14.1, 7.1 Hz, 3H).

[0108] Example 4: Preparation of compound 4

[0109]

[0110] The preparation method is similar to that in Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester, to obtain compound 4 in white solid, with a yield of 43.6%.

[0111] 1H NMR (600 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.51 (s, 1H), 8.10 (s, 1H), 7.95 (d, J = 6.9 Hz, 1H), 7.75 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.30 - 7.12 (m, 2H), 7.02 (dd, J = 26.9, 8.3 Hz, 2H), 6.89 (t, J = 7.4 Hz, 1H), 6.47 (d, J = 9.4 Hz, 1H), 5.85 (d, J = 9.5 Hz, 1H), 5.05 - 4.92 (m, 1H), 4.56 (dt, J = 13.5, 6.9 Hz, 1H), 4.30 - 4.10 (m, 2H), 3.80 - 3.70 (m, 1H), 3.64 (dt, J = 10.4, 5.3 Hz, 1H), 2.77 - 2.55 (m, 2H), 2.02 (d, J = 12.8 Hz, 2H), 1.80 (s, 2H), 1.60 - 1.41 (m, 6H).

[0112] Example 5: Preparation of compound 6

[0113]

[0114] The preparation method is similar to Example 1, except that 1-tert-butoxycarbonyl- pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of 2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propan-2-ol to obtain compound 6 as a white solid with a yield of 63.5%.

[0115] 1 H NMR (600 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.54 (s, 1H), 8.02 (s, 1H), 7.96 (s, 1H), 7.75 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.28 - 7.13 (m, 2H), 7.04 (dd, J = 22.9, 8.5 Hz, 2H), 6.91 (t, J = 7.6 Hz, 1H), 6.40 (d, J = 9.4 Hz, 1H), 5.88 (s, 1H), 4.95 (t, J = 5.4 Hz, 1H), 4.77 (s, 1H), 4.23 (s, 1H), 4.17 (t, J = 11.4, 10.4 Hz, 1H), 4.11 (s, 2H), 3.73 (s, 1H), 3.62 (s, 1H), 2.76 - 2.57 (m, 2H), 2.13 - 1.91 (m, 2H), 1.89 - 1.71 (m, 2H), 1.12 (s, 6H).

[0116] Example 6: Preparation of compound 7.

[0117]

[0118] The preparation method is similar to example 1, except that 1-tert-butoxycarbonyl- pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of 1- cyclopropylpyrazole-4-boronic acid pinacol ester, to obtain compound 7 as a white solid with a yield of 56.0%.

[0119] 1 H NMR (600 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.52 (s, 1H), 8.13 (s, 1H), 7.94 (s, 1H), 7.76 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.28 - 7.17 (m, 2H), 7.13 - 7.00 (m, 2H), 6.93 (t, J = 7.2 Hz, 1H), 6.58 (s, 1H), 5.97 - 5.83 (m, 1H), 4.99 (s, 1H), 4.31 - 4.16 (m, 2H), 3.88 - 3.59 (m, 3H), 2.78 - 2.60 (m, 2H), 2.07 - 1.96 (m, 2H), 1.88 - 1.74 (m, 2H), 1.30 - 1.11 (m, 4H).

[0120] Example 7: Preparation of compound 8

[0121]

[0122] The preparation method is similar to example 1, except that 1-tert-butoxycarbonyl- pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of 1- cyclopropylpyrazole-4-boronic acid pinacol ester, to obtain compound 7 as a white solid with a yield of 56.0%.

[0123] 1H NMR (600 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.54 (s, 1H), 8.12 (s, 1H), 7.97 (s, 1H), 7.79 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.29 - 7.16 (m, 2H), 7.11 - 7.00 (m, 2H), 6.93 (t, J = 7.2 Hz, 1H), 6.52 (d, J = 9.6 Hz, 1H), 5.89 (dt, J = 9.0, 4.2 Hz, 1H), 5.00 (d, J = 5.4 Hz, 1H), 4.84 - 4.76 (m, 1H), 4.30 - 4.15 (m, 2H), 3.79 - 3.61 (m, 2H), 2.72 - 2.60 (m, 2H), 2.17 - 2.11 (m, 3H), 2.05 - 1.97 (m, 5H), 1.85 - 1.81 (m, 2H), 1.71 - 1.66 (m, 2H).

[0124] Example 8: Preparation of compound 5

[0125]

[0126] The preparation method is similar to that of Example 1, except that 1-tert- butyloxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazole-1-ethanol to obtain compound 5 as a white solid with a yield of 47.3%.

[0127] 1 H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.07 (s, 1H), 7.95 (s, 1H), 7.78 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.28 - 7.16 (m, 2H), 7.10 - 7.01 (m, 2H), 6.93 (t, J = 7.8 Hz, 1H), 6.45 (d, J = 9.6 Hz, 1H), 5.93 - 5.87 (m, 1H), 4.98 (q, J = 5.4 Hz, 2H), 4.29 - 4.15 (m, 4H), 3.84 - 3.72 (m, 3H), 3.67 - 3.60 (m, 1H), 2.75 - 2.61 (m, 2H), 2.11 - 2.00 (m, 2H), 1.89 - 1.74 (m, 2H).

[0128] Example 9: Preparation of compound 9

[0129]

[0130] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with an equimolar amount of 4,4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-ylpyrazole to obtain compound 9 as a white solid with a yield of 51.4%.

[0131] 1 H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.52 (s, 1H), 8.25 (s, 1H), 7.96 (s, 1H), 7.91 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.22 (t, J = 9.2 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1H), 6.50 (d, J = 9.4 Hz, 1H), 5.94 - 5.82 (m, 1H), 5.68 (p, J = 7.2 Hz, 1H), 5.06 - 4.88 (m, 5H), 4.27 - 4.11 (m, 2H), 4.02 (q, J = 7.2 Hz, 1H), 3.82 - 3.71 (m, 1H), 3.69 - 3.51 (m, 1H), 2.77 - 2.57 (m, 2H), 2.13 - 2.00 (m, 2H), 1.87 - 1.72 (m, 2H).

[0132] Example 10: Preparation of compound 10

[0133]

[0134] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with an equimolar amount of 4,4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-ylpyrazole to obtain compound 9 as a white solid with a yield of 51.4%.

[0135] 1H NMR (600 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.50 (s, 1H), 8.15 (s, 1H), 7.95 (s, 1H), 7.79 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.26 - 7.13 (m, 2H), 7.02 (dd, J = 24.2, 8.7 Hz, 2H), 6.90 (t, J = 7.7 Hz, 1H), 6.47 (d, J = 9.7 Hz, 1H), 5.94 - 5.77 (m, 1H), 4.97 (s, 1H), 4.55 - 4.40 (m, 1H), 4.28 - 4.12 (m, 2H), 4.05 - 3.89 (m, 2H), 3.77 - 3.69 (m, 1H), 3.64 (dt, J = 11.0, 5.8 Hz, 1H), 3.48 (t, J = 11.7 Hz, 2H), 2.75 - 2.54 (m, 2H), 2.17 - 1.89 (m, 6H), 1.86 - 1.70 (m, 2H).

[0136] Example 11: Preparation of compound 13

[0137]

[0138] The preparation method is similar to that of Example 1, except that 1-tert- butyloxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with equimolar amount of N,N'-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinan-2-yl)-1H- pyrazole-1-ethanamine to obtain compound 13 as a white solid with a yield of 44.3%.

[0139] 1 H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.51 (s, 1H), 8.10 (s, 1H), 7.93 (s, 1H), 7.73 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.28 - 7.10 (m, 2H), 7.02 (dd, J = 23.1, 8.1 Hz, 2H), 6.89 (t, J = 7.5 Hz, 1H), 6.40 (d, J = 8.9 Hz, 1H), 5.93 - 5.79 (m, 1H), 4.97 (s, 1H), 4.41 - 4.23 (m, 2H), 4.24 - 4.06 (m, 2H), 3.80 - 3.66 (m, 1H), 3.66 - 3.53 (m, 1H), 2.81 - 2.57 (m, 4H), 2.18 (s, 6H), 2.01 (s, 2H), 1.85 - 1.66 (m, 2H).

[0140] Example 12: Preparation of compound 14

[0141]

[0142] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with an equimolar amount of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carbonitrile to obtain compound 14 as a white solid with a yield of 57.5%.

[0143] 1 H NMR (600 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.53 (s, 1H), 8.20 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.27 - 7.16 (m, 2H), 7.11 - 7.00 (m, 2H), 6.92 (t, J = 7.4 Hz, 1H), 6.62 - 6.40 (m, 1H), 5.97 - 5.76 (m, 1H), 4.99 (s, 1H), 4.51 (t, J = 6.6 Hz, 2H), 4.30 - 4.08 (m, 2H), 3.81 - 3.69 (m, 1H), 3.67 - 3.56 (m, 1H), 3.15 (t, J = 6.6 Hz, 2H), 2.77 - 2.62 (m, 2H), 2.06 - 1.94 (m, 2H), 1.89 - 1.72 (m, 2H).

[0144] Example 13: Preparation of compound 15

[0145]

[0146] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with an equimolar amount of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carbonitrile to obtain compound 14 as a white solid with a yield of 57.5%.

[0147] 1H NMR (600 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.59 - 8.49 (m, 2H), 8.12 (s, 1H), 8.01 - 7.98 (m, 1H), 7.82 (t, J = 60.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.29 - 7.20 (m, 2H), 7.09 (dd, J = 8.4, 2.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.60 (d, J = 9.6 Hz, 1H), 5.92 (d, J = 9.0 Hz, 1H), 4.93 (t, J = 5.4 Hz, 1H), 4.27 - 4.15 (m, 2H), 3.80 - 3.73 (m, 1H), 3.68 - 3.58 (m, 1H), 2.74 - 2.58 (m, 2H), 2.08 - 1.95 (m, 2H), 1.89 - 1.72 (m, 2H).

[0148] Example 14: Preparation of compound 16

[0149]

[0150] The preparation method is similar to that of Example 1, except that 1-tert- butyloxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 1-(2-morpholinoethyl)-1H-pyrazole-4-boronic acid pinacol ester to obtain compound 17 as a white solid with a yield of 56.7%.

[0151] 1 H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.52 (s, 1H), 8.12 (s, 1H), 7.94 (s, 1H), 7.75 (s, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.04 (dd, J = 23.8, 7.8 Hz, 2H), 6.90 (t, J = 8.5, 7.8 Hz, 1H), 6.40 (d, J = 8.1 Hz, 1H), 5.93 - 5.84 (m, 1H), 4.98 (s, 1H), 4.32 (s, 2H), 4.27 - 4.13 (m, 2H), 3.81 - 3.70 (m, 1H), 3.69 - 3.61 (m, 1H), 3.54 (s, 4H), 2.77 (s, 2H), 2.71 - 2.57 (m, 2H), 2.43 (s, 4H), 2.08 - 1.99 (m, 2H), 1.87 - 1.71 (m, 2H).

[0152] Example 15: Preparation of compound 17

[0153]

[0154] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 1-[2-(1-pyrrolidine)ethyl]-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole to obtain compound 18 in white solid with a yield of 55.3%.

[0155] 1 H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.52 (s, 1H), 8.13 (s, 1H), 7.95 (s, 1H), 7.75 (s, 1H), 7.52 (s, 1H), 7.19 (d, J = 39.0 Hz, 2H), 7.04 (dd, J = 24.2, 6.5 Hz, 2H), 6.90 (s, 1H), 6.47 - 6.30 (m, 1H), 5.96 - 5.78 (m, 1H), 5.09 - 4.90 (m, 1H), 4.44 - 4.05 (m, 4H), 3.82 - 3.69 (m, 1H), 3.69 - 3.55 (m, 1H), 2.96 - 2.56 (m, 5H), 2.44 - 2.25 (m, 3H), 2.15 - 1.91 (m, 2H), 1.91 - 1.72 (m, 2H), 1.72 - 1.44 (m, 4H).

[0156] Example 16: Preparation of compound 18

[0157]

[0158] The preparation method is similar to that of Example 1, except that 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 1-[2-(1-pyrrolidine)ethyl]-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole to obtain compound 18 in white solid with a yield of 55.3%.

[0159] 1H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.53 (s, 1H), 8.10 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.29 - 7.10 (m, 2H), 7.04 (dd, J = 28.4, 8.4 Hz, 2H), 6.89 (s, 1H), 6.39 (d, J = 9.7 Hz, 1H), 5.96 - 5.80 (m, 1H), 4.97 (s, 1H), 4.42 - 4.05 (m, 4H), 3.87 - 3.71 (m, 1H), 3.70 - 3.59 (m, 1H), 3.01 - 2.77 (m, 2H), 2.77 - 2.52 (m, 6H), 2.14 - 1.94 (m, 2H), 1.91 - 1.63 (m, 2H), 0.94 (s, 6H).

[0160] Example 17: Preparation of compound 11

[0161]

[0162] The preparation method is similar to that of Example 1, except that (S)-2-(1- amino-2-hydroxyethyl)phenol in step 6) is replaced by equimolar amount of (S)-2-(1- amino-2-hydroxyethyl)-4-fluorophenol; 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced by equimolar amount of 2-methyl-1-(4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propan-2-ol. Compound 11 is obtained as a white solid with a yield of 53.4%.

[0163] 1 H NMR (600 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.51 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 7.73 (s, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.11 - 6.98 (m, 3H), 6.94 (d, J = 11.6 Hz, 1H), 6.43 (s, 1H), 5.85 (s, 1H), 5.02 (s, 1H), 4.76 (s, 1H), 4.22 (s, 1H), 4.14 (s, 1H), 4.10 (s, 2H), 3.73 (s, 1H), 3.64 (s, 1H), 2.74 - 2.58 (m, 2H), 2.08 - 1.94 (m, 2H), 1.86 - 1.76 (m, 2H), 1.12 (s, 6H).

[0164] Example 18: Preparation of compound 12

[0165]

[0166] The preparation method is similar to that of Example 1, except that (S)-2-(1-amino-2-hydroxyethyl)phenol in step 6) is replaced with an equal molar amount of (S)-2-(1-amino-2-hydroxyethyl)-3-fluorophenol; 1-tert-butoxycarbonyl-pyrazole-4-boronic acid pinacol ester in step 12) is replaced with an equal molar amount of 2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propan-2-ol. Compound 12 is obtained as a white solid in a yield of 41.1%.

[0167] 1 H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.79 (s, 1H), 7.96 (s, 1H), 7.91 (s, 1H), 7.67 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 6.3 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 7.9 Hz, 1H), 6.78 (t, J = 10.0 Hz, 1H), 6.44 (s, 1H), 6.18 (s, 1H), 5.09 (t, J = 5.6 Hz, 1H), 4.75 (s, 1H), 4.45 (s, 1H), 4.15 (s, 2H), 4.07 (s, 1H), 3.68 - 3.56 (m, 2H), 2.86 (s, 2H), 2.15 - 1.94 (m, 2H), 1.94 - 1.80 (m, 2H), 1.10 (s, 6H).

[0168] Test Example 1: Experiment of kinase inhibitory activity of the compound of the present application

[0169] Materials and methods:

[0170] Preparation of compound working solution: The test compound was diluted with dimethyl sulfoxide (DMSO), and 50 nL of the compound at different concentrations (500 μM, 166.67 μM, 55.56 μM, 18.52 μM, 6.173 μM, 2.058 μM, 0.686 μM, 0.229 μM, 0.076 μM and 0.025 μM) in a 384-well LDV plate was transferred to the experimental plate by a liquid workstation ECHO, and the final concentration of the compound in the experimental plate was 5000 nM, 1667 nM, 556 nM, 185.19 nM, 61.73 nM, 20.58 nM, 6.86 nM, 2.29 nM, 0.76 nM and 0.25 nM.

[0171] Experimental process:

[0172] The experiment was performed in 384-well plates (Corning, product number 4512) with a total reaction volume of 20 μΐ per well. The reaction mixture in each well contained 1 x kinase buffer (1 mM MOPS (pH 7.2), 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 2.5 mM β-Glycerol-Phosphate, 0.05 mM DTT), 2 μΜ MBP protein and 40 μΜ ATP, 5 μΐ ADP-Glo TM reagent, 10 μΐ kinase detection reagent, and 5 nM HPK1 or GLK. The 5 nM HPK1 or GLK, MBP protein, ATP reaction was performed for 60 min, then 5 μΐ ADP-Glo TM reagent was added and incubated for 60 min at room temperature. The signal was read using a Multilabel Reader (EnVision).

[0173] Data processing: The luminescence signal value was converted to the inhibition rate.

[0174] Inhibition rate % = (Max - sample value) / (Max - Min) x 100;

[0175] Min represents the value measured in the well without enzyme but with DMSO and substrate, and Max represents the value measured in the well with DMSO, enzyme and substrate.

[0176] IC 50 Value calculation: The concentration of the test compound at which the enzyme activity was inhibited by 50% was calculated by curve fitting in Excel XLFit (version 5.3.1.3). 50

[0177] The results are shown in Table 1.

[0178] Table 1 In vitro kinase inhibition activity of the compounds of the examples

[0179]

[0180]

[0181] As can be seen from Table 1, the compounds listed in the examples all exhibit excellent inhibitory activity against HPK1 kinase, and some of the compounds also exhibit very high selectivity against GLK kinase (more than 100-fold).

[0182] Test Example 2: In vivo drug metabolism study in mice

[0183] ​The compound provided in the foregoing part of the example was administered to mice in the form of an aqueous solution of polyethylene glycol 400 (70%). Blood samples of about 40 μL were collected at 5 min, 15 min, 30 min, 1, 2, 4, 8, 24 h after administration in the intravenous group and at 15 min, 30 min, 1, 2, 4, 8, 24 h after administration in the oral group into heparinized Eppendorf tubes, and were temporarily stored on ice until centrifugation. After centrifugation of the whole blood at 8000 rpm for 6 min, the plasma was collected and transferred to a 96-well plate, and was stored at -20°C until LC-MS / MS detection.

[0184] The pharmacokinetic parameters after administration to mice were calculated by non-compartment model using WinNonlin software.

[0185] Peak concentration C max : measured value;

[0186] Area under the concentration-time curve AUC0-t value: calculated by trapezoidal rule; AUC 0-∞ = AUC 0-t + Ct / ke, Ct is the blood concentration at the last measurable time point, and ke is the elimination rate constant;

[0187] Elimination half-life t 1 / 2 = 0.693 / ke;

[0188] Absolute bioavailability F = Dose iv AUC 0-t，ig / Dose ig AUC 0-t，iv x 100%.

[0189] The results are shown in Table 2.

[0190] Table 2: Pharmacokinetic properties of compound 6 in mice

[0191]

[0192]

[0193] As can be seen from Table 2, after intragastrical administration to mice, compound 6 has high blood peak concentration and exposure, ideal bioavailability, and good overall pharmacokinetic properties.

[0194] Test Example 3: Anti-tumor activity of the compound of the application in a mouse subcutaneous tumor model

[0195] The pharmacodynamic effect of the compound provided in the application was evaluated by a standard murine model of transplanted tumors. After culture and collection, the mouse colon cancer cells MC38 were subcutaneously inoculated into the back of 5-6-week-old female mice (C57BL / 6, Shanghai Jihui Experimental Animal Co., Ltd.). When the tumor volume reached about 100 mm3 At the same time, the animals were randomly divided into a solvent control group (70% PEG-400 aqueous solution) and a dosing group (6 animals in each group). Subsequently, the animals were intraperitoneally injected or orally administered with the compound of the example or the antibody in combination with the compound of the example (corresponding doses, the compound dissolved in 70% PEG-400 aqueous solution and the antibody dissolved in PBS solution) at any time from 0 to 7 days after inoculation of tumor cells, and once or twice a day during the experiment.

[0196] The experimental index was to investigate the effect of the compound of the example on tumor growth, and the specific index was T / C% or tumor inhibition rate TGI (%).

[0197] The tumor diameter was measured twice a week with a vernier caliper, and the tumor volume (V) was calculated according to the following formula:

[0198] V = 1 / 2 x a x b 2 ; wherein a and b represent the length and width, respectively.

[0199] T / C (%) = (T - T0) / (C - C0) x 100; wherein T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0200] Tumor inhibition rate (TGI) (%) = 100 - T / C (%).

[0201] When the tumor regressed, the tumor inhibition rate (TGI) (%) = 100 - (T - T0) / T0 x 100

[0202] If the tumor volume was smaller than the initial volume, i.e. T < T0 or C < C0, it was defined as partial regression of tumor (PR); if the tumor completely disappeared, it was defined as complete regression of tumor (CR).

[0203] The comparison between the tumor volumes of the two groups was performed by two-tailed Student's t-test, and P < 0.05 was defined as statistically significant difference.

[0204] The results are shown in Table 3.

[0205] Table 3 In vivo tumor inhibition effect of compound 6

[0206]

[0207] As can be seen from Table 3, the tumor inhibition effect of compound 6 alone was not obvious (tumor inhibition rate 9%), the PD-1 antibody alone had a certain tumor inhibition effect (tumor inhibition rate 45%), and the combination of compound 6 and PD-1 antibody produced obvious synergistic effect, and the tumor inhibition rate increased to 75%.

[0208] The compound having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof provided by the present application exhibits excellent inhibitory activity on HPK1 kinase, good selectivity on GLK kinase, and good pharmacokinetic properties and in vivo efficacy. More specifically, for example, compound 23 has excellent HPK1 kinase inhibitory activity (IC 50 less than 1 nM) and good GLK kinase selectivity (IC 50 more than 100 nM).

[0209] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as the disclosed content of the present application, and all fall within the protection scope of the present application.

Claims

1. A macrocyclic compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound has the structure shown in formula (I): The compound is selected from any one of the following compounds:

2. A method for preparing the macrocyclic compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The method comprises: contacting a compound represented by formula (A) with a compound represented by formula (B), Wherein, in formula (A), R 11 、R 12 、R 13 、R 14 The definition is the same as that in claim 1; X is Br; In formula (B), the definition of R2 is the same as that in claim 1; and B is a boron element.

3. Use of the macrocyclic compound having the structure represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in combination with a PD-1 antibody in the preparation of a medicament for preventing and / or treating colon cancer.

4. A pharmaceutical composition comprising an active ingredient and excipients, wherein the active ingredient comprises the macrocyclic compound having a structure represented by formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, wherein The content of the active ingredient is 1-99.9% by weight.

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