NSD3 inhibitors and preparation methods and applications thereof

Abstract

The present invention discloses a NSD3 inhibitor and a preparation method and application thereof. The present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof. The compound of the present invention can have good NSD3 inhibition and good NSD3 / NSD2 selectivity, which is of great significance for targeted cancer therapy.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a NSD3 inhibitor and a preparation method and application thereof. Background Art

[0002] Histone methylation is one of the most important epigenetic modification mechanisms. Histone methylation and its regulator, histone methyltransferase, abnormal expression is closely related to the occurrence of genetic diseases, autoimmune diseases, aging, cancer and other diseases, especially in the occurrence and development of tumors. Nuclear receptor-binding SET domain (NSD) belongs to a histone lysine methyltransferase (HKMTase), which is divided into three categories: NSD1, NSD2 and NSD3. NSD protein mainly uses S-adenosyl-L-methionine (SAM) as a methyl donor to promote target gene transcription by methylating lysine 36 of histone H3 (H3K36) (Nat Rev Mol Cell Biol. 2012; 13(2): 115–126., Epigenomics. 2015; 7(5): 863–874. and Proc Natl Acad Sci US A. 2010; 107(39): 16952–16957.).

[0003] NSD3 belongs to the NSD family. NSD3 was identified in 2001 by Genomics.2001;74(1):79–88. and Genomics.2001;76(1–3):5–8. From a structural point of view, NSD3 has three subtypes: long, short and whistle.

[0004] 1) The NSD3-long phenotype contains two PWWP (the conserved motif Pro-Trp-Trp-Pro) domains, five PHD (finger plant homeodomain) fingers domains and a SET-related domain. SET, as the catalytic center of NSD3-long, is crucial for its targeted methylation of histones H3 and H4 (J Biomol Screen. 2014; 19(6): 928-935. and BMC Struct Biol. 2014; 14: 25.).

[0005] 2) NSD3-short contains only one PWWP domain, which is necessary for binding to H3K36.4 (PLoS One. 2011; 6(6): e18919.).

[0006] 3) NSD3-Whistle, the shortest isoform, also contains a PWWP domain and a SET-related domain. NSD3-whistle plays an important role as a transcriptional repressor by promoting the dimethylation of H3K4 and H3K27 (Cell. 2010; 142(6): 967–980. and Biochem Biophys Res Commun. 2006; 345(1): 318–323.).

[0007] Multiple studies (J Med Chem. 2021; 64 (20): 14913-14929.) have shown that NSD3 methyltransferase is mutated, amplified, and overexpressed in a variety of human cancers, including multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, breast cancer, prostate cancer, and lung cancer, and is a driver of a variety of cancers. In breast cancer, in lung cancer, Proc Natl Acad Sci US A. 2005; 102 (27): 9625-9630. Using high-resolution gene expression profiling, NSD3 was shown to be an important target for 8p11-12 amplification in non-small cell lung cancer (NSCLC). A recent study (Nature. 2021; 590 (7846): 504-508.) showed that NSD3 is the main driver in about 20% of lung squamous cell carcinoma cells. Although NSD3 is an important therapeutic target for cancer, there are few reports of high-affinity NSD3 inhibitors targeting the SET domain on the market due to the unique autoinhibitory loop on the SET domain that can prevent drug binding (Protein Sci. 2016; 25(5): 1021-1029). In addition, due to the high conservation of the SET catalytic domain in different histone lysine methyltransferases, it is difficult to achieve ligand selectivity. Therefore, the discovery of novel, highly active and selective NSD3 inhibitors is of great significance for targeted cancer therapy. Summary of the invention

[0008] The present invention provides a NSD3 inhibitor and a preparation method and application thereof. The compound of the present invention can have good NSD3 inhibition and good NSD3 / NSD2 selectivity, which is of great significance for cancer targeted therapy.

[0009] The present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof,

[0010]

[0011] in,

[0012] R 1are independently deuterium, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy or C3-C 10 Cycloalkyl; wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted by one or more R 1-1 replace;

[0013] R 1-1 are independently halogen, C1-C6 alkyl or C1-C6 alkoxy;

[0014] n is 0, 1, 2 or 3;

[0015] A is N or CR 2 , where R 2 is H, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 3-12 membered heterocycloalkyl;

[0016] B is CH or N;

[0017] R 3 for or one or more R 3-1 Substituted C1-C6 alkyl;

[0018] Each R 4 , R 5 and R 6 are independently H, C1-C6 alkyl or C3-C 10 Cycloalkyl; wherein the C1-C6 alkyl is optionally substituted by one or more R 4-1 replace;

[0019] R 7 is a 3-12 membered heterocycloalkyl group; wherein the 3-12 membered heterocycloalkyl group is optionally substituted by one or more halogens, C1-C6 alkyl groups or C1-C6 alkoxy groups;

[0020] Each R 3-1 and R 4-1 are independently halogen, hydroxy, C1-C6 alkoxy, -N(R 8 2. C3-C 10 Cycloalkyl, 3-12 membered heterocycloalkyl or 4-10 membered heteroaryl; wherein C3-C 10 Cycloalkyl, 3-12 membered heterocycloalkyl and 4-10 membered heteroaryl are optionally substituted with one or more R 3-2 replace;

[0021] R 3-2 are independently halogen, -N(R 8 )2, hydroxy, C1-C6 alkyl or C1-C6 alkoxy;

[0022] R 8 are independently H or C1-C6 alkyl;

[0023] The chirality at “*” is R, S or a mixture thereof;

[0024] The compound of formula I satisfies one or two of the following conditions:

[0025] (1) A is CR 2 :

[0026] (2) B is CH.

[0027] In one embodiment, the halogen is independently fluorine, chlorine, bromine or iodine.

[0028] In a certain embodiment, the C1-C6 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0029] In a certain embodiment, the C1-C6 alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy, preferably methoxy.

[0030] In one embodiment, the C3-C 10 Cycloalkyl is C3-C7 cycloalkyl, for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0031] In one embodiment, the 3-12 membered heterocycloalkyl is a 5-10 membered heterocycloalkyl, such as morpholinyl, piperazinyl, piperidinyl, tetrahydropyrrole, tetrahydrofuranyl or

[0032] In one embodiment, the heteroatom in the 3-12 membered heterocycloalkyl group is N, O or S; the number of the heteroatoms is 1, 2 or 3.

[0033] In a certain embodiment, the 3-12 membered heterocycloalkyl is a monocyclic or polycyclic ring, and the polycyclic ring may be a bridged ring, a fused ring or a spiro ring; the polycyclic ring may be a bicyclic ring or a tricyclic ring; preferably, each 3-12 membered heterocycloalkyl is independently a 5-6 membered monocyclic heterocycloalkyl or an 8-10 membered bicyclic heterocycloalkyl.

[0034] In one embodiment, the 4-10 membered heteroaryl is a 5-6 membered heteroaryl.

[0035] In one embodiment, the heteroatom in the 4-10 membered heteroaryl group is N, O or S; the number of the heteroatoms is 1, 2 or 3.

[0036] In one embodiment, n is 1 or 2.

[0037] In one scenario, R 1are independently halogen, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted by one or more R 1-1 replace;

[0038] Preferably, R 1 independently halogen, C1-C4 alkoxy; wherein the C1-C4 alkoxy is optionally substituted by one or more R 1-1 replace.

[0039] In one scenario, R 1 The substitution position is the meta or para position of the benzene ring; when R 1 When the substitution position is the meta position of the benzene ring, R 1 is a halogen; when R 1 When the substitution position is the para position of the benzene ring, R 1 is C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted by one or more R 1-1 replace.

[0040] In one scenario, R 1-1 Optionally, it is a C1-C6 alkoxy group or a C1-C6 alkyl group, and preferably a C1-C6 alkoxy group.

[0041] In one scenario, R 3 for or one or more R 3-1 Substituted C1-C6 alkyl, preferably

[0042] In one scheme, each R 4 and R 5 are independently H or C1-C6 alkyl; wherein the C1-C6 alkyl is optionally replaced by one or more R 4-1 replace.

[0043] In one scenario, R 4-1 It is a C1-C6 alkoxy group.

[0044] In one scenario, R 7 is a 5-10 membered heterocycloalkyl group optionally substituted by one or more C1-C6 alkyl groups, preferably a 5-6 membered monocyclic heterocycloalkyl group optionally substituted by one or more C1-C6 alkyl groups or an 8-10 membered bicyclic spirocyclic heterocycloalkyl group optionally substituted by one or more C1-C6 alkyl groups, for example

[0045] In one scenario, R 7 The heterocycloalkyl group is connected to the carbonyl group via the N atom in the heterocycloalkyl group, and the heterocycloalkyl group contains at least 1 or 2 N atoms.

[0046] In one scenario, R 3-1 are independently C1-C6 alkoxy, C3-C 10 Cycloalkyl, 3-12 membered heterocycloalkyl or 4-10 membered heteroaryl; wherein C3-C 10 Cycloalkyl, 3-12 membered heterocycloalkyl and 4-10 membered heteroaryl are optionally substituted with one or more R 3-2 replace.

[0047] In one scenario, R 3-2 It is a C1-C6 alkyl group.

[0048] In one scenario, R 8 For H.

[0049] In one embodiment, the chirality at "*" is R.

[0050] In a certain scheme, when A is CR 2 When R 2 It is H or C1-C6 alkoxy, preferably C1-C6 alkoxy, further preferably C1-C3 alkoxy, such as methoxy.

[0051] In a certain scheme, when A is N, CR 2 Does not exist.

[0052] In one scenario, R 1 is F, methoxy or

[0053] In one scenario, R 3 for

[0054] In one embodiment, the compound of formula I is a compound of formula II,

[0055]

[0056] Among them, A, B, R 1 , R 4 and R 5 is defined as above.

[0057] In one embodiment, the compound of formula I is a compound of formula III-1, a compound of formula III-2 or a compound of formula III-3.

[0058]

[0059] Where R 1 , R 4 and R 5 is defined as above.

[0060] In one embodiment, the compound of formula I is selected from any one of the following compounds:

[0061]

[0062]

[0063] In one embodiment, the compound of formula I is selected from any one of the following compounds:

[0064] Compounds that elute first in the column The compound that elutes first is a compound with a relatively shorter retention time among the two isomers; preferably, a compound with a retention time of 1.392 min under the following conditions: SFC supercritical fluid chromatography; chromatographic column: Daicel CHIRALCEL IB-N, 250 mm × 30 mm ID, 10 μm; mobile phase: CO2 / MeOH [0.2% NH3] = 55 / 45; flow rate: 70 g / min;

[0065] Compounds that elute late in the column The compound with the later peak is a compound with a relatively longer retention time among the two isomers; preferably, a compound with a retention time of 1.850 min under the following conditions: SFC supercritical fluid chromatography; chromatographic column: Daicel CHIRALCEL IB-N, 250mm×30mm ID, 10μm; mobile phase: CO2 / MeOH[0.2%NH3]=55 / 45; flow rate: 70g / min.

[0066] In another aspect, the present invention also provides a compound of formula IV,

[0067]

[0068] Among them, A, B, R 1 , R 3 and n are as defined above.

[0069] In a certain embodiment, the compound of formula IV is selected from any one of the following compounds:

[0070]

[0071]

[0072] In another aspect, a method for preparing the compound of formula I as described above is also provided, comprising the following steps: in a solvent, in the presence of an acid, a compound of formula IV undergoes the following reaction to obtain a compound of formula I,

[0073]

[0074] Among them, A, B, R 1 , R 3 and n are as defined above.

[0075] The reaction conditions may be conventional conditions for such reactions in the art.

[0076] In one embodiment, the acid is one or more of hydrochloric acid, trifluoroacetic acid and hydrobromic acid.

[0077] In one embodiment, the solvent is a halogenated alkane solvent and / or an alcohol solvent.

[0078] In one embodiment, the halogenated alkane solvent is dichloromethane, chloroform or 1,2-dichloroethane, preferably dichloromethane.

[0079] In one embodiment, the alcohol solvent is methanol, ethanol or isopropanol.

[0080] In a certain embodiment, when the acid is trifluoroacetic acid, the feed ratio of the compound of formula IV to trifluoroacetic acid is 0.01 to 1 mmol / mL, preferably 0.01 to 0.1 mmol / mL.

[0081] In one embodiment, when the acid is trifluoroacetic acid, the volume ratio of the solvent to trifluoroacetic acid is (1:10) to (10:1).

[0082] In a certain embodiment, when the acid is hydrochloric acid, the hydrochloric acid is a 1,4-dioxane hydrochloric acid solution; the concentration of the hydrochloric acid 1,4-dioxane hydrochloric acid solution is 0.5 to 10 mol / L, preferably 1 to 5 mol / L, for example 4 mol / L.

[0083] In a certain embodiment, when the acid is hydrochloric acid, the molar ratio of the compound of formula IV to hydrochloric acid is 1:(10-1000), preferably 1:(100-200).

[0084] In one embodiment, the reaction temperature of the reaction is 0 to 60°C, preferably 15 to 30°C.

[0085] In a certain embodiment, the reaction time of the reaction is such that no product is generated, preferably 2 to 24 hours, more preferably 10 to 20 hours.

[0086] In one embodiment, the reaction further comprises the following post-treatment steps: concentration, alkalization (preferably alkalization with saturated NaHCO3 solution or ammonia-methanol solution), extraction with an organic solvent (preferably ethyl acetate), concentration, and purification by chromatography (MeOH / DCM) to obtain a compound of formula I.

[0087] On the other hand, the present invention also provides a pharmaceutical composition, which comprises the compound of formula I or a pharmaceutically acceptable salt thereof as described above, and at least one pharmaceutical excipient.

[0088] On the other hand, the present invention also provides a use of the compound of formula I or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above in the preparation of a drug for treating tumors.

[0089] In a certain embodiment, the tumor is preferably lung cancer, breast cancer, pancreatic cancer, bladder cancer, head and neck squamous cell carcinoma, pelvic high-grade serous carcinoma, osteosarcoma, acute myeloid leukemia or acute granulocytic leukemia.

[0090] On the other hand, the present invention also provides a use of the compound of formula I or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above in the preparation of a medicament for preventing and / or treating NSD3-related diseases.

[0091] In a certain embodiment, the NSD3-related disease is a tumor, such as lung cancer, preferably lung squamous cell carcinoma.

[0092] On the other hand, the present invention also provides a use of the compound of formula I or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as described above, in the preparation of an NSD3 inhibitor. In the application, the NSD3 inhibitor can be used in mammals; it can also be used in vitro, mainly for experimental purposes, such as providing a standard sample or a control sample for comparison, or preparing a kit according to conventional methods in the art to provide a rapid test for the effect of inhibiting NSD3.

[0093] the term

[0094] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of the present invention, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and coupled with each other. The group definitions and compound structures after such combinations and couplings shall fall within the scope of the description of the present invention.

[0095] Certain chemical groups defined herein are preceded by a simplified symbol to indicate the total number of carbon atoms present in the group. For example, a C1-C6 alkyl or C 1-6 Alkyl refers to an alkyl group as defined below having a total of 1, 2, 3, 4, 5 or 6 carbon atoms.

[0096] As used herein, numerical ranges such as 0 to 10, 1-6, 1-3, etc., defined in a substituent group indicate that the integers within the range, such as 1-6, are 1, 2, 3, 4, 5, or 6.

[0097] The term "plurality" refers to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably 2, 3 or 4.

[0098] The term "alkoxy" refers to a group R X -O-, R X The same definition as the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like.

[0099] The term "halogen" refers to F, Cl, Br, I.

[0100] The term "alkyl" refers to a linear or branched, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6 or C1-C4). Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0101] The term "cycloalkyl" refers to a cycloalkyl radical having a specified number of carbon atoms (e.g., C3 to C 10 or C3-C7), cyclic, saturated, monovalent hydrocarbon group, which is a monocyclic ring. Cycloalkyl includes but is not limited to: wait.

[0102] The term "heterocycloalkyl" refers to a cyclic, saturated, monovalent group having a specified number of ring atoms (e.g., 3 to 12 members or 5 to 10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), a specified heteroatom type (one or more of N, O, and S), which is a monocyclic or polycyclic ring, the polycyclic ring may be a bridged ring, a cyclic ring, or a spirocyclic ring, the polycyclic ring may be a bicyclic or tricyclic ring. Heterocycloalkyl is connected to the rest of the molecule through a carbon atom or a heteroatom. Heterocycloalkyl includes, but is not limited to: wait.

[0103] The term "heteroaryl" refers to a cyclic, unsaturated, monovalent group having a specified number of ring atoms (e.g., 4-10 or 5-6 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatoms (one or more of N, O, and S), which is monocyclic or polycyclic. The heteroaryl group is attached to the rest of the molecule through a carbon atom or a heteroatom. Heteroaryl includes, but is not limited to, pyridyl, thiazolyl, oxazolyl, thienyl, pyrazolyl, indolyl, pyrrolyl, quinolyl, etc.

[0104] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than active pharmaceutical ingredients, which are generally divided into two categories: excipients and additives. For details, please refer to the "Pharmacopoeia of the People's Republic of China (2020 Edition)" and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0105] The term "treating" refers to removing the cause or alleviating the symptoms.

[0106] The term "prevention" refers to reducing the risk of developing a disease.

[0107] The term "patient" refers to any animal, usually a mammal, such as a human, that needs to be treated or prevented. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0108] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0109] The reagents and raw materials used in the present invention are commercially available.

[0110] The positive and progressive effect of the present invention is that the compounds of the present invention have good NSD3 inhibition and good NSD3 / NSD2 selectivity, which is of great significance for targeted cancer therapy. DETAILED DESCRIPTION

[0111] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0112] In this disclosure, 1H NMR spectra were measured using a Bruker nuclear magnetic resonance instrument, using CD3OD containing 0.1% TMS as solvent; or CDCl3 containing 0.1% TMS as solvent; or DMSO-d6 containing 0.03% TMS as solvent; LRMS spectra were measured on an ABTriple 4600 mass spectrometer, HPLC preparation was measured on a SHIMADZU LC-20AP instrument, and HPLC purity was measured on a SHIMADZU LC-30AP or Waters 1525 instrument; the product was separated by column chromatography (silica gel: 200-300 mesh or C18 reverse phase column (100 g). All reactions were carried out under air atmosphere unless otherwise specified; the reactions were tracked by TLC or LC-MS.

[0113] Solvents and reagents were handled as follows:

[0114] Preparation-grade acetonitrile and deionized water were used for HPLC preparation; other reagents and drugs were purchased from the manufacturers and used directly unless otherwise specified.

[0115] Boc tert-Butyloxycarbonyl

[0116] Con. Concentration

[0117] DCM Dichloromethane

[0118] DME 1,2-Dichloroethane

[0119] DMF N,N-dimethyl formamide N,N-dimethylformamide

[0120] DMSO Dimethylsulfoxide

[0121] DIPEA N,N-Diisopropylethylamine

[0122] EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0123] ESI Electrospray ionization

[0124] equiv equivalent

[0125] EtOH

[0126] HATU Bis(dimethylamino)methylene-triazolo[4,5-b]pyridine 3-oxide, hexafluorophosphate

[0127] HOAT N-Hydroxy-7-azabenzotriazole

[0128] HPLC High Performance Liquid Chromatography

[0129] LC-MS Liquid chromatography-mass spectrometry

[0130] LRMS Low Resolution Mass Spectrometer

[0131] LC Liquid Chromatography

[0132] Me Methyl

[0133] MeCN Acetonitrile

[0134] MeOH Methanol

[0135] MS

[0136] MW Microwave

[0137] NMM N-Methylmorpholine

[0138] NMP N-Methylpyrrolidone

[0139] PMB p-Methoxybenzyl

[0140] 1 H NMR Nuclear Magnetic Resonance Spectroscopy

[0141] rt room temperature

[0142] TFA Trifluoroacetic acid

[0143] THF Tetrahydrofuran

[0144] TLC Thin-Layer chromatography

[0145] TMS Trimethylsilane

[0146] Intermediates

[0147] Intermediate A: 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)benzoic acid methyl ester

[0148]

[0149] Step 1: 1-benzyl-3-{[(tert-butoxy)carbonyl]amino}piperidine-3-carboxylic acid (3.0 g, 9.0 mmol), dimethylamine hydrochloride (1.47 g, 18.0 mmol), HATU (6.84 g, 18.0 mmol) and DIPEA (3.49 g, 27.0 mmol) were added to a 100 mL round-bottom flask containing DMF (40 mL) at room temperature, and the reaction mixture was stirred at room temperature for 1 hour. The reaction solution was vacuum dried, diluted with water (20 mL), and the filtrate was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 0-80 / 20), and the solvent was removed by vacuum concentration to obtain tert-butyl (1-benzyl-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate as a white solid (2 g, yield 56%). MS m / z (ESI): 362.2 [M+H] +

[0150] Step 2: Add N-[1-benzyl-3-(dimethylcarbamoyl)piperidin-3-yl]carbamic acid tert-butyl ester (2g, 5.5mmol) and MeOH (20mL) to a 50mL three-necked flask, and add 10% wet palladium carbon (590mg, 0.55mmol) at room temperature. The reaction solution was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth and rinsed with methanol (3mL x 3). The filtrates were combined and concentrated in vacuo to give a crude product. The solvent was removed by vacuum concentration to give: tert-butyl (3-(dimethylcarbamoyl)piperidin-3-yl)carbamate as a white solid product (1.7g, yield 96%). MS m / z(ESI):272.2[M+H] +

[0151] Step 3: tert-butyl (3-(dimethylcarbamoyl)piperidin-3-yl)carbamate 1.2 g, 4.4 mmol), methyl 2-fluoro-5-(3-fluorophenyl)benzoate (2.05 g, 8.8 mmol) and DIPEA (2.84 g, 22 mmol) were added to a 100 mL round-bottom flask containing DMF (10 mL) at room temperature, and the reaction mixture was stirred at 100 ° C for 20 hours. LCMS detection showed that it contained 80% of the product. The reaction solution was vacuum dried, diluted with water (20 mL), and the filtrate was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 0-80 / 20), and the solvent was removed by vacuum concentration to obtain intermediate A: methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)benzoate as a white solid (1.0 g, yield 38.64%). MS m / z (ESI): 484.2 [M+H] + Intermediate B: methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)benzoate

[0152]

[0153] Step 1: Compound 7-bromo-4-chloroimidazopyridine (2.5 g, 10.8 mmol, 1 eq) was dissolved in (4-methoxyphenyl)methylamine (9.34 g, 53.8 mmol, 5 mL), and then stirred at 120 ° C for 16 hours. The reaction solution was filtered and dried. The crude product was separated by chromatography column (120 g, 0-78.5%, dichloromethane / ethyl acetate, 100 ml / min). Compound 7-bromo-N-methoxybenzyl-4-aminopyridine (3.3 g, yield: 92.1%) was obtained as a brown solid.

[0154] 1 H NMR (400MHz, CDCl3) δppm 10.41(s,1H),7.96(s,1H),7.84(s,1H),7.30-7.27(m,2H),6.87-6.82(m,1H),5.74(t,J=5.3Hz,1H),4.71(d,J=5.3Hz,2H),3.77(s,3H).

[0155] MS m / z(ESI):331.3[M+H] +

[0156] Step 2: 7-bromo-N-methoxybenzyl-4-aminopyridine (3.3 g, 9.9 mmol) and 4-dimethylaminopyridine (122 mg, 0.1 mmol), di-tert-butyl dicarbonate (6.48 g, 29.7 mmol, 6.83 mL) were dissolved in tetrahydrofuran (100 mL), stirred and reacted at 20 ° C under nitrogen protection for 16 hours. The reaction solution was diluted with 100 ml of ethyl acetate and washed three times with 100 ml of water, washed once with 100 ml of saturated ammonium chloride, and washed once with 100 ml of brine, dried over anhydrous sodium sulfate, filtered, and dried. The crude product was separated by chromatography column (40 g, 0-57% ethyl acetate / petroleum ether, 40 ml / min). 7-Bromo-4-[(4-methoxyphenyl)methylamino]imidazolium tert-butyl[4,5-c]pyridine-1-carboxylate (2.5 g, yield: 58.3%) was obtained as a light yellow solid.

[0157] 1 H NMR (400MHz, CDCl3) δppm 10.41(s,1H),7.96(s,1H),7.83(s,1H),7.32-7.27(m,2H),6.87-6.82(m,2H),5.73(t,J=5.6Hz,1H),4.71(d,J=5.4Hz,2H),3.80(s,3H).

[0158] MS m / z(ESI):435.4[M+H] +

[0159] Step 3: 7-bromo-4-[(4-methoxyphenyl)methylamino]imidazol tert-butyl ester [4,5-c]pyridine-1-carboxylate (600mg, 1.38mmol), and cuprous bromide (260mg, 1.81mmol) were dissolved in a methanol solution of sodium methoxide (5M, 12.00mL) and dimethylacetamide (12mL), and the nitrogen was replaced three times and stirred at 140°C for 2 hours. After cooling, it was diluted with water (50mL), extracted with ethyl acetate three times (50mL x 3), washed once with saturated brine (100mL), dried, filtered, and after spin drying, the crude product was separated by a chromatography column (12g, 0-100% ethyl acetate / petroleum ether, 30ml / min). The product 7-methoxy-N-(4-methoxyphenyl)methyl-4-aminopyridine (50mg, yield: 12.7%) was obtained as a white solid. 1H NMR (400MHz, CDCl3) δppm 7.87(s,1H),7.53(s,1H),7.34(d,J=8.6Hz,2H),6.85(d,J=8.6Hz,2H),5.52(s,1H),4.71(d,J=1.6Hz,2H),3.91(s,3H),3.79(s,3H).MS m / z(ESI):285.1[M+H] +

[0160] Example

[0161] Example 1: 3-amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide

[0162]

[0163] Step 1: At room temperature, tert-butyl N-{1-[2-(chloromethyl)-4-(3-fluorophenyl)phenyl]-3-(methylcarbamoyl)piperidin-3-yl}carbamate (50 mg, 0.11 mmol) and tert-butyl (tert-butyloxycarbonyl)(9H-purine-6-yl)carbamate (43 mg, 0.13 mmol) were dissolved in DMF (4 mL), and K2CO3 (45.5 mg, 0.33 mmol) was added. The mixture was heated at room temperature for 16 hours. The reaction solution was slowly added to 15 mL of water, extracted with ethyl acetate (15 mL x 2), and the organic phases were combined and washed with water (10 mL) and saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound tert-butyl(tert-butoxycarbonyl)(9-((4-(3-(tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-yl)methyl)-9H-purin-6-yl)carbamate (35 mg, yield 41%) was obtained as a light yellow solid by chromatography column separation (4 g, 0-50% EtOAc / PE, 20 mL / min). MS m / z (ESI): 775.3 [M+H] +

[0164] Step 2: At room temperature, tert-butyl (tert-butoxycarbonyl) (9-((4-(3-(tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-yl)methyl)-9H-purin-6-yl)carbamate (35 mg, 0.045 mmol) was dissolved in DCM (3 mL), and then TFA (1.0 mL, 13.5 mol) was added and stirred at 25 ° C for 16 hours. The reaction solution was concentrated, saturated Na2CO3 solution (10 mL) was added, and dichloromethane was extracted (10 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Prep-TLC separation (12% MeOH / DCM) gave compound E1: 3-amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N-methylpiperidine-3-carboxamide (11 mg, yield 51.5%) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ8.20(s,1H),8.10(s,1H),8.08(s,1H),7.55(dd,J=10.0,5.0Hz,1H),7.40-7.36(m,1H),7.32-7.19(m,6H),7 .09-7.05(m,1H),5.45(s,2H),3.09(d,J=10.0Hz,1H),2.78-2.69(m,2H),2.61-2.57(m,4H),1.95-1.77(m,2H)1.86-1.48(m,2H).MS m / z(ESI):475.2[M+H] +

[0165] Example 2: 3-amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidin-3-carboxylate

[0166]

[0167] Step 1: At room temperature, tert-butyl N-[3-(dimethylcarbamoyl)piperidin-3-yl]carbamate (500mg, 1.84mmol), methyl 2-fluoro-5-(3-fluorophenyl)benzoate (457mg, 1.84mmol) and K2CO3 (764mg, 5.53mmol) were added to a 100mL round-bottom flask containing DMF (10mL) in sequence, and the reaction mixture was stirred at 100°C for 16 hours. The reaction solution was vacuum dried, diluted with water (20mL), and the filtrate was extracted with ethyl acetate (50mL x 3). The combined organic phase was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 0-80 / 20), and the solvent was removed by vacuum concentration to obtain 4-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-carboxylic acid methyl ester as a white solid (420 mg, yield 41%). MS m / z (ESI): 500.2 [M+H] +

[0168] Step 2: At room temperature, methyl 2-(3-{[(tert-butoxy)carbonyl]amino}-3-(dimethylcarbamoyl)piperidin-1-yl)-5-(3-fluorophenyl)benzoate (400 mg, 0.80 mmol) and LiCl (407 mg, 9.61 mmol) were dissolved in THF (4 mL) and MeOH (4 mL) solution, and NaBH4 (757 mg, 20.02 mmol) was added, and a large number of bubbles were generated. The reaction solution was stirred at room temperature for 16 hours. Under an ice bath, H2O was added dropwise to the reaction solution to quench, diluted with 30 mL of water, and extracted with ethyl acetate (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (EtOAc / PE=0-90%) to give tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate as a colorless viscous oil (300 mg, yield 79%). MS m / z (ESI): 472.2 [M+H] +

[0169] Step 3: Under ice bath, add thionyl chloride (98.40 mg, 0.827 mmol) to tert-butyl N-[3-(dimethylcarbamoyl)-1-[4-(3-fluorophenyl)-2-(hydroxymethyl)phenyl]piperidin-3-yl]carbamate (300 mg, 0.636 mmol) in anhydrous DCM (5.0 mL). The reaction solution was stirred at room temperature for 1 hour. The reaction solution was diluted with DCM (30 mL), washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give E2: tert-butyl (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate as a white solid (250 mg, yield: 72%).

[0170] MS m / z(ESI):486.2[M+H] + (Methanol sample, methoxyl MS)

[0171] Step 4: At room temperature, tert-butyl N-{1-[2-(chloromethyl)-4-(3-fluorophenyl)phenyl]-3-(dimethylcarbamoyl)piperidin-3-yl}carbamate (150 mg, 0.306 mmol) and tert-butyl (tert-butyloxycarbonyl) (9H-purine-6-yl)carbamate (103.3 mg, 0.306 mmol) were dissolved in DMF (4 mL), and K2CO3 (84.6 mg, 0.612 mmol) was then added. The mixture was heated at room temperature for 16 hours. The reaction solution was slowly added to 20 mL of water, extracted with ethyl acetate (20 mL x 2), and the organic phase was combined and washed with water (30 mL x 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound tert-butyl(tert-butoxycarbonyl)(9-((4-(3-(tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-yl)methyl)-9H-purin-6-yl)carbamate (75 mg, yield 31%) was obtained as a light yellow solid by chromatography column separation (4 g, 0-50% EtOAc / PE, 20 mL / min). MS m / z (ESI): 789.3 [M+H] +

[0172] Step 5: Tert-butyl (tert-butoxycarbonyl) (9-((4-(3-(tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-yl)methyl)-9H-purin-6-yl)carbamate (75 mg, 0.0948 mmol) was dissolved in HCl in 1,4-dioxane (4N, 3 mL) at room temperature and then stirred at 25 ° C for 16 hours. The reaction solution was concentrated, saturated NaHCO3 solution (10 mL) was added, and ethyl acetate was extracted (10 mL x 4). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Prep-TLC separation (12% MeOH / DCM) gave compound E2: 3-amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide (26.34 mg, yield 56.86%) as a white solid.

[0173] 1 H NMR(400MHz, DMSO-d6)δ8.23(s,1H),8.13(s,1H),7.63(dd,J=8.3,2.2Hz,1H),7.49-7.40(m,1H),7.35-7.27(m,4H),7.23(s,2H),7.13(t d,J=8.6,2.1Hz,1H),5.60-5.45(m,2H),3.13(d,J=11.3Hz,2H),2.95-2.81(m,3H),2.66-2.54(m,2H),2.35(s,2H),1.98-1.58(m,5H).MS m / z(ESI):489.2[M+H] +

[0174] Example 3: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N-methylpiperidine-3-carboxamide

[0175]

[0176] Step 1: Dissolve tert-butyl (3-(methylcarbamoyl)piperidin-3-yl)carbamate (5.0 g, 19.43 mmol) and methyl (5-bromo-2-fluorobenzoate (5.43 g, 23.32 mmol) in DMF (50 mL) and DIPEA (7.62 g, 58.29 mmol), purge with nitrogen three times, and react at 100 ° C for 16 hours. LCMS showed that the starting material had been consumed and the product appeared. DMF (50 mL) was spin-dried, diluted with 50 mL of water, extracted with ethyl acetate (50 mL x 3), and the organic phases were combined and washed with saturated brine (50 mL x 3). 3), then dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by chromatography (EtOAc / PE=0-30%) to give methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)benzoate (1.1 g, yield: 12.04%) as a white solid. MS m / z (ESI): 470.4 [M+H] +

[0177] Step 2: At room temperature, methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)benzoate (870 mg, 1.85 mmol), (3-fluorophenyl)boronic acid (310 mg, 2.22 mmol) and potassium carbonate (511 mg, 3.70 mmol) were added to 1,4-dioxane (5 mL) and water (2 mL), and Pd(dppf)Cl2 (270 mg, 0.37 mmol) was added. After nitrogen was replaced three times in the reaction flask, the temperature was raised to 80 ° C and stirred for 2 h. The reaction solution was cooled to room temperature and filtered through celite. The filtrate was diluted with water (80 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (EtOAc / PE=0-3%) to give methyl 4-(3-((tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-carboxylate (820 mg, yield: 82%) as a white solid. MS m / z (ESI): 486.2 [M+H] +

[0178] Step 3: At room temperature, 4-(3-((tert-butoxycarbonyl)amino)-3-(methylcarbamoyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-carboxylic acid methyl ester (800mg, 0.80mmol) and LiCl (838.10mg, 19.77mmol) were dissolved in THF (8mL) and MeOH (8mL) solution, and NaBH4 (1558.22mg, 41.19mmol) was added, and a large number of bubbles were generated. The reaction solution was stirred at room temperature for 16h. In an ice bath, H2O was added dropwise to the reaction solution to quench, diluted with 60mL of water, and extracted with ethyl acetate (60mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (EtOAc / PE=0-90%) to give tert-butyl (1-(3'-fluoro-3-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (560 mg, yield: 74.3%) as a white solid. MS m / z (ESI): 458.2 [M+H] +

[0179] Step 4: Under ice bath, add thionyl chloride (189.29 mg, 1.5910 mmol) to tert-butyl (1-(3'-fluoro-3-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (560 mg, 1.2239 mmol) in anhydrous DCM (10 mL). The reaction solution was stirred at room temperature for 1 h. The reaction solution was diluted with DCM (30 mL), washed with saturated sodium bicarbonate (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give tert-butyl (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (480 mg) as a white solid, which was used directly in the next step without further purification. MS m / z(ESI):472.1[M+H] + (Methanol sample, methoxyl MS)

[0180] Step 5: 7-methoxy-N-(4-methoxyphenyl)methyl-4-aminopyridine (80 mg, 0.2177 mmol), tert-butyl (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (103.62 mg, 0.22 mmol) and potassium carbonate (90.26 mg, 0.65 mmol) were added to DMF (2 mL) at room temperature. The reaction solution was stirred at room temperature for 16 h. The reaction was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was separated and purified by chromatography (EtOAc / PE=0-90%) to give: tert-butyl (1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-)[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (100 mg, yield: 56.91%) as a white solid. MS m / z (ESI): 724.3 [M+H] +

[0181] Step 6: Tert-butyl (1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-)[1,1'-biphenyl]-4-yl)-3-(methylcarbamoyl)piperidin-3-yl)carbamate (90 mg, 0.12 mmol) was dissolved in TFA (1.5 mL) and dichloromethane (1.5 mL) and reacted at room temperature for 16 hours. The reaction solution was spin dried. The crude product was purified by prep-TLC (0-10% ethyl acetate / petroleum ether) to give E3: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N-methylpiperidine-3-carboxamide (24.30 mg, yield: 40.2%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ8.17(s,1H),8.08(d,J=4.5Hz,1H),7.58(dd,J=8.3,1.9Hz,1H),7.40(dd,J=15.1,7.3 Hz,1H),7.34(s,1H),7.28(d,J=8.3Hz,1H),7.18(t,J=9.5Hz,2H),7.11(dd,J=12.5,4.5Hz,1H),6.80(s,1H),5 .78(s,2H),5.77-5.64(m,2H),3.64(s,3H),3.15(d,J=11.2Hz,1H),2.96(d,J=10.9Hz,1H),2.77(d,J=11.1Hz ,1H),2.68(d,J=9.9Hz,1H),2.64(d,J=4.6Hz,3H),1.99(t,J=10.2Hz,2H),1.68(s,1H),1.54(d,J=9.5Hz,1H). 19 F NMR(377MHz,DMSO-d6)δ-112.70(s,1H).MS m / z(ESI):504.2[M+H] +

[0182] Example 4: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide

[0183]

[0184] Step 1: 7-methoxy-N-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine (60 mg, 0.21 mmol), tert-butyl (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate (103.4 mg, 0.21 mmol) and potassium carbonate (87.5 mg, 0.63 mmol) were added to DMF (2 mL) at room temperature. The reaction solution was stirred at room temperature for 16 h. The reaction was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain the crude product. The crude product was purified by Prep-TLC (DCM:MeOH=12:1). Tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (44 mg, yield: 18.7%) was a white solid. MS m / z (ESI): 738.3 [M+H] +

[0185] Step 2: At room temperature, tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (44 mg, 0.06 mmol) was dissolved in anhydrous DCM (2 mL), and TFA (4 mL) was added. The reaction solution was stirred at room temperature for 16 h. After vacuum concentration to remove the solvent, 3 mL of ammonia-methanol solution (7 mol / L) was added for alkalization, stirred for 3 min, and vacuum concentrated to obtain a crude product. The crude product was purified by Prep-TLC (DCM:MeOH=8:1) to give E4: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide (11.09 mg, yield: 35.7%) as a white solid. 1H NMR(400MHz, DMSO-d6)δ8.17(s,1H),7.60(dd,J=8.3,1.9Hz,1H),7.44-7.35(m,3H),7.18(t,J=10.2Hz,2H),7.11(t,J=8.6Hz, 1H), 6.80 (s, 1H), 6.11 (s, 2H), 5.76 (q, J = 16.3Hz, 2H), 3.67 (s, 3H), 3.27-2.69 (m, 10H), 2.05-1.84 (m, 3H), 1.78-1.70 (m, 1H). 19 F NMR(377MHz,DMSO)δ-112.68(s,1F).MS m / z(ESI):518.3[M+H] +

[0186] Example 5: (R)-3-amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide

[0187]

[0188] 3-Amino-1-(3-((6-amino-9H-purin-9-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide (21 mg, 0.043 mmol) was separated by SFC (Column: Daicel CHIRALCEL IB-N, 250 mm×30 mm ID, 10 μm; Mobile phase: CO2 / MeOH[0.2% NH3(7M Solution in MeOH)]=55 / 45; Flow rate: 70 g / min) to give white solid isomer A (rt=1.392 min, 3.03 mg, purity 98%, yield 14.4%, ee 100%) and white solid isomer B (rt=1.850 min, 2.49 mg, purity 97%, yield 12%, ee 99%).

[0189] Isomer A:E5-1

[0190] 1H NMR (400MHz, DMSO) δ8.23(s,1H),8.13(s,1H),7.62(d,J=8.3Hz,1H),7.44(dd,J=14.4,7.7Hz,1H),7.35–7.27(m,4H),7.23(s,2H), 7.17–7.09(m,1H),5.60–5.45(m,2H),3.12(d,J=11.5Hz,2H),2.95–2.81(m,3H),2.66–2.54(m,2H),2.34(s,2H),1.99–1.57(m,5H).

[0191] MS m / z(ESI):489.3[M+H] +

[0192] Isomer B:E-5

[0193] 1 H NMR (400MHz, DMSO) δ8.23(s,1H),8.13(s,1H),7.63(dd,J=8.3,2.2Hz,1H),7.49–7.40(m,1H),7.35–7.27(m,4H),7.23(s,2H),7.13(t d,J=8.6,2.1Hz,1H),5.60–5.45(m,2H),3.13(d,J=11.3Hz,2H),2.95–2.81(m,3H),2.66–2.54(m,2H),2.35(s,2H),1.98–1.58(m,5H).

[0194] MS m / z(ESI):489.3[M+H] +

[0195] Example 6: 3-amino-1-[2-({4-amino-7-methoxyimidazole[4,5-c]pyridin-1-yl}methyl)-4-(3-fluorophenyl)phenyl]-N-(2-methoxyethyl)-N-methylpiperidine-3-carboxamide

[0196]

[0197] Step 1: Dissolve the compound 1-benzyl-3-((tert-butoxycarbonyl)amino)piperidine-3-carboxylic acid (2.2 g, 6.6 mmol) and 2-methoxy-N-methylethane-1-amine (0.76 g, 8.5 mmol) in DMF (25 mL), then add DIPEA (2.56 g, 19.7 mmol) and HATU (3.0 g, 7.9 mmol), and stir the mixture at room temperature for 1 hour. The reaction solution is slowly added to 100 mL of water, extracted with ethyl acetate (60 mL x 2), and the organic phases are combined and washed with water (100 mL x 3) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was separated by chromatography column (20 g, 0-20% EtOAc / PE, 30 mL / min) to obtain the compound tert-butyl (1-benzyl-3-((2-methoxyethyl)(methyl)carbamoyl)piperidin-3-yl)carbamate (1.05 g, yield 39.4%) as a white solid. MS m / z (ESI): 406.2 [M+H] +

[0198] Step 2: At room temperature, the compound tert-butyl (1-benzyl-3-((2-methoxyethyl)(methyl)carbamoyl)piperidin-3-yl)carbamate (1.05 g, 2.6 mmol) was dissolved in MeOH (25 mL), and Pd / C (350 mg, 10%) was added. The above mixture was stirred at room temperature for 16 hours. The reaction solution was filtered through diatomaceous earth and concentrated to give the compound tert-butyl (3-((2-methoxyethyl)(methyl)carbamoyl)piperidin-3-yl)carbamate (770 mg, yield 76.92%) as a white solid. MS m / z (ESI): 316.2 [M+H] +

[0199] Step 3: At room temperature, tert-butyl N-{3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-3-yl}carbamate (770 mg, 2.44 mmol) and methyl 2-fluoro-5-(3-fluorophenyl)benzoate (727.2 mg, 2.93 mmol) were dissolved in DMF (15 mL), and then K2CO3 (674.82 mg, 4.88 mmol) was added. The mixture was heated in an oil bath at 120°C for 16 hours. The reaction solution was slowly added to 70 mL of water, extracted with ethyl acetate (30 mL x 2), and the organic phases were combined and washed with water (50 mL x 3) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound was separated by chromatography column (20 g, 0-50% EtOAc / PE, 30 mL / min) to give methyl 2-(3-{[(tert-butoxy)carbonyl]amino}-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-1-yl)-5-(3-fluorophenyl)benzoate (740 mg, yield 50.18%) as a white solid. MS m / z (ESI): 544.3 [M+H] +

[0200] Step 4: Methyl 2-(3-{[(tert-butoxy)carbonyl]amino}-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-1-yl)-5-(3-fluorophenyl)benzoate (740 mg, 1.36 mmol) was dissolved in THF (8 mL) and MeOH (6 mL) at room temperature, cooled to 0°C in an ice bath, and then NaBH4 (1.03 g, 27.22 mmol) and LiCl (577 mg, 13.61 mmol) were added. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with saturated ammonium chloride solution, concentrated to remove MeOH and THF, 30 mL of water was added, and extracted with ethyl acetate (30 mL x 2). The organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound N-{1-[4-(3-fluorophenyl)-2-(hydroxymethyl)phenyl]-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-3-yl}carbamic acid tert-butyl ester (410 mg, yield 55.5%) was obtained by chromatography column separation (12 g, 0-50% EtOAc / PE, 30 mL / min) as a white solid. MS m / z (ESI): 516.3 [M+H] +

[0201] Step 5: tert-Butyl N-{1-[4-(3-fluorophenyl)-2-(hydroxymethyl)phenyl]-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-3-yl}carbamate (200 mg, 0388 mmol) was dissolved in anhydrous DCM (4 mL), cooled to 0°C, and a solution of SOCl2 (92.3 mg, 0.776 mmol) in DCM (1 mL) was slowly added dropwise. The mixture was then reacted at 0°C for 1 hour. DCM (20 mL) was added to the reaction solution, and saturated NaHCO3 solution (15 mL) was added under ice bath. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the compound N-{1-[2-(chloromethyl)-4-(3-fluorophenyl)phenyl]-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-3-yl}carbamic acid tert-butyl ester (207 mg, yield 89.9%) as a light yellow solid. MS m / z (ESI): 530.3 [M+H] + (in MeOH)

[0202] Step 6: At room temperature, tert-butyl N-{1-[2-(chloromethyl)-4-(3-fluorophenyl)phenyl]-3-[(2-methoxyethyl)(methyl)carbamoyl]piperidin-3-yl}carbamate (207 mg, 0.388 mmol), 7-methoxy-N-(2-methoxy-5-methylphenyl)-1H-imidazo[4,5-c]pyridin-4-amine (110.2 mg, 0.388 mmol) and K2CO3 (107.14 mg, 0.775 mmol) were dissolved in DMF (6 mL). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was slowly added to 30 mL of water, extracted with ethyl acetate (20 mL x 2), and the organic phases were combined and washed with water (40 mL x 3) and saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was separated by chromatography column (12 g, 0-60% EtOAc / PE, 30 mL / min) to give the compound tert-butyl N-{1-[4-(3-fluorophenyl)-2-({7-methoxy-4-[(2-methoxy-5-methylphenyl)amino]imidazo[4,5-c]pyridin-1-yl}methyl)phenyl]-3-[(2-methylethyl)(methyl)carbamoyl]piperidin-3-yl}carbamate (32 mg, yield 9.49%) as a white solid. 1H NMR (400MHz, DMSO) δ8.21(s,1H),7.63-7.54(m,2H),7.45-7.37(m,2H),7.30(d,J=8.4Hz,1H),7.26(d,J=8.5Hz,2H ),7.23-7.17(m,2H),7.11(t,J=7.6Hz,1H),6.97(s,1H),6.81(d,J=8.5Hz,2H),6.67-6.59(m,1H),5.73(d,J=18.0H z,1H),5.46(d,J=16.0Hz,1H),4.60-4.47(m,2H),3.71(s,3H),3.69(s,3H),3.53-3.37(m,4H),3.24-3.15(m,6H), 3.06(d,J=13.3Hz,1H),2.96(s,1H),2.88-2.82(m,1H),2.21–2.06(m,2H),1.76-1.60(m,2H),1.12-1.06(m,1H).MS m / z(ESI):782.2[M+H] +

[0203] Step 7: Tert-butyl N-{1-[4-(3-fluorophenyl)-2-({7-methoxy-4-[(2-methoxy-5-methylphenyl)amino]imidazo[4,5-c]pyridin-1-yl}methyl)phenyl]-3-[(2-methylethyl)(methyl)carbamoyl]piperidin-3-yl}carbamate (32 mg, 0.0409 mmol) was dissolved in DCM (1 mL), TFA (1 mL) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, saturated NaHCO3 solution (15 mL) was added, and ethyl acetate was extracted (20 mL x 3). The organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Prep-TLC separation (12% MeOH / DCM) gave compound E6: 3-amino-1-[2-({4-amino-7-methoxyimidazo[4,5-c]pyridin-1-yl}methyl)-4-(3-fluorophenyl)phenyl]-N-(2-methoxyethyl)-N-methylpiperidine-3-carboxamide (6.02 mg, yield 25.18%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ8.15(s,1H),7.60(d,J=6.4Hz,1H),7.41(dd,J=14.5,8.0Hz,1H),7.34(s, 1H),7.31(d,J=8.3Hz,1H),7.23-7.15(m,2H),7.13-7.07(m,1H),6.81(s,1H),5.80-5.68(m,4H),3 .64(s,3H),3.47(s,3H),3.32(s,2H),3.22(s,3H),3.15(d,J=11.8Hz,1H),2.94(d,J=10.9Hz,2H) ,2.69-2.60(m,1H),2.34-2.15(m,2H),2.02-1.87(m,1H),1.85-1.75(m,2H),1.73-1.65(m,1H).MS m / z(ESI):562.2[M+H]

[0204] Example 7: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide

[0205]

[0206] Step 1: 4-bromo-2-fluorophenol (5 g, 26.2 mmol), 1-bromo-2-methoxyethane (4.4 g, 31.4 mmol) and potassium carbonate (10.9 g, 78.6 mmol) were added to DMF (2 mL) at room temperature. The reaction solution was stirred at room temperature for 16 h. The reaction was diluted with H2O (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 4-bromo-2-fluoro-1-(2-methoxyethoxy)benzene (6.1 g, yield: 93.5%) as a light yellow oil. The crude product was not purified and used directly in the next step. MS m / z (ESI): 249.0 / 251.0 [M+H] +

[0207] Step 2: At room temperature, 4-bromo-2-fluoro-1-(2-methoxyethoxy)benzene (3 g, 12.0 mmol), B2Pin2 (4.6 g, 18.0 mmol) and potassium phosphate (7.6 g, 36.0 mmol) were added to DME (50 mL), and Pd(dppf)Cl2·DCM (0.98 g, 1.2 mmol) was added. After the reaction flask was replaced with nitrogen three times, the temperature was raised to 80 °C and stirred under nitrogen atmosphere for 16 h. The reaction solution was cooled to room temperature and filtered through diatomaceous earth. The filtrate was diluted with water (80 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by chromatography (EtOAc / PE=0-10%) to give 2-(3-fluoro-4-(2-methoxyethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.7 g, yield: 47.5%) as a white solid. 1 H NMR (400MHz, DMSO) δ7.43(d,J=8.1Hz,1H),7.33(dd,J=11.8,1.4Hz,1H),7.18(t,J=8.3Hz,1H),4.21-4.19(m,2H),3.69-3.67(m,2H),1.28(s,12H).

[0208] MS m / z(ESI):319.1[M+Na]

[0209] Step 3: At room temperature, methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)benzoate (170 mg, 0.4 mmol), 2-(3-fluoro-4-(2-methoxyethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborin (124.7 mg, 0.4 mmol) and potassium carbonate (97.0 mg, 0.7 mmol) were added to 1,4-dioxane (4 mL) and water (0.5 mL), and Pd(dppf)Cl2 (25.5 mg, 0.04 mmol) was added. After nitrogen was replaced three times in the reaction flask, the temperature was raised to 80 °C and stirred for 2 h under nitrogen atmosphere. The reaction solution was cooled to room temperature and filtered through celite. The filtrate was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a crude product. The crude product was purified by chromatography (EtOAc / PE=0-60%) to give 4-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-3-carboxylic acid methyl ester (100 mg, yield: 43.2%) as a brown viscous oil. MS m / z(ESI):574.2[M+H] +

[0210] Step 4: At room temperature, 4-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-3-carboxylic acid methyl ester (100 mg, 0.2 mmol) and lithium chloride (73.9 mg, 1.7 mmol) were dissolved in THF (1.5 mL) and MeOH (1.5 mL), and NaBH4 (164.8 mg, 4.4 mmol) was added in three batches under ice bath. The reaction solution was stirred at room temperature for 16 h. Under ice bath, water was added dropwise to the reaction solution to quench. Dilute with water (30 mL) and extract with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by Prep-TLC (100% EtOAc) to give tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-(hydroxymethyl)-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (80 mg, yield: 83.7%) as a colorless oil. MS m / z (ESI): 546.3 [M+H] +

[0211] Step 5: Dissolve tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-(hydroxymethyl)-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (90 mg, 0.2 mmol) in anhydrous dichloromethane (2 mL) at room temperature. Add thionyl chloride (25.5 mg, 0.2 mmol) dropwise under ice bath. Stir the reaction solution at room temperature for 1 h. Dilute the mixture with dichloromethane (30 mL) and add saturated sodium bicarbonate (30 mL). Extract the separated aqueous phase with dichloromethane (30 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give tert-butyl (1-(3-(chloromethyl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate (80 mg, yield: 77.4%) as a light yellow solid. The crude product was not purified and used directly in the next step.

[0212] Step 6: 7-methoxy-N-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine (40 mg, 0.14 mmol), tert-butyl (1-(3-(chloromethyl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate (79.4 mg, 0.14 mmol) and potassium carbonate (58.3 mg, 0.42 mmol) were added to DMF (2 mL) at room temperature. The reaction solution was stirred at room temperature for 16 h. The reaction was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain the crude product. The crude product was purified by Prep-TLC (100% EtOAc) to give tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (25 mg, yield: 13.8%) as a white solid. MS m / z (ESI): 812.2 [M+H] +

[0213] Step 7: Dissolve tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (125 mg, 0.15 mmol) in anhydrous DCM (1 mL) at room temperature, and then add TFA (2 mL). The reaction solution was stirred at room temperature for 16 h. After the reaction solution was concentrated in vacuo to remove the solvent, 20 mL of saturated sodium bicarbonate was added to alkalize, stirred for 3 min, and extracted with dichloromethane (15 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by Prep-TLC (DCM:MeOH=8:1) to give E7: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide (6.88 mg, yield: 37.3%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ8.14 (s, 1H), 7.53 (dd, J = 8.3, 2.1Hz, 1H), 7.34 (s, 1H), 7. 29-7.23(m,2H),7.17(t,J=8.8Hz,1H),7.07(d,J=8.6Hz,1H),6.76(s,1H),5.75(s ,2H),5.71(s,2H),4.17-4.14(m,2H),3.66-3.64(m,5H),3.29(s,3H),3.27-2.73( m,9H),2.60(t,J=10.3Hz,1H),2.24(s,2H),1.97-1.88(m,1H),1.84-1.64(m,3H). 19 F NMR(377MHz,DMSO-d6)δ-134.06(s,1F).MS m / z(ESI):592.3[M+H] +

[0214] Example 8: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide

[0215]

[0216] Step 1: A mixed solution of methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)benzoate (520 mg, 1.07 mmol), (3-fluoro-4-methoxyphenyl)boronic acid (218.92 mg, 1.29 mmol), Pd(dppf)Cl2 (77.9 mg, 0.107 mmol) and K2CO3 (296.74 mg, 2.15 mmol) in 1,4-dioxane (10 mL) and H2O (2 mL) was heated in an oil bath at 80°C for 2 hours under nitrogen protection at room temperature. The reaction solution was concentrated, added to 40 mL of water, extracted with ethyl acetate (30 mL x 2), and the organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was separated by chromatography column (20 g, 0-20% EtOAc / PE, 30 mL / min) to give compound 4-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-3-carboxylic acid methyl ester (500 mg, yield 87.95%) as a white solid. MS m / z (ESI): 530.2 [M+H] +

[0217] Step 2: At room temperature, methyl 4-(3-((tert-butoxycarbonyl)amino)-3-(dimethylcarbamoyl)piperidin-1-yl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-3-carboxylate (100 mg, 0.189 mmol) was dissolved in THF (3 mL) and MeOH (2 mL), cooled to 0°C in an ice bath, and then NaBH4 (142.85 g, 3.78 mmol) and LiCl (80 mg, 1.89 mmol) were added. The reaction solution was quenched with saturated ammonium chloride solution, concentrated to remove MeOH and THF, 20 mL of water was added, and ethyl acetate was extracted (20 mL x 2). The organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-(hydroxymethyl)-4'-methoxy-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (90 mg, yield 90.3%) was obtained by chromatography column separation (12 g, 0-50% EtOAc / PE, 30 mL / min) as a white solid. MS m / z (ESI): 502.2 [M+H]+

[0218] Step 3: tert-Butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-3-(hydroxymethyl)-4'-methoxy-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (90 mg, 0.179 mmol) was dissolved in anhydrous DCM (3 mL), cooled to 0°C, and a solution of SOCl2 (42.69 mg, 0.359 mmol) in DCM (1 mL) was slowly added dropwise. The mixture was then reacted at 0°C for 1 hour. DCM (20 mL) was added to the reaction solution, and saturated NaHCO3 solution (15 mL) was added under ice bath. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the compound tert-butyl (1-(3-(chloromethyl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate (93 mg, yield 99.7%) as a light yellow solid. MS m / z (ESI): 516.3 [M+H] + (in MeOH)

[0219] Step 4: At room temperature, tert-butyl (1-(3-(chloromethyl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-4-yl)-3-(dimethylcarbamoyl)piperidin-3-yl)carbamate (93 mg, 0.179 mmol), 7-methoxy-N-(2-methoxy-5-methylphenyl)-1H-imidazo[4,5-c]pyridin-4-amine (50.84 mg, 0.179 mmol) and K2CO3 (49.42 mg, 0.358 mmol) were dissolved in DMF (3 mL). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was slowly added to 20 mL of water, extracted with ethyl acetate (15 mL x 2), and the organic phases were combined and washed with water (30 mL x 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-4'-methoxy-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (40 mg, yield 29.14%) was obtained as a white solid by chromatography column separation (4 g, 0-50% EtOAc / PE, 20 mL / min) and Prep-TLC (EtOAc). MS m / z (ESI): 768.3 [M+H] +

[0220] Step 5: Dissolve tert-butyl (3-(dimethylcarbamoyl)-1-(3'-fluoro-4'-methoxy-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-[1,1'-biphenyl]-4-yl)piperidin-3-yl)carbamate (40 mg, 0.0521 mmol) in DCM (1 mL), add TFA (1 mL), and stir at room temperature for 16 hours. The reaction solution was concentrated, saturated NaHCO3 solution (15 mL) was added, and extracted with ethyl acetate (20 mL x 3). The organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Prep-TLC separation (12% MeOH / DCM) gave compound E8: 3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-4'-methoxy-[1,1'-biphenyl]-4-yl)-N,N-dimethylpiperidine-3-carboxamide (10.52 mg, 36.85% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ8.14 (s, 1H), 7.53 (dd, J = 8.3, 2.2Hz, 1H), 7.34 (s, 1H), 7.30-7. 22(m,2H),7.16(t,J=8.8Hz,1H),7.11-7.05(m,1H),6.75(s,1H),5.75(s,2H),5.71(d, J=2.8Hz,2H),3.81(s,3H),3.64(s,3H),3.28-3.00(m,4H),2.97-2.84(m,3H),2.65-2. 52(m,3H),2.24(s,2H),1.99-1.87(m,1H),1.86-1.64(m,3H).MSm / z(ESI):548.3[M+H] +

[0221] Example 9: (3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)piperidin-3-yl)(piperidin-1-yl)methanone

[0222]

[0223] Step 1: 1-benzyl-3-{[(tert-butoxy)carbonyl]amino}piperidine-3-carboxylic acid (1000 mg, 2.99 mmol), piperidine (305 mg, 3.59 mmol) and DMF (15 mL) were stirred at room temperature for 2 min, and then EDCI (1146 mg, 5.98 mmol), HOBT (808 mg, 5.98 mmol) and DIPEA (1546 mg, 11.96 mmol) were added at room temperature and stirred at room temperature for 16 hours. The reaction solution was slowly added to 80 mL of water, extracted with ethyl acetate (50 mL x 2), and the organic phases were combined and washed with water (80 mL x 3) and saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The mixture was separated by chromatography (25 g, 0-20% EtOAc / PE, 30 mL / min) to give the compound N-{1-benzyl-3-[(piperidin-1-yl)carbonyl]piperidin-3-yl}carbamic acid tert-butyl ester (760 mg, yield 37.98%) as a colorless oil. MS m / z (ESI): 402.3 [M+H] +

[0224] Step 2: At room temperature, tert-butyl N-{1-benzyl-3-[(piperidin-1-yl)carbonyl]piperidin-3-yl}carbamate (760 mg, 1.89 mmol) was dissolved in MeOH (6 mL) and Pd / C (400 mg, 10%) was added. The mixture was stirred at room temperature for 5 hours. The reaction solution was filtered through diatomaceous earth and concentrated to obtain the compound tert-butyl N-{3-[(piperidin-1-yl)carbonyl]piperidin-3-yl}carbamate (600 mg, yield 61.08%) as a white solid. MS m / z (ESI): 312.3 [M+H] +

[0225] Step 3: At room temperature, tert-butyl N-{3-[(piperidin-1-yl)carbonyl]piperidin-3-yl}carbamate (500 mg, 1.61 mmol) and methyl 2-fluoro-5-(3-fluorophenyl)benzoate (399 mg, 1.61 mmol) were dissolved in DMSO (10 mL), and then DIPEA (415 mg, 3.21 mmol) was added. The mixture was heated in an oil bath at 120°C for 16 hours. The reaction solution was slowly added to 70 mL of water, extracted with ethyl acetate (40 mL x 2), the organic phases were combined and washed with water (80 mL x 3) and saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was separated by chromatography (20 g, 0-50% EtOAc / PE, 40 mL / min) to give compound 4-(3-((tert-butoxycarbonyl)amino)-3-(piperidine-1-carbonyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-carboxylic acid methyl ester (340 mg, yield 35.31%), as a light yellow solid. MS m / z(ESI):540.3[M+H] +

[0226] Step 4: At room temperature, 4-(3-((tert-butoxycarbonyl)amino)-3-(piperidine-1-carbonyl)piperidin-1-yl)-3'-fluoro-[1,1'-biphenyl]-3-carboxylic acid methyl ester (300 mg, 0.556 mmol) was dissolved in EtOH (6 mL), and NaBH4 (420.59 mg, 11.117 mmol) and LiCl (471.29 mg, 11.17 mmol) were added. The mixture was heated and stirred in a 50°C oil bath for 1 hour. The reaction solution was quenched with saturated ammonium chloride solution, concentrated to remove EtOH, 30 mL of water was added, and ethyl acetate was extracted (30 mL x 2). The organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The compound (1-(3'-fluoro-3-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamic acid tert-butyl ester (180 mg, yield 56.95%) was obtained by chromatography column separation (12 g, 0-40% EtOAc / PE, 30 mL / min) as a white solid. MS m / z (ESI): 512.3 [M+H] +

[0227] Step 5: Dissolve tert-butyl (1-(3'-fluoro-3-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamate (100 mg, 0.19 mmol) in anhydrous DCM (3 mL) at room temperature, cool to 0°C, and slowly add a solution of SOCl2 (46.33 mg, 0.39 mmol) in DCM (1 mL). Then react at room temperature for 1 hour. Add DCM (20 mL) to the reaction solution, and add saturated NaHCO3 solution (15 mL) under ice bath. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the compound (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamic acid tert-butyl ester (100 mg, yield 96.52%) as a light yellow solid. MS m / z (ESI): 526.3 [M+H] + (Chlorine turns into methoxy MS)

[0228] Step 6: Dissolve tert-butyl (1-(3-(chloromethyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamate (100 mg, 0.189 mmol), 7-methoxy-N-(2-methoxy-5-methylphenyl)-1H-imidazo[4,5-c]pyridin-4-amine (53.65 mg, 0.189 mmol) and K2CO3 (52 mg, 0.38 mmol) in DMF (3 mL) at room temperature. The reaction solution was reacted at room temperature for 16 hours. The reaction solution was slowly added to 20 mL of water, extracted with ethyl acetate (20 mL x 2), and the organic phases were combined and washed with water (30 mL x 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was separated by chromatography column (4 g, 0-50% EtOAc / PE, 20 mL / min) and Prep-TLC to give tert-butyl (1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-)[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamate (35 mg, yield 23.85%) as a white solid. MS m / z (ESI): 778.3 [M+H] +

[0229] Step 7: Dissolve tert-butyl (1-(3'-fluoro-3-((7-methoxy-4-((4-methoxybenzyl)amino)-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-)[1,1'-biphenyl]-4-yl)-3-(piperidine-1-carbonyl)piperidin-3-yl)carbamate (33 mg, 0.0424 mmol) in DCM (1 mL) at room temperature, add TFA (1 mL), and stir at room temperature for 16 hours. The reaction solution was concentrated, saturated NaHCO3 solution (15 mL) was added, and extracted with ethyl acetate (20 mL x 3). The organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Prep-TLC separation (10% MeOH / DCM) gave E9: compound ((3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)piperidin-3-yl)(piperidin-1-yl)methanone (11.73 mg, 47.17% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ8.16 (s, 1H), 7.59 (dd, J = 8.3, 2.1Hz, 1H), 7.43-7.38 (m, 1H), 7.35-7. 30(m,2H),7.18(dd,J=9.5,8.3Hz,2H),7.10(td,J=8.7,2.1Hz,1H),6.81(s,1H),5.76(s,2H), 5.72(s,2H),3.69(d,J=48.5Hz,7H),3.14(d,J=11.6Hz,1H),2.94(t,J=13.2Hz,2H),2.62(t, J=10.4Hz,1H),1.94(d,J=11.4Hz,1H),1.83-1.66(m,3H),1.58(d,J=4.5Hz,2H),1.48(s,4H). 19 F NMR(377MHz, DMSO-d6)δ-112.71(s,1H).HPLC:Purity:95.08%(214nm),95.21%(254nm).MS m / z(ESI):558.3[M+H] +

[0230] Example 10: (3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)piperidin-3-yl)(morpholinyl)methanone

[0231] Example 10 can be prepared following procedures similar to those described in Example 9. E10: 1 H NMR (400MHz, DMSO) δ8.14(s,1H),7.60(d,J=8.3Hz,1H),7.41(dd,J=14.7,7.2Hz ,1H),7.36-7.28(m,2H),7.19(t,J=9.0Hz,2H),7.10(t,J=8.4Hz,1H),6.81(s,1H ),5.74(d,J=14.9Hz,4H),3.74(d,J=85.8Hz,7H),3.57(s,4H),3.14(d,J=11.5H z,1H),2.93(t,J=12.8Hz,2H),2.63(s,1H),1.91(s,1H),1.75(d,J=31.1Hz,3H). 19 F NMR (377MHz, DMSO-d6) δ-112.70 (s, 1H). HPLC: Purity: 95.00% (214nm), 95.36% (254nm); MS m / z (ESI): 560.3[M+H] +

[0232] Example 11: (3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)piperidin-3-yl)(6-azaspiro[2,5]octan-6-yl)methanone

[0233] Example 11 can be prepared following procedures similar to those described in Example 9. E11: 1 H NMR (400MHz, DMSO-d6) δ8.15 (s, 1H), 7.60 (dd, J = 8.3, 2.2Hz, 1H), 7.45-7.36 (m, 1H), 7.35-7.2 8(m,2H),7.22-7.15(m,2H),7.10(td,J=8.9,2.3Hz,1H),6.83-6.78(m,1H),5.75(s,2H),5.73 (s,2H),4.20-3.45(m,4H),3.63(s,3H),3.15(d,J=11.7Hz,1H),3.02-2.90(m,2H),2.66-2.57 (m,1H),2.33(s,2H),2.03-1.90(m,1H),1.86-1.64(m,3H),1.36-1.28(m,4H),0.32(s,4H).MS m / z(ESI):562.2[M+H]+

[0234] Example 12: (3-amino-1-(3-((4-amino-7-methoxy-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-3'-fluoro-[1,1'-biphenyl]-4-yl)piperidin-3-yl)(6-azaspiro[2,5]octan-6-yl)methanone

[0235] Example 12 can be prepared following procedures similar to those described in Example 9. E12: 1 H NMR (400MHz, DMSO-d6) δ8.15 (s, 1H), 7.60 (dd, J = 8.3, 2.2Hz, 1H), 7.43-7.38 (m, 1H), 7.3 4-7.31(m,2H),7.22-7.17(m,2H),7.13-7.08(m,1H),6.81(d,J=1.5Hz,1H),5.80(s,2H), 5.73(s,2H),4.04-3.48(m,7H),3.15(d,J=11.5Hz,1H),2.97-2.89(m,2H),2.67-2.60(m ,1H),2.31(s,4H),2.18(s,3H),1.98-1.93(m,1H),1.86-1.75(m,2H),1.71-1.67(m,1H). 19 F NMR(377MHz,DMSO-d6)δ-112.71(s,1F).MS m / z(ESI):573.3[M+H] +

[0236] Biological test cases

[0237] A 96-well filter plate-based radioactive assay was used to screen compounds that inhibit NSD1 / 2 / 3 enzyme activity. In vitro screening of compounds that inhibit NSD1 / 2 / 3, with chaetocin as a reference compound.

[0238] Experimental reagents and consumables:

[0239] NSD2 (Active Motif, Catalog No. 31476)

[0240] NSD1 (Active Motif, Catalog No. 31475)

[0241] NSD3 (Active Motif, Product No. M1059)

[0242] GF / B Plate (Millipore, Catalog No. MSFBN6B50)

[0243] SAM (Sigma, Catalog No. A7007)

[0244] [3H]-SAM (PerkinElmer, Catalog No. NET1551MC)

[0245] Chaetocin (BPS, Product No. 27221)

[0246] Control compound: (Prepared with reference to Example 78 of WO2021028854A1)

[0247] Experimental methods:

[0248] This experiment is based on the isotope detection experiment. The substrate nucleosome and [3H]-SAM react under the action of enzyme activity in the enzyme solution (containing NSD1 / 2 / 3), and the nucleosome is methylated. The added compound can inhibit nucleosome methylation, and then an excess of SAM is added to terminate the above reaction. The IC50 value of the compound NSD1 / 2 / 3 inhibitory enzyme activity can be calculated based on the instrument reading.

[0249] Prepare 1x assay buffer (modified Tris buffer) and transfer the compound containing 100% DMSO to the assay plate using Echo. Prepare NSD1 / 2 / 3 enzyme solution in 1x assay buffer, add nucleosomes to 1x assay buffer to prepare substrate solution, add [3H]-SAM to 1x assay buffer to prepare [3H]-SAM solution, add SAM to 1x assay buffer to prepare SAM solution, transfer 10μL enzyme solution to the assay plate, and transfer 10μL assay buffer to the assay plate as a low value control, incubate at room temperature for 15 minutes, add 10μL [3H]-SAM solution to each well of the 96-well plate, and start the reaction. For NSD1, NSD2, and NSD3, incubate at room temperature for 240 minutes, 180 minutes, and 240 minutes, respectively, and add 15μL ice-cold SAM solution to each well to terminate the reaction. 40 μL of the reaction mixture was transferred to a GF / B plate that had been treated with 0.5% PEI for 15 minutes, and then the plate was washed three times with double distilled water and dried by vacuum pump. The plate was read on a Microbeta instrument.

[0250] Experimental data processing method:

[0251] Paste the data into Excel and calculate the inhibition rate using equation (1):

[0252] Equation (1): Inhibition rate % = (maximum value - signal value) / (maximum value - minimum value) * 100

[0253] Paste the data into XL-Fit and calculate the IC50 value using equation (2)

[0254] Equation (2): Y = lowest value + (highest value - lowest value) / (1 + (IC50 / X) * slope)

[0255] Where Y is the percentage inhibition and X is the concentration of the compound.

[0256] Through the NSD1 / 2 / 3 enzyme activity assay experiment, compounds with better test activity were screened. The specific test results are shown in Table 1.

[0257] Table 1

[0258]

[0259] The compounds of the present invention have good NSD3 inhibitory activity, especially compounds E9 and E11 show excellent NSD3 inhibitory activity. The compounds of the present invention also have selective inhibitory activity of NSD3 targets, and have obvious NSD3 selective inhibitory activity compared with NSD2.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, in, R 1 are independently halogen, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted by one or more R 1-1 replace; R 1-1 are independently C1-C6 alkoxy; n is 0, 1, 2 or 3; A is CR 2 , where R 2 is methoxy; B is CH; R 3 for Each R 4 and R 5 are independently H or C1-C6 alkyl; wherein the C1-C6 alkyl is optionally replaced by one or more R 4-1 replace; R 7 is a 5-10 membered heterocycloalkyl group; wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more C1-C6 alkyl groups; Wherein, the heteroatom in the 5-10 membered heterocycloalkyl group is N, O or S; the number of heteroatoms is 1, 2 or 3; R 7 The heterocycloalkyl group is connected to the carbonyl group through the N atom in the heterocycloalkyl group, and the heterocycloalkyl group contains at least 1 or 2 N atoms; Each R 4-1 are independently C1-C6 alkoxy; The chirality at "*" is R, S or a mixture thereof.

2. The compound of formula I according to claim 1, characterized in that The compound of formula I satisfies one or more of the following conditions: (1) The halogen is independently fluorine, chlorine, bromine or iodine; (2) The C1-C6 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; (3) The C1-C6 alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy.

3. The compound of formula I according to claim 1, characterized in that The compound of formula I satisfies one or more of the following conditions: (1) The C1-C6 alkyl group is a methyl group; (2) the C1-C6 alkoxy group is a methoxy group; and (3) The 5-10 membered heterocycloalkyl group is morpholinyl, piperazinyl, piperidinyl, tetrahydropyrrolyl or 4. The compound of formula I according to claim 1, characterized in that The 5-10 membered heterocycloalkyl group is independently a 5-6 membered monocyclic heterocycloalkyl group or an 8-10 membered bicyclic heterocycloalkyl group.

5. The compound of formula I according to claim 1, characterized in that The compound of formula I satisfies one or more of the following conditions: (1) n is 1 or 2; (2)R 1 The substitution position is the meta or para position of the benzene ring; when R 1 When the substitution position is the meta position of the benzene ring, R 1 is a halogen; when R 1 When the substitution position is the para position of the benzene ring, R 1 is C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted by one or more R 1-1 replace; (3) The chirality at "*" is R.

6. The compound of formula I according to claim 1, characterized in that The compound of formula I satisfies one or more of the following conditions: (1)R 1 independently halogen, C1-C4 alkoxy; wherein the C1-C4 alkoxy is optionally substituted by one or more R 1-1 replace; (2)R 1-1 is a C1-C6 alkoxy group; (3)R 3 for (4)R 7 It is a 5-6 membered monocyclic heterocycloalkyl group optionally substituted by one or more C1-C6 alkyl groups or an 8-10 membered bicyclic spirocyclic heterocycloalkyl group optionally substituted by one or more C1-C6 alkyl groups.

7. The compound of formula I according to claim 1, characterized in that R 7 for 8. The compound of formula I according to claim 1, characterized in that The compound of formula I satisfies one or both of the following conditions: (1)R 1 is F, methoxy or and (2)R 3 for 9. A compound of formula I according to any one of claims 1 to 8, characterized in that The compound of formula I is a compound of formula II, Among them, A, B, R 1 , R 4 and R 5 The definition as described in any one of claims 1 to 8.

10. The compound of formula I according to claim 1, characterized in that The compound of formula I is selected from any one of the following compounds:

11. A compound of formula IV, in, A, B, R 1 , R 3 and n are as defined in any one of claims 1-10.

12. The compound of formula IV according to claim 11, characterized in that The compound of formula IV is selected from any one of the following compounds:

13. A method for preparing a compound of formula I, comprising the following steps: in a solvent, in the presence of an acid, a compound of formula IV undergoes the following reaction to obtain a compound of formula I, in, A, B, R 1 , R 3 and n are as defined in any one of claims 1-10.

14. The method for preparing the compound of formula (I) according to claim 13, characterized in that: The preparation method of the compound of formula I satisfies one or more of the following conditions: (1) The acid is selected from one or more of hydrochloric acid, trifluoroacetic acid and hydrobromic acid; (2) The solvent is a halogenated alkane solvent and / or an alcohol solvent; The halogenated alkane solvent is dichloromethane, chloroform or 1,2-dichloroethane; The alcohol solvent is methanol, ethanol or isopropanol; (3) When the acid is trifluoroacetic acid, the feed ratio of the compound of formula IV to trifluoroacetic acid is 0.01 to 1 mmol / mL; (4) When the acid is trifluoroacetic acid, the volume ratio of the solvent to trifluoroacetic acid is (1:10) to (10:1); (5) When the acid is hydrochloric acid, the hydrochloric acid is a 1,4-dioxane hydrochloride solution; the concentration of the 1,4-dioxane hydrochloride solution is 0.5 to 10 mol / L; (6) When the acid is hydrochloric acid, the molar ratio of the compound of formula IV to hydrochloric acid is 1:(10-1000); (7) The reaction temperature of the reaction is 0 to 60° C.; (8) The reaction time of the reaction is 2 to 24 hours.

15. The method for preparing the compound of formula (I) according to claim 13, characterized in that: The preparation method of the compound of formula I satisfies one or more of the following conditions: (1) The solvent is dichloromethane; (2) When the acid is trifluoroacetic acid, the feed ratio of the compound of formula IV to trifluoroacetic acid is 0.01 to 0.1 mmol / mL; (3) When the acid is hydrochloric acid, the hydrochloric acid is a 1,4-dioxane hydrochloric acid solution; The concentration of the hydrochloric acid 1,4-dioxane solution is 1 to 5 mol / L; (4) When the acid is hydrochloric acid, the molar ratio of the compound of formula IV to hydrochloric acid is 1:(100-200); (5) The reaction temperature of the reaction is 15 to 30° C.; (6) The reaction time of the reaction is 10 to 20 hours.

16. The method for preparing the compound of formula (I) according to claim 13, characterized in that: When the acid is hydrochloric acid, the hydrochloric acid is a 1,4-dioxane hydrochloric acid solution; the concentration of the 1,4-dioxane hydrochloric acid solution is 4 mol / L.

17. A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, and at least one pharmaceutical excipient.

18. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating tumors.

19. The use according to claim 18, characterized in that The tumor is lung cancer, breast cancer, pancreatic cancer, bladder cancer, head and neck squamous cell carcinoma, pelvic high-grade serous carcinoma, osteosarcoma, acute myeloid leukemia or acute granulocytic leukemia.

20. Use of a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, or a pharmaceutical composition according to claim 17, in the preparation of a medicament for preventing and / or treating NSD3-related diseases.

21. The use according to claim 20, characterized in that The NSD3-related disease is a tumor.

22. The use according to claim 20, characterized in that The NSD3-related disease is lung cancer.

23. The use according to claim 20, characterized in that The NSD3-related disease is lung squamous cell carcinoma.

24. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 17 in the preparation of a NSD3 inhibitor.

Images