2,4-Disubstituted-5-fluoropyrimidine derivatives, their preparation methods and uses

Abstract

The present invention discloses 2,4-disubstituted-5-fluoropyrimidine derivatives, and their general structural formula is shown as Formula I: #imgabs0# wherein, R1 and R2 are each independently selected from: #imgabs1# R4 is selected from: #imgabs2# The present invention also discloses a preparation method of the compounds and their use in the preparation of drugs for anti-renal interstitial fibrosis.

Description

Technical Field

[0001] Renal interstitial fibrosis is a chronic and progressive process that affects the normal function of the kidneys in aging and chronic kidney disease, thus endangering human health. Among the currently known pathological mechanisms, the NF-κB, TGF-β / Smad3, Wnt / β-catenin, Notch, and p53 signaling pathways play important roles in the occurrence and development of renal interstitial fibrosis. From the existing drug research results, it can be seen that the strategy of drug discovery targeting one or two of the above channels has not led to the successful marketing of drugs in the field of research and development of therapeutic drugs for renal interstitial fibrosis (Nat Rev Drug Discov, 2016, 15(8):568; Am J Kidney Dis. 2022, doi:10.1053 / j.ajkd.2021.11.010). The research results show that although the above-mentioned pathways have been proven to be important links in the disease process, due to the complex interactions between the signal pathways, inhibiting one or two of them alone will activate other pathways related to inflammation and fibrosis (Front Cell Dev Biol. 2021, 9:696542). This complex situation is an important reason why there has been no breakthrough in the research and development of therapeutic drugs for renal interstitial fibrosis. Therefore, searching for highly effective therapeutic drugs for renal interstitial fibrosis solely targeting these downstream targets is not an ideal strategy.

[0002] During the pathogenesis of renal interstitial fibrosis, oxidative stress, inflammation, and fibrosis are key links. Searching for multi-channel drugs targeting the above key links simultaneously is expected to avoid the interactions between pathways and improve the drug efficacy. However, from the perspective of drug design, it is very difficult to design a single-structure inhibitor that can simultaneously inhibit the above five pathways. It is necessary to explore from another perspective to open up new research fields and provide innovative ideas for the research and development of therapeutic drugs for renal interstitial fibrosis.

[0003] John He et al. published an article pointing out (Nat Med. 2012, 18(4):580) that homeodomain-interacting protein kinase 2 (HIPK2) is highly expressed in the lesions of renal interstitial tubules and participates in the fibrosis and inflammation processes through the regulation of []. Therefore, inhibiting HIPK2 can simultaneously regulate fibrosis and inflammation to achieve the purpose of treating the disease.

[0004] The HIPK2 inhibitors reported in the literature research are classified into three categories ( Figure 1):HIPK2 kinase inhibitors, HIPK2 / SMAD interaction inhibitors, and HIPK2 expression inhibitors. TBID (Plos one.2014,9(2):e89176) is a HIPK2 kinase inhibitor that inhibits the kinase activity of HIPK2 by occupying the ATP-binding pocket. Except for TBID, other compounds with HIPK2 kinase inhibitory activity were discovered through off-target effect studies, such as TAE-226 (Nat Med.2012,18(4):580), CTx-0294857 (J.Proteome Res.2013,12:3104), Sorafenib (Plos one.2015,10(2):e0117757), GW-5074, Compound C (Biochem.J.2007,408:297). BT173 (J Am Soc Nephrol.2017,28:2133) is a HIPK2 / SMAD interaction inhibitor that can exert anti-fibrotic effects by blocking the protein-protein interaction between HIPK2 and Smad3, but has no inhibitory activity on the kinase activity of HIPK2. The HIPK2 expression inhibitor phosphate niclosamide (Kidney International.2017,92:612) can inhibit the transcription of HIPK2 by interfering with the binding of Smad3 to the promoter sequence of the HIPK2 gene, thereby inhibiting multiple downstream signaling pathways of HIPK2.

[0005] Patent Invention 201911152583.9 and PCT / CN2020 / 129308 disclose a urea-based benzimidazole derivative and its preparation method and application.

[0006]

[0007] Patent Invention CN106565673 discloses a 5-fluoropyrimidine heterocyclic compound with Wnt signaling pathway inhibitory activity and uses it for the treatment or prevention of diseases caused by Wnt signaling pathway disorders.

[0008]

[0009] Patent Invention CN105017159 discloses a 5-fluoro-2,4-disubstituted amino pyrimidine derivative and its preparation method and anti-tumor application, but does not report its inhibitory activity on HIPK2 kinase and anti-renal interstitial fibrosis activity.

[0010] Therefore, so far, no HIPK2 inhibitor has been successfully used in clinical practice. Given the significant clinical need for renal interstitial fibrosis disease and the promising prospects of HIPK2 inhibitors, there is an urgent need in this field to develop effective HIPK2 inhibitors. Summary of the Invention

[0011] The present invention relates to the field of pharmaceutical chemical synthesis, and specifically relates to a class of 2,4-disubstituted-5-fluoropyrimidine derivatives, and their preparation methods and applications.

[0012] A 2,4-disubstituted-5-fluoropyrimidine derivative, the general formula of its structure is shown in Formula I:

[0013]

[0014] Wherein, R1 and R2 can be independently selected from:

[0015]

[0016] R4 is selected from:

[0017]

[0018] X and Y are independently selected from a nitrogen atom or a carbon atom; Z is selected from a hydrogen atom, a tert-butoxycarbonyl group, a C1-C5 alkyl group, a C1-C5 alkyl acyl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkyl sulfonyl group, a C1-C3 substituted phenyl group, a C1-C5 alkyl amide group.

[0019] Preferably, either of R1 and R2 is: R4 is Z is selected from a hydrogen atom, a C1-C5 alkyl group, a C1-C5 alkyl acyl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkyl sulfonyl group, a C1-C3 substituted phenyl group, a C1-C5 alkyl amide group.

[0020] Preferably, either of R1 and R2 is: R4 is Z is butyl, pentyl, isobutyl, isopentyl, and the other of R1 and R2 is: When X and Y are not both carbon atoms.

[0021] Preferably, either of R1 and R2 is: R4 is Z is selected from a C4-C5 alkyl group, and the other of R1 and R2 is not:

[0022] Preferably, either of R1 and R2 is: R4 is Z is hydrogen, and the other of R1 and R2 is not:

[0023] Preferably, either R1 or R2 is: The other of R1 and R2 is: Z is selected from a hydrogen atom, a C4-C5 alkyl group, a C1-C3 alkyl acyl group, a C1-C3 substituted phenyl group, a C1-C5 alkyl amide group.

[0024] The present invention also provides another 2,4-disubstituted-5-fluoropyrimidine derivative, and its structural general formula is shown as Formula II or Formula III:

[0025]

[0026] Wherein, R3 is:

[0027] R4 is selected from:

[0028]

[0029] X and Y are each independently selected from a nitrogen atom or a carbon atom; Z is selected from a hydrogen atom, a tert-butoxycarbonyl group, a C1-C5 alkyl group, a C1-C5 alkyl acyl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkyl sulfonyl group, a C1-C3 substituted phenyl group, a C1-C5 alkyl amide group.

[0030] Preferably, the C1-C5 alkyl group includes a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, an isobutyl group, an isopentyl group, an isopropyl group.

[0031] Preferably, R3 is: R4 is Z is selected from a hydrogen atom, a C1-C3 alkyl group, a C1-C5 alkyl acyl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkyl sulfonyl group, a C1-C3 substituted phenyl group, a C1-C5 alkyl amide group.

[0032] Preferably, in R3, when R4 is Z is not a tert-butoxycarbonyl group.

[0033] Preferably, when R4 in Formula III is Z is not a tert-butoxycarbonyl group, a C4-C5 alkyl group, a C4-C5 alkyl acyl group, a C4-C5 alkyl amide group.

[0034] Preferably, the C1-C5 alkyl acyl group includes a formyl group, an acetyl group, a propionyl group, a butyryl group, a valeryl group, a 3,3-dimethyl-1-butyryl group, an isovaleryl group.

[0035] Preferably, the C1-C5 alkyl amide group includes a formamide group, an acetamide group, a propionamide group, a butyramide group, a valeramide group, an N-tert-butylcarbamoyl group, an isovaleramide group.

[0036] Preferably, the C1-C3 alkyl hydroxy groups include hydroxymethyl, 1-hydroxyethyl, and 1-hydroxypropyl.

[0037] Preferably, the C1-C3 alkylsulfonyl groups include sulfonylmethyl, sulfonylethyl, and sulfonylpropyl.

[0038] Preferably, the C1-C3 substituted phenyl groups include benzyl, phenethyl, and phenylpropyl.

[0039] Preferably, the pharmaceutically acceptable salts of the above compounds are hydrochloride, sulfate, phosphate, perchlorate, mesylate, trifluoromethanesulfonate, formate, acetate, propionate, butyrate, maleate, succinate, trifluoroacetate, suberate, salicylate, DL-aspartate, D-aspartate, L-aspartate, DL-glutamate, D-glutamate, L-glutamate, glycerate, stearate, DL-tartrate, D-tartrate, L-tartrate, (±)-mandelate, (R)-(-)-mandelate, (S)-(+)-mandelate, citrate, mucate, malonate, benzoate, DL-malate, (±)-lactate, L-(+)-lactate, D-(+)-lactate, pamoate, D-α-galacturonate, glycerate, DL-cysteinate, D-cysteinate, L-cysteinate, (4S)-hydroxy-L-prolinate, cyclopropane-1,1-dicarboxylate, 2,2-dimethylmalonate, tyrosinate, prolinate, fumarate, 1-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate, 3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, L-pyroglutamate, p-toluenesulfonate, benzenesulfonate, ethanesulfonate, (±)-camphorsulfonate, naphthalenesulfonate, 1R-(-)-camphorsulfonate, 1S-(+)-camphorsulfonate, 1,5-naphthalenedisulfonate, 1,2-ethanedisulfonate, 1,3-propanedisulfonate, 3-(N-morpholino)propanesulfonate, biphenylsulfonate, hydroxyethylsulfonate, 1-hydroxy-2-naphthalenesulfonate, potassium dihydrogen phosphate, potassium hydrogen phosphate, dipotassium phosphate, potassium phosphate, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, sodium dihydrogen phosphate, calcium phosphate, tricalcium phosphate, hexafluorophosphate, vinyl phosphate, 2-hydroxyethyl phosphate, and phenyl phosphate.

[0040] Preferably, specifically the following compounds:

[0041] tert-Butyl 4-(4-((2-(2-amino-1H-benzo[d]imidazol-1-yl)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6a);

[0042] tert-Butyl 4-(4-((2-((1H-benzo[d]imidazol-2-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6h);

[0043] tert-Butyl 4-(4-((5-fluoro-2-(isoquinolin-3-ylamino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6b);

[0044] tert-Butyl 4-(4-((5-fluoro-2-(((tetrahydrofuran-2-yl)methyl)amino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6c);

[0045] tert-Butyl 4-(4-((5-fluoro-2-((pyridin-2-ylmethyl)amino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6d);

[0046] tert-Butyl 4-(4-((5-fluoro-2-((4-(methoxycarbonyl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6e);

[0047] 1-(5-Fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 6f);

[0048] 1-(4-((4-(4-ethylpiperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 6g);

[0049] N 2 -(1H-benzo[d]imidazol-2-yl)-N 4 -(4-(4-ethylpiperazin-1-yl)phenyl)-5-fluoropyrimidine-2,4-diamine (Compound 6g-1);

[0050] 1-(5-Fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 7a);

[0051] 5-Fluoro-N 2 -(isoquinolin-3-yl)-N 4 -(4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine (Compound 7b);

[0052] 5-Fluoro-N 4 -(4-piperazin-1-yl)phenyl)-N 2 -((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine (Compound 7c);

[0053] 5-Fluoro-N 4 -(4-(piperazin-1-yl)phenyl)-N 2 -(pyridin-2-ylmethyl)pyrimidine-2,4-diamine (Compound 7d);

[0054] Methyl 4-((5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)amino)benzoate (Compound 7e);

[0055] 1-(5-Fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 8a);

[0056] 5-Fluoro-N 4 -(4-(4-isopentylpiperazin-1-yl)phenyl)-N 2 -isoquinolin-3-yl)pyrimidine-2,4-diamine (Compound 8b);

[0057] 5-Fluoro-N 4 -(4-(4-isopentylpiperazin-1-yl)phenyl)-N 2 -((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine (Compound 8c);

[0058] 5-Fluoro-N 4 -(4-(4-isopentylpiperazin-1-yl)phenyl)-N 2 -(pyridin-2-ylmethyl)pyrimidine-2,4-diamine (Compound 8d);

[0059] Methyl 4-((5-fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)amino)benzoate (Compound 8e);

[0060] tert-Butyl 4-(5-(2-(2-amino-1H-benzo[d]imidazol-1-yl)-5-fluoropyrimidin-4-yl)pyridin-2-yl)piperazine-1-carboxylate (Compound 11h);

[0061] 1-(5-Fluoro-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 12h);

[0062] 1-(5-Fluoro-4-(6-(4-isopentylpiperazin-1-yl)pyridin-3-yl)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 13h);

[0063] N 6-(5-Fluoro-4-((4-morpholinophenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23a);

[0064] N 6 -(5-Fluoro-4-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23b);

[0065] N 6 -(4-((4-(diethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23c);

[0066] N 6 -(4-((4-(dipropylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23d);

[0067] N 6 -(5-Fluoro-4-((4-(piperidin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23e);

[0068] tert-Butyl 4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 23f);

[0069] N 6 -(4-((4-(dimethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23i);

[0070] tert-Butyl 4-(5-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-2-yl)piperazine-1-carboxylate (Compound 23g);

[0071] tert-Butyl 4-(5-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-3-yl)piperazine-1-carboxylate (Compound 23h);

[0072] N 6 -(5-Fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24a);

[0073] N 6 -(5-Fluoro-4-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24b);

[0074] N 6 -(5-Fluoro-4-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24c);

[0075] N 6 -(5-Fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25a);

[0076] 1-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethanone (Compound 25b);

[0077] N 6 -(5-Fluoro-4-((4-(4-isopropylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25c);

[0078] 2-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethanol (Compound 25d);

[0079] N6 -(4-((4-(4-(ethylsulfonyl)piperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25e);

[0080] N 6 -(5-fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25f);

[0081] N 6 -(4-((4-(4-benzylpiperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25g);

[0082] 1-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)-3,3-dimethylbutan-1-one (Compound 25h);

[0083] N-(tert-butyl)-4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxamide (Compound 25i);

[0084] N 6 -(5-fluoro-2-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29a);

[0085] N 6 -(5-fluoro-2-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29b);

[0086] N 6 -(5-fluoro-2-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-N 2 ,N 2-Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29c);

[0087] N 6 -(2-((4-(Dimethylamino)phenyl)amino)-5-fluoropyrimidin-4-yl)-N 2 ,N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29d);

[0088] N 6 -(5-Fluoro-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N 2 ,N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29e);

[0089] 1-(4-(4-((4-((2-(Dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-2-yl)amino)phenyl)piperazin-1-yl)ethanone (Compound 29f);

[0090] N 6 -(5-Fluoro-2-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29g);

[0091] N 6 ,N 6 -(5-Fluoropyrimidine-2,4-diyl)bis(N2,N2-dimethyl-1H-benzo[d]imidazole-2,6-diamine) (Compound 29h).

[0092] The present invention also discloses a preparation method of 2,4-disubstituted-5-fluoropyrimidine derivatives, which is characterized by comprising the following steps:

[0093]

[0094] 2,4-Dichloro-5-fluoro-pyrimidine reacts with different amine compounds successively to obtain Compound I or II, and the intermediate generated by the reaction of 2,4-dichloro-5-fluoro-pyrimidine with borate ester continues to react with 2-aminobenzimidazole to obtain Compound III.

[0095] The present invention includes the application of the above 2,4-disubstituted-5-fluoropyrimidine derivatives in the preparation of drugs for treating fibrosis.

[0096] Preferably, the fibrosis is renal interstitial fibrosis.

[0097] The present invention includes 2,4-disubstituted-5-fluoropyrimidine derivatives or pharmaceutically acceptable salts thereof as anti-fibrotic drugs.

[0098] The present invention provides a pharmaceutical composition comprising an effective amount of a compound, a stereoisomer of the compound, a tautomer, an N-oxide, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof, and a pharmaceutically acceptable excipient.

[0099] The pharmaceutically acceptable excipients include solvents, diluents, other liquid excipients, dispersants or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders or lubricants, etc.

[0100] The pharmaceutical composition can be in liquid, solid, semi-solid, gel or spray form.

[0101] The compounds or pharmaceutically acceptable compositions of the present invention can be administered by any suitable means, and the above-mentioned compounds and pharmaceutically acceptable compositions can be administered to humans or other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically, etc. according to the severity of the disease.

[0102] The term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present invention. The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammalian being treated therewith.

[0103] On the other hand, the compounds of the present invention include compounds defined by the present invention labeled with various isotopes, for example, those compounds in which radioactive isotopes such as 3H, 14C and 18F are present, or those compounds in which non-radioactive isotopes such as 2H and 13C are present. Description of the Drawings

[0104] Figure 1 Compounds reported in the literature to have HIPK2 inhibitory activity.

[0105] Figure 2 Effect of compound 7a in Example 16 on the expression of α-SMA and Fn 1 in NRK-49F cells.

[0106] Figure 3 Effect of compound 7a in Example 17 on the expression of P65 in the inflammatory signaling pathway

[0107] Figure 4 HE staining (1×) of renal tissues of mice in each group in Example 18, the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0108] Figure 5 For the HE staining of the kidney tissues of mice in each group in Example 18 (200×), the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0109] Figure 6 For the injury score of the kidney tissues of mice in each group in Example 18, the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0110] Figure 7 For the Masson staining of the kidney tissues of mice in each group in Example 18 (×1), the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0111] Figure 8 For the Masson staining of the kidney tissues of mice in each group in Example 18 (200×), the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0112] Figure 9 For the Masson staining score of the kidney tissues of mice in each group in Example 18, the left side is the Sham group, the middle is the UUO group, and the right side is the UUO + compound 7a group.

[0113] Figure 10 For the kidney tissue staining and injury score of the compound 25f group in Example 18. Detailed implementation method

[0114] Conditions and reagents: a. K2CO3, DMSO, 90 °C, 5 h; b. Fe, NH4Cl, EtOH, H2O, reflux, 5 h; c. H2O, MeOH, 60 °C, 12 h; d. Corresponding amines, Cs2CO3, Xantphos, Pd2(dba)3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h; e. TFA, DCM, r.t., 4 h; f. 3-Methyl butanal, DCE, AcOH, NaHB(OCOCH3)3, 60 °C, 16 h.

[0115] Using p-fluoronitrobenzene and 1-Boc-piperazine as starting materials, dimethyl sulfoxide as the reaction solvent, a nucleophilic substitution reaction occurs under the condition of refluxing for 5 h to obtain intermediate 3. Then, the nitro group is reduced to an amino group using iron powder and ammonium chloride to obtain intermediate 4. Intermediate 4 and 2,4-dichloro-5-fluoropyrimidine undergo a nucleophilic substitution reaction in methanol to obtain compound 5. Compound 5 and various different amines undergo a Buchwald–Hartwig coupling reaction using Cs2CO3 as the base, Xantphos as the ligand, Pd2(dba)3 as the catalyst, and 1,4-dioxane as the solvent to obtain the target compounds 6a-e, 6h. Compounds 6a-e are deprotected with trifluoroacetic acid at room temperature to obtain the target compounds 7a-e. Compounds 7a-e undergo a Borch reduction reaction with isovaleraldehyde to obtain the target compounds 8a-e.

[0116]

[0117] Conditions and reagents: a. K2CO3, DMSO, 90 °C, 5 - 18 h; b. NH4Cl, Fe, H2O, EtOH, reflux, 5 h; c. H2O, MeOH, 60 °C, 12 h; d. Cs2CO3, Xantphos, Pd2(dba)3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h.

[0118] Using p-fluoronitrobenzene, 1-methylpiperazine or 1-ethylpiperazine as starting materials, dimethyl sulfoxide as the reaction solvent, the reaction is carried out at 90 °C to obtain intermediates 3f-g. Using iron powder as the reducing agent, ethanol and water as the reaction solvents, an appropriate amount of ammonium chloride is added, and the reaction is carried out under reflux conditions for 5 hours to obtain the reduction products 4f-g. Then, they undergo a nucleophilic substitution reaction with 2,4-dichloro-5-fluoropyrimidine in methanol to obtain compounds 5f-g. Compounds 5f-g and 2-aminobenzimidazole undergo a Buchwald–Hartwig coupling reaction using Cs2CO3 as the base, Xantphos as the ligand, Pd2(dba)3 as the catalyst, and 1,4-dioxane as the solvent to obtain the target compounds 6f-g and compound 6g-1.

[0119]

[0120] Conditions and reagents: a. K2CO3, Pd(dppf)Cl2, H2O:THF = 1:5, reflux, 5 h; b. Xantphos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h; c. TFA, DCM, r.t., 4 h; d. Trimethyl acetaldehyde, DCE, AcOH, NaHB(OCOCH3)3, 60 °C, 16 h.

[0121] 2-(4-BOC-piperazinyl)pyridine-5-boronic acid pinacol ester reacted with 2,4-dichloro-5-fluoropyrimidine for 5 h under Suzuki reaction conditions (using H2O:THF = 1:5 as the solvent, palladium acetate as the catalyst, and potassium carbonate as the base) to obtain Compound 10. Compound 10 underwent Buchwald–Hartwig coupling reaction with 2-aminobenzimidazole to obtain the target compound 11h. Compound 11h was deprotected with Boc using trifluoroacetic acid at room temperature to obtain the target compound 12h. Compound 12h underwent Bausch reduction reaction with isovaleraldehyde to obtain the target compound 13h.

[0122] Conditions and reagents: a. Corresponding amines, DMSO, K2CO3, 90 °C, 5 h or diethylamine, DMSO, K2CO3, 50 °C, 18 h; b. Fe, NH4Cl, H2O, EtOH, reflux, 5 h; c. DCM, Et3N, reflux, 4 h; d. H2, Pd / C; MeOH, 18 h; e. MeOH, H2O, 60 °C, 12 h; f. 17, Pd2(dba)3, Xanphos, Cs2CO3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h.

[0123] First, the benzimidazole fragment was synthesized. Using compounds 14 and 15 as starting materials, the benzimidazole fragment 16 containing a nitro group was obtained by cyclization. Then, the nitro group was reduced to an amino group by hydrogen reduction to synthesize the benzimidazole fragment 17 containing an amino group. Then, using 18a-c as starting materials, the corresponding aliphatic amino groups were introduced through nucleophilic substitution reactions to obtain the aromatic ring-linked aliphatic heterocyclic fragment with a nitro group at the para position. Then, the nitro group was reduced by the method of iron powder reduction to obtain the aromatic ring-linked aliphatic heterocyclic fragment 20a-h with an amino group at the para position. 20i was not synthesized by this method and was directly purchased from Energy Chemical. 20a-i and the starting material 21 underwent nucleophilic substitution reactions in methanol as the solvent without any basic environment to obtain compounds 22a-i. Compounds 22a-i and compound 17 underwent Buchwald–Hartwig coupling reactions using Pd2(dba)3 as the catalyst, Xanphos as the ligand, Cs2CO3 to provide a basic environment, and 1,4-dioxane as the solvent to obtain the target compounds 23a-i. Among them, 23f-h were deprotected to obtain the target compounds 24a-c.

[0124]

[0125] Conditions and reagents: g. DCM, TAF, r.t., 4 h; h. for 25a, methyl iodide, ACN, Et3N, -10 °C, 10 min; for 25b, acetic anhydride, ACN, Et3N, r.t., 2 h; for 25c, 2-iodopropane, ACN, K2CO3, 65 °C, 16 h; for 25d, 2-bromoethan-1-ol, ACN, Et3N, r.t., 16 h; for 25e, ethanesulfonyl chloride, ACN, Et3N, r.t., 1 h; for 25f, trimethyl acetaldehyde, AcOH, DCE, NaHB(OCOCH3)3, 60 °C, 16 h; for 25g, (bromomethyl)benzene, ACN, Et3N, -10 °C, 10 min; for 25h, 3,3-dimethylbutanoyl chloride, ACN, Et3N, -10 °C, 10 min; for 25i, 2-isocyanato-2-methylpropane, ACN, Et3N, -10 °C, 10 min.

[0126] 24a reacts with iodomethane in acetonitrile as the solvent and triethylamine as the catalyst at -10 °C to obtain compound 25a. 24a reacts with acetic anhydride in acetonitrile as the solvent and triethylamine as the catalyst at room temperature to obtain compound 25b. 24a reacts with 2-iodopropane in acetonitrile as the solvent at 65 °C to obtain compound 25c. 24a reacts with 2-bromoethanol in acetonitrile as the solvent and triethylamine as the catalyst at room temperature for 16 h to obtain compound 25d. 24a reacts with ethanesulfonyl chloride in acetonitrile as the solvent and triethylamine as the catalyst at room temperature for 1 h to obtain compound 25e. 24a reacts with trimethylacetaldehyde in dichloroethane as the solvent, with glacial acetic acid and sodium trimethoxyborohydride as the catalysts at 60 °C for 16 h to obtain compound 25f. 24a reacts with benzyl bromide in acetonitrile as the solvent and triethylamine as the catalyst at -10 °C for 10 minutes to obtain compound 25g. 24a reacts with 3,3-dimethylbutanoyl chloride in acetonitrile as the reaction solvent and triethylamine as the catalyst at -10 °C for 10 minutes to obtain compound 25h. 24a reacts with tert-butyl cyanate in acetonitrile as the reaction solvent and triethylamine as the catalyst at -10 °C for 10 minutes to obtain compound 25i.

[0127] Conditions and reagents: a. 17, MeOH, H2O, 60 °C, 12 h; b. (Boc)2O, DMAP, Et3N, DCM, r.t., 24 h; c1. Corresponding amines, Pd2(dba)3, Xanphos, Cs2CO3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h; c2. 17, Pd2(dba)3, Xanphos, Cs2CO3, 1,4-dioxane, N2 protection, sealed tube, 120 °C, 12 h; d. DCM, TAF, r.t., 4 h; e. for 29f, Acetic anhydride, ACN, Et3N, r.t., 2 h; for 29g Trimethylacetaldehyde, AcOH, DCE, NaBH(OCOCH3)3, 60 °C, 16 h.

[0128] First, a nucleophilic substitution reaction occurs between compound 17 and raw material 12 in methanol as the solvent without any basic reagent to obtain compound 26. Then, it reacts with di-tert-butyl dicarbonate using DMAP and triethylamine as catalysts and dichloromethane as the reaction solvent at room temperature for 24 hours to obtain compound 27. Compound 27 reacts with the corresponding amine or compound 17 respectively under the conditions of using Pd2(dba)3 as the catalyst, Xanphos as the ligand, Cs2CO3 to provide a basic environment, and 1,4-dioxane as the solvent to undergo Buchwald–Hartwig coupling reaction to obtain the target compounds 28a - e and 28f. The Boc protection groups of 28a - e and 28f are removed to obtain the target compounds 29a - e and 29h. 29b reacts with acetic anhydride using acetonitrile as the solvent and triethylamine as the catalyst at room temperature for 2 hours to obtain compound 29f. 29b reacts with trimethylacetaldehyde using dichloroethane as the reaction solvent,

[0129] using glacial acetic acid and sodium trimethoxyborohydride as catalysts and reacting at 60 °C for 16 hours to obtain compound 29g.

[0130] Example 1: Preparation of tert-butyl 4-(4-((2-(2-amino-1H-benzo[d]imidazol-1-yl)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6a)

[0131] Compound 5 was synthesized in the previous work of the laboratory, and its structure was confirmed by 1H NMR and HRMS. 1 1H NMR(500MHz,DMSO-d6)δ9.83(s,1H),8.23(d,J=3.5Hz,1H),7.51(d,J=9.1Hz,2H),6.97(d,J=9.1Hz,2H),3.52 - 3.41(m,4H),3.14 - 3.03(m,4H),1.42(s,9H).HRMS(ESI)m / z calcd for[C 19 H 23 ClFN5O2+H] + :408.1597;found:408.1601[M+H] + 。

[0132] Compound 5 (2.03 g, 5 mmol), 2-aminobenzimidazole (20 mmol), Xantphos (0.046 g, 0.08 mmol), Pd2(dba)3 (0.018 g, 0.02 mmol), and cesium carbonate (4.864 g, 15 mmol) were added to a 100 mL sealed tube containing a stir bar. 30 mL of dioxane (dried over 4A molecular sieves) was added, the air in the flask was displaced with nitrogen, the lid was screwed on, and the mixture was sonicated for dissolution. It was placed in an oil bath at 120 °C and reacted for 12 h. After cooling to room temperature, the reaction solution was evaporated to dryness, extracted with dichloromethane / water, and the aqueous layer was washed 3 times with dichloromethane. The organic layer was dried over anhydrous Na2SO4 for 4 h.

[0133] It was filtered by suction, the organic solvent was evaporated to dryness, and a yellow-green solid was obtained. It was separated and purified by silica gel column chromatography with 200 - 300 mesh (DCM:MeOH = 40:1) to obtain 1.169 g of a white solid powder with a yield of 47%. 1 1H NMR (500 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.39 (d, J = 3.4 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.60 (s, 2H), 7.44 - 7.38 (m, 2H), 7.17 - 7.12 (m, 1H), 7.07 - 7.00 (m, 3H), 6.82 - 6.75 (m, 1H), 3.50 (t, J = 5.1 Hz, 4H), 3.14 (t, J = 5.1 Hz, 4H), 1.44 (s, 9H). 13 13C NMR (125 MHz, DMSO-d6) δ 154.51, 154.35, 149.20, 143.37, 139.86, 139.70, 131.92, 129.53, 125.63, 123.19, 119.23, 116.73, 115.26, 115.18, 79.46, 49.09, 28.55. HRMS (ESI) m / z calcd for [C 26 H 29 FN8O2 + H]+ + : 505.2476; found: 505.2491.

[0134] Preparation of tert-butyl 4-(4-((2-((1H-benzo[d]imidazol-2-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6h)

[0135] Using the method of Example 1 above, with 2-aminobenzimidazole as the raw material, Compound 6h was prepared, which was a white solid powder with a yield of 16%. 11H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H), 10.95 (s, 1H), 9.54 (s, 1H), 8.16 (d, J = 3.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.43 - 7.15 (m, 2H), 7.02 (dd, J = 9.3, 3.4 Hz, 4H), 3.51 (t, J = 5.1 Hz, 4H), 3.14 (t, J = 5.1 Hz, 4H), 1.43 (s, 9H). 13 13C NMR (125 MHz, DMSO-d6) δ 154.37, 154.20, 150.96, 150.87, 149.47, 148.25, 142.63, 140.66, 140.13, 139.97, 130.78, 124.32, 116.68, 79.49, 49.06, 28.55. HRMS (ESI) m / z calcd for 26 H 29 [C + H

[0136] 8FN8O2 + H]+

[0137] : 505.2476; found: 505.2515. 1 1H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 9.34 (s, 1H), 9.05 (s, 1H), 8.34 (s, 1H), 8.15 (d, J = 3.6 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.62 (s, 1H), 7.60 - 7.54 (m, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 8.5 Hz, 2H), 3.53 (t, J = 4.9 Hz, 4H), 3.14 (t, J = 5.1 Hz, 4H), 1.45 (s, 9H). 1313C NMR (125 MHz, DMSO-d6) δ 154.96, 154.36, 151.45, 149.20, 148.07, 140.71, 140.55, 137.94, 131.10, 130.69, 128.08, 126.25, 125.07, 124.77, 124.56, 116.71, 104.44, 79.48, 49.25, 28.55. HRMS (ESI) m / z calcd for 28 H 30 FN7O2 + H] + : 516.2523; found: 516.2554. Preparation of tert-butyl 4-(4-((5-fluoro-2-(((tetrahydrofuran-2-yl)methyl)amino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6c)

[0138] Using the method of Example 1 above, with 2-methylaminotetrahydrofuran as the raw material, Compound 6c was prepared as a white solid powder with a yield of 35%. 1 1H NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H), 7.85 (d, J = 3.9 Hz, 1H), 7.70 - 7.63 (m, 2H), 6.93 - 6.87 (m, 2H), 6.61 (s, 1H), 3.97 (p, J = 6.4 Hz, 1H), 3.79 - 3.71 (m, 1H), 3.60 (q, J = 7.4 Hz, 1H), 3.46 (t, J = 5.0 Hz, 4H), 3.30 - 3.15 (m, 2H), 3.03 (t, J = 5.1 Hz, 4H), 1.92 - 1.72 (m, 3H), 1.56 (ddt, J = 12.3, 7.9, 6.4 Hz, 1H), 1.42 (s, 9H). 13 13C NMR (125 MHz, DMSO-d6) δ 158.71, 154.31, 147.12, 132.35, 122.14, 116.70, 79.40, 77.57, 67.46, 49.51, 45.93, 29.12, 28.52, 25.54. HRMS (ESI) m / z calcd for 24 H 33 FN6O3 + H] + : 473.2676; found: 473.2723.

[0139] Preparation of tert-butyl 4-(4-((5-fluoro-2-((pyridin-2-ylmethyl)amino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6d)

[0140] Using the method of Example 1 above, 2-methylaminopyridine was used as the raw material to prepare Compound 6d, which is a white solid powder with a yield of 13%. 1 H NMR (500 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.54 - 8.50 (m, 1H), 7.87 (d, J = 3.8 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H), 7.51 (s, 2H), 7.28 (t, J = 8.4 Hz, 2H), 7.22 (t, J = 6.2 Hz, 1H), 6.88 - 6.73 (m, 2H), 4.48 (d, J = 6.1 Hz, 2H), 3.46 (t, J = 4.9 Hz, 4H), 3.01 (t, J = 5.1 Hz, 4H), 1.42 (d, J = 2.0 Hz, 9H). 13 C NMR (125 MHz, DMSO-d6) δ 160.54, 158.61, 154.31, 149.17, 147.03, 136.92, 132.18, 122.17, 120.86, 116.62, 79.41, 49.47, 47.22, 28.53. HRMS (ESI) m / z calcd for [C 25 H 30 FN7O2 + H] + : 480.2523; found: 480.2578.

[0141] Preparation of tert-butyl 4-(4-((5-fluoro-2-((4-(methoxycarbonyl)phenyl)amino)pyridin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 6e)

[0142] Using the method of Example 1 above, methyl p-aminobenzoate was used as the raw material to prepare Compound 6e, which is a white solid powder with a yield of 55%. 1 H NMR (500 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.33 (s, 1H), 8.10 (d, J = 3.6 Hz, 1H), 7.82 - 7.74 (m, 4H), 7.54 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 3.79 (s, 3H), 3.49 (t, J = 5.1 Hz, 4H), 3.09 (t, J = 5.1 Hz, 4H), 1.43 (s, 9H). 1313C NMR(125 MHz, DMSO-d6) δ 166.49, 155.37, 155.35, 154.30, 150.74, 148.12, 146.12, 140.49, 140.38, 140.23, 131.18, 130.42, 124.18, 121.34, 117.59, 116.70, 79.44, 52.06, 49.41, 28.52. HRMS(ESI) m / z calcd for [C 27 H 31 FN6O4 + H] + : 523.2469; found: 523.2527.

[0143] Preparation of tert-butyl 4-(5-(2-(2-amino-1H-benzo[d]imidazol-1-yl)-5-fluoropyrimidin-4-yl)pyridin-2-yl)piperazine-1-carboxylate (Compound 11h)

[0144] 2-(4-BOC-piperazinyl)pyridine-5-boronic acid pinacol ester was reacted with 2,4-dichloro-5-fluoropyrimidine for 5 h under Suzuki reaction conditions (using H2O:THF = 1:5 as the solvent, palladium acetate as the catalyst, and potassium carbonate as the base). The solvent was evaporated, and the residue was slurried with water and filtered to obtain Compound 10, which was directly used in the next step.

[0145] Using the method of Example 1 above, with 2-aminobenzimidazole as the raw material, Compound 11h was prepared as a white solid powder with a yield of 53%. 1 1H NMR(500 MHz, DMSO-d6) δ 8.83 - 8.77 (m, 2H), 8.13 (q, J = 4.9, 4.0 Hz, 2H), 7.63 (s, 2H), 7.21 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.98 (t, J = 7.8 Hz, 1H), 6.94 (dd, J = 9.3, 3.1 Hz, 1H), 3.67 - 3.62 (m, 4H), 3.46 - 3.40 (m, 4H), 1.44 (s, 9H). 13 13C NMR(125 MHz, DMSO-d6) δ 159.70, 154.34, 153.29, 152.92, 151.37, 151.26, 149.88, 149.79, 147.12, 146.86, 143.53, 137.90, 137.83, 131.92, 123.63, 119.79, 117.23, 117.17, 115.70, 114.22, 107.10, 79.61, 44.32, 28.53. HRMS(ESI) m / z calcd for [C25 H 27 FN8O2+H] + :491.2319; found:491.2361.

[0146] Example 2: Preparation of 1-(5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 7a)

[0147] Compound 6a (1.2 g, 15 mmol) was added to 60 mL of DCM / TFA = 2 / 1 in a 150 mL eggplant-shaped flask and stirred at room temperature for 4 h. The pH was adjusted to alkaline with NaOH, washed with water three times, and the organic layer was evaporated to dryness to obtain 0.8 g of a white solid powder with a yield of 83%. 1 HNMR (500 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.37 (d, J = 3.4 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.65 (s, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 7.7 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2H), 6.78 (t, J = 7.7 Hz, 1H), 3.07 (dd, J = 6.3, 3.7 Hz, 4H), 2.86 (dd, J = 6.4, 3.7 Hz, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 154.54, 152.42, 150.04, 143.93, 143.37, 141.91, 139.74, 139.57, 131.93, 128.82, 125.67, 123.19, 119.20, 115.97, 115.26, 115.23, 50.04, 46.06. HRMS (ESI) m / z calcd for [C 21 H 21 FN8+H] + :405.1951; found:405.1975.

[0148] 5-fluoro-N 2 -(isoquinolin-3-yl)-N 4 -(4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine Preparation (Compound 7b)

[0149] Using the method of Example 2 above, with compound 6b as the raw material, compound 7b was prepared, a white solid powder with a yield of 83%. 11H NMR (500 MHz, DMSO-d6) δ 9.56 (s, 1H), 9.35 - 9.31 (m, 1H), 9.07 (s, 1H), 8.34 (s, 1H), 8.17 (d, J = 3.8 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.3 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.01 - 6.95 (m, 2H), 3.29 (s, 1H), 3.11 (t, J = 4.9 Hz, 4H), 2.92 (dd, J = 6.0, 3.6 Hz, 4H). 13 13C NMR (125 MHz, DMSO-d6) δ 154.98, 151.45, 150.81, 149.18, 142.20, 140.73, 140.57, 140.23, 137.95, 130.73, 128.05, 126.28, 125.05, 124.82, 124.77, 116.08, 104.43, 49.06, 45.86. HRMS (ESI) m / z calcd for 23 H 22 FN7 + H] + : 416.1999; found: 416.2017.

[0150] 5-Fluoro-N 4 -(4-piperazin-1-yl)phenyl)-N 2 -((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine Preparation (Compound 7c)

[0151] Using the method of Example 2 above, with compound 6c as the raw material, compound 7c was prepared, which is a white solid powder with a yield of 75%. 11H NMR (500 MHz, Chloroform-d) δ 7.76 (d, J = 3.2 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 6.91 (s, 1H), 6.87 (d, J = 8.5 Hz, 2H), 5.33 (t, J = 6.0 Hz, 1H), 4.04 (dt, J = 11.3, 5.1 Hz, 1H), 3.85 (q, J = 7.3 Hz, 1H), 3.73 (q, J = 7.3 Hz, 1H), 3.53 (dt, J = 14.2, 5.0 Hz, 1H), 3.31 (dt, J = 13.2, 6.2 Hz, 1H), 3.10–3.05 (m, 4H), 3.00 (t, J = 4.8 Hz, 4H), 2.16 (s, 1H), 1.94 (dt, J = 16.6, 6.4 Hz, 1H), 1.86 (hept, J = 6.0 Hz, 2H), 1.58 (dq, J = 15.2, 7.8 Hz, 1H). 13 13C NMR (125 MHz, Chloroform-d) δ 158.56, 158.53, 150.34, 150.26, 148.35, 139.84, 139.68, 130.76, 121.92, 116.55, 77.92, 67.97, 50.89, 46.10, 45.89, 28.74, 25.78. HRMS (ESI) m / z calcd for 19 H 25 FN6O + H] + : 373.2152; found: 373.2188.

[0152] 5-Fluoro-N 4 -(4-(piperazin-1-yl)phenyl-N 2 -(pyridin-2-ylmethyl)pyrimidine-2,4-diamine Preparation (Compound 7d)

[0153] Using the method of Example 2 above, with compound 6d as the raw material, compound 7d was prepared, which was a white solid powder with a yield of 75%. 1 1H NMR (500 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.52 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 3.9 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.65 - 7.40 (m, 2H), 7.40 - 7.26 (m, 2H), 7.20 (t, J = 6.2 Hz, 1H), 6.79 (s, 2H), 4.52 (d, J = 6.1 Hz, 2H), 3.03 (t, J = 4.8 Hz, 4H), 2.91 (t, J = 4.8 Hz, 4H). 13CNMR(125MHz,DMSO-d6)δ160.52,158.65,149.13,147.58,141.47,139.54,136.92,131.73,122.17,120.94,116.04,49.62,47.25,45.48.HRMS(ESI)m / z calcd for[C 20 H 22 FN7+H]+:380.1999;found:380.2026.

[0154] Preparation of methyl 4-((5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)amino)benzoate (Compound 7e)

[0155] Using the method of Example 2 above, with Compound 6e as the raw material, Compound 7e was prepared as a white solid powder with a yield of 72%. 1 H NMR(400MHz,DMSO-d6)δ9.63(s,1H),9.30(d,J=1.7Hz,1H),8.10(d,J=3.7Hz,1H),7.83-7.72(m,4H),7.55-7.47(m,2H),6.99-6.90(m,2H),3.80(s,3H),3.10-3.03(m,4H),2.92-2.85(m,4H). 13 C NMR(101MHz,DMSO-d6)δ166.51,155.40,155.37,150.78,150.67,148.88,146.14,140.34,140.15,130.49,130.42,124.24,121.34,117.59,115.96,52.10,50.11,45.93.HRMS(ESI)m / z calcd for[C 22 H 23 FN6O2+H]+:423.1945;found:423.1968.

[0156] Preparation of 1-(5-fluoro-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 12h)

[0157] Using the method of Example 2 above, with Compound 11h as the raw material, Compound 12h was prepared as a white solid powder with a yield of 92%. 11H NMR (400 MHz, DMSO-d6) δ 8.80 (dd, J = 15.8, 3.0 Hz, 2H), 8.19 - 8.07 (m, 2H), 7.65 (s, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 9.2 Hz, 1H), 3.58 (t, J = 5.0 Hz, 4H), 2.78 (d, J = 10.0 Hz, 4H). 13 13C NMR (101 MHz, DMSO-d6) δ 159.91, 154.35, 153.14, 152.89, 151.30, 150.57, 149.96, 149.86, 146.84, 146.57, 143.50, 137.58, 131.89, 123.56, 119.73, 116.60, 115.65, 114.25, 106.72, 45.76, 45.68. HRMS (ESI) m / z calcd for 20 12 19 [C + 19

[0158] Example 3: Preparation of 1-(5-fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 8a)

[0159] Compound 7a (0.2 g, 0.5 mmol) and acetic acid (0.03 g, 0.5 mmol) were added to a eggplant-shaped flask containing 10 mL of DCE, and the mixture was stirred at 60 °C for 20 min. Then, isovaleraldehyde (0.22 g, 2.5 mmol) and sodium triacetoxyborohydride (0.3 g, 1.5 mmol) were added to the reaction solution, and the mixture was stirred at 60 °C for 13 h. The reaction solution was extracted with dichloromethane and water, and the aqueous layer was washed with dichloromethane three times until there was no fluorescence at 254 nm in the aqueous layer. The organic layers were combined, dried by evaporation, and further separated and purified by silica gel column chromatography (200 - 300 mesh, DCM:MeOH:Et3N = 20:1:1) to obtain 0.1 g of a white solid powder with a yield of 50%. 11H NMR (500 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.38 (d, J = 3.4 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.64 (s, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 7.7 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 7.01 - 6.96 (m, 2H), 6.81 - 6.74 (m, 1H), 3.15 (t, J = 5.0 Hz, 4H), 2.53 - 2.47 (m, 5H), 2.32 (t, J = 7.6 Hz, 2H), 1.66 - 1.54 (m, J = 6.8 Hz, 1H), 1.35 (q, J = 7.2 Hz, 2H), 0.89 (d, J = 6.7 Hz, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 154.51, 149.39, 143.36, 139.77, 139.60, 131.91, 128.89, 125.63, 123.19, 119.20, 115.98, 115.23, 56.55, 53.30, 48.90, 35.85, 26.32, 23.11. HRMS (ESI) m / z calcd for 26 H 31 [C + 18H26FN8 + H]+: 475.2734; found: 475.2762.

[0160] 5-Fluoro-N 4 -(4-(4-isopentylpiperazin-1-yl)phenyl)-N 2 -isoquinolin-3-yl)pyrimidine-2,4-diamine Preparation (Compound 8b)

[0161] Using the method of Example 3 above, with compound 7b as the raw material, compound 8b was prepared as a white solid powder with a yield of 72%. 11H NMR(500 MHz, DMSO-d6) δ 9.47 (s, 1H), 9.32 (s, 1H), 9.06 (s, 1H), 8.33 (s, 1H), 8.15 (d, J = 3.7 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.56 (dd, J = 7.9, 5.6 Hz, 3H), 7.48 (d, J = 8.3 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 3.17 (t, J = 4.9 Hz, 4H), 2.55 (t, J = 4.9 Hz, 4H), 2.36 (t, J = 7.6 Hz, 2H), 1.63 (dp, J = 13.3, 6.7 Hz, 1H), 1.39 (q, J = 7.4 Hz, 2H), 0.91 (d, J = 6.6 Hz, 6H). 13 13C NMR(125 MHz, DMSO-d6) δ 151.40, 149.19, 137.94, 130.67, 128.04, 126.30, 125.07, 124.74, 115.96, 104.43, 56.56, 53.45, 49.05, 39.54, 35.87, 26.36, 23.13. HRMS(ESI) m / z calcd for 28 1H 32 FN7 + H] + : 486.2781; found: 486.2826.

[0162] 5-Fluoro-N 4 -(4-(4-Isopentylpiperazin-1-yl)phenyl)-N 2 -((Tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine Preparation (Compound 8c)

[0163] Using the method of Example 3 above, with compound 7c as the raw material, compound 8c was prepared, which was a white solid powder with a yield of 36%. 11H NMR (500 MHz, Chloroform-d) δ 7.81 (d, J = 3.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.60 (s, 1H), 5.17 (d, J = 6.2 Hz, 1H), 4.08 (p, J = 6.4 Hz, 1H), 3.89 (q, J = 7.3 Hz, 1H), 3.77 (q, J = 7.3 Hz, 1H), 3.57 (dt, J = 10.4, 4.9 Hz, 1H), 3.35 (dt, J = 13.2, 6.1 Hz, 1H), 3.20 (t, J = 4.9 Hz, 4H), 2.63 (t, J = 4.9 Hz, 4H), 2.42 (t, J = 7.9 Hz, 2H), 1.98 (dt, J = 12.1, 6.6 Hz, 1H), 1.92 (d, J = 6.7 Hz, 1H), 1.89 (d, J = 7.1 Hz, 1H), 1.64 (dt, J = 13.9, 7.0 Hz, 2H), 1.45 (q, J = 7.5 Hz, 2H), 0.94 (d, J = 6.6 Hz, 6H). 13 13C NMR (125 MHz, Chloroform-d) δ 158.50, 150.27, 147.91, 130.56, 121.75, 116.54, 68.06, 57.05, 53.37, 49.64, 45.87, 35.91, 28.73, 26.73, 25.84, 22.76. HRMS (ESI) m / z calcd for 24 C 35 21H + FN6O + H] 4 : 443.2935; found: 443.2966. 5-Fluoro-N 2 -(4-(4-Isopentylpiperazin-1-yl)phenyl)-N

[0164] -(Pyridin-2-ylmethyl)pyrimidine-2,4-diamine Preparation (Compound 8d) 11H NMR (400 MHz, Chloroform-d) δ 8.59 (dd, J = 5.3, 2.9 Hz, 1H), 7.88 - 7.81 (m, 1H), 7.63 (ddq, J = 9.6, 5.4, 1.8 Hz, 1H), 7.46 (dd, J = 9.1, 3.0 Hz, 2H), 7.32 (d, J = 7.9 Hz, 1H), 7.22 – 7.14 (m, 1H), 6.94 - 6.85 (m, 2H), 6.62 (dd, J = 11.6, 4.0 Hz, 1H), 5.91 - 5.78 (m, 1H), 3.20 (dt, J = 6.2, 3.2 Hz, 4H), 2.68 – 2.61 (m, 4H), 2.44 (td, J = 7.7, 3.2 Hz, 2H), 1.63 (dp, J = 13.3, 6.7 Hz, 1H), 1.45 (dd, J = 9.5, 6.1 Hz, 2H), 1.27 (d, J = 4.2 Hz, 2H), 0.94 (dd, J = 6.6, 1.3 Hz, 6H). 13 13C NMR (101 MHz, Chloroform-d) δ 154.96, 149.07, 147.77, 139.84, 139.68, 136.54, 130.43, 126.07, 121.59, 121.46, 120.27, 116.57, 116.08, 56.94, 53.19, 49.44, 35.62, 29.69, 29.43, 26.72, 23.35, 22.73. HRMS (ESI) m / z calcd for 25 12 32 C + 25

[0165] Preparation of Methyl 4 - ((5 - fluoro - 4 - ((4 - (4 - isopentylpiperazin - 1 - yl)phenyl)amino)pyrimidin - 2 - yl)amino)benzoate (Compound 8e)

[0166] Using the method of Example 3 above, with Compound 7e as the starting material, Compound 8e was prepared as a white solid powder with a yield of 57%. 11H NMR (500 MHz, Chloroform-d) δ 7.98–7.91 (m, 3H), 7.63 - 7.57 (m, 2H), 7.48 - 7.43 (m, 2H), 7.42 (s, 1H), 6.99 - 6.94 (m, 2H), 6.80 (d, J=2.9 Hz, 1H), 3.90 (s, 3H), 3.24 (t, J=5.0 Hz, 4H), 2.65 (t, J=4.9 Hz, 4H), 2.44 (dd, J=9.3, 6.6 Hz, 2H), 1.70 - 1.58 (m, J=6.8 Hz, 1H), 1.46 (q, J=7.4 Hz, 2H), 0.95 (d, J=6.6 Hz, 6H). 13 13C NMR (125 MHz, Chloroform-d) δ 166.96, 148.81, 144.36, 139.73, 139.58, 130.76, 129.25, 123.30, 122.84, 117.34, 116.37, 57.04, 53.32, 51.81, 49.41, 35.91, 26.72, 22.77. HRMS (ESI) m / z calcd for 27 1H 33 [C + 19H26FN6O2 + H]+: 493.2727; found: 493.2754.

[0167] Preparation of 1-(5-Fluoro-4-(6-(4-isopentylpiperazin-1-yl)pyridin-3-yl)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 13h)

[0168] Using the method of Example 3 above, with Compound 12h as the raw material, Compound 13h was prepared as a yellow solid powder with a yield of 54%. 1 1H NMR (400 MHz, Chloroform-d) δ 9.08 (d, J=2.5 Hz, 1H), 8.52 (d, J=3.4 Hz, 1H), 8.36 (d, J=8.0 Hz, 1H), 8.30 (dd, J=9.1, 2.5 Hz, 1H), 7.44 (d, J=7.8 Hz, 1H), 7.25 (t, J=7.6 Hz, 1H), 7.14 (t, J=7.7 Hz, 1H), 6.86 (s, 2H), 6.78 (d, J=9.2 Hz, 1H), 3.82 - 3.75 (m, 4H), 2.60 (t, J=5.1 Hz, 4H), 2.48 - 2.39 (m, 2H), 1.72 - 1.58 (m, J=6.6 Hz, 1H), 1.46 (q, J=7.1 Hz, 2H), 0.95 (d, J=6.6 Hz, 6H).13 C NMR (101 MHz, Chloroform-d) δ 159.84, 153.77, 153.07, 150.49, 150.35, 150.25, 145.50, 145.23, 142.52, 137.64, 131.93, 123.73, 120.49, 116.14, 114.49, 106.10, 57.01, 53.05, 44.68, 35.85, 26.67, 22.76. HRMS (ESI) m / z calcd for [C 25 H 29 FN8 + H] + : 461.2577; found: 461.2616.

[0169] Example 4: Preparation of 1-(5-fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 6f)

[0170] Using p-fluoronitrobenzene and 2f-g as starting materials, a nucleophilic substitution reaction occurred under reflux for 5 h in dimethyl sulfoxide as the solvent to obtain intermediate 3f-g. Then, the nitro group was reduced by the method of iron powder reduction to obtain intermediate 4f-g with an amino group at the para position. Intermediate 4g-f and 2,4-dichloro-5-fluoropyrimidine underwent a nucleophilic substitution reaction in methanol to obtain compound 5f-g. The first three steps of the synthesis were relatively simple, and a solid precipitated directly after the reaction was completed. Therefore, the product was directly obtained by suction filtration without NMR confirmation and directly used in the next step. Compound 5f (2 g, 6 mmol), 2-aminobenzimidazole (1.655 g, 12 mmol), Pd2(dba)3 (0.285 g, 0.28 mmol), cesium carbonate (3.038 g, 9.3 mmol), and Xantphos (0.180 g, 0.31 mmol) were added to a 120 mL pressure bottle, 50 mL of dioxane dried over 4A molecular sieve was added, and it was added to a 100 mL sealed tube containing a magnetic stirrer. 30 mL of dioxane (dried over 4A molecular sieve) was added, the air in the bottle was replaced with nitrogen, the lid was screwed on, and it was placed in an ultrasonic bath for dissolution. It was reacted in an oil bath at 120 °C for 12 h. After cooling to room temperature, the reaction solution was concentrated under reduced pressure, extracted with dichloromethane / water three times, and the organic layer was dried with anhydrous Na2SO4 for 4 h. Filtered by suction, the organic solvent was concentrated under reduced pressure to obtain a yellow-green solid, which was separated and purified by silica gel column chromatography (200 - 300 mesh, DCM:MeOH = 40:1) to obtain a white solid powder with a yield of 46%. 1HNMR(500MHz,DMSO-d6)δ9.86(s,1H),8.39(dd,J=3.5,1.6Hz,1H),7.97(d,J=8.0Hz,1H),7.60(s,2H),7.41 -7.36(m,2H),7.14(d,J=7.8Hz,1H),7.07-6.98(m,3H),6.77(t,J=7.7Hz,1H),3.21-3.15(m,4H),2.49(s,4H),2.25(d,J=1.6Hz,3H). 13 C NMR(125MHz,DMSO-d6)δ154.49,149.34,143.36,141.66,139.81,139.61,131.91,128.95,125.66,123.19,119.20,116.07,115.25,115.21,55.02,48.72,46.20.HRMS(ESI)m / z calcd for[C 22 H 23 FN8+H] + :419.2108;found:419.2134.

[0171] Preparation of 1-(4-((4-(4-ethylpiperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine (Compound 6g)

[0172] Using the method of Example 4 above, with Compound 5g as the raw material, Compound 6g was prepared, which is a white solid powder with a yield of 54%. 1 H NMR(400MHz,DMSO-d6)δ9.79(s,1H),8.36(d,J=3.5Hz,1H),7.99(d,J=8.0Hz,1H),7.55(s,2H),7.39(d,J=8.5Hz,2H),7.15(d,J=7.7Hz,1H),7.04(t,J=7.6Hz,1H),6.99(d,J=8.6Hz,2H),6.78(t,J=7.7Hz,1H),3.20-3.15(m,4H),2.56(t,J=5.0Hz,4H),2.42(q,J=7.2Hz,2H),1.06(t,J=7.2Hz,3H). 1313C NMR (101 MHz, DMSO-d6) δ 154.50, 149.35, 143.36, 139.80, 139.60, 131.90, 128.95, 125.66, 123.20, 119.20, 116.03, 115.23, 52.73, 52.09, 48.79, 12.39. HRMS (ESI) m / z calcd for [C 23 H 25 FN8+H] + : 433.2264; found: 433.2289. N 2 -(1H-Benzimidazol-2-yl)-N 4 -(4-(4-Ethylpiperazin-1-yl)phenyl)-5-fluoropyrimidine-2,4-diamine (Compound 6g-1)

[0173] Using the method of Example 4 above, with Compound 5g as the raw material, Compound 6g-1 was prepared, which is a white solid powder with a yield of 30%. 1 1H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 9.57 (s, 1H), 8.17 (s, 1H), 7.52 (d, J = 8.3 Hz, 3H), 7.38 - 7.34 (m, 1H), 7.02 (s, 2H), 6.97 (d, J = 8.5 Hz, 3H), 3.40 (s, 4H), 2.38 (q, J = 7.3 Hz, 4H), 1.04 (t, J = 7.3 Hz, 5H). 13 13C NMR (125 MHz, DMSO-d6) δ 154.19, 151.19, 151.10, 149.60, 148.62, 142.59, 140.62, 129.79, 115.89, 52.93, 52.14, 48.66, 12.46. HRMS (ESI) m / z calcd for [C 23 H 25 FN8+H] + : 433.2264; found: 433.2289.

[0174] Example 5: N,N-Dimethyl-6-nitro-1H-benzimidazol-2-amine (Compound 16)

[0175] Dissolve dichloromethylenedimethylammonium chloride (19.44 g, 120 mmol) in 300 mL of DCM in a 500 mL eggplant-shaped flask, add 4-nitro-o-phenylenediamine (15.30 g, 100 mmol), place the eggplant-shaped flask at room temperature and stir, and place a reflux condenser on the eggplant-shaped flask. Add triethylamine (30.30 g, 300 mmol) to the constant-pressure dropping funnel, seal the mouth of the constant-pressure dropping funnel with a balloon, insert it onto the reflux condenser, and seal all the joints with sealing film. Slowly add triethylamine dropwise to the eggplant-shaped flask, and finish dropping in 20 min. Raise the temperature to reflux and continue the reaction for 4 h. Rotavaporize the reaction solution to obtain a yellowish-white solid. Recrystallize with methanol to obtain 18.1 g of a yellowish-white solid. The yield is 88%. 1 H NMR (500 MHz, DMSO-d6) δ 13.91 (s, 1H), 8.16 (dd, J = 8.7, 2.0 Hz, 1H), 8.11 (d, J = 1.9 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 3.28 (s, 6H). HRMS (ESI) m / z calcd for [C9H 10 N4O2+H] + : 207.0877; found: 207.0877 [M+H] + .

[0176] Example 6: N 2 , N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 17)

[0177] Dissolve N,N-dimethyl-5-nitro-1H-benzo[d]imidazole-2-amine (2.06 g, 10 mmol) in 40 mL of MeOH in a 100 mL special reaction vessel for a hydrogenator, and add 0.20 g of 10% palladium on carbon (containing about 55% water). Stir and react at room temperature under a hydrogen environment of 0.4 MPa for 18 h. Filter off the palladium on carbon by suction, rotavaporize the filtrate to obtain a gray solid. Recrystallize with methanol to obtain 1.45 g of a gray solid. The yield is 82%. 1 H NMR (500 MHz, DMSO-d6) δ 7.05 (d, J = 8.4 Hz, 1H), 6.69 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 3.18 (s, 6H). HRMS (ESI) m / z calcd for [C9H 12 N4+H] + : 177.1135; found: 117.1107 [M+H] + .

[0178] Example 7: 4-(4-nitrophenyl)morpholine (Compound 19a)

[0179] p-Fluoronitrobenzene (7.05 g, 50 mmol) and morpholine (5.22 g, 60 mmol) were added to a 250 mL eggplant-shaped flask, and potassium carbonate (10.35 g, 75 mmol) was added. The reaction was carried out at 90 °C for 5 h. Then it was cooled to room temperature. After cooling to room temperature, it was extracted with ethyl acetate. The organic layer was washed with water twice and then washed with saturated NaCl solution once. The organic layer was dried over anhydrous Na2SO4 and left for 4 h. It was filtered by suction, and the organic solvent was rotary evaporated to obtain a yellow solid. Recrystallization from ethyl acetate gave 9.5 g of yellow crystals 19a with a yield of 91%. 1 HNMR (500 MHz, DMSO-d6) δ 8.08 (d, J = 9.5 Hz, 2H), 7.04 (d, J = 9.5 Hz, 2H), 3.76–3.70 (m, 4H), 3.44–3.38 (m, 4H). HRMS (ESI) m / z calcd for [C 10 H 12 N2O3+H] + : 209.0921; found: 209.0921 [M+H] + .

[0180] 1-(4-Nitrophenyl)pyrrole (Compound 19b)

[0181] Using the method of Example 7 above, with Compound 18b as the raw material, Compound 19b was prepared with a yield of 90% as a yellow solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.04 (d, J = 9.4 Hz, 2H), 6.60 (d, J = 9.4 Hz, 2H), 3.37 (t, J = 6.7 Hz, 4H), 2.04–1.94 (m, 4H). HRMS (ESI) m / z calcd for [C 10 H 12 N2O2+H] + : 193.0972; found: 193.0972 [M+H] + .

[0182] N,N-Diethyl-4-nitroaniline (Compound 19c)

[0183] Using the method of Example 7 above, with Compound 18c as the raw material, Compound 19c was prepared with a yield of 88% as a yellow solid. 11H NMR (500 MHz, DMSO-d6) δ 8.03 (d, J = 9.5 Hz, 2H), 6.75 (d, J = 9.5 Hz, 2H), 3.48 (q, J = 7.1 Hz, 4H), 1.14 (t, J = 7.1 Hz, 6H). HRMS (ESI) m / z calcd for [C 10 H 14 N2O2+H] + : 195.1128; found: 195.1131 [M+H] + .

[0184] 4-Nitro-N,N-dipropylaniline (Compound 19d)

[0185] Using the method of Example 7 above, with Compound 18d as the starting material, Compound 19d was prepared in a yield of 87%, as a yellow solid. 1 1H NMR (500 MHz, DMSO-d6) δ 8.01 (d, J = 9.4 Hz, 2H), 6.75 (d, J = 9.5 Hz, 2H), 3.47–3.34 (m, 4H), 1.66–1.46 (m, 4H), 0.91 (t, J = 7.3 Hz, 6H). HRMS (ESI) m / z calcd for [C 12 H 18 N2O2+H] + : 223.1441; found: 223.1445 [M+H] + .

[0186] 1-(4-Nitrophenyl)piperidine (Compound 19e)

[0187] Using the method of Example 7 above, with Compound 18e as the starting material, Compound 19e was prepared in a yield of 90%, as a yellow solid. 1 1H NMR (500 MHz, DMSO-d6) δ 8.02 (d, J = 9.5 Hz, 2H), 6.98 (d, J = 9.6 Hz, 2H), 3.54–3.44 (m, 4H), 1.69–1.60 (m, 2H), 1.57 (m, 4H). HRMS (ESI) m / z calcd for [C 11 H 14 N2O2+H] + : 207.1128; found: 207.1133 [M+H] + .

[0188] tert-Butyl 4-(4-nitrophenyl)piperazine-1-carboxylate (Compound 19f)

[0189] Using the method of Example 7 above, compound 19f was prepared from compound 18f with a yield of 92%, as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ8.07(d,J=9.4Hz,2H),7.01(d,J=9.5Hz,2H),3.48(m,8H),1.43(s,9H).HRMS(ESI)m / z calcd for[C 15 H 21 N3O4+Na] + :330.1426;found:330.1424[M+Na] + .

[0190] tert-Butyl 4-(5-nitropyridin-2-yl)piperazine-1-carboxylate (Compound 19g)

[0191] Using the method of Example 7 above, compound 19g was prepared from compound 18g with a yield of 90%, as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ8.97(d,J=2.7Hz,1H),8.25(dd,J=9.6,2.8Hz,1H),6.95(t,J=11.9Hz,1H),3.91–3.66(m,4H),3.61–3.38(m,4H),1.43(s,9H).HRMS(ESI)m / zcalcd for[C 14 H 20 N4O4+Na] + :331.1377;found:331.1382[M+Na] + .

[0192] tert-Butyl 4-(5-nitropyridin-2-yl)piperazine-1-carboxylate (Compound 19h)

[0193] Using the method of Example 7 above, compound 19h was prepared from compound 18h with a yield of 80%, as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ8.24(d,J=2.9Hz,1H),8.16(d,J=9.2Hz,1H),7.46(dd,J=9.2,3.0Hz,1H),3.51(dd,J=14.0,5.6Hz,8H),1.43(s,9H).HRMS(ESI)m / zcalcd for[C 14 H 20 N4O4+Na] +: 309.1557; found: 309.1571 [M+H] + .

[0194] Example 8: 4-Morpholinoaniline (Compound 20a)

[0195] 4-Morpholinonitrobenzene (6.24 g, 30 mmol), iron powder (8.40 g, 150 mmol), and ammonium chloride (7.95 g, 150 mmol) were added to a 250 mL eggplant-shaped flask. 120 mL of a solvent with ethanol / water = 5 / 1 was added and the mixture was refluxed for 5 h. Then it was allowed to cool to room temperature. After cooling to room temperature, it was filtered through a funnel filled with diatomaceous earth. A small amount of sodium sulfite was added to the filtrate to prevent the product from being oxidized by oxygen in the air. Ethanol in the solvent was evaporated by rotary evaporation at low temperature. The mixture was extracted with a solvent of dichloromethane / methanol = 10 / 1 and water, and the aqueous solution was washed 3 times with a solvent of dichloromethane / methanol = 10 / 1. The organic layers were combined, dried over anhydrous Na2SO4 and left for 4 h. Sodium sulfate was filtered off, and the organic solvent was evaporated to dryness to obtain a gray oil. A total of 4.5 g was obtained, with a yield of 84%. Since this product is easily oxidized in air, it was directly used in the next step. Compounds 20b - 20h were prepared by the same method as 20a and were also directly used in the next step without structural confirmation.

[0196] Example 9: 2-Chloro-5-fluoro-N-(4-morpholinophenyl)pyrimidin-4-amine (Compound 22a)

[0197] 4-Morpholinoaniline (3.56 g, 20 mmol) and 2,4-dichloro-5-fluoropyrimidine (4.65 g, 28 mmol) were added to a 250 mL eggplant-shaped flask. 120 mL of a solvent with methanol / water = 1 / 1 was added and the reaction was carried out at 60 °C for 12 h. Then it was left at room temperature overnight. White solid precipitated in the flask. The solid was filtered to obtain an off-white solid, which was recrystallized from methanol to obtain 5.0 g of an off-white solid, with a yield of 81%. 1 H NMR (500 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.24 (d, J = 3.5 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 9.1 Hz, 2H), 3.89–3.53 (m, 4H), 3.22–2.92 (m, 4H). HRMS (ESI) m / z calcd for [C 14 H 14 ClFN4O + H] + : 309.0913; found: 309.0923 [M+H] + .

[0198] 2-Chloro-5-fluoro-N-(4-(pyrrol-1-yl)phenyl)pyrimidin-4-amine (Compound 22b)

[0199] Using the method of Example 9 above, compound 22b was prepared from compound 20b with a yield of 77%, as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.71 (s, 1H), 8.17 (d, J = 3.6 Hz, 1H), 7.40 (d, J = 8.9 Hz, 2H), 6.53 (d, J = 9.0 Hz, 2H), 3.21 (t, J = 6.5 Hz, 4H), 2.06–1.78 (m, 4H). HRMS (ESI) m / z calcd for [C 14 H 14 ClFN4+H] + : 293.0964; found: 293.0976 [M+H] + .

[0200] N 1 -(2-Chloro-5-fluoropyrimidin-4-yl)-N 4 ,N 4 -diethylbenzene-1,4-diamine (compound 22c)

[0201] Using the method of Example 9 above, compound 22c was prepared from compound 20c with a yield of 81%, as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.19 (d, J = 3.6 Hz, 1H), 7.38 (d, J = 9.1 Hz, 2H), 6.67 (d, J = 9.1 Hz, 2H), 3.34 (t, J = 7.0 Hz, 4H), 1.09 (t, J = 7.0 Hz, 6H). HRMS (ESI) m / z calcd for [C 14 H 16 ClFN4+H] + : 295.1120; found: 295.1131 [M+H] + .

[0202] N 1 -(2-Chloro-5-fluoropyrimidin-4-yl)-N 4 ,N 4 -dipropylbenzene-1,4-diamine (compound 22d)

[0203] Using the method of Example 9 above, compound 22d was prepared from compound 20d with a yield of 75%, as a yellow oil. 11H NMR (500 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.18 (d, J = 3.6 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 6.64 (d, J = 9.1 Hz, 2H), 3.29–3.16 (m, 4H), 1.61–1.46 (m, 4H), 0.89 (t, J = 7.4 Hz, 6H). HRMS (ESI) m / z calcd for [C 16 H 20 ClFN4 + H] + : 323.1433; found: 323.1454 [M + H] + .

[0204] 2-Chloro-5-fluoro-N-(4-piperidin-1-yl)phenyl)pyrimidin-4-amine (Compound 22e)

[0205] Using the method of Example 9 above, with Compound 20e as the starting material, Compound 22e was prepared with a yield of 83%, as a white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.22 (d, J = 3.5 Hz, 1H), 7.46 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 3.18–3.05 (m, 4H), 1.66–1.56 (m, 4H), 1.56–1.47 (m, 2H). HRMS (ESI) m / z calcd for [C 15 H 16 ClFN4 + H] + : 307.1120; found: 307.1135 [M + H] + .

[0206] tert-Butyl 1-(4-((2-chloro-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-4-carboxylate (Compound 22f)

[0207] Using the method of Example 9 above, with Compound 20f as the starting material, Compound 22f was prepared with a yield of 85%, as a white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.23 (d, J = 3.5 Hz, 1H), 7.51 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 9.1 Hz, 2H), 3.52–3.41 (m, 4H), 3.14–3.03 (m, 4H), 1.42 (s, 9H). HRMS (ESI) m / z calcd for [C 19 H 23ClFN5O2+H] + : 408.1597; found: 408.1601 [M+H] + .

[0208] tert-Butyl 4-(5-((2-chloro-5-fluoropyrimidin-4-yl)amino)pyridin-2-yl)piperazine-1-carboxylate (Compound 22g)

[0209] Using the method of Example 9 above, with Compound 20g as the raw material, Compound 22g was prepared with a yield of 75%, a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.34 (d, J = 2.7 Hz, 1H), 8.26 (d, J = 3.4 Hz, 1H), 7.78 (dd, J = 9.1, 2.7 Hz, 1H), 6.91 (d, J = 9.1 Hz, 1H), 3.46 (m, 8H), 1.43 (s, 9H). HRMS (ESI) m / z calcd for [C 18 H 22 ClFN6O2+H] + : 409.1550; found: 409.1557 [M+H] + .

[0210] tert-Butyl 4-(6-((2-chloro-5-fluoropyrimidin-4-yl)amino)pyridin-3-yl)piperazine-1-carboxylate (Compound 22h)

[0211] Using the method of Example 9 above, with Compound 20h as the raw material, Compound 22h was prepared with a yield of 72%, a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.31 (d, J = 3.3 Hz, 1H), 8.11 (d, J = 2.9 Hz, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.49 (dd, J = 9.1, 3.0 Hz, 1H), 3.57–3.41 (m, 4H), 3.28–3.01 (m, 4H), 1.42 (s, 9H). HRMS (ESI) m / z calcd for [C 18 H 22 ClFN6O2+H] + : 409.1550; found: 409.1581 [M+H] + .

[0212] N 1 -(2-chloro-5-fluoropyrimidin-4-yl)-N 4 ,N 4-Dimethylbenzene-1,4-diamine (Compound 22i)

[0213] Using the method of Example 9 above, with Compound 20i as the raw material, Compound 22i was prepared with a yield of 79%, as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.20 (d, J = 3.6 Hz, 1H), 7.42 (d, J = 9.0 Hz, 2H), 6.75 (d, J = 9.1 Hz, 2H), 2.89 (s, 6H). HRMS (ESI) m / z calcd for [C 12 H 12 ClFN4 + H] + : 267.0807; found: 267.0817 [M + H] + .

[0214] Example 10: N 6 -(5-Fluoro-4-((4-morpholinophenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23a)

[0215] Compound 22a (2.53 g, 8 mmol), Compound 9 (1.4 g, 8 mmol), Pd2(dba)3 (0.18 g, 0.02 mmol), Xanphos (0.46 g, 0.08 mmol), and cesium carbonate (4.89 g, 15 mmol) were added to a 120 mL pressure-resistant flask. 50 mL of dioxane was added, a magnetic stirrer was added, and the air in the flask was replaced with nitrogen. After sealing, the mixture was sonicated to dissolve the raw materials in the solvent as much as possible. The reaction was carried out in an oil bath at 120 °C for 12 h. Then it was cooled to room temperature. After cooling to room temperature, the reaction solution was concentrated by rotary evaporation, extracted with dichloromethane / water, and the aqueous layer was washed twice with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and left for 4 h. Filtration was carried out by suction, and the organic solvent was concentrated by rotary evaporation to obtain a black oil, which was separated and purified by silica gel column chromatography (DCM:MeOH:Et3N = 200:1:1) to obtain 2.34 g of a white solid with a yield of 65%. 11H NMR (500 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.07 (s, 1H), 8.83 (s, 1H), 7.98 (d, J = 3.7 Hz, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.57 (s, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.9 Hz, 2H), 3.83–3.67 (m, 4H), 3.11–3.04 (m, 4H), 3.03 (s, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.17, 156.76, 156.74, 150.10, 150.01, 147.44, 141.70, 140.36, 140.21, 139.75, 134.03, 131.69, 122.63, 115.80, 113.18, 66.62, 49.43, 38.56. HRMS (ESI) m / z calcd for 23 H 25 [C + 14H18FN8O + H] + : 449.2208; found: 449.2211 [M + H]

[0216] N 6 -(5-Fluoro-4-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23b)

[0217] Using the method of Example 10 above, with Compound 22b as the raw material, Compound 23b was prepared with a yield of 62%, as an off-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.92 (s, 1H), 8.74 (s, 1H), 7.94 (d, J = 3.4 Hz, 1H), 7.59 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 6.51 (d, J = 8.4 Hz, 2H), 3.21 (m, 4H), 3.03 (s, 6H), 1.94 (m 4H). 1313C NMR (125 MHz, DMSO-d6) δ 170.77, 157.31, 156.76, 150.29, 150.20, 144.83, 141.76, 139.89, 139.81, 139.75, 133.96, 127.96, 123.37, 112.83, 111.87, 47.53, 38.02, 24.80. HRMS (ESI) m / z calcd for [C 23 H 25 FN8+H] + : 433.2259; found: 443.2261 [M+H] + .

[0218] N 6 -(4-((4-(Diethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23c)

[0219] Using the method of Example 10 above, with Compound 22c as the raw material, Compound 23c was prepared with a yield of 65%, as an off-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 13.10 (s, 1H), 9.16 (s, 1H), 9.05 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.51 (m, 3H), 7.20 (d, J = 8.6 Hz, 1H), 6.66 (s, 2H), 3.32 (d, J = 5.3 Hz, 4H), 3.22 (s, 6H), 1.08 (t, J = 6.9 Hz, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 155.99, 151.44, 150.54, 150.44, 142.19, 140.25, 139.46, 137.34, 131.32, 125.66, 123.88, 114.96, 112.27, 111.05, 102.42, 44.30, 39.29, 12.91. HRMS (ESI) m / z calcd for [C 23 H 27 FN8+H] + : 435.2415; found: 435.2420 [M+H] + .

[0220] N 6 -(4-((4-(Dipropylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2, N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23d)

[0221] Using the method of Example 10 above, with Compound 22d as the raw material, Compound 23d was prepared with a yield of 61%, as an off-white solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.22 (d, J = 29.3 Hz, 1H), 8.93 (s, 1H), 8.73 (d, J = 32.5 Hz, 1H), 7.92 (s, 1H), 7.55 (m, 3H), 7.12 (m, 1H), 6.98 (m, 1H), 6.58 (d, J = 6.9 Hz, 2H), 3.28–3.14 (m, 4H), 3.02 (s, 6H), 1.63–1.42 (m, 4H), 0.88 (t, J = 7.3 Hz, 6H). (Tautomerism) 13 C NMR (125 MHz, DMSO-d6) δ 157.70, 157.11, 156.80, 150.25, 150.17, 144.81, 144.40, 141.72, 141.70, 139.84, 139.74, 139.57, 139.41, 134.78, 133.25, 130.23, 127.78, 123.41, 114.35, 113.79, 112.06, 111.79, 108.15, 107.60, 101.83, 52.72, 38.64, 20.54, 11.73. HRMS (ESI) m / z calcd for [C 25 H 31 FN8 + H] + : 463.2728; found: 463.2732 [M + H] + .

[0222] N 6 -(5-Fluoro-4-((4-(piperidin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 , N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23e)

[0223] Using the method of Example 10 above, with Compound 22e as the raw material, Compound 23e was prepared with a yield of 70%, as an off-white solid. 11H NMR (500 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.80 (s, 1H), 7.98 (d, J = 3.0 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.58 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 3.12–3.05 (m, 4H), 3.04 (s, 6H), 1.63 (s, 4H), 1.52 (d, J = 5.0 Hz, 2H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.35, 156.77, 150.10, 150.02, 148.19, 141.69, 140.32, 140.17, 139.74, 133.88, 131.12, 122.62, 116.51, 113.15, 113.09, 50.62, 38.55, 25.78, 24.25. HRMS (ESI) m / z calcd for 24 C 27 22 + H + FN8 + H]

[0224] tert-Butyl 4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (Compound 23f)

[0225] Using the method of Example 10 above, with Compound 22f as the starting material, Compound 23f was prepared with a yield of 69%, as an off-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.80 (s, 1H), 7.99 (d, J = 3.2 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.60 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.5 Hz, 2H), 3.48 (m, 4H), 3.05 (m, 10H), 1.44 (s, 9H). 1313C NMR (125 MHz, DMSO-d6) δ 156.79, 156.74, 150.10, 150.01, 147.29, 141.73, 140.29, 139.78, 134.16, 132.04, 122.60, 116.79, 113.19, 111.39, 104.43, 79.36, 49.55, 38.61, 28.55. HRMS (ESI) m / z calcd for [C 28 H 34 FN9O2 + H] + : 548.2892; found: 548.2898 [M + H] + .

[0226] tert-Butyl 4-(5-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-2-yl)piperazine-1-carboxylate (Compound 23g)

[0227] Using the method of Example 10 above, with Compound 22g as the raw material, Compound 23g was prepared with a yield of 49%, as a light yellow solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.17 (s, 1H), 8.90 (s, 1H), 8.53 (s, 1H), 7.99 (d, J = 10.7 Hz, 2H), 7.58 (s, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H), 3.02 (s, 6H), 1.43 (s, 9H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.07, 156.60, 156.58, 155.74, 154.41, 150.27, 150.18, 141.81, 141.16, 140.43, 140.28, 139.85, 136.89, 135.94, 132.38, 127.51, 112.95, 107.44, 79.47, 55.39, 45.51, 38.57, 28.55. HRMS (ESI) m / z calcd for [C 27 H 33 FN 10 O2 + H] + : 549.2845; found: 549.2851 [M + H] + .

[0228] tert-Butyl 4-(6-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-2-yl)piperazine-1-carboxylate (Compound 23h)

[0229] Using the method of Example 10 above, with Compound 22h as the raw material, Compound 23h was prepared with a yield of 45%, as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.03 (s, 1H), 8.26–7.99 (m, 3H), 7.64 (s, 1H), 7.35 (dd, J = 9.0, 2.5 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 3.49 (t, 4H), 3.10 (t, 4H), 3.06 (s, 6H), 1.43 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 156.76, 156.52, 149.34, 149.23, 145.16, 143.69, 141.64, 141.42, 141.23, 139.19, 136.46, 134.10, 125.96, 116.05, 113.38, 111.56, 104.59, 79.51, 52.45, 49.08, 38.60, 28.52. HRMS (ESI) m / z calcd for [C 27 H 33 FN 10 O2 + H] + : 549.2845; found: 549.2849 [M + H] + .

[0230] N 6 -(4-((4-(dimethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 ,N-dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 23i)

[0231] Using the method of Example 10 above, with Compound 22i as the raw material, Compound 23i was prepared with a yield of 55%, as a white solid. 11H NMR (500 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.83 (s, 1H), 7.98 (d, J = 3.2 Hz, 1H), 7.64 (s, 1H), 7.62 (d, J = 3.5 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 3.04 (s, 6H), 2.86 (s, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.20, 156.79, 150.28, 150.20, 147.40, 141.78, 140.05, 139.90, 139.83, 134.12, 129.14, 123.14, 113.07, 111.32, 104.43, 40.99, 38.56. HRMS (ESI) m / z calcd for 21 H 23 [C + H + FN8 + H]

[0232] Example 11: N 6 -(5-Fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 , N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24a)

[0233] 23f (8.2 g, 15 mmol) was added to 45 mL of DCM / TAF = 2 / 1 in a 100 mL eggplant-shaped flask and stirred at room temperature for 4 h. The reaction solution was evaporated to dryness, and triethylamine and methanol were added and evaporated to dryness several times to obtain a black-gray oily substance. It was extracted with dichloromethane and water, and the aqueous layer was washed with dichloromethane 5 times until the aqueous layer had no fluorescence at 254 nm. The organic layers were combined, evaporated to dryness, and the resulting black-gray oily substance was further separated and purified by silica gel column chromatography (DCM:MeOH:Et3N = 40:1:1) to obtain 3.50 g of a white solid with a yield of 52%. 11H NMR (500 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.04 (s, 1H), 8.80 (s, 1H), 7.97 (d, J = 3.3 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.56 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 4.34 (s, 1H), 3.03 (s, 6H), 3.02–2.97 (m, 4H), 2.86 (m, 4H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.40, 156.82, 156.80, 150.10, 150.01, 148.05, 141.68, 140.35, 140.19, 139.73, 133.88, 131.43, 122.63, 115.94, 113.41, 50.25, 45.98, 38.55. HRMS (ESI) m / z calcd for 23 C 26 21H + FN9 + H] + : 448.2368 found: 448.2372 [M + H]

[0234] N 6 -(5-Fluoro-4-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24b)

[0235] Using the method of Example 11 above, with Compound 23g as the raw material, Compound 24b was prepared with a yield of 47%, as a grayish-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.03 (d, J = 44.5 Hz, 1H), 9.10 (s, 1H), 8.82 (d, J = 30.5 Hz, 1H), 8.47 (d, J = 16.6 Hz, 1H), 7.97 (t, J = 11.2 Hz, 2H), 7.55 (d, J = 34.2 Hz, 1H), 7.12 (s, 1H), 7.01 (s, 1H), 6.77 (s, 1H), 3.38–3.31 (m, 4H), 3.02 (s, 6H), 2.89–2.64 (m, 4H). (Tautomerism). 1313C NMR (125 MHz, DMSO-d6) δ 162.74, 156.59, 150.28, 141.81, 141.34, 140.31, 134.65, 132.47, 126.83, 114.46, 113.54, 108.16, 107.59, 106.86, 101.55, 46.85, 45.93, 38.55. HRMS (ESI) m / z calcd for [C 22 H 25 FN 10 +H] + : 449.2320; found: 449.2323 [M+H] + .

[0236] N 6 -(5-Fluoro-4-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 24c)

[0237] Using the method of Example 11 above, with Compound 23h as the raw material, Compound 24c was prepared with a yield of 49%, as an off-white solid. 1 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.99 (s, 1H), 8.06 (dd, J = 13.3, 6.3 Hz, 3H), 7.60 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 3.59 (m, 4H), 3.36 (s, 1H), 3.04 (m, 10H). 13 13C NMR (101 MHz, DMSO-d6) δ 157.50, 156.79, 149.33, 149.23, 144.56, 144.37, 141.58, 141.40, 141.32, 141.22, 139.13, 135.66, 133.67, 125.17, 116.16, 113.23, 49.78, 45.74, 38.54. HRMS (ESI) m / z calcd for [C 22 H 25 FN 10 +H] + : 449.2320; found: 449.2321 [M+H] + .

[0238] Example 12: N 6-(5-Fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25a)

[0239] A 25 mL eggplant-shaped flask containing 10 mL of ACN was placed in a -10 °C cold trap. 24a (0.45 g, 1 mmol) and triethylamine (0.15 g, 1.5 mmol) were added to the eggplant-shaped flask. Methyl iodide (0.17 g, 1.2 mmol) was dissolved in 2 mL of ACN and cooled to -10 °C, and then slowly added dropwise to the eggplant-shaped flask. The mixture was stirred at -10 °C for 10 min. The reaction solution was extracted with dichloromethane and water. The aqueous layer was washed with dichloromethane three times until there was no fluorescence at 254 nm in the aqueous layer. The organic layers were combined, dried by evaporation, and a colorless oil was obtained. Further separation and purification were carried out by silica gel column chromatography (DCM:MeOH:Et3N = 80:1:1) to obtain 0.20 g of a white solid with a yield of 44%. 1 H NMR (500 MHz, DMSO-d6) δ 9.21 (s, 1H), 9.18 (s, 1H), 8.04 (d, J = 3.5 Hz, 1H), 7.76 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 3.41 (s, 4H), 3.25 (s, 4H), 3.19 (s, 6H), 2.75 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 152.45, 150.29, 146.31, 142.05, 140.15, 136.73, 132.48, 132.15, 123.07, 116.67, 114.80, 111.20, 102.78, 52.82, 46.67, 42.75, 39.15. HRMS (ESI) m / z calcd for 24 H 28 FN9 + H] + : 462.2524 found: 462.2535 [M + H] + .

[0240] Example 13: 1-(4-(4-((2-((2-dimethylamino)-1H-benzo[d]imidazole-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethanone (Compound 25b)

[0241] 24a (0.45 g, 1 mmol), triethylamine (0.15 g, 1.5 mmol) and acetic anhydride (0.18 g, 1.5 mmol) were added to a eggplant-shaped flask containing 10 mL of ACN, and the mixture was stirred at room temperature for 2 h. The reaction solution was extracted with dichloromethane and water, and the aqueous layer was washed with dichloromethane three times until there was no fluorescence at 254 nm in the aqueous layer. The organic layers were combined, dried by rotary evaporation to obtain a colorless oil, and further separated and purified by silica gel column chromatography (DCM:MeOH:Et3N = 80:1:1) to obtain 0.35 g of a white solid with a yield of 71%. 1 1H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H), 9.09 (s, 1H), 8.87 (s, 1H), 7.99 (d, J = 3.5 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.60 (s, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 3.58 (m, 4H), 3.11–3.00 (m, 10H), 2.05 (s, 3H). 13 13C NMR (125 MHz, DMSO-d6) δ 168.69, 156.66, 150.08, 150.00, 147.20, 141.72, 140.38, 140.25, 139.77, 134.32, 131.99, 122.61, 116.71, 113.26, 49.44, 46.01, 38.61, 21.67. HRMS (ESI) m / z calcd for 25 C 28 H + FN9O + H] + .

[0242] Example 14: N 6 -(5-Fluoro-4-((4-(4-isopropylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25c)

[0243] 24a (0.45 g, 1 mmol), potassium carbonate (0.41 g, 3 mmol) and 2-iodopropane (0.51 g, 3 mmol) were added to a eggplant-shaped flask containing 10 mL of ACN, and stirred at 65 °C for 16 h. The reaction solution was extracted with dichloromethane and water, and the aqueous layer was washed with dichloromethane three times until there was no fluorescence at 254 nm in the aqueous layer. The organic layers were combined, dried by rotary evaporation to obtain a colorless oil, and further separated and purified by silica gel column chromatography (DCM:MeOH:Et3N = 80:1:1) to obtain 0.26 g of a white solid with a yield of 53%. 1 H NMR (500 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.83 (s, 1H), 7.98 (s, 1H), 7.65 (d, J = 7.4 Hz, 2H), 7.58 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.88 (d, J = 7.3 Hz, 2H), 3.10 (s, 4H), 3.04 (s, 6H), 2.76 (s, 1H), 2.65 (s, 4H), 1.04 (s, 6H). 13 C NMR (125 MHz, DMSO-d6) δ 157.08, 156.73, 150.10, 150.02, 147.41, 141.71, 140.35, 140.31, 140.18, 139.75, 134.11, 131.47, 122.66, 116.05, 113.10, 54.39, 49.34, 48.54, 38.57, 18.53. HRMS (ESI) m / z calcd for 26 H 32 [C + H + FN9 + H]

[0244] 2-(4-(4-((2-((2-(Dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethan-1-ol (Compound 25d)

[0245] Using the method of Example 13 above, with compound 24a and 2-bromoethanol as raw materials, compound 25d was prepared with a yield of 70%, a grayish-white solid. 11H NMR (500 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.84 (s, 1H), 7.98 (d, J = 3.7 Hz, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.58 (s, 1H), 7.13 (d, J = 9.9 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 9.0 Hz, 2H), 4.51 (s, 1H), 3.56 (t, J = 6.2 Hz, 2H), 3.14–3.05 (m, 4H), 3.03 (s, 6H), 2.59 (s, 4H), 2.47 (t, J = 6.1 Hz, 2H). 13 13C NMR (125 MHz, DMSO-d6) δ 156.98, 156.72, 156.57, 150.10, 147.38, 141.71, 140.33, 140.17, 139.79, 134.26, 131.48, 122.71, 116.05, 113.28, 111.42, 104.52, 60.48, 58.70, 53.47, 48.95, 38.62. HRMS (ESI) m / z calcd for 25 C 30 29H34FN9O + H + : 492.2630 found: 492.2636 [M + H] + .

[0246] N 6 -(4-((4-(4-(Ethylsulfonyl)piperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 25e)

[0247] Using the method of Example 13 above, with compound 24a and ethanesulfonyl chloride as raw materials, compound 25e was prepared with a yield of 68%, as a grayish-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.08 (s, 1H), 8.82 (s, 1H), 7.99 (d, J = 3.7 Hz, 1H), 7.69 (d, J = 8.9 Hz, 2H), 7.57 (s, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.9 Hz, 2H), 3.35–3.31 (m, 4H), 3.22–3.14 (m, 4H), 3.14–3.08 (m, 2H), 3.04 (s, 6H), 1.25 (t, J = 7.3 Hz, 3H).13 C NMR(125MHz,DMSO-d6)δ157.10,156.74,156.69,150.02,149.63,146.87,146.54,141.78,140.37,140.17,139.41,133.82,132.44,132.32,132.25,122.58,116.93,112.77,49.62,45.67,42.90,38.56,7.95.HRMS(ESI)m / z calcd for[C 25 H 30 FN9O2S+H] + :540.2300found:540.2310[M+H] + .

[0248] N 6 -(5-Fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine(compound 25f)

[0249] N 6 -(5-Fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine(0.45g,1mmol)and acetic acid(0.06g,1mmol)were added to a eggplant-shaped flask containing 10 mL of DCE,and the mixture was stirred at 60 °C for 20 min.Then,pivalaldehyde(0.26g,3mmol)and sodium triacetoxyborohydride(0.64g,3mmol)were added to the reaction solution,and the mixture was stirred at 60 °C for 16 h.The reaction solution was extracted with dichloromethane and water,and the aqueous layer was washed with dichloromethane three times until there was no fluorescence at 254 nm in the aqueous layer.The organic layers were combined,rotary evaporated to dryness to obtain a colorless oil,which was further separated and purified by silica gel column chromatography(DCM:MeOH:Et3N = 80:1:1)to obtain 0.316 g of a white solid with a yield of 61%. 11H NMR (500 MHz, DMSO-d6) δ 11.01 (s, 1H), 9.04 (s, 1H), 8.81 (s, 1H), 7.97 (d, J = 3.3 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.57 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 3.08 (m, 4H), 3.03 (s, 6H), 2.51 (m, 4H), 2.34 (t, J = 7.4 Hz, 2H), 1.61 (m, 1H), 1.36 (m, 2H), 0.90 (d, J = 6.6 Hz, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.23, 156.78, 156.76, 150.10, 150.01, 147.46, 141.69, 140.33, 140.19, 139.74, 134.05, 134.01, 131.42, 122.63, 116.00, 113.26, 56.47, 53.31, 49.22, 38.56, 35.76, 26.31, 23.09. HRMS (ESI) m / z calcd for 28 H 36 FN9 + H] + : 518.3150 found: 518.3155 [M + H] + .

[0250] N 6 -(4 - ((4 - (4 - benzylpiperazin - 1 - yl)phenyl)amino)-5 - fluoropyrimidin - 2 - yl)-N 2 ,N 2 -dimethyl - 1H - benzimidazole - 2,6 - diamine (Compound 25g)

[0251] Using the method of Example 12 above, with compound 24a and bromobenzyl as raw materials, compound 25g was prepared with a yield of 70%, as a white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.07 (s, 1H), 8.85 (s, 1H), 7.98 (d, J = 3.4 Hz, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.58 (s, 1H), 7.30 (m, 5H), 7.13 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 3.08 (m, 4H), 3.52 (s, 2H), 3.01 (s, 6H), 2.51 (m, 4H).13 C NMR (125 MHz, DMSO-d6) δ 157.14, 156.82, 150.08, 149.99, 147.45, 141.68, 140.31, 140.18, 139.73, 138.54, 133.97, 131.45, 129.39, 128.67, 127.45, 122.62, 116.07, 113.13, 113.07, 62.53, 53.05, 49.28, 38.54. HRMS (ESI) m / z calcd for [C 30 H 32 FN9 + H] + : 538.2837 found: 538.2842 [M + H] + .

[0252] 1-(4-(4-((2-((2-(Dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)-3,3-dimethylpropan-1-one (Compound 25h)

[0253] Using the method of Example 12 above, with compound 24a and 3,3-dimethylbutanoyl chloride as raw materials, compound 25h was prepared with a yield of 73%, as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.93 (s, 1H), 8.00 (d, J = 3.7 Hz, 1H), 7.69 (t, J = 8.8 Hz, 2H), 7.64 (s, 1H), 7.21 (dd, J = 8.5, 1.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 3.67 (t, J = 16.6 Hz, 4H), 3.05 (d, J = 10.6 Hz, 10H), 2.28 (s, 2H), 1.01 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 169.86, 156.60, 156.11, 150.10, 149.98, 147.21, 141.99, 140.35, 139.54, 134.66, 131.96, 122.62, 116.66, 113.47, 111.31, 104.18, 49.58, 44.09, 38.66, 31.49, 30.25. HRMS (ESI) m / z calcd for [C 29 H 36 FN9O + H] +:546.3100 found:546.3106 [M+H] + .

[0254] N-(tert-Butyl)-4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxamide (Compound 25i)

[0255] Using the method of Example 12 above, with Compound 24a and tert-butyl isocyanate as raw materials, Compound 25i was prepared with a yield of 68%, as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.08 (s, 1H), 8.84 (s, 1H), 7.99 (s, 1H), 7.68 (d, J = 8.1 Hz, 2H), 7.58 (s, 1H), 7.13 (d, J = 6.7 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 8.1 Hz, 2H), 5.92 (s, 1H), 3.42 (m, 4H), 3.04 (m, 10H), 1.28 (s, 9H). 13 C NMR (125 MHz, DMSO-d6) δ 157.54, 157.40, 156.81, 150.13, 150.05, 147.44, 141.73, 140.39, 140.23, 139.78, 133.89, 131.87, 122.65, 116.54, 113.08, 50.44, 49.52, 44.14, 38.57, 29.70. HRMS (ESI) m / z calcd for [C 28 H 35 FN 10 O+H] + :547.3052 found:547.3060 [M+H] + .

[0256] N 6 -(2-Chloro-5-fluoropyrimidin-4-yl)-N 2 ,N 2 -Dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 26)

[0257] Using the method of Example 9 above, with Compound 12 as the raw material, Compound 26 was prepared with a yield of 88%, as a white solid. 11H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 10.13 (s, 1H), 8.31 (s, 1H), 7.85 (s, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 3.24 (s, 6H). HRMS (ESI) m / z calcd for [C 13 H 12 ClFN6+H] + : 307.0869; found: 307.0875 [M+H] + .

[0258] tert-Butyl 6-((tert-butoxycarbonyl)(2-chloro-5-fluoropyrimidin-4-yl)amino)-2-(dimethylamino)-1H-benzo[d]imidazole-1-carboxylate (Compound 27)

[0259] N6-(2-Chloro-5-fluoropyrimidin-4-yl)-N2,N2-dimethyl-1H-benzo[d]imidazole-2,6-diamine (3.06 g, 10 mmol) was added to 100 mL of DCM in a 250 mL eggplant-shaped flask, and (Boc)2O (21.81 g, 100 mmol), triethylamine (5.05 g, 50 mmol) and DMAP (0.49 g, 4 mmol) were added. The reaction was carried out at room temperature for 24 h. The reaction solution was evaporated to dryness to obtain an orange-red oily substance. It was extracted with dichloromethane and water. The organic layer was washed 3 times with an aqueous solution of saturated citric acid, once with an aqueous solution of saturated sodium bicarbonate, and finally once with saturated brine. The organic layer was dried over anhydrous Na2SO4 and left for 4 h. It was filtered by suction, the organic solvent was evaporated to dryness, and the orange-red oily substance was obtained. It was placed in an oil pump and dried for 2 h to obtain 4.72 g of an orange-red solid with a yield of 93%. 1 1H NMR (500 MHz, DMSO-d6) δ 8.96 (s, 1H), 7.53 (s, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 2.99 (s, 6H), 1.58 (s, 9H), 1.42 (s, 9H). HRMS (ESI) m / z calcd for [C 23 H 28 ClFN6O4+H] + : 507.1917; found: 507.1923 [M+H] + .

[0260] tert-Butyl 6-((tert-butoxycarbonyl)(5-fluoro-2-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-2-(dimethylamino)-1H-benzo[d]imidazole-1-carboxylate (Compound 28a)

[0261] 6-((tert-Butoxycarbonyl)(2-chloro-5-fluoropyrimidin-4-yl)amino)-2-(dimethylamino)-1H-benzo[d]imidazole-1-carboxylic acid butyl ester (2.53 g, 5 mmol), 4-(pyrrolidin-1-yl)aniline (0.81 g, 5 mmol), Pd2(dba)3 (0.18 g, 0.02 mmol), Xanphos (0.46 g, 0.08 mmol), and cesium carbonate (4.89 g, 15 mmol) were added to a 120 mL pressure-resistant flask. 50 mL of dioxane was added, and a magnetic stir bar was added. The air in the flask was replaced with nitrogen, and after sealing, the mixture was sonicated to dissolve the starting materials as much as possible in the solvent. The reaction was carried out in an oil bath at 120 °C for 12 h. Then it was allowed to cool to room temperature. After cooling to room temperature, the reaction solution was rotary evaporated, extracted with dichloromethane / water, and the aqueous layer was washed twice with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and left for 4 h. It was filtered by suction, and the organic solvent was rotary evaporated to obtain a black oil. It was separated and purified by silica gel column chromatography (DCM:MeOH:Et3N = 200:1:1), and 2.34 g of a black oil was obtained with a yield of 74%. The structure was not confirmed and directly used for the next step. Compounds 28b - 28f were prepared by the same method as 28a and also directly used for the next step without structure confirmation.

[0262] N 6 -(5-Fluoro-2-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29a)

[0263] Using the method of Example 11 above, with Compound 28a as the starting material, Compound 29a was prepared with a yield of 50%, as a grayish-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.19 (d, 1H), 9.01 (d, 1H), 8.67 (s, 1H), 7.93 (d, J = 3.1 Hz, 1H), 7.47 (m, 3H), 7.25 (m, 1H), 7.09 (m, 1H), 6.40 (m, 2H), 3.15 (m, 4H), 3.05 (s, 6H), 1.93 (m, 4H). 1313C NMR (125 MHz, DMSO-d6) δ 157.56, 156.61, 150.63, 150.53, 144.43, 143.62, 141.60, 141.10, 140.22, 139.66, 134.69, 132.46, 131.50, 130.57, 130.49, 121.26, 121.16, 114.38, 113.97, 111.95, 109.99, 108.20, 104.60, 48.08, 38.52, 25.32. HRMS (ESI) m / z calcd for [C 23 H 25 FN8+H] + : 433.2259; found: 443.2258 [M+H] + .

[0264] N 6 -(5-Fluoro-2-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29b)

[0265] Using the method of Example 11 above, with Compound 28b as the raw material, Compound 29b was prepared with a yield of 45%, as a grayish-white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.08 (s, 1H), 8.84 (s, 1H), 7.97 (d, J = 3.8 Hz, 1H), 7.50 (m, 3H), 7.21 (s, 1H), 7.12 (s, 1H), 6.74 (s, 2H), 3.33 (s, 1H), 3.06 (s, 6H), 2.96–2.86 (m, 4H), 2.85–2.71 (m, 4H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.76, 156.37, 150.76, 150.68, 146.73, 141.75, 140.35, 140.34, 140.21, 140.19, 139.80, 133.81, 120.17, 116.31, 114.35, 50.92, 46.19, 38.51. HRMS (ESI) m / z calcd for [C 23 H 26 FN9+H] + : 448.2368 found: 448.2370 [M+H] + .

[0266] N6 -(5-Fluoro-2-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29c)

[0267] Using the method of Example 11 above, with Compound 28c as the starting material, Compound 29c was prepared with a yield of 65%, as a light purple solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.07 (s, 1H), 8.82 (s, 1H), 8.27 (d, J = 2.0 Hz, 1H), 7.96 (d, J = 3.7 Hz, 1H), 7.94–7.85 (m, 1H), 7.47 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.65 (d, J = 9.1 Hz, 1H), 3.34–3.22 (m, 4H), 3.05 (s, 6H), 2.95–2.64 (m, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 157.77, 156.53, 155.47, 150.90, 150.81, 142.05, 140.18, 140.09, 140.03, 139.34, 131.39, 130.37, 129.43, 115.27, 106.99, 46.82, 45.68, 38.53. HRMS (ESI) m / z calcd for [C 22 H 25 FN 10 +H] + : 449.2320 found: 449.2321 [M+H] + .

[0268] N 6 -(2-((4-(Dimethylamino)phenyl)amino)-5-fluoropyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29d)

[0269] Using the method of Example 11 above, with Compound 28d as the starting material, Compound 29d was prepared with a yield of 65%, as a light purple solid. 11H NMR (500 MHz, DMSO-d6) δ 11.37 (s, 1H), 9.04 (s, 1H), 8.73 (s, 1H), 7.95 (d, 1H), 7.51 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.61 (d, J = 8.1 Hz, 2H), 3.06 (s, 6H), 2.80 (s, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 157.37, 156.53, 150.67, 150.59, 146.15, 141.69, 140.35, 140.19, 139.74, 131.81, 131.67, 120.76, 115.36, 113.48, 111.16, 107.34, 41.28, 38.58. HRMS (ESI) m / z calcd for 20 H 22 [C + H + FN9 + H]

[0270] N 6 -(5-Fluoro-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29e)

[0271] Using the method of Example 11 above, with Compound 28e as the raw material, Compound 29e was prepared with a yield of 68%, as a light purple solid. 1 1H NMR (500 MHz, DMSO-d6) δ 11.37 (s, 1H), 9.04 (s, 1H), 8.73 (s, 1H), 7.95 (d, 1H), 7.51 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.61 (d, J = 8.1 Hz, 2H), 3.06 (s, 6H), 2.80 (s, 6H). 1313C NMR (125 MHz, DMSO-d6) δ 157.37, 156.53, 150.67, 150.59, 146.15, 141.69, 140.35, 140.19, 139.74, 131.81, 131.67, 120.76, 115.36, 113.48, 111.16, 107.34, 41.28, 38.58. HRMS (ESI) m / z calcd for [C 20 H 22 FN9 + H] + : 449.2320 found: 449.2319 [M + H] + .

[0272] N 6 ,N 6 -(5-Fluoropyrimidine-2,4-diyl)bis(N2,N2-dimethyl-1H-benzo[d]imidazole-2,6-diamine) (Compound 29h)

[0273] Using the method of Example 11 above, with Compound 28f as the raw material, Compound 29h was prepared with a yield of 50%, as a white solid. 1 1H NMR (500 MHz, DMSO-d6) δ 8.11 (d, J = 4.1 Hz, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.34 (m, 2H), 7.27 (d, J = 8.6 Hz, 1H), 3.18 (s, 6H), 3.16 (s, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 152.64, 152.55, 151.81, 151.53, 151.42, 140.72, 138.74, 133.43, 132.38, 130.94, 130.77, 128.38, 127.52, 119.24, 117.67, 111.44, 111.20, 107.04, 105.32, 39.50, 39.48. HRMS (ESI) m / z calcd for [C 22 H 23 FN 10 + H] + : 447.2164 found: 447.2169 [M + H] + .

[0274] 1-(4-(4-((4-((2-(Dimethylamino)-1H-benzo[d]imidazole-6-yl)amino)-5-fluoropyrimidin-2-yl)amino)phenyl)piperazin-1-yl)propan-1-one (Compound 29f)

[0275] Using the method of Example 13 above, compound 29b was used as the raw material to prepare compound 29f with a yield of 60%, which was a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.92 (s, 1H), 8.00 (d, J = 3.7 Hz, 1H), 7.58 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.9 Hz, 2H), 3.77–3.33 (m, 4H), 3.10 (s, 6H), 3.06–2.83 (m, 4H), 2.04 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 168.66, 156.28, 150.64, 150.53, 145.77, 142.05, 140.50, 140.32, 139.61, 134.48, 132.17, 120.17, 117.08, 115.88, 111.24, 107.13, 50.35, 49.92, 38.69, 21.66. HRMS (ESI) m / z calcd for [C 25 H 28 FN9O+H] + : 490.2474 found: 490.2475 [M+H] + .

[0276] N 6 -(5-Fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine (Compound 29g)

[0277] Using the method of Example 13 above, compound 29b was used as the raw material to prepare compound 29g with a yield of 65%, which was a white solid. 11H NMR (500 MHz, DMSO-d6) δ 11.51 (s, 1H), 9.19 (s, 1H), 8.92 (s, 1H), 8.00 (d, J = 3.0 Hz, 1H), 7.58 (s, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 2H), 3.18 (d, J = 68.6 Hz, 14H), 2.89 (s, 2H), 1.66–1.47 (m, 3H), 0.91 (d, J = 5.8 Hz, 6H). 13 13C NMR (125 MHz, DMSO-d6) δ 156.28, 155.93, 150.61, 150.52, 144.91, 141.83, 140.49, 140.34, 139.88, 134.61, 132.41, 120.26, 116.81, 116.05, 111.24, 106.91, 55.09, 51.77, 47.54, 38.79, 33.00, 26.26, 22.77. HRMS (ESI) m / z calcd for 28 C 36 H + FN9 + H] + .

[0278] Example 15: Perform the inhibitory activity experiment of HIPK2 on the synthesized compound and the proliferation inhibition experiment on rat renal fibroblasts (NRK-49F) for the synthesized compound

[0279] I. Perform the inhibitory activity experiment of HIPK2 on the synthesized compound

[0280] 1. Experimental materials

[0281] HIPK2 kinase (Carna); MBP, ATP (Promega), ADP-Glo TM detection kit, Cytation 5.

[0282] 2. Experimental method

[0283] (1) Add 1 μL of the compound solution (or 5% DMSO), 2 μL of the HIPK2 solution, and 2 μL of the mixed solution of ATP and substrate into a 384-well plate, and incubate at room temperature for 60 min;

[0284] (2) Add 5 μL of ADP-Glo TM reagent, and incubate at room temperature for 40 min;

[0285] (3) Add 10 μL of kinase elimination reagent and incubate at room temperature for 30 min;

[0286] (4) Record the fluorescence intensity.

[0287] II. Perform a proliferation inhibition experiment on the synthesized compound using rat renal fibroblasts (NRK-49F)

[0288] 1. Experimental materials

[0289] (1) Cell line: Rat renal fibroblasts (NRK-49F)

[0290] (2) Reagents and instruments: 96-well plates (Corning); fetal bovine serum (Gibco), F12 medium, DMEM medium (BI); biological safety cabinet, carbon dioxide incubator (ESCO); Cytation 5 multi-functional imager (Bio-Tek).

[0291] 2. Experimental methods

[0292] (1) Cell culture: NRK-49F cells are cultured conventionally. During the experiment, cells in the logarithmic growth phase with a viable cell ratio higher than 90% are used for the experiment.

[0293] (2) Cell growth detection: Digest the cells, dilute the cells into a cell suspension with a concentration of 3 - 5×104 cells / mL, add 100 μL of the cell suspension to each of the 60 wells in the middle of the 96-well plate (3 - 5×103 cells per well), and add 100 μL of PBS to each of the 36 wells around; place the 96-well plate in an incubator at 37°C and 5% CO2 until the cells adhere (enter the logarithmic growth phase); after aspirating the medium in the middle 60 wells, add 100 μL of the medium containing different concentrations of the compound to each well, with 3 replicates in each group; place the 96-well plate in the incubator and culture for 48 h; discard the medium containing the compound and PBS, add 100 μL of the basal medium containing 10% CCK-8 to each well of the 96-well plate, incubate the 96-well plate in the incubator for 1 - 4 h, and then measure the OD value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0294] 3. Experimental results

[0295] Table 1 Inhibitory activities of the target compound against HIPK2 kinase and NRK-49F cell proliferation

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302] illustrate: a The maximum concentration of the compound is 10 μM, and the data in brackets are the inhibition rates at the maximum concentration, for example, >10 (2.7%) means the inhibition rate at a concentration of 10 μM; b The maximum concentration of the compound was 100 μM, and the data in brackets are the inhibition rates at the maximum concentration; c Active Wnt inhibitor disclosed in patent CN106565673 d Patent CN105017159 discloses 5-fluoro-2,4-disubstituted pyrimidine derivatives.

[0303] The above data results show that the HIPK2 inhibitory activity experimental results of most target compounds are consistent with the proliferation inhibition experimental results of renal fibroblasts (NRK-49F), and the activity is stronger than the marketed anti-fibrosis drug pirfenidone, and is better than the compounds in the comparative literature, with obvious activity advantages.

[0304] Example 16: Effects of target compounds on fibrosis

[0305] 1. Experimental Materials

[0306] (1) Cell line: Rat kidney fibroblasts (NRK-49F)

[0307] (2) Antibodies: P65, p-P65, α-SMA, Fn1, Collagen1, and GAPDH were purchased from CST.

[0308] (3) Drugs and reagents: Ultrasensitive ECL chemiluminescence reagent was purchased from Abbkine; goat anti-rabbit secondary antibody, developer, fixer, RIPA lysis buffer, BSA, BCA protein concentration detection kit, and prestained protein marker were all purchased from Nanjing Enjing Biotechnology Co., Ltd.

[0309] (4) Instruments and consumables: 6-well cell culture plates were purchased from Corning; clean bench (ESCO); carbon dioxide incubator (ESCO); fluorescent inverted biological microscope (Optec); desktop high-speed centrifuge (SCILOGEX); micro vertical electrophoresis tank (Bio-RAD); transfer electrophoresis tank (Bio-RAD); electrophoresis instrument (Junyi Co., Ltd.); decolorization shaker (Huali Da Co., Ltd.); Cytation 5 microplate reader (Bio-tek).

[0310] 2. Experimental Methods

[0311] (1) Protein sample preparation

[0312] Cells in the logarithmic growth phase of NRK-49F were used for the experiment. The cells were digested, counted, and diluted into a cell suspension of 2×105 cells / mL. Then, 2 mL of the cell suspension was inoculated into each well of a 6-well plate and cultured in an incubator at 37°C with 5% CO2 for 24 hours. The fresh medium was replaced, and a blank control group and an experimental group (stimulated with TGF-β) were set up. Different concentrations of compound 7a were added. After 24 hours of treatment, the culture medium was discarded, and each well of cells was washed three times with 2 mL of pre-cooled PBS at 4°C. Then, the culture dish was placed on ice. 100 μL of RIPA lysis buffer (containing phosphatase inhibitors and PMSF) was added to lyse the cells for 30 minutes, and the cells needed to be shaken back and forth frequently to ensure sufficient lysis. After lysis, the cells were quickly scraped to one side of the well with a cell scraper, and then the liquid in the well was transferred to a 1.5 mL centrifuge tube with a pipette (the whole operation needed to be carried out on ice). Finally, the mixture was centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was transferred to a 0.5 mL centrifuge tube. The protein concentration in the supernatant was measured using a protein quantification assay kit, and the supernatant was stored at -20°C. The sample protein needed to be denatured before loading onto the electrophoresis gel.

[0313] (2) SDS-PAGE electrophoresis

[0314] Align the clean glass plates and place them in the clamp, then vertically clamp them on the rack to prepare for pouring the gel. Aspirate 5 mL of separating gel and slowly pour it along the glass. Stop pouring when the gel surface reaches the height of the middle line of the green band, and then add a layer of anhydrous ethanol on top of the gel for liquid sealing. After the gel solidifies, pour off the water on the upper layer of the gel and blot it dry with absorbent paper. Fill the remaining space with stacking gel and then insert the comb into the stacking gel. After the stacking gel solidifies, gently pull out the comb vertically upward. Rinse the stacking gel with water and then place it in the electrophoresis tank. Load 30 - 50 μg of protein, and load the sample after adding enough electrophoresis buffer. Electrophoresis was first carried out at 70 V for 20 minutes. When the protein aggregated to the boundary of the separating gel, the voltage was changed to 130 V, and electrophoresis was terminated when the bromophenol blue just ran out, and then transfer membrane was carried out.

[0315] (3) Transfer membrane

[0316] Carefully pry open the glass plate with a gel prying plate, carefully spread the gel evenly on three pieces of filter paper, and remove the air bubbles. Stick the soaked PVDF membrane on the place of the target band required, and do not move it again after carefully removing the air bubbles. Then stack three pieces of filter paper on the PVDF membrane, and note that the filter papers above and below both sides of the gel cannot be in direct contact to avoid forming a short circuit and damaging the membrane transfer equipment. After confirmation, cover with a sponge pad and close the membrane transfer plate. Place the membrane transfer template in the way that the PVDF membrane is on the positive electrode and the gel is on the negative electrode, add the membrane transfer buffer in the membrane transfer tank, and place the membrane transfer tank in an ice-water mixture. Transfer the membrane at 80V for 45 minutes. After the membrane transfer is completed, take out the PVDF membrane, soak it with TBS and then put it into the blocking solution, and block it overnight at 4°C. After blocking, take out the membrane from the blocking solution, and after sucking off the residual blocking solution with filter paper, place the membrane protein side down on the liquid surface of the primary antibody solution diluted to a specific concentration, and lift the four corners of the membrane to drive out the residual air bubbles; incubate at room temperature for 2 hours. After the incubation of the primary antibody, wash the PVDF membrane with TBST on a horizontal shaker for 10 minutes each time, and repeat 3 times. Incubate the PVDF membrane with the secondary antibody (diluted 1:5000) in the same way as the primary antibody incubation, and incubate at room temperature for 1 hour. Subsequently, wash the PVDF membrane with TBST on a horizontal shaker for 10 minutes each time again, and repeat 3 times, and then perform chemiluminescence development.

[0317] (4) Chemiluminescence development

[0318] Mix solution A and solution B of the ECL luminescent solution in a ratio of 1:1, and let it stand at room temperature for 1 minute. Stick the PVDF membrane with the protein side up on the flat plate. After 1 minute, apply the ECL luminescent solution for development and fixation. Using GAPDH as the internal reference, analyze the net optical density value of the target band with Adobe Photoshop CS5 software.

[0319] (5) Statistical processing

[0320] Scan the developed band with a scanner, obtain the gray value of the result with ImageJ software, and plot a graph with Graphpad software.

[0321] 3. Experimental results

[0322] The results obtained are as Figure 2 shown. Among them, (A) and (C) detect the expression of α-SMA and Fn 1 in NRK-49F cells treated with different concentrations of compound 7a by Western blot; (B) and (D) are the quantitative analysis of the Western blot data. # indicates p < 0.0001 compared with the negative control group (0 / -), indicates p < 0.001 compared with the negative control group (0 / -), *** indicates p < 0.001 compared with the positive control group (0 / +), and **** indicates p < 0.0001 compared with the positive control group (0 / +).

[0323] 4. Experimental conclusions

[0324] It can be seen from Figure 2 that after adding 10 ng / mL of TGF-β, the levels of α-SMA and Fn1 in the cells increased significantly; after treatment with 7a, the levels of α-SMA and Fn1 in the cells decreased significantly. Therefore, 7a can inhibit the expression of molecules related to its downstream fibrosis signaling pathway by inhibiting HIPK2.

[0325] Example 17: Effects of the compound on inflammation-related pathways

[0326] 1. Experimental materials

[0327] Antibodies: P65, p-P65, and GAPDH were all purchased from CST. Others were the same as in Example 16.

[0328] 2. Experimental method (the same as in Example 16)

[0329] 3. Experimental results

[0330] The results obtained are as Figure 3 shown. Among them, (A) detected the expression of P-P65 and P65 in NRK-49F cells treated with different concentrations of compound 7a by Western blot; (B) and (C) were quantitative analyses of the Western blot data in the middle left. *** indicates p < 0.001 compared with the positive control group (0 / +), and **** indicates p < 0.0001 compared with the positive control group (0 / +).

[0331] As shown in the figure, when treated with different concentrations of 7a, at low concentrations, P65 increased; at high concentrations, P65 decreased significantly compared with low concentrations and was not much different from the control group. However, the phosphorylation level of P65 decreased significantly.

[0332] 4. Experimental conclusions

[0333] 7a can inhibit the expression of molecules related to its downstream inflammation signaling pathway by inhibiting HIPK2.

[0334] Example 18: Effects of the compound on the mouse model of renal interstitial fibrosis with unilateral ureteral ligation

[0335] The unilateral ureteral obstruction model (UUO model) is a classic model of renal interstitial fibrosis, which simulates the entire dynamic process from acute kidney injury to renal interstitial fibrosis. It is generally believed that 3 days after ureteral ligation is the acute phase of injury, mainly characterized by cell edema, death, and infiltration of inflammatory cells; the 3rd to 7th day is the subacute phase, mainly characterized by renal interstitial edema, tubular dilation, and infiltration of inflammatory cells, and extracellular matrix deposition begins to appear; after 7 days, renal interstitial edema and tubular dilation gradually disappear, replaced by renal interstitial atrophy, significant increase in interstitial extracellular matrix deposition, and deterioration of fibrosis, entering the chronic phase. The purpose of this study: To observe the efficacy of the target compound (HIPK2-inhibitor) of the present invention on the 14-day UUO model to clarify the therapeutic effect of the target compound on renal interstitial fibrosis.

[0336] 1. Experimental materials

[0337] 1.1 Drugs: Target compound (HIPK2-inhibitor); Character: Yellow solid. Provider: School of Pharmacy, Central South University. The target compound was dissolved with double-distilled water as the solvent (adjust the pH to about 4.5).

[0338] 1.2 Experimental animals: C57-BL6 mice, male, weighing 22 - 25 g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., Experimental Animal Production License Number: SCXK(Xiang)2015 - 0017.

[0339] 2. Experimental methods

[0340] 2.1 Experimental design

[0341] SPF-grade male mice were divided into sham operation group (Sham), unilateral ureteral obstruction group (UUO), and UUO + target compound (75 mg / kg) group. After anesthesia, the back hair was shaved, and disinfected with povidone iodine. Except for the sham operation group, a small skin incision was made in the upper left of the midline of the back of all mice to expose the posterior peritoneum, then the peritoneum was cut open, the left kidney and ureter were extruded, the ureter was ligated with 3-0 suture near the renal hilum and below the ureter, the ureter was cut between the two ligated sites, and the kidney was pushed back into the abdominal cavity, and the peritoneum and back skin were sutured. The mice in the sham operation group were only given laparotomy and abdominal closure. Starting from the 2nd day after model construction, the UUO + target compound (75 mg / kg) group was given the target compound by gavage at a dose of 75 mg / kg / 6 mL, once a day, for 14 consecutive days, and the mice were sacrificed on the 14th day after surgery.

[0342] 2.2 Detection indicators

[0343] 2.2.1 Sample preparation and analysis of test samples: Weigh an appropriate amount of compound 7a according to the body weight of the mice, adjust the PH, and use double-distilled water as the solvent to prepare a corresponding concentration of the medicinal solution.

[0344] 2.2.2 Effects on renal histopathology: After 14 days of drug administration, all mice were anesthetized and euthanized with CO2, bled immediately, and half of the left kidney was taken, fixed in formalin, dehydrated, embedded in paraffin, sectioned, and subjected to HE staining and Masson staining respectively. For the tissue sections stained with HE, pathological scoring was performed using the renal injury scoring criteria, and for the tissue sections stained with Masson, pathological scoring was performed using the renal fibrosis scoring criteria. The specific scoring criteria and area calculation methods are as follows:

[0345] HE staining: The renal tissue lesions were classified into renal interstitial fibrosis, renal interstitial inflammation, tubular vacuolar degeneration, tubular atrophy, tubular dilation, renal interstitial edema, red blood cell casts, and protein casts. Each type of the above-mentioned lesions was scored. Among them, normal was recorded as "0" points; mild was recorded as "1" point; moderate was recorded as "2" points; severe was recorded as "3" points, and the scores of each animal were added up to obtain the total score. The scores of each type and the total score were statistically analyzed respectively.

[0346] Masson staining: Calculated according to the area of extracellular matrix deposition, 0 - 25%, 25 - 50%, 50 - 75%, and greater than 75% were recorded as 1, 2, 3, and 4 points respectively.

[0347] 2.3 Statistical methods

[0348] The software used for statistics was SPSS 20.0. If the variances were homogeneous, one-way analysis of variance (One-Way ANOVA) was used for statistical analysis. If the variances were not homogeneous, the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P < 0.05), Dunnett’s Test (non-parametric method) was used for comparative analysis. The statistical results were expressed as P < 0.05 indicating statistical significance, and P < 0.01 indicating very significant differences in the tests.

[0349] 3. Experimental results

[0350] 3.1. Effects on the pathological score of renal HE staining

[0351] As Figures 4 - 6 and Figure 10As shown, compared with the sham operation group (Sham), the infiltration of inflammatory cells, tubular atrophy, and renal interstitial fibrosis in the kidneys of mice in the UUO group were obvious on the 14th day, and the pathological scores of HE staining were significantly increased (P < 0.05); compared with the UUO group, the infiltration of inflammatory cells, tubular atrophy, and renal interstitial fibrosis in the kidneys of the compound group (only the data of 7a and 25f are listed) were significantly reduced, while the pathological changes of tubular dilation were obvious, and the pathological scores of HE staining in the compound group (only the data of 7a and 25f are listed) were decreased (P < 0.05). The other target compound groups also showed obvious reduction in the infiltration of inflammatory cells, tubular atrophy, and renal interstitial fibrosis in the kidneys.

[0352] 3.2 Effect on the pathological score of renal masson staining

[0353] As Figures 7 - 9 and Figure 10 shown, compared with the sham operation group (Sham), the deposition of renal interstitial fibrosis in the kidneys of mice in the UUO group was obvious on the 14th day, and the pathological scores of masson staining were significantly increased (P < 0.01); compared with the UUO group, the deposition of renal interstitial fibrosis in the kidneys of the compound group (only the data of 7a and 25f are listed) was significantly reduced, and the pathological scores of masson staining were significantly decreased (P < 0.05). The other target compound groups also showed obvious reduction in the deposition of renal interstitial fibrosis in the kidneys, and the pathological scores of masson staining were significantly decreased.

[0354] 4. Conclusion:

[0355] The 2,4-disubstituted-5-fluoropyrimidine derivatives of the present invention can alleviate renal injury and the deposition of renal interstitial fibrosis.

Claims

1. A 2,4-disubstituted-5-fluoropyrimidine derivative, characterized in that, Its structural general formula is shown in Formula I: Wherein, R1 and R2 are each independently selected from: R4 is selected from: X and Y are each independently selected from a nitrogen atom or a carbon atom; Z is selected from a hydrogen atom, a tert-butoxycarbonyl group, a C1-C5 alkyl group, a formyl group, an acetyl group, a propionyl group, a butyryl group, a valeryl group, a 3,3-dimethyl-1-butyryl group, an isovaleryl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkylsulfonyl group, a formamido group, an acetamido group, a propionamido group, a butyramido group, a valeramido group, an N-tert-butylaminocarbonyl group, an isovaleramido group, a benzyl group, a phenethyl group, a phenylpropyl group; when R1 is and R2 is X and Y are not simultaneously carbon atoms; When both R1 and R2 are R4 is not selected from R1 and R2 are not both When R1 is and R2 is then R4 is selected from 2. The 2,4-disubstituted-5-fluoropyrimidine derivative according to claim 1, wherein Either R1 or R2 is: R4 is Z is selected from a hydrogen atom, a C1-C5 alkyl group, a formyl group, an acetyl group, a propionyl group, a butyryl group, a valeryl group, a 3,3-dimethyl-1-butyryl group, an isovaleryl group, a C1-C3 alkyl hydroxy group, a C1-C3 alkylsulfonyl group, a benzyl group, a phenethyl group, a phenylpropyl group, a formamido group, an acetamido group, a propionamido group, a butyramido group, a valeramido group, an N-tert-butylaminocarbonyl group, an isovaleramido group.

3. The 2,4-disubstituted-5-fluoropyrimidine derivative according to claim 1, wherein, Either R1 or R2 is: R4 is Z is butyl, pentyl, isobutyl, isopentyl, and the other of R1 and R2 is: When, X and Y are not both carbon atoms at the same time.

4. The 2,4-disubstituted-5-fluoropyrimidine derivative according to any one of claims 1-3, characterized in that, C1-C5 alkyl is selected from methyl, ethyl, propyl, butyl, pentyl, isobutyl, isopentyl, isopropyl.

5. A 2,4-disubstituted-5-fluoropyrimidine derivative, characterized in that, Its structural general formula is shown in Formula II or Formula III: Among them, R3 is selected from: R4 is selected from: X and Y are each independently selected from a nitrogen atom or a carbon atom; Z is selected from a hydrogen atom, tert-butoxycarbonyl, C1-C5 alkyl, formyl, acetyl, propionyl, butyryl, valeryl, 3,3-dimethyl-1-butyryl, isovaleryl, C1-C3 alkyl hydroxy, C1-C3 alkyl sulfonyl, benzyl, phenethyl, phenylpropyl, formamido, acetamido, propionamido, butyramido, valeramido, N-tert-butylaminocarbonyl, isovaleramido.

6. The 2,4-disubstituted-5-fluoropyrimidine derivative according to claim 5, wherein In R3, when R4 is , Z is not tert-butoxycarbonyl.

7. The 2,4-disubstituted-5-fluoropyrimidine derivative according to claim 5, wherein, When R4 in formula III is Z is not tert-butoxycarbonyl, C4-C5 alkyl, butyryl, valeryl, isovaleryl, butyramide, valeramide, or isovaleramide.

8. The 2,4-disubstituted-5-fluoropyrimidine derivative according to any one of claims 5 to 7, characterized in that, C1-C5 alkyl is selected from methyl, ethyl, propyl, butyl, pentyl, isobutyl, isopentyl, isopropyl.

9. A 2,4-disubstituted-5-fluoropyrimidine derivative, characterized in that, Specifically, it is one of the following compounds: tert-butyl 4-(4-((5-fluoro-2-((4-(methoxycarbonyl)phenyl)amino)pyridin-4-yl)amino)phenyl)piperazine-1-carboxylate; 1-(5-fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine; 1-(4-((4-(4-ethylpiperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine; N 2 -(1H-benzo[d]imidazol-2-yl)-N 4 -(4-(4-ethylpiperazin-1-yl)phenyl)-5-fluoropyrimidine-2,4-diamine; 1-(5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine; 5-fluoro-N 2 -(isoquinolin-3-yl)-N 4 -(4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine; 5-Fluoro-N 4 -(4-piperazin-1-yl)phenyl-N 2 -((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine; 5-Fluoro-N 4 -(4-(piperazin-1-yl)phenyl)-N 2 -(pyridin-2-ylmethyl)pyrimidine-2,4-diamine; methyl 4-(5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)aminobenzoate; 1-(5-fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine; 5-Fluoro-N 4 -(4-(4-Isopentylpiperazin-1-yl)phenyl)-N 2 -((Tetrahydrofuran-2-yl)methyl)pyrimidine-2,4-diamine; 5-Fluoro-N 4 -(4-(4-Isopentylpiperazin-1-yl)phenyl)-N 2 -(pyridin-2-ylmethyl)pyrimidine-2,4-diamine; 1-(5-fluoro-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrimidin-2-yl)-1H-benzo[d]imidazol-2-amine; N 6 -(5-Fluoro-4-((4-morpholinophenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(4-((4-(diethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(4-((4-(Dipropylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((4-(piperidin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(4-((4-(Dimethylamino)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; tert-butyl 4-(5-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-2-yl)piperazine-1-carboxylate; tert-butyl 4-(5-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)pyridin-3-yl)piperazine-1-carboxylate; N 6 -(5-Fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; 1-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethanone; N 6 -(5-Fluoro-4-((4-(4-isopropylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; 2-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)ethanol; N 6 -(4-((4-(4-(ethylsulfonyl)piperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-4-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(4-((4-(4-Benzylpiperazin-1-yl)phenyl)amino)-5-fluoropyrimidin-2-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; 1-(4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazin-1-yl)-3,3-dimethylbutan-1-one; N-(tert-butyl)-4-(4-((2-((2-(dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxamide; N 6 -(5-Fluoro-2-((4-(pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-2-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-2-((6-(piperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(2-((4-(dimethylamino)phenyl)amino)-5-fluoropyrimidin-4-yl)-N 2 ,N 2 ,N-dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 -(5-Fluoro-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; 1-(4-(4-((4-((2-(Dimethylamino)-1H-benzo[d]imidazol-6-yl)amino)-5-fluoropyrimidin-2-yl)amino)phenyl)piperazin-1-yl)ethanone; N 6 -(5-Fluoro-2-((4-(4-isopentylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-N 2 ,N 2 -dimethyl-1H-benzo[d]imidazole-2,6-diamine; N 6 , N 6 -(5-Fluoropyrimidine-2,4-diyl)bis(N2,N2-dimethyl-1H-benzo[d]imidazole-2,6-diamine).

10. A method for preparing a 2,4-disubstituted-5-fluoropyrimidine derivative according to any one of claims 1-4, characterized in that, Comprising the following steps:

11. A method for preparing a 2,4-disubstituted-5-fluoropyrimidine derivative according to any one of claims 5-8, characterized in that, Comprising the following steps:

12. A pharmaceutical composition, characterized in that, Containing the 2,4-disubstituted-5-fluoropyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, as well as a pharmaceutically acceptable carrier and excipient.

13. Use of the 2,4-disubstituted-5-fluoropyrimidine derivative according to any one of claims 1-9 or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating fibrosis; the fibrosis refers to renal interstitial fibrosis.

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