Highly selective activator of OCT4

Abstract

The present invention relates to a highly selective activator that can be used for the expression of OCT4 and downstream genes, a pharmaceutical composition thereof, and a preparation method thereof. The activator has the following formula: #imgabs0# wherein m1, m2, A2, and A3 are described herein.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an OCT4 highly selective activator, a pharmaceutical composition and a preparation method thereof, and an application thereof in regulating Oct4 and its associated genes. Background Art

[0002] Regenerative medicine refers to an emerging science that uses a variety of new technical disciplines to rebuild aging or dysfunctional tissues and organs, and treats related diseases through a variety of medical means. The important research directions of regenerative medicine are the mechanisms of normal tissue characteristics and functions, the biological basis of post-traumatic repair, and the regeneration mechanisms of tissues and organs and the differentiation mechanisms of various stem cells, so as to ultimately obtain effective biological treatment methods. Among them, embryonic stem cells (ESCs, referred to as ES, EK or ESC cells) are the most popular cell types in early regenerative medicine research. However, the acquisition and use of this cell are both ethically controversial, because embryonic stem cell research requires the destruction of embryos, and embryos are the life forms in the uterus when humans are not yet formed. This ethical controversy has greatly hindered the advancement and application of regenerative medicine.

[0003] In 2006, Shinya Yamanaka's team invented a "cocktail" method consisting of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into stem cells with differentiation pluripotency. Such stem cells are called induced pluripotent stem cells (Takahashi K, et al., Cell, 2006, 126 (4) pp. 663-676; Takahashi K and Yamanaka S, Cell, 2007, 131 (5) pp. 861-872). These stem cells have differentiation potential similar to embryonic stem cells, and can form the three most basic germ layers of human development: ectoderm, mesoderm and endoderm, and finally form a variety of adult cells. This invention broke through the ethical restrictions on the use of human embryonic stem cells in medicine and greatly expanded the application potential of stem cell technology in clinical medicine.

[0004] In the study of induced pluripotent stem cells and embryonic stem cells, Oct4 has been shown to be a major regulatory gene for reprogramming and inducing cell plasticity (Malik, V et al., Nat. Commun. 2019, 10, 3477). The protein encoded by the Oct4 gene plays a key role in embryonic development and stem cell pluripotency, and alternative splicing leads to multiple transcript variants. The protein encoded by Oct4 belongs to the POU domain family of transcription factors and is located in Chromosome 17: 35,825,200-35,829,401. The hallmark feature of the POU transcription factor family is the POU domain, which consists of two structurally independent subdomains: a POU-specific (POU) region consisting of 75 highly conserved amino acids and a 60-amino acid carboxyl-terminal homology domain (POUh). The expression of Oct4 is regulated by the cis-acting elements and chromatin structure methylation upstream of the Oct4 gene at the transcriptional level (Klemm JD, et al., Cell, 1994, 77: 21-32; Brehm A, et al., Mol Cell Biol 1997; 17: 154-62). Yeom et al. analyzed the expression of the LacZ reporter gene under the control of an 18Kb fragment from the Oct4 genomic locus and identified two elements, which they named the proximal enhancer (PE) and the distal enhancer (DE) that may need to be regulated. They identified the precise binding sites of transcription factors in these two enhancers (Yeom Y, et al. Integrated Ann Indexes 1996; 122: 881-94). POU domain transcription factors bind to specific octamer DNA and regulate cell type-specific differentiation pathways. Among them, in the process of iPSC formation, Oct4 containing POU domain and Sox2 containing HMG domain are transcription factors that are crucial for maintaining the pluripotency of pluripotent cells (Nichols, J., et al., Cell, 1998, 95, 379-391; Avilion, A., et al., 2003, Genes Dev. 17, 126-140), and at least part of their function in pluripotent cells is to drive the transcription of target genes through the synergistic interaction between the two (Tomioka, M., et al. Nucleic Acids Res. 2002; 30, 3202-3213). These findings indicate that developmental transitions can be controlled by Oct4.The currently widely used reprogramming methods mostly overexpress Oct4 through viruses or other types of vectors (Takahashi K, et al., Cell, 2006, 126 (4): 663-676; Takahashi K and Yamanaka S, Cell, 2007, 131 (5): 861-872); Yu J, et al. Science. 2007; 318: 1917-1920). Such methods have potential clinical risks in the clinical use of induced pluripotent stem cells (iPSCs), such as the tumorigenicity risks caused by the use of viral vectors; in addition, the complex GMP production process of the vector also brings complexity to the clinical supervision of induced pluripotent stem cells. Furthermore, such products will be expensive due to the use of vectors.

[0005] Based on the above reasons, the present invention designs and uses benzimidazole derivatives and aminopyridine derivatives to achieve expression regulation of its downstream genes through chemical regulation of Oct4 promoter, thereby avoiding the use of viruses or other vectors to regulate Oct4, and further achieving safe and convenient chemical small molecules to initiate biological expression functions. Summary of the invention

[0006] The present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, ester, optical isomer or prodrug thereof, a pharmaceutical composition comprising the compound of formula (I) and its use as a highly selective OCT4 activator for cell reprogramming.

[0007] The present invention provides a compound of formula (I):

[0008]

[0009] in:

[0010] A1 is

[0011] m1 and m2 are 0 or 1 respectively;

[0012] A2 is C1-C6 alkylene, C2-C6 alkenylene, -O(CH2)q-, -NR1-, -SO2-, -(CH2) V NHS(O)2- or bond, wherein q is 1 or 2 or 3 or 4, V is 0 or 1 or 2, and R1 is selected from H or C1-C4 alkyl;

[0013] A3 is C1-C6 alkyl; C2-C6 alkenyl; C4-C6 cycloalkyl, one of the carbon atoms of which may be substituted by a N, O, or S heteroatom; Z and Z1 are N or CR2, R2 is selected from H, halogen, C1-C4 alkyl or cyano; Z3 is N, O, S or C=O, when the bond between Z4 and Z5 is a single bond, Z4 is N or CH, Z5 is CH2 or C=O, when the bond between Z4 and Z5 is a double bond, Z4 is C, Z5 is CH;

[0014] and pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof.

[0015] In certain embodiments, the compounds disclosed herein have formula (II) or formula (III):

[0016]

[0017] in:

[0018] A2 is -CH2-, -CH=CH-, -C(CH3)=CH-, -O(CH2)-, -O(CH2)2-, -NH-, -N(CH3)-, -NHS(O)2-, -(CH2)2NHS(O)2- or is absent.

[0019] A3 is -CH3, butenyl,

[0020]

[0021] In some embodiments: m1 is 0, m2 is 1;

[0022] A2 is -N(CH3)-;

[0023] A3 is

[0024] In some embodiments: m1 is 1, m2 is 0;

[0025] A2 is -CH2-, -SO2-, -(CH2)2NHS(O)2- or a bond;

[0026] A3 is -CH3,

[0027] In some embodiments: m1 is 1, m2 is 1;

[0028] A2 is -CH2-, -NH-, -C(CH3)=CH- or a bond;

[0029] A3 is -CH3, -C(CH3)=CH-CH3,

[0030]

[0031] In some embodiments: m1 is 0, m2 is 0;

[0032] A2 is -CH2-, -CH=CH-, -O(CH2)-, -O(CH2)2- or a bond;

[0033] A3 is

[0034] In some embodiments, the compound is:

[0035]

[0036] The present invention relates to a pharmaceutical composition, which comprises any one of the above compounds and its pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0037] The present invention relates to the use of any one of the above-mentioned compounds and / or its pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof in the preparation of a drug that is a highly selective activator of OCT4 for induced pluripotent stem cells.

[0038] The present invention relates to the use of a drug for preparing a highly selective OCT4 activator for inducing pluripotent stem cells. The diseases treated by the highly selective OCT4 activator for inducing pluripotent stem cells include cancer, heart disease, stroke, diabetes, obesity, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myocardial infarction, muscular dystrophy, CMT-1A, spinal cord injury, traumatic brain injury, tooth loss, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, Crohn's disease and genetic diseases and other similar diseases.

[0039] The present invention relates to a method for obtaining induced pluripotent stem cells in a subject suffering from a disease, the method comprising administering to the subject a therapeutically effective amount of any one of the above-mentioned compounds and / or its pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof.

[0040] In the method, the subject suffering from the disease refers to humans suffering from cancer, heart disease, stroke, diabetes, obesity, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myocardial infarction, muscular dystrophy, CMT-1A, spinal cord injury, traumatic brain injury, tooth loss, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, Crohn's disease and genetic diseases and other similar diseases.

[0041] The present invention relates to a method for activating OCT4 function, which comprises contacting any one of the above compounds and / or its pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof with an OCT4 target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shown are the results of aminopyridine derivative small molecules on the basal expression of OCT4 and the expression of its downstream gene Nanog (CK is the control group);

[0043] Figure 2 Shown are the results of benzimidazole derivative small molecules on the basal expression of OCT4 and the expression of its downstream gene Nanog (CK is the control group);

[0044] Figure 3 The immunofluorescence staining images of aminopyridine derivatives and benzimidazole derivatives promoting mesenchymal cells to express Rex l gene and form pluripotent stem cell clone-like cell clusters are shown (CK is the control group) DETAILED DESCRIPTION

[0045] In the present invention, the following definitions are applicable:

[0046] The term "alkyl" herein refers to a straight or branched chain saturated hydrocarbon containing 1 to 12 carbon atoms. Examples of (C1-C6) alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

[0047] The term "alkenyl" refers to a straight or branched unsaturated hydrocarbon containing 2 to 12 carbon atoms, containing at least one C=C double bond in the chain. Examples of alkenyl include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl or hexenyl.

[0048] The term "alkylene" refers to a divalent alkyl group. Any of the monovalent alkyl groups may be an alkylene group by removing the second hydrogen atom from the alkyl group. As defined herein, the alkylene group may also be a C1-C6 alkylene group. The alkylene group may further be a C1-C4 alkylene group. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc.

[0049] The term "alkenylene" refers to a divalent alkenyl group. Any of the monovalent alkenyl groups may be an alkenyl group by removing the second hydrogen atom from the alkenyl group. As defined herein, the alkenyl group may further be a C2-C6 alkenyl group. Typical alkenyl groups include, but are not limited to: -CH=CH-, -CH=C(CH3)-, -CH=CHCH2-, -CH=CHCH2CH2-, -CH=CHCH2CH2CH2-, -CH=CHCH2CH2CH2-, -CH=CHCH2CH2CH2-, etc.

[0050] The term "cycloalkyl" refers to a monocyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. The cycloalkyl may further be a C4-C6 cycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0051] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0052] The term "cyano" refers to a substituent having a carbon atom connected to a nitrogen atom by a triple bond (ie, C≡N).

[0053] The term "substituted" as used herein refers to the replacement of any one or more hydrogen atoms on the designated atom or group with a group selected from the designated range, provided that the normal valency of the designated atom is not exceeded.

[0054] The compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures. In these cases, a single enantiomer or diastereomer, i.e., an optically active configuration, can be obtained by asymmetric synthesis or by resolving a racemate or a diastereomeric mixture. For the resolution of a racemate or a diastereomeric mixture, conventional methods can be used, such as crystallization in the presence of a resolving agent or chromatography using, for example, a chiral high pressure liquid chromatography (HPLC) column. In addition, these compounds include compounds with chiral centers in R- and S- configurations. These compounds also include crystalline forms, including polymorphs and inclusion compounds. Similarly, the term "salt" also includes all isomers, racemates, other mixtures, R- and S- configurations, tautomers, and crystalline forms of the salts of the compound.

[0055] "Pharmaceutically acceptable salt" refers to a salt of a free acid or base of a compound represented by Formula (I), Formula (II) or Formula (III) that is non-toxic, biologically tolerable or otherwise biologically suitable for administration to a subject for treatment. See generally: SMBerge, et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferably, pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with patient tissues without undue toxicity, irritation or allergic reaction. The compound of Formula (I), Formula (II) or Formula (III) may have sufficient acidic groups, sufficient basic groups or both types of functional groups, and react accordingly with some inorganic or organic bases, and inorganic and organic acids to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromides, hydroiodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

[0056] "Solvates" such as "hydrates" are formed by the interaction of a solvent with a compound. The term "compound" includes solvates of the compound, including hydrates. Similarly, "salts" include solvates of salts, such as hydrates. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemihydrates.

[0057] "Prodrug" may refer to a precursor of a given compound, which, after administration to a subject, undergoes chemical or physiological processes in vivo (e.g., solvolysis, enzymatic decomposition) or under physiological conditions (e.g., conversion of the prodrug to a compound of formula (I) under physiological pH conditions) to obtain the compound. "Pharmaceutically acceptable prodrugs" are non-toxic, biologically tolerated, or otherwise biologically suitable prodrugs for administration to a subject. Exemplary procedures for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrugs," edited by H. Bundgaard, Elsevier, 1985.

[0058] "Active metabolites" refer to pharmaceutically active products metabolized in vivo by compounds of formula (I), (II) or (III) or their salts. Prodrugs and active metabolites of compounds can be determined using conventional techniques known or available in the art. See, for example, Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86(7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991)

[0059] A "therapeutically effective amount" refers to an amount of a compound disclosed herein that is sufficient to effect treatment (as defined below) of a disease or disorder in a mammal (preferably a human) when administered to the mammal (preferably a human). The amount of a disclosed compound that constitutes a "therapeutically effective amount" will vary with the compound, the disorder and its severity, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and the disclosure herein.

[0060] The term "treatment" refers to administering to an individual at least one compound described herein and / or at least one pharmaceutically acceptable salt thereof to slow down (reduce) undesirable physiological changes or diseases, such as the development or spread of inflammation or cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the severity of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the condition, and alleviating (whether partially or completely) the disease, whether detected or undetectable. "Treatment" also means that survival can be prolonged compared to those expected survival if not receiving treatment. Individuals in need of treatment include individuals who have symptoms of or suffer from these diseases.

[0061] Pharmaceutical composition

[0062] The present invention provides a pharmaceutical composition comprising one or more compounds described herein or their pharmaceutically acceptable salts or esters as active ingredients, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers and adjuvants. The pharmaceutical composition can be administered alone or together with other therapeutic agents. Such compositions are prepared in a manner known in the pharmaceutical field.

[0063] The pharmaceutical composition can be administered in single or multiple doses by any of the acceptable modes for pharmaceutical agents having similar utilities, such as those described in the patents and patent applications incorporated herein by reference, including rectal, buccal, intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, as an inhalant, or by implanted or coated devices such as stents, for example, or arterial insertion of pillar polymers.

[0064] The present invention also provides a kit comprising a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0065] "Pharmaceutically acceptable carrier or excipient" refers to a non-toxic, biologically tolerable and other biologically suitable substance for administration to an individual, such as an inert substance, which is added to a pharmacological composition or used as a vehicle, carrier or diluent to facilitate administration of the active ingredient and is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various types of sugars or starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0066] Uses of compounds and compositions thereof

[0067] The present invention provides the use of a compound and a composition thereof, which are mainly used as a highly selective activator of OCT4, for activating the function of OCT4, and achieving expression regulation of its downstream genes through chemical regulation of the Oct4 promoter, thereby inducing pluripotent stem cells in subjects suffering from diseases, thereby achieving the purpose of treating the diseases, wherein the diseases include cancer, heart disease, stroke, diabetes, obesity, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myocardial infarction, muscular dystrophy, CMT-1A, spinal cord injury, traumatic brain injury, tooth loss, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, Crohn's disease and genetic diseases and other similar diseases.

[0068] List of abbreviations used in the following examples and elsewhere herein:

[0069] Atm: atmosphere; Boc: tert-butyloxycarbonyl; (Boc)2O: di-tert-butyl dicarbonic anhydride; CH2Cl2: dichloromethane; Cs2CO3: cesium carbonate; Cu(I)Br: copper bromide; Cu2SO4: cuprous sulfate; DCM: dichloromethane; DMAC: N,N- dimethylacetamide; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; DIOX: 1,4-dioxane; dioxane: dioxane; EA: acetic acid; Et3N: triethylamine; ETOH: ethanol; EtOAc: ethyl acetate; FA: formic acid; g: gram; h: hour; H2: hydrogen; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HBr: hydrobromic acid; (Hbim)BF4: 1-butylimidazole tetrafluoroborate; H2O: water; HAc: acetic acid; H2O2: hydrogen peroxide; H2SO4: fuming sulfuric acid; KCN: potassium cyanide; K2CO3: potassium carbonate; Li: lithium; MCA: chloroacetic acid; MeCN: acetonitrile; MeOH: methanol; mg: milligram; ml: milliliter; mmol: millimole; mol: mole; m / z: mass-to-charge ratio; N2: nitrogen; NaBH3CN: sodium cyanoborohydride; NaNO2: sodium nitrite; NaOH: sodium hydroxide; NaOMe: sodium methoxide; Na2SO4: sodium sulfate; Ni(OAc)2 4H2O: nickel diacetate tetrahydrate; Pd-C: palladium on carbon; PH: acidity; TBN: tert-butyl nitroso; TEA: triethanolamine; THF: tetrahydrofuran; TLC: thin layer chromatography; Xylene: xylene.

[0070] The following is a detailed description of the technical means used by the present invention to achieve the intended purpose of the invention with the aid of the accompanying drawings and embodiments of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following embodiments are commercially available products unless otherwise specified.

[0071] Universal synthesis

[0072] The general synthetic routes described in this application can be varied by replacing the starting materials with other raw materials having similar structures, thereby obtaining different products accordingly. The following synthetic route descriptions give multiple examples of how the starting materials can be varied to obtain the corresponding products.

[0073] General Method A-1-n

[0074]

[0075] Where R is wherein q is 1 or 2 or 3 or 4, Z and Z1 are N or CR2 respectively, and R2 is selected from H, halogen, C1-C4 alkyl or cyano.

[0076] General Method A-2-n

[0077]

[0078] Wherein R is C2-C6 alkenyl; Wherein, q is 1 or 2 or 3 or 4, Z and Z1 are N or CR2 respectively, R2 is selected from H, halogen, C1-C4 alkyl or cyano, R1 is selected from H or C1-C4 alkyl, and Z3 is N, O, S or C=O.

[0079] General Method A-4-n

[0080]

[0081] Where R is wherein q is 1 or 2 or 3 or 4, Z and Z1 are N or CR2 respectively, and R2 is selected from H, halogen, C1-C4 alkyl or cyano.

[0082] General Method A-6-n

[0083]

[0084] Wherein R is a C4-C6 cycloalkyl group, one of the carbon atoms of which may be substituted by a N, O, or S heteroatom; or R=o is wherein q is 1 or 2 or 3 or 4, Z and Z1 are N or CR2 respectively, and R2 is selected from H, halogen, C1-C4 alkyl or cyano.

[0085] General Method B-1-n

[0086]

[0087] wherein R is defined as in general method A-1-n.

[0088] General Method B-3-n

[0089]

[0090] wherein R is defined as in general method A-2-n.

[0091] General Method B-4-n

[0092]

[0093] wherein R is defined as in general method A-4-n.

[0094] General Method B-6-n

[0095]

[0096] wherein R is defined as in general method A-6-n.

[0097] Intermediate synthesis

[0098] Intermediate Ⅰ-3: tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate

[0099] Synthesis route:

[0100]

[0101] Step 1: Mix 6-methyl-5-nitropyridin-2-amine I-1 (15.3 g, 0.1 mol) with di-tert-butyl dicarbonic anhydride (26.2 g, 0.12 mol) and K2CO3 (69 g, 0.5 mol), add tetrahydrofuran (200 ml), and stir at room temperature for 3 hours. Add ethyl acetate and water to the reaction mixture, separate the organic layer, wash with water, dry with sodium sulfate, and remove the solvent by distillation under reduced pressure to obtain (6-methyl-5-nitropyridin-2-yl)carbamic acid tert-butyl ester I-2 (23.3 g, 92%). Liquid phase mass spectrum m / z = 254.1 [M+H] +

[0102] Step 2: The above-mentioned (6-methyl-5-nitropyridin-2-yl)carbamic acid tert-butyl ester I-2 (12.7 g, 0.05 mol) was stirred with 10% Pd-C (1 g) and MeOH (100 mL) under a hydrogen atmosphere (1 atm) at room temperature for 2 h. The mixture was filtered and the solvent was removed by distillation under reduced pressure to obtain the intermediate (5-amino-6-methylpyridin-2-yl)carbamic acid tert-butyl ester I-3 (11 g, 98%). Liquid phase mass spectrum m / z = 224.1 [M+H] +

[0103] Intermediate II-3: 1H-benzo[d]imidazol-6-ol

[0104]

[0105] Synthesis route:

[0106]

[0107] Step 1: Intermediate 1H-benzo[d]imidazole-6-diazo II-2

[0108] Water (2 ml) and 20% H2SO4 aqueous solution (1 ml) were added to 1H-benzo[d]imidazole-6-amine (67 mg, 0.5 mmol). A sodium nitrite aqueous solution (42 mg, 0.6 mmol) solution (0.5 ml) and acetonitrile (2 ml) were added to the mixture under ice cooling, and the mixture was stirred for 30 minutes. A solution of Cu2SO4 (67 mg, 0.3 mmol) in a 20% HBr aqueous solution (0.5 ml) at room temperature was added to the obtained reaction mixture, and the mixture was stirred at 80°C for 30 minutes. Ethyl acetate and water were added to the reaction mixture. The organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain the hydrogen sulfate salt of IⅠ-2 (85 mg, 70%), liquid phase mass spectrum m / z = 145.1 [m] +.

[0109] Step 2: Intermediate 1H-benzo[d]imidazol-6-ol II-3

[0110] The aqueous solution (1 ml) of hydrogen sulfate of 1H-benzo[d]imidazole-6-diazide (48 mg, 0.2 mmol) was added dropwise to a 40% aqueous sulfuric acid solution (5 ml) at 100°C, and the mixture was stirred for 10 minutes. NaOH was added to the obtained reaction mixture until the pH was about 3, ethyl acetate was added to the reaction mixture, the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform: methanol = 50:1) to obtain IⅠ-3 (19 mg, 69%), liquid phase mass spectrum m / z = 134.1 [m+H] +.

[0111] Example 1: 3-(((6-amino-2-methylpyridin-3-yl)oxy)methyl)benzonitrile A-1-1

[0112]

[0113] Step 1: (3-Cyanophenyl)lithium methoxide Ⅰ-5-1

[0114] Mix (3-cyanophenyl)methanol (20 mg, 0.15 mol) and metallic lithium (12 mg, 1.73 mmol), add tetrahydrofuran (3 mL) and stir at 20°C for 3 h. Filter the mixture and remove the solvent by distillation under reduced pressure to obtain (3-cyanophenyl)methanol lithium Ⅰ-5-1 (20 mg, 96%).

[0115] Step 2: 3-(((6-amino-2-methylpyridin-3-yl)oxy)methyl)benzonitrile

[0116] Mix (3-cyanophenyl)methoxide lithium Ⅰ-5-1 (20 mg, 0.14 mol) and 5-bromo-6-methylpyridin-2-amine Ⅰ-6 (32 mg, 0.17 mol), add cuprous bromide (28 mg, 0.2 mol), methanol (1 mL), N,N-dimethylformamide (3 mL), heat in an oil bath at 110°C and stir for 1 h. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (dichloromethane: methanol = 40: 1) to obtain 3-(((6-amino-2-methylpyridin-3-yl)oxy)methyl)benzonitrile A-1-1 (26 g, 78%). Liquid phase mass spectrum m / z = 240.1 [M+H] +

[0117] Example 2: 6-methyl-5-phenethoxypyridin-2-amine A-1-2

[0118]

[0119] Step 1: 2-phenylethoxide lithium Ⅰ-5-2

[0120] 2-Phenylethanol (18 mg, 0.15 mol) was mixed with metallic lithium (12 mg, 1.73 mmol), tetrahydrofuran (3 mL) was added and stirred at 20°C for 3 h. The mixture was filtered and the solvent was removed by distillation under reduced pressure to obtain 2-phenylethoxide lithium I-5-2 (18 mg, 94%).

[0121] Step 2: 6-Methyl-5-phenethoxypyridin-2-amine A-1-2

[0122] Mix 2-phenylethoxide lithium I-5-2 (18 mg, 0.14 mol) with 5-bromo-6-methylpyridin-2-amine (32 mg, 0.17 mol), add cuprous bromide (28 mg, 0.2 mol), methanol (1 mL), N,N-dimethylformamide (3 mL), and stir at 110 ° C in an oil bath for 1 h. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate it under reduced pressure. The residue is purified by silica gel column chromatography (dichloromethane: methanol = 40: 1) to obtain 6-methyl-5-phenylethoxypyridin-2-amine (22 g, 69%). Liquid phase mass spectrum m / z = 228.1 [M+H] +

[0123] Example 3: N-(6-amino-2-methylpyridin-3-yl)thiophene-3-carboxamide A-2-1

[0124]

[0125] Step 1: Tert-butyl (6-methyl-5-(thiophene-3-carboxamido)pyridin-2-yl)carbamate I-8-1

[0126] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), thiophene-2-carboxylic acid (15 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain tert-butyl (6-methyl-5-(thiophene-3-carboxamido)pyridin-2-yl)carbamate I-8-1 (16 mg, 48%). Liquid phase mass spectrum m / z = 334.1 [M+H] +

[0127] Step 2: N-(6-amino-2-methylpyridin-3-yl)thiophene-3-carboxamide

[0128] To tert-butyl (6-methyl-5-(thiophene-3-carboxamido)pyridin-2-yl)carbamate (16 mg, 0.05 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)thiophene-3-carboxamide A-2-1 (11 mg, 94%). Liquid phase mass spectrum m / z = 234.0 [M+H] +

[0129] Example 4: (E) N-(6-amino-2-methylpyridin-3-yl)-2-methyl-2-enamide A-2-2

[0130]

[0131] Step 1: tert-Butyl (E)-(6-methyl-5-(2-methyl-2-enamido)pyridin-2-yl)carbamate I-8-2

[0132] tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), (E)-2-methyl-2-enoic acid (12 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to give (E)-(6-methyl-5-(2-methyl-2-enamido)pyridin-2-yl)carbamic acid tert-butyl ester I-8-2 (19 mg, 62%). HPLC mass spectrum m / z = 306.1 [M+H] +

[0133] Step 2: (E) N-(6-amino-2-methylpyridin-3-yl)-2-methyl-2-enamide

[0134] To tert-butyl (E)-(6-methyl-5-(2-methyl-2-enamido)pyridin-2-yl)carbamate (19 mg, 0.06 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain (E) N-(6-amino-2-methylpyridin-3-yl)-2-methyl-2-enamide A-2-2 (12 mg, 97%). Liquid phase mass spectrum m / z = 206.1 [M+H] +

[0135] Example 5: N-(6-amino-2-methylpyridin-3-yl)-2,3-dihydrobenzofuran-2-carboxamide A-2-3

[0136]

[0137] Step 1: tert-Butyl (5-(2,3-dihydrobenzofuran-2-carboxamide)-6-methylpyridin-2-yl)carbamate I-8-3

[0138] tert-Butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) was mixed with benzofuran-2-carboxylic acid (19 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to give (5-(2,3-dihydrobenzofuran-2-carboxamide)-6-methylpyridin-2-yl)carbamic acid tert-butyl ester I-8-3 (20 mg, 55%). HPLC mass spectrum m / z = 370.2 [M+H] +

[0139] Step 2: N-(6-amino-2-methylpyridin-3-yl)-2,3-dihydrobenzofuran-2-carboxamide

[0140] To tert-butyl (5-(2,3-dihydrobenzofuran-2-carboxamide)-6-methylpyridin-2-yl)carbamate (20 mg, 0.055 mmol) was added a solution of HCl in dioxane (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)-2,3-dihydrobenzofuran-2-carboxamide A-2-3 (11 mg, 74%). Liquid phase mass spectrum m / z = 270.1 [M+H] +

[0141] Example 6: N-(6-amino-2-methylpyridin-3-yl)-3,4-dichlorobenzamide A-2-4

[0142]

[0143] Step 1: tert-Butyl (5-(3,4-dichlorobenzamide)-6-methylpyridin-2-yl)carbamate I-8-4

[0144] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), 3,4-dichlorobenzoic acid (23 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain tert-butyl (5-(3,4-dichlorobenzamide)-6-methylpyridin-2-yl)carbamate I-8-4 (20 mg, 51%). Liquid phase mass spectrum m / z = 396.1 [M+H] +

[0145] Step 2: N-(6-amino-2-methylpyridin-3-yl)-3,4-dichlorobenzamide A-2-4

[0146] To tert-butyl (5-(3,4-dichlorobenzamide)-6-methylpyridin-2-yl)carbamate (20 mg, 0.05 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)-3,4-dichlorobenzamide A-2-4 (14 mg, 86%). Liquid phase mass spectrum m / z = 295.0 [M+H] +

[0147] Example 7: N-(6-amino-2-methylpyridin-3-yl)benzothiophene-2-carboxamide A-2-5

[0148]

[0149] Step 1: tert-Butyl (5-(Benzothiophene-2-carboxamide)-6-methylpyridin-2-yl)carbamate I-8-5

[0150] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), benzothiophene-2-carboxylic acid (21 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain tert-butyl (5-(benzothiophene-2-carboxamide)-6-methylpyridin-2-yl)carbamate I-8-5 (22 mg, 57%). Liquid phase mass spectrum m / z = 384.1 [M+H]

[0151] Step 2: N-(6-amino-2-methylpyridin-3-yl)benzothiophene-2-carboxamide

[0152] To tert-butyl (5-(benzothiophene-2-carboxamide)-6-methylpyridin-2-yl)carbamate (22 mg, 0.057 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)benzothiophene-2-carboxamide A-2-5 (11 mg, 68%). Liquid phase mass spectrum m / z = 284.1 [M+H] +

[0153] Example 8: N-(6-amino-2-methylpyridin-3-yl)-2-phenylacetamide A-2-6

[0154]

[0155] Step 1: tert-Butyl (6-methyl-5-(2-phenylacetamido)pyridin-2-yl)carbamate I-8-6

[0156] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), 2-phenylacetic acid (16 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain tert-butyl (6-methyl-5-(2-phenylacetamide)pyridin-2-yl)carbamate I-8-6 (18 mg, 53%). Liquid phase mass spectrum m / z = 342.2 [M+H] +

[0157] Step 2: N-(6-amino-2-methylpyridin-3-yl)-2-phenylacetamide:

[0158] To tert-butyl (6-methyl-5-(2-phenylacetamide)pyridin-2-yl)carbamate (18 mg, 0.05 mmol), add HCl in dioxane (5%, 5 mL), heat and stir at 70°C for 3 h, add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it over sodium sulfate, and concentrate under reduced pressure. The residue is chromatographed on a silica gel column (ethyl acetate: hexane = 3: 7), and the solvent is removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)-2-phenylacetamide A-2-6 (12 mg, 94%). Liquid phase mass spectrum m / z = 242.1 [M+H] +

[0159] Example 9: N-(6-amino-2-methylpyridin-3-yl)benzamide A-2-7

[0160]

[0161] Step 1: Intermediate (5-benzamido-6-methylpyridin-2-yl)carbamic acid tert-butyl ester I-8-7

[0162] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol), 2-benzoic acid (15 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain tert-butyl (5-benzamido-6-methylpyridin-2-yl)carbamate I-8-7 (15 mg, 46%). Liquid phase mass spectrum m / z = 328.2 [M+H] +

[0163] Step 2: N-(6-amino-2-methylpyridin-3-yl)benzamide

[0164] To tert-butyl (5-benzamido-6-methylpyridin-2-yl)carbamate (15 mg, 0.046 mmol), add HCl in dioxane (5%, 5 mL), heat and stir at 70°C for 3 h, add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate under reduced pressure. The residue is chromatographed on a silica gel column (ethyl acetate: hexane = 3: 7), and the solvent is removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)benzamide A-2-7 (10 mg, 96%). Liquid phase mass spectrum m / z = 227.1 [M+H] +

[0165] Example 10: 2-(6-amino-2-methylpyridin-3-yl)isoindol-1-one A-3

[0166]

[0167] Its synthetic route is:

[0168]

[0169] Step 1: Tert-butyl (6-methyl-5-(1-oxoisoindole-2-yl)pyridin-2-yl)carbamate I-10

[0170] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) with 2-formylbenzoic acid (18 mg, 0.12 mmol), add formic acid (0.2 ml), triethylamine (1 ml), ethanol (1 ml), and heat the mixture to 80 ° C and stir for 60 minutes. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: ethyl acetate = 20:1) to obtain tert-butyl (6-methyl-5-(1-oxoisoindole-2-yl)pyridin-2-yl)carbamate I-10 (20 mg, 59%). Liquid phase mass spectrum m / z = 340.4 [M+H] +

[0171] Step 2: 2-(6-amino-2-methylpyridin-3-yl)isoindol-1-one

[0172] To tert-butyl (6-methyl-5-(1-oxoisoindol-2-yl)pyridin-2-yl)carbamate (20 mg, 0.059 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 2-(6-amino-2-methylpyridin-3-yl)isoindol-1-one A-3 (12 mg, 85%). Liquid phase mass spectrum m / z = 240.1 [M+H] +

[0173] Example 11: 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea A-4-1

[0174]

[0175] Step 1: p-Nitrophenyl p-toluene aminocarbonate I-13-1

[0176] p-Toluidine (21 mg, 0.2 mmol) was mixed with 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol), triethanolamine (0.2 ml), dichloromethane (1 ml), tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: hexane = 5: 1) to obtain p-nitrophenyl p-toluene aminocarbonate I-13-1 (42 mg, 77%). Liquid phase mass spectrum m / z = 273.1 [M+H] +

[0177] Step 2: Tert-butyl (6-methyl-5-(3-(p-tolyl)ureido)pyridin-2-yl)carbamate I-14-1

[0178] By p-nitrophenyl p-toluene carbamate (27mg, 0.1mmol) and (5-amino-6-methylpyridin-2-yl) tert-butyl carbamate (26mg, 0.12mmol) and K2CO3 (0.2g, 1.45mmol) mixed, add acetonitrile (3ml) and be heated to 40 ℃ and stir for 4 hours. After the reaction is completed (monitored by TLC), the reaction mixture is separated and concentrated in a vacuum. The crude reaction mixture thus obtained is washed with dichloromethane, then washed with EtOAc, and finally washed with MeOH (each 1mL). Finally, the reaction product is recrystallized using EtOAc (under warm conditions) to obtain pure form of (6-methyl-5-(3-(p-tolyl) urea) pyridin-2-yl) tert-butyl carbamate I-14-1 (28mg, 80%) liquid phase mass spectrum m / z=357.2[M+H]+

[0179] Step 3: 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea:

[0180] To tert-butyl (6-methyl-5-(3-(p-tolyl)ureido)pyridin-2-yl)carbamate (28 mg, 0.08 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea A-4-1 (16 mg, 78%). Liquid phase mass spectrum m / z = 257.1 [M+H] +

[0181] Example 12: 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea A-4-2

[0182]

[0183] Step 1: 4-nitrophenyl (3-bromophenyl) aminocarbonate I-13-2

[0184] 3-Bromoaniline (34 mg, 0.2 mmol) was mixed with 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol), triethanolamine (0.2 ml), dichloromethane (1 ml), tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: hexane = 5: 1) to obtain 4-nitrophenyl (3-bromophenyl) aminocarbonate I-13-2 (46 mg, 68%). Liquid phase mass spectrum m / z = 338.1 [M+H] +

[0185] Step 2: Tert-butyl (5-(3-(3-bromophenyl)ureido)-6-methylpyridin-2-yl)carbamate I-14-2

[0186] 4-nitrophenyl (3-bromophenyl) aminocarbonate (34mg, 0.1mmol) and (5-amino-6-methylpyridin-2-yl) tert-butyl carbamate (26mg, 0.12mmol) were mixed with K2CO3 (0.2g, 1.45mmol), acetonitrile (3ml) was added and heated to 40°C and stirred for 4 hours. After the reaction was completed (monitored by TLC), the reaction mixture was separated and concentrated in a vacuum. The crude reaction mixture thus obtained was washed with dichloromethane, then with EtOAc, and finally with MeOH (each 1mL). Finally, the reaction product was recrystallized using EtOAc (under warm conditions) to obtain pure form of (5-(3-(3-bromophenyl) urea)-6-methylpyridin-2-yl) tert-butyl carbamate I-14-2 (34mg, 81%) liquid phase mass spectrum m / z=421.1[M+H]+

[0187] Step 3: 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea

[0188] To tert-butyl (5-(3-(3-bromophenyl)ureido)-6-methylpyridin-2-yl)carbamate (34 mg, 0.08 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 1-(6-amino-2-methylpyridin-3-yl)-3-(p-tolyl)urea A-4-2 (20 mg, 78%). Liquid phase mass spectrum m / z = 321.0 [M+H] +

[0189] Example 13: 6-amino-N,2-dimethyl-N-phenylnicotinamide A-5

[0190]

[0191] Its synthetic route is:

[0192]

[0193] Step 1: tert-Butyl (5-bromo-6-methylpyridin-2-yl)carbamate I-15

[0194] 5-Bromo-6-methylpyridin-2-amine (37 mg, 0.2 mmol) was mixed with di-tert-butyl dicarbonic anhydride (52 mg, 0.24 mmol) and K2CO3 (69 mg, 0.5 mmol), tetrahydrofuran (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, washed with water, dried over sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain tert-butyl (5-bromo-6-methylpyridin-2-yl)carbamate I-15 (50 mg, 88%). Liquid phase mass spectrum m / z = 287.0 [M+H] +

[0195] Step 2: Tert-butyl (6-methyl-5-(methyl(phenyl)carbamoyl)pyridin-2-yl)carbamate I-17

[0196] Mix tert-butyl (5-bromo-6-methylpyridin-2-yl)carbamate (50 mg, 0.175 mmol), N-methyl-N-phenylformamide (27 mg, 0.175 mmol), sodium methoxide (11 mg, 0.2 mmol), and nickel diacetate tetrahydrate (25 mg, 0.1 mmol), add 1,4-dioxane (8 mL), and heat and stir in an oil bath at 110°C for 12 h under helium protection. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate it under reduced pressure. The residue is chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent is removed by distillation under reduced pressure to obtain tert-butyl (6-methyl-5-(methyl(phenyl)carbamoyl)pyridin-2-yl)carbamate Ⅰ-17 (50 mg, 84%). Liquid phase mass spectrum m / z = 342.2 [M+H] +

[0197] Step 3: 6-amino-N,2-dimethyl-N-phenylnicotinamide:

[0198] To tert-butyl (6-methyl-5-(methyl(phenyl)carbamoyl)pyridin-2-yl)carbamate (50 mg, 0.147 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 6-amino-N,2-dimethyl-N-phenylnicotinamide A-5 (27 mg, 76%). Liquid phase mass spectrum m / z = 242.1 [M+H] +

[0199] Example 14: 6-methyl-N5-(tetrahydro-2H-pyran-4-yl)pyridine-2,5-diamine A-6-1

[0200]

[0201] Step 1: tert-Butyl (6-methyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyridin-2-yl)carbamate I-18-1

[0202] By (5-amino-6-methylpyridine-2-yl) tert-butyl carbamate (22mg, 0.1mmol) and tetrahydro-4H-pyran-4-one (12mg, 0.12mmol) mix, add methanol (2ml), stir 2 hours at room temperature, then add sodium cyanoborohydride (20mg, 0.3mmol), stir 2 hours at room temperature.Then add the NaOH aqueous solution (10mL, 0.3mol / L) in the mixture.Gained mixture is extracted with DCM (20mL x 3).The organic phase merged is at anhydrous Na2SO4 upper drying, filter and concentrate in a vacuum. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent was distilled off under reduced pressure to obtain tert-butyl (6-methyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyridin-2-yl)carbamate Ⅰ-18-1 (23 mg, 75%). Liquid phase mass spectrum m / z = 308.2 [M+H] +

[0203] Step 2: 6-Methyl-N5-(tetrahydro-2H-pyran-4-yl)pyridine-2,5-diamine

[0204] To tert-butyl (6-methyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyridin-2-yl)carbamate (23 mg, 0.075 mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain 6-methyl-N5-(tetrahydro-2H-pyran-4-yl)pyridine-2,5-diamine A-6-1 (12 mg, 78%). Liquid phase mass spectrum m / z = 208.1 [M+H] +

[0205] Example 15: 6-Methyl-N 5 -(Pyridin-4-ylmethyl)pyridine-2,5-diamine A-6-2

[0206]

[0207] Step 1: tert-Butyl (6-methyl-5-((pyridin-4-ylmethyl)amino)pyridin-2-yl)carbamate I-18-2

[0208] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) with isonicotinaldehyde (13 mg, 0.12 mmol), add methanol (2 ml), stir at room temperature for 2 hours, then add sodium cyanoborohydride (20 mg, 0.3 mmol), stir at room temperature for 2 hours. Then add aqueous NaOH solution (10 mL, 0.3 mol / L) to the mixture. The resulting mixture was extracted with DCM (20 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent was removed by distillation under reduced pressure to obtain tert-butyl (6-methyl-5-((pyridin-4-ylmethyl)amino)pyridin-2-yl)carbamate Ⅰ-18-2 (24 mg, 76%) liquid phase mass spectrum m / z = 314.2 [M+H] +

[0209] Step 2: 6-Methyl-N 5 -(Pyridin-4-ylmethyl)pyridine-2,5-diamine:

[0210] To tert-butyl (6-methyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyridin-2-yl)carbamate (23 mg, 0.075 mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain 6-methyl-N5-(tetrahydro-2H-pyran-4-yl)pyridine-2,5-diamine A-6-2 (12 mg, 78%). Liquid phase mass spectrum m / z = 208.1 [M+H] +

[0211] Example 16: N 5 -Cyclobutyl-6-methylpyridine-2,5-diamine A-6-3

[0212]

[0213] Step 1: tert-Butyl (5-(cyclobutylamino)-6-methylpyridin-2-yl)carbamate I-18-3

[0214] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) with cyclobutanone (8 mg, 0.12 mmol), add methanol (2 ml), stir at room temperature for 2 hours, then add sodium cyanoborohydride (20 mg, 0.3 mmol), stir at room temperature for 2 hours. Then add NaOH aqueous solution (10 mL, 0.3 mol / L) to the mixture. The resulting mixture is extracted with DCM (20 mL x 3). The combined organic phase is dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product is purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent is removed by distillation under reduced pressure to obtain tert-butyl (5-(cyclobutylamino)-6-methylpyridin-2-yl)carbamate Ⅰ-18-3 (20 mg, 72%) liquid phase mass spectrum m / z = 278.2 [M+H] +

[0215] Step 2: N 5 -Cyclobutyl-6-methylpyridine-2,5-diamine:

[0216] To tert-butyl (5-(cyclobutylamino)-6-methylpyridin-2-yl)carbamate (20 mg, 0.072 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 6-methyl-N5-(tetrahydro-2H-pyran-4-yl)pyridine-2,5-diamine A-6-3 (10 mg, 78%). Liquid phase mass spectrum m / z = 178.1 [M+H] +

[0217] Example 17: 2-(6-amino-2-methylpyridin-3-yl)isoindole-1,3-dione A-7

[0218]

[0219] The synthesis route is:

[0220]

[0221] Step 1: tert-Butyl (5-(1,3-dioxoisooctanol-2-yl)-6-methylpyridin-2-yl)carbamate Ⅰ-20

[0222] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) and isobenzofuran-1,3-dione (18 mg, 0.12 mmol), add N,N-dimethylacetamide (2 ml), stir at room temperature for 24 hours, then add xylene (1 ml), stir in an oil bath at 140°C for 48 hours. After completion (monitored by TLC), separate the insoluble catalyst by filtration, wash with acetone and dry. Concentrate the organic layer under reduced pressure to obtain the desired product, wash with water, and recrystallize in ethanol. The crude product is purified by silica gel column chromatography (CH2Cl2 / n-hexane=1:1), and the solvent is removed by distillation under reduced pressure to obtain tert-butyl (5-(1,3-dioxoisooctanol-2-yl)-6-methylpyridin-2-yl)carbamate I-20 (24 mg, 68%). Liquid phase mass spectrum m / z=354.1[M+H]+

[0223] Step 2: 2-(6-amino-2-methylpyridin-3-yl)isoindole-1,3-dione

[0224] To tert-butyl (5-(1,3-dioxoisooctanol-2-yl)-6-methylpyridin-2-yl) carbamate (24 mg, 0.068 mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain 2-(6-amino-2-methylpyridin-3-yl)isoindole-1,3-dione A-7 (12 mg, 70%). Liquid phase mass spectrum m / z = 254.1 [M+H] +

[0225] Example 18: 6-Methyl-N 5 -phenylpyridine-2,5-diamine A-8

[0226]

[0227] The synthesis route is:

[0228]

[0229] Step 1: tert-Butyl (6-methyl-5-(anilino)pyridin-2-yl)carbamate I-22

[0230] By (5-amino-6-methylpyridine-2-yl) tert-butyl carbamate (22mg, 0.1mmol) and iodobenzene (24mg, 0.12mmol), (N, N-bipyridyl imidazole subunit) copper dibromide (10mg, 0.023mmol), cesium carbonate (100mg, 0.3mmol) mixed, add 1,4-dioxane (5ml), oil bath 170 ℃ stirring 12 hours. Then add NaOH aqueous solution (10mL, 0.3mol / L) to the mixture. The resulting mixture is extracted with DCM (20mL x 3). The combined organic phase is dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1), and the solvent was distilled off under reduced pressure to obtain tert-butyl (6-(methyl-5-(anilino)pyridin-2-yl)carbamate I-22 (25 mg, 84%). HPLC mass spectrum m / z = 300.2 [M+H] +

[0231] Step 2: 6-Methyl-N 5 -phenylpyridine-2,5-diamine:

[0232] To tert-butyl (6-(methyl-5-(anilino)pyridin-2-yl)carbamate (25 mg, 0.084 mmol) was added a solution of HCl in dioxane (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain 6-methyl-N 5 -phenylpyridine-2,5-diamine A-8 (12 mg, 72%) HPLC mass spectrum m / z = 200.1 [M+H] +

[0233] Example 19: (E)-6-Methyl-5-phenylvinylpyridin-2-amine A-9

[0234]

[0235] The synthesis route is:

[0236]

[0237] Step 1: tert-Butyl (E)-(6-methyl-5-phenylvinylpyridin-2-yl)carbamate I-24

[0238] By (5-amino-6-methylpyridin-2-yl) carbamic acid tert-butyl ester (22mg, 0.1mmol) and styrene (13mg, 0.12mmol), bis (dibenzylideneacetone)-palladium (0) (5mg, 0.0087mmol) mixed, add tert-butyl nitroso (0.5ml), chloroacetic acid (0.5ml), acetic acid (3ml) oil bath 50 ℃ stirring 2 hours. Then add NaOH aqueous solution (10mL, 0.3mol / L) to the mixture. The resulting mixture is extracted with DCM (20mL x 3). The combined organic phase is dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1), and the solvent was distilled off under reduced pressure to obtain tert-butyl (E)-(6-methyl-5-phenylvinylpyridin-2-yl)carbamate I-24 (22 mg, 71%). HPLC mass spectrum m / z = 311.2 [M+H] +

[0239] Step 2: (E)-6-Methyl-5-phenylvinylpyridin-2-amine:

[0240] To tert-butyl (E)-(6-methyl-5-phenylvinylpyridin-2-yl)carbamate (22 mg, 0.071 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain (E)-6-methyl-5-phenylvinylpyridin-2-amine A-9 (13 mg, 88%). Liquid phase mass spectrum m / z = 211.1 [M+H] +

[0241] Example 20: N-(6-amino-2-methylpyridin-3-yl)thiophene-2-sulfonamide A-10

[0242]

[0243] The synthesis route is:

[0244]

[0245] Step 1: tert-Butyl (6-methyl-5-(thiophene-2-sulfonyl)pyridin-2-yl)carbamate I-26

[0246] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (22 mg, 0.1 mmol) and thiophene-2-sulfonyl chloride (22 mg, 0.12 mmol), add triethylamine (0.5 ml) and dichloromethane (3 ml), and stir at room temperature for 24 hours under nitrogen protection. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate it under reduced pressure. The residue is purified by silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent is removed by distillation under reduced pressure to obtain tert-butyl (6-methyl-5-(thiophene-2-sulfonyl)pyridin-2-yl)carbamate I-26 (19 mg, 52%). Liquid phase mass spectrum m / z = 370.1 [M+H] +

[0247] Step 2: N-(6-amino-2-methylpyridin-3-yl)thiophene-2-sulfonamide

[0248] To tert-butyl (6-methyl-5-(thiophene-2-sulfonyl)pyridin-2-yl)carbamate (19 mg, 0.052 mmol), a solution of HCl in dioxane (5%, 5 mL) was added, and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain N-(6-amino-2-methylpyridin-3-yl)thiophene-2-sulfonamide A-10 (10 mg, 72%). Liquid phase mass spectrum m / z = 270.0 [M+H] +

[0249] Example 21: N-(2-((6-amino-2-methylpyridin-3-yl)amino)ethyl)methanesulfonamide A-11

[0250]

[0251] The synthesis route is:

[0252]

[0253] Step 1: tert-Butyl (5-((2-aminoethyl)amino)-6-methylpyridin-2-yl)carbamate I-28

[0254] Mix tert-butyl (5-amino-6-methylpyridin-2-yl)carbamate (44 mg, 0.2 mmol) and 2-bromoethane-1-amine (29 mg, 0.24 mmol), add water (5 ml), and stir in an oil bath at 95°C for 18 hours. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it over sodium sulfate, and concentrate under reduced pressure. The mixture is purified by flash chromatography on silica gel (CH2Cl2 methanol-ammonia water) to obtain the desired product. The solvent is removed by distillation under reduced pressure to obtain tert-butyl (5-((2-aminoethyl)amino)-6-methylpyridin-2-yl)carbamate I-28 (38 mg, 72%). Liquid phase mass spectrum m / z=267..2 [M+H]+

[0255] Step 2: Tert-butyl (6-methyl-5-((2-(methylsulfonylamino)ethyl)amino)pyridin-2-yl)carbamate I-30

[0256] Mix tert-butyl (5-((2-aminoethyl)amino)-6-methylpyridin-2-yl)carbamate (38 mg, 0.144 mmol) and methanesulfonyl chloride (28 mg, 0.16 mmol), add dichloromethane (5 ml), and stir in an ice-water bath for 24 hours. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it over sodium sulfate, and concentrate it under reduced pressure. The residue is purified by silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent is removed by distillation under reduced pressure to obtain tert-butyl (6-methyl-5-((2-(methylsulfonylamino)ethyl)amino)pyridin-2-yl)carbamate I-30 (20 mg, 40%), liquid phase mass spectrum m / z = 255.1 [M+H] +

[0257] Step 3: N-(2-((6-amino-2-methylpyridin-3-yl)amino)ethyl)methanesulfonamide

[0258] To tert-butyl (6-methyl-5-((2-(methylsulfonylamino)ethyl)amino)pyridin-2-yl)carbamate (20 mg, 0.057 mmol) was added a solution of HCl in dioxane (5%, 5 mL), and the mixture was heated and stirred at 70°C for 3 h. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent was removed by distillation under reduced pressure to obtain N-(2-((6-amino-2-methylpyridin-3-yl)amino)ethyl)methanesulfonamide A-11 (11 mg, 78%). Liquid phase mass spectrum m / z = 245.1 [M+H] +

[0259] Example 22: 3-((1H-benzo[d]imidazol-6-yl)oxymethyl)benzonitrile B-1-1

[0260]

[0261] Steps: 1H-benzo[d]imidazol-6-ol II-3 (9 mg, 0.07 mmol) and 3-(bromomethyl)benzonitrile (20 mg, 0.1 mmol) were mixed with K2CO3 (138 mg, 1 mmol), acetonitrile (2 ml) was added, and the mixture was heated to 40°C and stirred for 10 minutes. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol = 40: 1) to give 3-((1H-benzo[d]imidazol-6-yl)oxymethyl)benzonitrile B-1-1 (10 mg, 57%). Liquid phase mass spectrum m / z = 249.3 [m+H] +

[0262] Example 23: 6-Phenoxy-1H-benzo[d]imidazole B-1-2

[0263]

[0264] Steps: 1H-benzo[d]imidazole-6-ol II-3 (9 mg, 0.07 mmol) was mixed with (2-bromoethyl)benzene (22 mg, 0.12 mmol) and K2CO3 (138 mg, 1 mmol), acetonitrile (2 ml) was added, and the mixture was heated to 40°C and stirred for 10 minutes. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol = 40: 1) to give 6-phenoxy-1H-benzo[d]imidazole B-1-2 (12 mg, 70%). Liquid phase mass spectrum m / z = 238.1 [m+H] +

[0265] Example 24: 2-(1H-Benzo[d]imidazol-6-yl)isoindol-1-one B-2

[0266]

[0267] Its synthetic route:

[0268]

[0269] Steps: 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) was mixed with 2-formylbenzoic acid (18 mg, 0.12 mmol), formic acid (0.2 ml), triethylamine (1 ml), and ethanol (1 ml) were added, and the mixture was heated to 80°C and stirred for 60 minutes. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: ethyl acetate = 20:1) to obtain 2-(1H-benzo[d]imidazol-6-yl)isoindol-1-one B-2 (13 mg, 54%). HPLC mass spectrum m / z = 249.1 [m+H] +

[0270] Example 25: N-(1H-benzo[d]imidazol-6-yl)thiophene-3-carboxamide B-3-1

[0271]

[0272] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) and thiophene-2-carboxylic acid (15 mg, 0.12 mmol) were mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain N-(1H-benzo[d]imidazole-6-yl)thiophene-3-carboxamide B-3-1 (9 mg, 37%). LC-MS m / z = 243.1 [m+H] +

[0273] Example 26: (E)-N-(1H-benzo[d]imidazol-6-yl)-2-methyl-2-enamide B-3-2

[0274]

[0275] Steps: 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) and (E)-2-methyl-2-enoic acid (12 mg, 0.12 mmol) were mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain (E)-N-(1H-benzo[d]imidazol-6-yl)-2-methyl-2-enoic acid B-3-2 (10 mg, 46%). LC-MS m / z = 215.1 [m+H]+

[0276] Example 27: N-(1H-benzo[d]imidazol-6-yl)-2,3-dihydrobenzofuran-2-carboxamide B-3-3

[0277]

[0278] Steps: 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) was mixed with benzofuran-2-carboxylic acid (19 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain N-(1H-benzo[d]imidazol-6-yl)-2,3-dihydrobenzofuran-2-carboxamide B-3-3 (12 mg, 41%). LC mass spectrum m / z = 279.1 [m+H] +

[0279] Example 28: N-(1H-benzo[d]imidazol-6-yl)-3,4-dichlorobenzamide B-3-4

[0280]

[0281] Steps: Mix 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) with 3,4-dichlorobenzoic acid (23 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), add N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml), and stir at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain N-(1H-benzo[d]imidazol-6-yl)-3,4-dichlorobenzamide B-3-4 (12 mg, 39%). HPLC mass spectrum m / z = 305.0 [m+H] +

[0282] Example 29: N-(1H-benzo[d]imidazol-6-yl)benzo[b]thiophene-2-carboxamide B-3-5

[0283]

[0284] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with benzothiophene-2-carboxylic acid (21 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain N-(1H-benzo[d]imidazole-6-yl)benzo[b]thiophene-2-carboxamide B-3-5 (13 mg, 43%). LC-MS m / z = 293.1 [m+H]+

[0285] Example 30: N-(1H-benzo[d]imidazol-6-yl)-2-phenylacetamide B-3-6

[0286]

[0287] Steps: 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) was mixed with 2-phenylacetic acid (16 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to obtain N-(1H-benzo[d]imidazol-6-yl)-2-phenylacetamide B-3-6 (11 mg, 43%). LC mass spectrum m / z = 251.11 [m+H] +

[0288] Example 31: 1-(1H-benzo[d]imidazol-6-yl)-3-(p-tolyl)urea B-4-1

[0289]

[0290] Step 1: p-Nitrophenyl p-toluene aminocarbonate II-9-1

[0291] p-Toluidine (21 mg, 0.2 mmol) was mixed with 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol), triethanolamine (0.2 ml), dichloromethane (1 ml), tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: hexane = 5: 1) to obtain p-nitrophenyl p-toluene aminocarbonate II-9-1 (42 mg, 77%). Liquid phase mass spectrum m / z = 273.1 [m + H] +

[0292] Step 2: 1-(1H-benzo[d]imidazol-6-yl)-3-(p-tolyl)urea

[0293] p-Nitrophenyl p-toluene carbamate (27 mg, 0.1 mmol) was mixed with 1H-benzo [d] imidazole-6-amine (16 mg, 0.12 mmol) and K2CO3 (0.2 g, 1.45 mmol), acetonitrile (3 ml) was added and heated to 40 ° C and stirred for 4 hours. After the reaction was completed (monitored by TLC), the reaction mixture was separated and concentrated in a vacuum. The crude reaction mixture thus obtained was washed with dichloromethane, then with EtOAc, and finally with MeOH (1 mL each). Finally, the reaction product was recrystallized using EtOAc (under warm conditions) to obtain pure 1- (1H-benzo [d] imidazole-6-yl) -3- (p-tolyl) urea B-4-1 (20 mg, 75%) liquid phase mass spectrum m / z = 266.1 [m + H] +

[0294] Example 32: 1-(1H-benzo[d]imidazol-6-yl)-3-(3-bromophenyl)urea B-4-2

[0295]

[0296] Step 1: 4-nitrophenyl (3-bromophenyl) aminocarbonate II-9-2

[0297] 3-Bromoaniline (34 mg, 0.2 mmol) was mixed with 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol), triethanolamine (0.2 ml), dichloromethane (1 ml), tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: hexane = 5: 1) to obtain 4-nitrophenyl (3-bromophenyl) aminocarbonate II-9-2 (46 mg, 68%). Liquid phase mass spectrum m / z = 338.1 [m + H] +

[0298] Step 2: 1-(1H-benzo[d]imidazol-6-yl)-3-(3-bromophenyl)urea

[0299] 4-nitrophenyl (3-bromophenyl) aminocarbonate (34mg, 0.1mmol) and 1H-benzo [d] imidazoles -6-amine (16mg, 0.12mmol) and K2CO3 (0.2g, 1.45mmol) are mixed, acetonitrile (3ml) is added and heated to 40 ℃ and stirred for 4 hours. After the reaction is completed (monitored by TLC), the reaction mixture is separated and concentrated in a vacuum. The crude reaction mixture obtained in this way is washed with dichloromethane, then washed with EtOAc, and finally washed with MeOH (each 1mL). Finally, EtOAc (under warm conditions) is used to recrystallize the reaction product to obtain pure form of 1- (1H-benzo [d] imidazoles -6- bases) -3- (3-bromophenyl) urea B-4-2 (22mg, 68%) liquid phase mass spectrum m / z=330.0 [m+H] +

[0300] Example 33: N-methyl-N-phenyl-1H-benzo[d]imidazole-6-carboxamide B-5

[0301]

[0302] Its synthetic route is:

[0303]

[0304] Step 1: 6-Nitro-1H-benzo[d]imidazole II-10

[0305] To a solution of H2O2 (240 mg, 2.2 mmol) in fuming sulfuric acid (500 μL) was added dropwise a solution of 1h-benzo[d]imidazole-6-amine (40 mg, 0.3 mmol) in concentrated sulfuric acid (100 μL), keeping the reaction temperature at 0°C. After stirring at 10-25°C for 3 h, the reaction mixture was brought to pH = 11-12 by adding 40% aqueous NaOH at 0-5°C. The resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over Na2SO4 and filtered. The solvent was removed by distillation under reduced pressure to give the desired 6-nitro-1H-benzo[d]imidazole IⅠ-10 (35 mg, 72%) LC-mS m / z = 163.0 [m+H] +

[0306] Step 2: 1H-Benzo[d]imidazole-6-carboxylic acid II-11

[0307] 6-Nitro-1H-benzo[d]imidazole (39 mg, 0.24 mmol) was mixed with KCN (195 mg, 3 mmol), 1-butylimidazole tetrafluoroborate (3 mg, 0.014 mmol), EtOH (2 ml), and water (2 ml) were heated to 80°C and stirred for 19 hours. Then 10 ml of water was added and the mixture was extracted with CH2Cl2 (3×5 ml) and ether (3×10 ml). It was acidified to pH 1-2 with hydrochloric acid and extracted with ether (3×10 ml). Magnesium sulfate (3 g) and activated carbon (1 g) were added and stirred for 5 h. The solid was filtered off, the filtrate was evaporated, and the residue was crystallized from the corresponding solvent to obtain the desired 1H-benzo[d]imidazole-6-carboxylic acid II-11 (14 mg, 40%) LC-mS m / z=162.0[m+H]+

[0308] Step 3: N-methyl-N-phenyl-1H-benzo[d]imidazole-6-carboxamide

[0309] 1H-Benzo[d]imidazole-6-carboxylic acid (16 mg, 0.1 mmol) was mixed with N-methylaniline (13 mg, 0.12 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol), and N,N-diisopropylethylamine (0.2 ml) and N,N-dimethylformamide (2 ml) were added, and stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent was distilled off under reduced pressure to obtain N-methyl-N-phenyl-1H-benzo[d]imidazole-6-carboxamide B-5 (16 mg, 65%). Liquid phase mass spectrum m / z = 251.1 [m+H] +

[0310] Example 34: N-(Tetrahydro-2H-pyran-4-yl)-1H-benzo[d]imidazol-6-amine B-6-1

[0311]

[0312] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with tetrahydro-4H-pyran-4-one (12 mg, 0.12 mmol), methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, and then sodium cyanoborohydride (20 mg, 0.3 mmol) was added and stirred at room temperature for 2 hours. Then, an aqueous NaOH solution (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent was removed by distillation under reduced pressure to obtain N-(tetrahydro-2H-pyran-4-yl)-1H-benzo[d]imidazole-6-amine B-6-1 (16 mg, 75%). Liquid phase mass spectrum m / z = 217.1 [m+H] +

[0313] Example 35: N-(pyridine-4-methyl)-1H-benzo[d]imidazol-6-amine B-6-2

[0314]

[0315] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with isonicotinaldehyde (13 mg, 0.12 mmol), methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, and then sodium cyanoborohydride (20 mg, 0.3 mmol) was added and stirred at room temperature for 2 hours. Then, an aqueous NaOH solution (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent was removed by distillation under reduced pressure to obtain N-(pyridine-4-methyl)-1H-benzo[d]imidazole-6-amine B-6-2 (14 mg, 64%). Liquid phase mass spectrum m / z = 224.1 [m+H] +

[0316] Example 36: n-Cyclobutyl-1H-benzo[d]imidazol-6-amine B-6-3

[0317]

[0318] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with cyclobutanone (8 mg, 0.12 mmol), methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, and then sodium cyanoborohydride (20 mg, 0.3 mmol) was added and stirred at room temperature for 2 hours. Then, an aqueous solution of NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1), and the solvent was removed by distillation under reduced pressure to obtain n-cyclobutyl-1H-benzo[d]imidazole-6-amine B-6-3 (13 mg, 72%). Liquid phase mass spectrum m / z = 187.1 [m+H] +

[0319] Example 37: 2-(1H-benzo[d]imidazol-6-yl)isoindoline-1,3-dione B-7

[0320]

[0321] Its synthetic route:

[0322]

[0323] Procedure: 1H-benzo[d]imidazol-6-amine (13 mg, 0.1 mmol) and isobenzofuran-1,3-dione (18 mg, 0.12 mmol) were mixed, N,N-dimethylacetamide (2 ml) was added, stirred at room temperature for 24 hours, and then xylene (1 ml) was added and stirred in an oil bath at 140°C for 48 hours. After completion (monitored by TLC), the insoluble catalyst was separated by filtration, washed with acetone and dried. The organic layer was concentrated under reduced pressure to obtain the desired product, washed with water, and recrystallized from ethanol. The crude product was purified by silica gel column chromatography (CH2Cl2 / n-hexane=1:1), and the solvent was removed by distillation under reduced pressure to obtain 2-(1H-benzo[d]imidazol-6-yl)isoindoline-1,3-dione B-7 (18 mg, 68%). Liquid phase mass spectrum m / z=263.1[m+H]+

[0324] Example 38: N-phenyl-1H-benzo[d]imidazol-6-amine B-8

[0325]

[0326] Its synthetic route:

[0327]

[0328] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with iodobenzene (24 mg, 0.12 mmol), (N,N-bipyridyl imidazole) copper dibromide (10 mg, 0.023 mmol), and cesium carbonate (100 mg, 0.3 mmol), 1,4-dioxane (5 ml) was added, and the mixture was stirred at 170 ° C in an oil bath for 12 hours. Then, an aqueous solution of NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mLx3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1), and the solvent was removed by distillation under reduced pressure to obtain N-phenyl-1H-benzo[d]imidazole-6-amine B-8 (17 mg, 82%). Liquid phase mass spectrum m / z = 209.1 [m+H] +

[0329] Example 39: (E)-6-phenylvinyl-1H-benzo[d]imidazole B-9

[0330]

[0331] Its synthetic route:

[0332]

[0333] Steps: 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) was mixed with styrene (13 mg, 0.12 mmol), bis(dibenzylideneacetone)-palladium(0) (5 mg, 0.0087 mmol), tert-butyl nitrosate (0.5 ml), chloroacetic acid (0.5 ml), acetic acid (3 ml) were added and stirred in an oil bath at 50°C for 2 hours. Then, an aqueous solution of NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1), and the solvent was removed by distillation under reduced pressure to obtain (E)-6-phenylvinyl-1H-benzo[d]imidazole B-9 (13 mg, 61%). Liquid phase mass spectrum m / z = 220.1 [m+H] +

[0334] Example 40: N-(1H-benzo[d]imidazol-6-yl)thiophene-2-sulfonamide B-10

[0335]

[0336] Its synthesis route:

[0337]

[0338] Steps: Mix 1H-benzo[d]imidazole-6-amine (13 mg, 0.1 mmol) and thiophene-2-sulfonyl chloride (22 mg, 0.12 mmol), add triethylamine (0.5 ml) and dichloromethane (3 ml), and stir at room temperature for 24 hours under nitrogen protection. Add ethyl acetate and water to the reaction mixture, separate the organic layer, dry it with sodium sulfate, and concentrate it under reduced pressure. The residue is purified by silica gel column chromatography (ethyl acetate: hexane = 3: 7), and the solvent is removed by distillation under reduced pressure to obtain N-(1H-benzo[d]imidazole-6-yl)thiophene-2-sulfonamide B-10 (13 mg, 45%). Liquid phase mass spectrum m / z = 279.0 [m+H] +

[0339] Example 41: N-(2-(1h-benzo[d]imidazol-6-yl)aminoethyl)methylsulfonamide B-11

[0340]

[0341] Its synthesis route:

[0342]

[0343] Step 1: N1-(1H-benzo[d]imidazol-6-yl)ethane-1,2-diamine ⅠI-19

[0344] 1H-Benzo[d]imidazol-6-amine (27 mg, 0.2 mmol) was mixed with 2-bromoethane-1-amine (29 mg, 0.24 mmol), water (5 ml) was added, and the mixture was stirred in an oil bath at 95°C for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The mixture was purified by flash chromatography on silica gel (CH2Cl2 methanol-ammonia water) to obtain the desired product. The solvent was removed by distillation under reduced pressure to obtain N1-(1H-benzo[d]imidazol-6-yl)ethane-1,2-diamine ⅠI-19 (22 mg, 61%). Liquid phase mass spectrum m / z = 176.1 [m+H] +

[0345] Step 2: N-(2-(1h-benzo[d]imidazol-6-yl)aminoethyl)methanesulfonamide

[0346] N1-(1H-benzo[d]imidazol-6-yl)ethane-1,2-diamine (23 mg, 0.13 mmol) was mixed with methanesulfonyl chloride (28 mg, 0.16 mmol), and dichloromethane (5 ml) was added. The mixture was stirred in an ice-water bath for 24 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7), and the solvent was removed by distillation under reduced pressure to obtain N-(2-(1H-benzo[d]imidazol-6-yl)aminoethyl)methylsulfonamide B-11 (11 mg, 32%), liquid phase mass spectrum m / z = 254.1 [m+H] +

[0347] Small molecule binding energy prediction:

[0348] The aminopyridine derivative molecules in Examples 1-21 and the benzimidazole derivative molecules in Examples 22-41 were molecularly docked with the OCT4 target protein using AutoDock Vina and LeDock software, respectively, to produce 10 docking conformations. The binding energy and ligand efficiency of the optimal docking results of each aminopyridine derivative molecule and each benzimidazole derivative molecule with the OCT4 target protein were calculated, and the aminopyridine derivative molecules and benzimidazole derivative molecules were screened based on the comprehensive docking results. The specific docking results are shown in Table 1 and Table 2, respectively: The second and third columns of the table are the binding free energy (binding energy) calculated by the autodock vina and LeDock molecular docking software, respectively. The more negative the value, the stronger the binding ability of the small molecule ligand to the target protein. The fourth column of the table represents the ligand efficiency (ligand efficiency) calculated by LeDock, and the larger the absolute value, the stronger the potential of the small molecule activity. In the present invention, the binding energy level of the compound of the present invention is predicted according to the independent algorithms of the two softwares, and the predicted value shows that the binding energy of the compound in the present invention is much greater than the threshold value 3 set according to the target feature of the present invention.

[0349] Table 1. Prediction of binding of aminopyridine derivative molecules used in the present invention to target proteins

[0350]

[0351] Table 2. Prediction of the binding energy of the benzimidazole derivative molecules used in the present invention and the target sequence

[0352]

[0353]

[0354] Verification of transcriptional expression differences caused by small molecules:

[0355] The purpose of the present invention is to use a highly selective activator to achieve the effect of activating the target gene. The important function of such an activator is to activate the expression of Oct4, thereby increasing the expression abundance of Oct4 on downstream genes. Therefore, when verifying the function of the small molecule of the present invention, in addition to verifying the increase in the Oct4 gene itself, the increase in the expression of Nanog, a downstream gene of the Oct4 gene, is also an indicator of functional verification of the compound in the present invention.

[0356] Human mesenchymal cells were cultured in T25 medium at 4 × 10 5 The cells were inoculated and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM-F12 medium), to which 50 nM of the above-mentioned aminopyridine derivative small molecules and benzimidazole derivative small molecules were added, respectively, and the culture conditions were 37°C and 5% carbon dioxide. On the 5th day, total RNA was extracted using RNeasy Mini or MicroKit (QIAGEN), and 1 mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler DiceReal Time System (TaKaRa) were used for Quantitative PCR labeling and reaction, and beta-Actin was used as an internal reference. All data were analyzed using the delta-Ct method. Each group of experiments was repeated in three groups, and variance statistics were performed. The primer sequences used to identify the encoding genes of different cell markers are shown in Table 3. The results are shown in Figure 1 and Figure 2 As shown, compared with the control group without small molecules, the above aminopyridine derivative small molecules and benzimidazole derivative small molecules significantly increased the basal expression of OCT4 and the expression of its downstream gene Nanog.

[0357] Table 3. QPCR primer sequences for compound-effect genes

[0358]

[0359]

[0360] Verification of early cell pluripotency induced by small molecules:

[0361] The Rex1 gene is highly expressed in undifferentiated embryonic stem cells, and is closest to the natural state of embryonic stem cells and pre-implantation blastocysts in the in vitro culture of pluripotent stem cells (Boroviak, T et al., Nat. Cell Biol. 2014.16, 516-528; Kalkan, T et al., Development. 2017, 144, 1221-1234). The expression of the Rex1 gene shows that the cells are in a natural pluripotency state similar to embryonic stem cells or pre-implantation blastocysts. Therefore, the present invention uses the expression of Rex1 as a terminal detection indicator of small molecule function.

[0362] Human mesenchymal cells were cultured in T25 medium at 4 × 10 5 The cells were inoculated and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM-F12 medium), to which 50 nM of the above-mentioned aminopyridine derivative small molecules and benzimidazole derivative small molecules were added respectively, and the culture conditions were 37° C. and 5% carbon dioxide. Immunofluorescence staining was performed on the 5th day: cells were fixed with 4% paraformaldehyde at room temperature for 40 minutes, washed twice with DPBS buffer; then permeabilized with 0.1% Triton X-100 for 5 minutes, washed twice with DPBS buffer; then incubated overnight at 4°C with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then anti-Rex1 primary antibody (Abcam, ab28141) diluted 1:200 in DPBS buffer containing 2% horse serum and 0.1% Triton X-100 was added, incubated at 37°C for 2 hours, washed three times with DPBS buffer, and then goat anti-rabbit IgG (H+L) diluted 1:1000 in DPBS buffer containing 2% horse serum and 0.1% Triton X-100 was added, and Alexa Fluor488 (Invitrogen A10037) was taken for photography. Figure 3 As shown, the chemical small molecules of the present invention can promote mesenchymal cells to express the Rex1 gene and allow the mesenchymal cells to form pluripotent stem cell clone-like cell clusters. Sequence Listing <110> Wuhan Ruijian Pharmaceutical Technology Co., Ltd. <120> Highly selective activator of OCT4 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccatgcattc aaactgaggt 20 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 cctttgtgtt cccaattcct t 21 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 acctcagcta caaacaggtg aa 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 aaaggctgggtaggtaggt 20 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <400> 5 ggccgaggac tttgattgca ca 22 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 gggcacgaag gctcatcatt caa 23

Claims

1. Compounds of formula (II) and pharmaceutically acceptable salts thereof: in: When m1 is 0 and m2 is 1, A2 is -N(CH3)-, A3 is When m1 is 1 and m2 is 0, A2 is -CH2-, -SO2-, -(CH2)2NHS(O)2- or a bond, A3 is -CH3, When m1 is 1 and m2 is 1, A2 is -NH or a bond, A3 is -C(CH3)=CH-CH3, When m1 is 0 and m2 is 0, A2 is -O(CH2)-, -O(CH2)2- or a bond, A3 is 2. The compound according to claim 1 and its pharmaceutically acceptable salt, wherein the compound is selected from:

3. A compound and a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 and / or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

5. Use of the compound according to any one of claims 1 to 3 and / or a pharmaceutically acceptable salt thereof in the preparation of a medicament for a highly selective activator of OCT4 of induced pluripotent stem cells.

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