CN115572285A - OCT4 high selectivity activator - Google Patents

OCT4 high selectivity activator Download PDF

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CN115572285A
CN115572285A CN202110686564.5A CN202110686564A CN115572285A CN 115572285 A CN115572285 A CN 115572285A CN 202110686564 A CN202110686564 A CN 202110686564A CN 115572285 A CN115572285 A CN 115572285A
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魏君
蔡萌
云轩
张利达
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Wuhan Iregene Pharmaceutical Technology Co ltd
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Abstract

The present invention relates to highly selective activators useful for OCT4 and downstream gene expression, pharmaceutical compositions thereof, methods of preparing the same, said activators having the formula:
Figure DDA0003124934450000011
wherein, m1, m2 and A 2 、A 3 Described herein.

Description

OCT4 high selectivity activator
Technical Field
The invention relates to the field of medicines, in particular to an OCT4 high-selectivity activator, a pharmaceutical composition and a preparation method thereof, and application of the OCT4 high-selectivity activator in regulation of Oct4 and related genes thereof.
Background
Regenerative medicine refers to an emerging science that utilizes a variety of novel technical disciplines to reconstruct tissues and organs that are aged or functionally lost, and to treat related diseases through a variety of medical means. Important research directions of regenerative medicine are mechanisms of normal tissue characteristics and functions, biological bases of post-traumatic repair, regeneration mechanisms of tissues and organs and differentiation mechanisms of various stem cells, so that an effective biological treatment method is finally obtained. Among them, embryonic stem cells (ESCs, abbreviated as ES, EK or ESC cells) are the most interesting cell type in early regenerative medicine research. However, the availability and use of this cell is highly ethical because the study of embryonic stem cells must be carried out to destroy the embryo, which is the life form of a human in the uterus when it is not yet formed. This ethical debate has greatly hindered the advancement and application of regenerative medicine.
In 2006, the mountain-crossing team developed a "cocktail" method consisting of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which successfully reprogrammed terminally differentiated dermal fibroblasts into stem cells with differentiation pluripotency, known as 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 a differentiation potential similar to that of embryonic stem cells (embryonic stem cells), and are capable of forming the three most basic germ layers of human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. The invention breaks through the ethical limit of using human embryonic stem cells in medicine, and greatly expands the application potential of stem cell technology in clinical medicine.
In studies of induced pluripotent and embryonic stem cells, oct4 has been shown to be a major regulator of reprogramming and induced 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, with alternative splicing leading to multiple transcript variants. Oct4 encodes a protein belonging to the POU domain family of transcription factors, located in Chromosome 17, 35,825,200-35,829,401. A 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 highly conserved 75 amino acids and a 60 amino acid carboxy-terminal homology domain (POUh). Oct4 expression is regulated at the transcriptional level by cis-acting elements upstream of the Oct4 gene and by methylation of chromatin structure (Klemm JD, et al, cell,1994, 77, 21-32, brehm A, et al, mol Cell Biol 1997. Yeom et al identified two elements by analyzing expression of the LacZ reporter under the control of the 18Kb fragment from the Oct4 genomic locus, they named it as the Proximal Enhancer (PE) and the Distal Enhancer (DE) that may need to be regulated, among which they identified the precise binding site for transcription factors (Yeom Y, et al, integrated Ann Indexes 122. POU domain transcription factors bind to specific octameric DNA and modulate cell type specific differentiation pathways. Wherein, during the formation of iPSC, POU domain-containing Oct4 and HMG domain-containing Sox2 are transcription factors essential for maintaining pluripotency of pluripotent cells (Nichols, J., et al., cell,1998, 95,379-391 Avilion, A., et al.,2003, genes Dev.17, 126-140), and their function in pluripotent cells, at least in part, is to drive transcription of target genes through synergistic interaction between the two (Tomioka, M., et al., nucleic Acids Res.2002;30, 3202-3213). These findings suggest that developmental transitions can be controlled by Oct 4. However, many reprogramming methods currently in wide use overexpress Oct4 by 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 present potential clinical risks in the clinical use of induced pluripotent stem cells (ipscs), such as potential tumorigenic risks due to the use of viral vectors; in addition, the complex GMP production process of the carrier also brings complexity to clinical supervision of induced pluripotent stem cells, and further, the cost of the product is high due to the use of the carrier.
Based on the reasons, the benzimidazole derivative and the aminopyridine derivative are designed and used in the invention, and the expression regulation of the downstream gene is realized through the chemical regulation of an Oct4 promoter. Thereby avoiding the regulation and control of Oct4 by using viruses or other vectors and further realizing the safe and simple chemical micromolecule starting biological expression function.
Disclosure of Invention
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 high selective activator of OCT4 for reprogramming of cells.
The present invention provides a compound of the structure of formula (I):
Figure BDA0003124934430000021
wherein:
A 1 is that
Figure BDA0003124934430000022
m1 and m2 are each 0 or 1;
A 2 is C1-C6 alkylene, C2-C6 alkenylene, -O (CH) 2 )q-、-NR 1 -、-SO 2 -、-(CH 2 ) V NHS(O) 2 -or a bond, wherein q is 1 or 2 or 3 or 4, v is 0 or 1 or 2, R1 is selected from H or C1-C4 alkyl;
A 3 is C1-C6 alkyl; C2-C6 alkenyl; C4-C6 cycloalkyl, wherein one carbon atom may be substituted by a N, O, S heteroatom;
Figure BDA0003124934430000031
z and Z 1 Are each N or CR 2 R2 is selected from H, halogen, C1-C4 alkyl or cyano;
Figure BDA0003124934430000032
Z 3 is N, O, S or C = O, when Z is 4 And Z 5 When the bond therebetween is a single bond, Z 4 Is N or CH, Z 5 Is CH 2 Or C = O, when Z 4 And Z 5 When the bond between them is a double bond, Z 4 Is C, Z 5 Is CH;
and pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof.
In certain embodiments, the presently disclosed compounds have formula (II) or formula (III):
Figure BDA0003124934430000033
wherein:
A 2 is-CH 2 -、-CH＝CH-、-C(CH 3 )＝CH-、-O(CH 2 )-、-O(CH 2 ) 2 -、-NH-、-N(CH 3 )-、-NHS(O) 2 -、-(CH 2 ) 2 NHS(O) 2 -or absent.
A 3 is-CH 3 Butenyl, butenyl,
Figure BDA0003124934430000034
Figure BDA0003124934430000035
Figure BDA0003124934430000041
In some embodiments: m1 is 0, m2 is 1;
A 2 is-N (CH) 3 )-；
A 3 Is that
Figure BDA0003124934430000042
In some embodiments: m1 is 1, m2 is 0;
A 2 is-CH 2 -、-SO 2 -、-(CH 2 ) 2 NHS(O) 2 -or a bond;
A 3 is-CH 3 、
Figure BDA0003124934430000043
In some embodiments: m1 is 1, m2 is 1;
A 2 is-CH 2 -、-NH-、-C(CH 3 ) = CH-or a bond;
A 3 is-CH 3 、-C(CH 3 )＝CH-CH 3 、
Figure BDA0003124934430000044
Figure BDA0003124934430000045
In some embodiments: m1 is 0, m2 is 0;
A 2 is-CH 2 -、-CH＝CH-、-O(CH 2 )-、-O(CH 2 ) 2 -or a bond;
A 3 is that
Figure BDA0003124934430000051
In some embodiments, the compound is:
Figure BDA0003124934430000052
the present invention relates to pharmaceutical compositions comprising a compound of any of the above, and pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs, or combinations thereof, and at least one pharmaceutically acceptable carrier or excipient.
The present invention relates to the use of a compound of any of the above and/or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, ester, optical isomer, prodrug or combination thereof for the manufacture of a medicament for the induction of a pluripotent stem cell OCT4 highly selective activator.
The invention relates to the use of an OCT4 high selectivity activator for inducing pluripotent stem cells in the preparation of a medicament for the treatment of diseases induced by pluripotent stem cells, including 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, edentulous, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, crohn's disease, and genetic diseases and other similar diseases.
The present invention relates to a method of obtaining induced pluripotent stem cells in a subject with a disease, the method comprising administering to the subject a therapeutically effective amount of a compound of any of the above and/or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, ester, optical isomer, prodrug or combination thereof.
In the method, the subject having the disease is a human having 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, edentulous, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, crohn's disease, and genetic and other similar diseases.
The present invention relates to a method of activating OCT4 function comprising contacting a compound of any of the above and/or pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs, or combinations thereof, with an OCT4 target protein.
Detailed Description
In the present invention, the following definitions are applicable:
the term "alkyl" herein refers to straight or branched chain saturated hydrocarbons containing from 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.
The term "alkenyl" refers to straight or branched chain unsaturated hydrocarbons containing 2 to 12 carbon atoms, containing at least one C = C double bond in the chain. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl or hexenyl.
The term "alkylene": refers to a divalent alkyl group. Any of the monovalent alkyl groups can be an alkylene group by abstraction of a second hydrogen atom from the alkyl group. Alkylene groups, as defined herein, may also be C1-C6 alkylene groups. The alkylene group may further be a C1-C4 alkylene group. Typical alkylene groups include, but are not limited to: -CH 2 -、-CH(CH 3 )-、-C(CH 3 ) 2 -、-CH 2 CH 2 -、-CH 2 CH(CH 3 )-、-CH2C(CH 3 ) 2 -、-CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 -and the like.
The term "alkenylene": refers to a divalent alkenyl group. Any of the monovalent alkenyl groups may be alkenylene by abstraction of a second hydrogen atom from the alkenyl group. Alkenylene may further be C2-C6 alkenylene, as defined herein. Typical alkenylene groups include, but are not limited to: -CH = CH-, -CH = C (CH) 3 )-、-CH＝CHCH 2 -、-CH＝CHCH 2 CH 2 -、-CH＝CHCH 2 CH 2 CH 2 -、-CH＝CHCH 2 CH 2 CH 2 CH 2 -and the like.
The term "cycloalkyl" refers to a monocyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. Cycloalkyl groups may further be C4-C6 cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "cyano" refers to a substituent having a carbon atom attached to a nitrogen atom through a triple bond (i.e., C ≡ N).
The term "substituted," as used herein, means that any one or more hydrogen atoms on the designated atom or group is replaced with a group selected from the designated ranges, provided that the designated atom's normal valence is not exceeded.
Compounds described herein include, but are not limited to: their optical isomers, racemates and other mixtures. In these cases, the individual enantiomers or diastereomers, i.e., optically active configurations, can be obtained by asymmetric synthesis or by resolution of the racemates or diastereomeric mixtures. Resolution of the racemates or diastereomeric mixtures may be accomplished by conventional methods, such as crystallization in the presence of a resolving agent or chromatography, e.g., using a chiral High Pressure Liquid Chromatography (HPLC) column. Furthermore, these compounds include compounds having chiral centers in the R-and S-configurations. These compounds also include crystalline forms, including polymorphs and clathrates. Similarly, the term "salt" also includes all isomers, racemates, other mixtures, R-and S-configurations, tautomers and crystal forms of the salts of the compounds.
"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 to be treated. See generally: s.m. berge, 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 contacting the tissues of a patient without undue toxicity, irritation or allergic response. The compounds of formula (I), formula (II) or formula (III) may have sufficient acidic groups, sufficient basic groups or both types of functional groups and react with some inorganic or organic bases, and inorganic and organic acids, respectively, to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ -hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.
"solvates" such as "hydrates" are formed by the interaction of a solvent with a compound. The term "compound" includes solvates, including hydrates, of the compound. Likewise, "salt" includes solvates of the salt, such as hydrates. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including the monohydrate and the hemihydrate.
"prodrug" may refer to a precursor of a given compound that, upon administration to a subject, yields the compound in vivo via a chemical or physiological process (e.g., solvolysis, enzymatic cleavage) or under physiological conditions (e.g., a prodrug is converted to a compound of formula (I) at physiological pH). A "pharmaceutically acceptable prodrug" is a prodrug that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. Exemplary procedures for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of produgs", elsevier,1985, by h.
"active metabolite" refers to a pharmaceutically active product of the metabolism of a compound of formula (I), formula (II) or formula (III) or a salt thereof in the body. Prodrugs and active metabolites of a compound can be determined by conventional techniques known or available in the art. See, e.g., 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 produgs (ElsevierPress, 1985); and Larsen, design and Application of precursors, drug Design and development (Krogsgaard-Larsen et al, eds., harwood Academic Publishers, 1991)
A "therapeutically effective amount" refers to an amount of a compound disclosed herein that, when administered to a mammal (preferably a human), is sufficient to effect treatment (as defined below) of a disease or condition in the mammal (preferably a human). The amount of the disclosed compounds that constitutes a "therapeutically effective amount" will vary with the compound, the condition 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.
The term "treating" refers to administering to an individual at least one compound and/or at least one pharmaceutically acceptable salt thereof as described herein to slow down (reduce) the development or spread of an undesired physiological change or disease, such as inflammation or cancer. The beneficial or desired clinical results for purposes of the present invention include, but are not limited to: alleviation of symptoms, reduction of the severity of a disease, stabilization (i.e., not worsening) of the state of a disease, delay or slowing of disease progression, amelioration or palliation of the disease, and remission (whether partial or total), whether detected or undetectable. "treatment" also means that survival can be extended compared to expected survival without treatment. Individuals in need of treatment include individuals with symptoms of or suffering from such diseases.
Pharmaceutical composition
The present invention provides pharmaceutical compositions comprising as active ingredient one or more compounds described herein, or pharmaceutically acceptable salts or esters thereof, together with one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, penetration enhancers, solubilizers, and adjuvants. The pharmaceutical composition may be administered alone or in combination with other therapeutic agents. Such compositions are prepared in a manner well known in the pharmaceutical art.
The pharmaceutical compositions may be administered in single or multiple doses by any acceptable means of administration of agents having similar utility, such as those described in those patents and patent applications incorporated herein by reference, including rectal, buccal, intranasal, and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or by implantation or coating devices such as stents, for example, or arterial insertion of a pillared polymer.
The invention also provides a kit comprising a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
"pharmaceutically acceptable carrier or excipient" refers to a substance that is non-toxic, biologically tolerable and otherwise biologically suitable for administration to a subject, e.g., an inert substance, that is added to a pharmacological composition or that serves as a vehicle, carrier or diluent to facilitate and be compatible with administration of the active ingredient. Examples of excipients include calcium carbonate, calcium phosphate, various types of sugars or starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Use of compounds and compositions thereof
The invention provides a compound and a composition thereof, which are mainly used as OCT4 high-selectivity activators for activating OCT4 functions, realizing expression regulation of genes downstream of the Oct4 promoter through chemical regulation of the Oct4 promoter, further inducing pluripotent stem cells in a subject suffering from diseases and realizing the purpose of treating diseases, wherein the diseases comprise cancer, heart disease, stroke, diabetes, obesity, alzheimer disease, parkinson disease, amyotrophic lateral sclerosis, myocardial infarction, muscular dystrophy, CMT-1A, spinal cord injury, traumatic brain injury, edentulous teeth, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, alopecia, blindness, deafness, crohn's disease, genetic diseases and other similar diseases.
A list of abbreviations used in the following examples and elsewhere herein:
atm: an atmosphere; boc: a tert-butoxycarbonyl group; (Boc) 2 O: di-tert-butyl dicarbonate anhydride; CH (CH) 2 Cl 2 : dichloromethane; cs 2 CO 3 : cesium carbonate; cu (I) Br: copper bromide; cu 2 SO 4 : cuprous sulfate; DCM: dichloromethane; DMAC: n, N-dimethylacetamide; DMF: n, N-dimethylformamide; DIEA: n, N-diisopropylethylamine; DIOX:1,4-Dioxane; a dioxane: dioxane; EA: acetic acid; et (Et) 3 N: triethylamine; ETOH: ethanol; etOAc: ethyl acetate; FA: formic acid; g: g; h: h; h 2 : hydrogen gas; HATU:2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate; HBr: hydrobromic acid; (Hbim) BF4: 1-butylimidazolium tetrafluoroborate; h 2 O: water; HAc: acetic acid; h 2 O 2 : hydrogen peroxide; h 2 SO 4 : fuming sulfuric acid; KCN: potassium cyanide; k is 2 CO 3 : potassium carbonate; li: lithium; MCA: chloroacetic acid; meCN: acetonitrile; meOH: methanol; mg: mg; ml: ml; mmol: millimole; mol: molar ratio; m/z: mass to charge ratio; n is a radical of hydrogen 2 : nitrogen gas; naBH 3 CN: sodium cyanoborohydride; naNO 2 : sodium nitrite; naOH: sodium hydroxide; naOMe: sodium methoxide; na (Na) 2 SO 4 : sodium sulfate; ni (OAc) 2 4H 2 O: nickel diacetate tetrahydrate; pd-C: palladium on carbon; pH: the pH value; TBN: nitroso-tert-butyl ester; TEA: triethanolamine; THF: tetrahydrofuran; TLC: thin layer chromatography; a xylylene group: xylene.
The technical means adopted by the invention to achieve the predetermined purpose are further described below with reference to the drawings and the embodiments of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
General Synthesis
The general synthetic routes described in the present application can be varied by replacing the starting materials with other materials having similar structures, resulting in different products accordingly. The following synthetic scheme description gives a number of examples of how the starting materials may be varied to give the corresponding products.
General procedure A-1-n
Figure BDA0003124934430000091
Wherein R is
Figure BDA0003124934430000092
Wherein q is 1 or 2 or 3 or 4, Z and Z 1 Are each N or CR 2 R2 is selected from H, halogen, C1-C4 alkyl or cyano.
General procedure A-2-n
Figure BDA0003124934430000101
Wherein R is C2-C6 alkenyl;
Figure BDA0003124934430000102
Figure BDA0003124934430000103
wherein q is 1 or 2 or 3 or 4, Z and Z 1 Are each N or CR 2 R2 is selected from H, halogen, C1-C4 alkyl or cyano, R1 is selected from H or C1-C4 alkyl, Z 3 Is N, O, S or C = O.
General procedure A-4-n
Figure BDA0003124934430000104
Wherein R is
Figure BDA0003124934430000105
Wherein q is 1 or 2 or 3 or 4, Z and Z 1 Are each N or CR 2 R2 is selected from H, halogen, C1-C4 alkyl or cyano.
General procedure A-6-n
Figure BDA0003124934430000106
Wherein R is C4-C6 cycloalkyl, wherein one carbon atom may be substituted by a heteroatom N, O, S; or R = O is
Figure BDA0003124934430000107
Wherein q is 1 or 2 or 3 or 4Z and Z 1 Are each N or CR 2 R2 is selected from H, halogen, C1-C4 alkyl or cyano.
General procedure B-1-n
Figure BDA0003124934430000111
Wherein R is as defined for R in general procedure A-1-n.
General procedure B-3-n
Figure BDA0003124934430000112
Wherein R is as defined for R in general procedure A-2-n.
General procedure B-4-n
Figure BDA0003124934430000113
Wherein R is as defined for R in general procedure A-4-n.
General procedure B-6-n
Figure BDA0003124934430000114
Wherein R is as defined for R in general procedure A-6-n.
Intermediate synthesis
Intermediate I-3: (5-amino-6-methylpyridin-2-yl) carbamic acid tert-butyl ester
The synthetic route is as follows:
Figure BDA0003124934430000115
step 1: 6-methyl-5-nitropyridin-2-amine I-1 (15.3g, 0.1mol) and di-tert-butyldicarbonate anhydride (26.2g, 0.12mol) were reacted with K 2 CO 3 (69g, 0.5 mol), tetrahydrofuran (200 ml) was added thereto, and the mixture was stirred at room temperatureStirring for 3 hours. To the reaction mixture were added ethyl acetate and water, the organic layer was separated, washed with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to give tert-butyl (6-methyl-5-nitropyridin-2-yl) carbamate I-2 (23.3 g, 92%) as a liquid phase mass spectrum m/z =254.1[ m + h ])]+
Step 2: tert-butyl (6-methyl-5-nitropyridin-2-yl) carbamate I-2 (12.7 g, 0.05mol) as described above and 10% Pd-C (1 g) and MeOH (100 mL) were stirred under hydrogen atmosphere (1 atm) at room temperature for 2h. The mixture is filtered and the solvent is removed by distillation under reduced pressure to obtain an intermediate (5-amino-6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-3 (11g, 98%) liquid phase mass spectrum m/z =224.1[ M ] +H ] +
Intermediate II-3: 1H-benzo [ d ] imidazol-6-ol
Figure BDA0003124934430000121
The synthetic route is as follows:
Figure BDA0003124934430000122
step 1: intermediate 1H-benzo [ d ] imidazole-6-diazo II-2
1H-benzo [ d ]]To imidazol-6-amine (67mg, 0.5 mmol) was added water (2 ml) and 20% H 2 Aqueous SO4 solution (1 ml). To the mixture were added a solution (0.5 ml) of an aqueous solution (42mg, 0.6 mmol) of sodium nitrite and acetonitrile (2 ml) under ice-cooling, and the mixture was stirred for 30 minutes. Adding Cu in 20% aqueous HBr solution (0.5 ml) at room temperature to the obtained reaction mixture 2 SO4 (67mg, 0.3 mmol) solution, and the mixture was stirred at 80 ℃ 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 give a hydrogen sulfate salt of II-2 (85mg, 70%), liquid phase mass spectrum m/z =145.1[ m ]]+。
Step 2: intermediate 1H-benzo [ d ] imidazole-6-ol II-3
An aqueous solution (1 ml) of the bisulfate salt of 1H-benzo [ d ] imidazole-6-diazonium (48mg, 0.2mmol) was added dropwise to a 40% aqueous solution (5 ml) of sulfuric acid at 100 ℃ and the mixture was stirred for 10 minutes. To the obtained reaction mixture was added NaOH to a PH of about 3, ethyl acetate was added to the reaction mixture, and 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 give II-3 (19mg, 69%), liquid phase mass spectrum m/z =134.1[ 2 ], [ m + H ] +.
Example 1:3- (((6-amino-2-methylpyridin-3-yl) oxy) methyl) benzonitrile A-1
Figure BDA0003124934430000123
Step 1: (3-cyanophenyl) lithium methoxide I-5-1
(3-cyanophenyl) methanol (20mg, 0.15mol) was mixed with metallic lithium (12mg, 1.73mmol), and tetrahydrofuran (3 mL) was added thereto, and the mixture was stirred at 20 ℃ for 3 hours, the mixture was filtered, and the solvent was distilled off under reduced pressure to obtain (3-cyanophenyl) lithium methoxide I-5-1 (20mg, 96%).
Step 2:3- (((6-amino-2-methylpyridin-3-yl) oxy) methyl) benzonitrile
(3-cyanophenyl) lithium methoxide I-5-1 (20mg, 0.14mol) was mixed with 5-bromo-6-methylpyridin-2-amine I-6 (32mg, 0.17mol), cuprous bromide (28mg, 0.2mol), methanol (1 mL), N, N-dimethylformamide (3 mL) were added, and the mixture was stirred in an oil bath at 110 ℃ for 1 hour. To the reaction mixture were added ethyl acetate and water, 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: methanol =40: 1) to give 3- (((6-amino-2-methylpyridin-3-yl) oxy) methyl) benzonitrile a-1-1 (26g, 78%) as a liquid phase mass spectrum m/z =240.1[ 2 ], [ m + h ] + ]
Example 2: 6-methyl-5-phenethyloxy pyridine-2-amine A-1-2
Figure BDA0003124934430000131
Step 1: 2-Phenylethanolic lithium I-5-2
2-Phenylethanol (18mg, 0.15mol) and metallic lithium (12mg, 1.73mmol) were mixed, tetrahydrofuran (3 mL) was added thereto, and the mixture was stirred at 20 ℃ for 3 hours, and the mixture was filtered, and the solvent was distilled off under reduced pressure to give lithium 2-phenylethanolate I-5-2 (18mg, 94%).
Step 2: 6-methyl-5-phenethyloxy pyridine-2-amine A-1-2
Lithium 2-phenylethanol I-5-2 (18mg, 0.14mol) and 5-bromo-6-methylpyridin-2-amine (32mg, 0.17mol) were mixed, cuprous bromide (28mg, 0.2mol), methanol (1 mL), N, N-dimethylformamide (3 mL) were added, and the mixture was stirred for 1h with heating in an oil bath at 110 ℃. To the reaction mixture were added ethyl acetate and water, 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: methanol = 40)
Example 3: n- (6-amino-2-methylpyridin-3-yl) thiophene-3-carboxamide A-2-1
Figure BDA0003124934430000132
Step 1: (6-methyl-5- (thiophene-3-formamido) pyridine-2-yl) carbamic acid tert-butyl ester I-8-1
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), thiophene-2-carboxylic acid (15mg, 0.12mmol) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (6-methyl-5- (thiophene-3-carboxamido) pyridin-2-yl) carbamic acid tert-butyl ester I-8-1 (1695%) as a liquid phase mass spectrum m/z =334.1[ M ] +H ] +
And 2, step: n- (6-amino-2-methylpyridin-3-yl) thiophene-3-carboxamide
To tert-butyl (6-methyl-5- (thiophene-3-carboxamido) pyridin-2-yl) carbamate (16956.05mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) thiophene-3-carboxamide A-2-1 (11mg, 94%) as a liquid phase mass spectrum m/z =234.0[ 2 ], + M + H ] +
Example 4: (E) N- (6-amino-2-methylpyridin-3-yl) -2-methyl-2-enamide A-2
Figure BDA0003124934430000141
Step 1: (E) - (6-methyl-5- (2-methyl-2-enamido) pyridin-2-yl) carbamic acid tert-butyl ester I-8-2
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), and (E) -2-methyl-2-enoic acid (12mg, 0.12mmol) were mixed with 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol), and N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (E) - (6-methyl-5- (2-methyl-2-enamido) pyridin-2-yl) carbamic acid tert-butyl ester I-8-2 (19mg, 62%) as a liquid phase mass spectrum m/z =306.1[ M + H ] +
And 2, step: (E) N- (6-amino-2-methylpyridin-3-yl) -2-methyl-2-enamide
To tert-butyl (E) - (6-methyl-5- (2-methyl-2-enamido) pyridin-2-yl) carbamate (19mg, 0.06mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3h, 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 the reduced pressure to give (E) N- (6-amino-2-methylpyridin-3-yl) -2-methyl-2-enamide A-2-2 (12mg, 97%) as a liquid phase mass spectrum m/z =206.1[ M + H ] +
Example 5: n- (6-amino-2-methylpyridin-3-yl) -2, 3-dihydrobenzofuran-2-carboxamide A-2-3
Figure BDA0003124934430000142
Step 1: (5- (2, 3-dihydrobenzofuran-2-carboxamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-3
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), benzofuran-2-carboxylate (19mg, 0.12mmol) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (5- (2, 3-dihydrobenzofuran-2-carboxamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-3 (20mg, 55%) as a liquid phase mass spectrum m/z =370.2[ M + H ] +
Step 2: n- (6-amino-2-methylpyridin-3-yl) -2, 3-dihydrobenzofuran-2-carboxamide
To tert-butyl (5- (2, 3-dihydrobenzofuran-2-carboxamide) -6-methylpyridin-2-yl) carbamate (20mg, 0.055mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70 ℃ for 3 hours, to the reaction mixture was added ethyl acetate and water, 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 the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) -2, 3-dihydrobenzofuran-2-carboxamide A-2-3 (11mg, 74%) as a liquid phase mass spectrum m/z =270.1[ m + H ] +
Example 6: n- (6-amino-2-methylpyridin-3-yl) -3, 4-dichlorobenzamide A-2-4
Figure BDA0003124934430000151
Step 1: (5- (3, 4-Dichlorobenzamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-4
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), 3, 4-dichlorobenzoic acid (23mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (5- (3, 4-dichlorobenzamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-4 (20mg, 51%) as a liquid phase mass spectrum m/z =396.1[ M ] +H ] +
Step 2: n- (6-amino-2-methylpyridin-3-yl) -3, 4-dichlorobenzamide A-2-4
To tert-butyl (5- (3, 4-dichlorobenzamide) -6-methylpyridin-2-yl) carbamate (20mg, 0.05mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was stirred at 70 ℃ for 3 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7) and the solvent was removed by distillation under the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) -3, 4-dichlorobenzamide A-2-4 (14mg, 86%) as a liquid phase mass spectrum m/z =295.0[ M + H ] +
Example 7: n- (6-amino-2-methylpyridin-3-yl) benzothiophene-2-carboxamide A-2-5
Figure BDA0003124934430000152
Step 1: (5- (benzothiophene-2-carboxamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-5
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), benzothiophene-2-carboxylate (21mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (5- (benzothiophene-2-carboxamide) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-8-5 (22mg, 57%) as a liquid phase mass spectrum m/z =384.1[ M + ] H ] ]
And 2, step: n- (6-amino-2-methylpyridin-3-yl) benzothiophene-2-carboxamides
To tert-butyl (5- (benzothiophene-2-carboxamide) -6-methylpyridin-2-yl) carbamate (22mg, 0.057 mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated at 70 ℃ and stirred for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) benzothiophene-2-carboxamide A-2-5 (11mg, 68%) as a liquid phase mass spectrum m/z =284.1[ m + h ] +
Example 8: n- (6-amino-2-methylpyridin-3-yl) -2-phenylacetamide A-2-6
Figure BDA0003124934430000161
Step 1: (6-methyl-5- (2-phenylacetamide) pyridin-2-yl) carbamic acid tert-butyl ester I-8-6
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), 2-phenylacetic acid (1695g, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (38mg, 0.1mmol) were mixed, and N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added thereto and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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)
Step 2: n- (6-amino-2-methylpyridin-3-yl) -2-phenylacetamide:
to tert-butyl (6-methyl-5- (2-phenylacetamide) pyridin-2-yl) carbamate (18mg, 0.05mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3: 7), and the solvent was removed by distillation under the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) -2-phenylacetamide A-2-6 (12mg, 94%) as a liquid phase mass spectrum m/z =242.1[ 2 ], + H ] +
Example 9: n- (6-amino-2-methylpyridin-3-yl) benzamide A-2-7
Figure BDA0003124934430000162
Step 1: intermediate (5-benzamido-6-methylpyridine-2-yl) carbamic acid tert-butyl ester I-8-7
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol), 2-benzoic acid (15mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, and N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added thereto and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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)
And 2, step: n- (6-amino-2-methylpyridin-3-yl) benzamide
To tert-butyl (5-benzamido-6-methylpyridin-2-yl) carbamate (15mg, 0.046 mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give N- (6-amino-2-methylpyridin-3-yl) benzamide A-2-7 (10mg, 96%) as a liquid phase mass spectrum m/z =227.1[ m ] +H ] +
Example 10:2- (6-amino-2-methylpyridin-3-yl) isoindol-1-one A-3
Figure BDA0003124934430000171
The synthetic route is as follows:
Figure BDA0003124934430000172
step 1: (6-methyl-5- (1-oxoisoindol-2-yl) pyridin-2-yl) carbamic acid tert-butyl ester I-10
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and 2-formylbenzoic acid (18mg, 0.12mmol) were mixed, formic acid (0.2 ml), triethylamine (1 ml) and ethanol (1 ml) were added, and the mixture was heated to 80 ℃ and stirred for 60 minutes. To the reaction mixture were added ethyl acetate and water, 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 give (6-methyl-5- (1-oxoisoindol-2-yl) pyridin-2-yl) carbamic acid tert-butyl ester I-10 (20mg, 59%) as a liquid phase mass spectrum m/z =340.4[ M ] +H ] +
Step 2
To tert-butyl (6-methyl-5- (1-oxoisoindol-2-yl) pyridin-2-yl) carbamate (20mg, 0.059mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70 ℃ for 3 hours, to the reaction mixture was added ethyl acetate and water, 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 the reduced pressure to give 2- (6-amino-2-methylpyridin-3-yl) isoindol-1-one A-3 (12mg, 85%) as a liquid phase mass spectrum m/z =240.1[ M ] +H ] +
Example 11:1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea A-4-1
Figure BDA0003124934430000173
Step 1: p-nitrophenyl p-toluidine aminocarbonate I-13-1
P-toluidine (21mg, 0.2mmol) and 4-nitrophenylcarbonyl chloride (48mg, 0.24mmol) were mixed, and triethanolamine (0.2 ml), methylene chloride (1 ml) and tetrahydrofuran (2 ml) were added thereto and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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: hexane =5
Step 2: (6-methyl-5- (3- (p-tolyl) ureido) pyridin-2-yl) carbamic acid tert-butyl ester I-14-1
P-nitrophenyl p-toluidine carbamate (27mg, 0.1mmol) and tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (26mg, 0.12mmol) were reacted with K 2 CO 3 (0.2g, 1.45mmol) and acetonitrile (3 ml) was added thereto, and the mixture was heated to 40 ℃ and stirred for 4 hours. After completion of the reaction (monitored by TLC), the reaction mixture was isolated and concentrated in vacuo. 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 form of tert-butyl (6-methyl-5- (3- (p-tolyl) ureido) pyridin-2-yl) carbamate I-14-1 (28mg, 80%) as a liquid phase mass spectrum m/z =357.2[ 2 ],. M + H +]+
And step 3:1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea:
to tert-butyl (6-methyl-5- (3- (p-tolyl) ureido) pyridin-2-yl) carbamate (28mg, 0.08mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was stirred at 70 ℃ for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure, whereby 1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea A-4-1 (1695% by mass) was obtained as a liquid phase mass spectrum m/z =257.1[ M ] +H ] +
Example 12:1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea A-4-2
Figure BDA0003124934430000181
Step 1: 4-Nitrophenyl (3-bromophenyl) aminocarbonate I-13-2
3-bromoaniline (34mg, 0.2mmol) and 4-nitrophenylcarbonyl chloride (48mg, 0.24mmol) were mixed, and triethanolamine (0.2 ml), methylene chloride (1 ml) and tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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: hexane =5: 1) to give 4-nitrophenyl (3-bromophenyl) aminocarbonate I-13-2 (46mg, 68%) liquid phase mass spectrum m/z =338.1[ M ] +H ] +
Step 2: (5- (3- (3-bromophenyl) ureido) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-14-2
4-Nitrophenyl (3-bromophenyl) aminocarbonate (34mg, 0.1mmol) and tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (26mg, 0.12mmol) were reacted with K 2 CO 3 (0.2g, 1.45mmol) and acetonitrile (3 ml) was added thereto, and the mixture was heated to 40 ℃ and stirred for 4 hours. After completion of the reaction (monitored by TLC), the reaction mixture was isolated and concentrated in vacuo. 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 form of tert-butyl (5- (3- (3-bromophenyl) ureido) -6-methylpyridin-2-yl) carbamate I-14-2 (34mg, 81%) as a liquid phase mass spectrum m/z =421.1[ 2 ] M + H ]]+
And 3, step 3:1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea
To tert-butyl (5- (3- (3-bromophenyl) ureido) -6-methylpyridin-2-yl) carbamate (34mg, 0.08mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give 1- (6-amino-2-methylpyridin-3-yl) -3- (p-tolyl) urea A-4-2 (20mg, 78%) as a liquid phase mass spectrum m/z =321.0[ M ] +H ] +
Example 13: 6-amino-N, 2-dimethyl-N-phenylnicotinamide A-5
Figure BDA0003124934430000182
The synthetic route is as follows:
Figure BDA0003124934430000191
step 1: (5-bromo-6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-15
5-bromo-6-methylpyridin-2-amine (37mg, 0.2mmol), di-tert-butyldicarbonate anhydride (52mg, 0.24mmol) and K 2 CO 3 (69mg, 0.5 mmol), tetrahydrofuran (5 mL) was added thereto, and the mixture was stirred at room temperature for 3 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, washed with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to give tert-butyl (5-bromo-6-methylpyridin-2-yl) carbamate I-15 (50mg, 88%) as a liquid phase mass spectrum m/z =287.0[ 2 ], [ M + H ], []+
And 2, step: (6-methyl-5- (methyl (phenyl) carbamoyl) pyridin-2-yl) carbamic acid tert-butyl ester I-17
Tert-butyl (5-bromo-6-methylpyridin-2-yl) carbamate (50mg, 0.175mmol) and N-methyl-N-phenylformamide (27mg, 0.175mmol) were mixed with sodium methoxide (11mg, 0.2mmol), nickel diacetate tetrahydrate (25mg, 0.1mmol), 1,4 dioxane (8 mL) was added thereto, and the mixture was heated and stirred in an oil bath under helium protection at 110 ℃ for 12h. To the reaction mixture were added ethyl acetate and water, 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 the reduced pressure to give tert-butyl (6-methyl-5- (methyl (phenyl) carbamoyl) pyridin-2-yl) carbamate I-17 (50mg, 84%) as a liquid phase mass spectrum m/z =342.2[ M + H ] +
And 3, step 3: 6-amino-N, 2-dimethyl-N-phenylnicotinamide:
to tert-butyl (6-methyl-5- (methyl (phenyl) carbamoyl) pyridin-2-yl) carbamate (50mg, 0.147mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure, to give 6-amino-N, 2-dimethyl-N-phenylnicotinamide A-5 (27mg, 76%) as a liquid phase mass spectrum m/z =242.1[ M ] +H ] +
Example 14: 6-methyl-N5- (tetrahydro-2H-pyran-4-yl) pyridine-2, 5-diamine A-6-1
Figure BDA0003124934430000192
Step 1: tert-butyl (6-methyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyridin-2-yl) carbamate I-18-1
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and tetrahydro-4H-pyran-4-one (12mg, 0.12mmol) were mixed, methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol) and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20mL × 3). The combined organic phases were dried over anhydrous Na 2 SO 4 Dried, 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 the reduced pressure to give tert-butyl (6-methyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyridin-2-yl) carbamate I-18-1 (23mg, 75%) liquid phase mass spectrum m/z =308.2[ M + H ] +]+
Step 2: 6-methyl-N5- (tetrahydro-2H-pyran-4-yl) pyridine-2, 5-diamine
To tert-butyl (6-methyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyridin-2-yl) carbamate (23mg, 0.075mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3H, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure, to give 6-methyl-N5- (tetrahydro-2H-pyran-4-yl) pyridine-2, 5-diamine a-6-1 (12mg, 78%) as a liquid phase mass spectrum m/z =208.1[ m + H ] +
Example 15: 6-methyl-N 5 - (pyridin-4-ylmethyl) pyridine-2, 5-diamine A-6-2
Figure BDA0003124934430000201
Step 1: (6-methyl-5- ((pyridin-4-ylmethyl) amino) pyridin-2-yl) carbamic acid tert-butyl ester I-18-2
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and isonicotinal (13mg, 0.12mmol) were mixed, methanol (2 ml) was added, and stirring was carried out at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol) and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20mL. Times.3). The combined organic phases were in anhydrous Na 2 SO 4 Dried, 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 the reduced pressure to give (6-methyl-5- ((pyridin-4-ylmethyl) amino) pyridin-2-yl) carbamic acid tert-butyl ester I-18-2 (24mg, 76%) liquid phase mass spectrum m/z =314.2[ M + H ] +]+
Step 2: 6-methyl-N 5 - (pyridin-4-ylmethyl) pyridine-2, 5-diamine:
to tert-butyl (6-methyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyridin-2-yl) carbamate (23mg, 0.075mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3H, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give 6-methyl-N5- (tetrahydro-2H-pyran-4-yl) pyridine-2, 5-diamine A-6-2 (12mg, 78%) as a liquid phase mass spectrum m/z =208.1[ M + H ] +
Example 16: n is a radical of 5 -cyclobutyl-6-methylpyridine-2, 5-diamine A-6-3
Figure BDA0003124934430000202
Step 1: (5- (cyclobutylamine) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-18-3
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and cyclobutanone (8mg, 0.12mmol) were mixed, methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol) and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20mL. Times.3). The combined organic phases were dried over anhydrous Na 2 SO 4 Dried, 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 the reduced pressure to give tert-butyl (5- (cyclobutylamine) -6-methylpyridin-2-yl) carbamate i-18-3 (20mg, 72%) as a liquid phase mass spectrum m/z =278.2[ m ] +h ]]+
And 2, step: n is a radical of hydrogen 5 -cyclobutyl-6-methylpyridine-2, 5-diamine:
to tert-butyl (5- (cyclobutylamine) -6-methylpyridin-2-yl) carbamate (20mg, 0.072mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated at 70 ℃ and stirred for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give 6-methyl-N5- (tetrahydro-2H-pyran-4-yl) pyridine-2, 5-diamine A-6-3 (10mg, 78%) as a liquid phase mass spectrum m/z =178.1[ M + H ] +
Example 17:2- (6-amino-2-methylpyridin-3-yl) isoindole-1, 3-dione A-7
Figure BDA0003124934430000211
The synthesis circuit is as follows:
Figure BDA0003124934430000212
step 1: tert-butyl (5- (1, 3-dioxoisooctanol-2-yl) -6-methylpyridin-2-yl) carbamate I-20
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and isobenzofuran-1, 3-dione (18mg, 0.12mmol) were mixed, and N, N-dimethylacetamide (2 ml) was added thereto, followed by stirring at room temperature for 24 hours, followed by addition of xylene (1 ml), and stirring in an oil bath at 140 ℃ for 48 hours. Upon completion (monitored by TLC), the insoluble catalyst was isolated by filtration, washed with acetone and dried. The organic layer was concentrated under reduced pressure to give the desired product, washed with water and recrystallized from ethanol, and the crude product was subjected to silica gel column Chromatography (CH) 2 Cl 2 N-hexane =1: 1) Purification and reduced pressure distillation to remove the solvent, thus obtaining tert-butyl (5- (1, 3-dioxyisooctanol-2-yl) -6-methylpyridin-2-yl) carbamate I-20 (24mg, 68%) liquid phase mass spectrum m/z =354.1[ M + [ H ] +]+
And 2, step: 2- (6-amino-2-methylpyridin-3-yl) isoindole-1, 3-dione
To tert-butyl (5- (1, 3-dioxoisooctanol-2-yl) -6-methylpyridin-2-yl) carbamate (24mg, 0.068mmol) was added a dioxane solution of HCl (5%, 5 mL), and the mixture was heated and stirred at 70 ℃ for 3 hours, to the reaction mixture was added ethyl acetate and water, 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 the reduced pressure to give 2- (6-amino-2-methylpyridin-3-yl) isoindole-1, 3-dione A-7 (12mg, 70%) as a liquid phase mass spectrum m/z =254.1[ M + H ] +
Example 18: 6-methyl-N 5 -phenylpyridine-2, 5-diamine A-8
Figure BDA0003124934430000213
The synthesis circuit is as follows:
Figure BDA0003124934430000214
step 1: (6-methyl-5- (anilino) pyridin-2-yl) carbamic acid tert-butyl ester I-22
Reacting (5-amino-Tert-butyl 6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) was mixed with iodobenzene (24mg, 0.12mmol), (N, N-bipyridinylimidazolidene) copper dibromide (10mg, 0.023mmol), and cesium carbonate (100mg, 0.3mmol), 1, 4-dioxane (5 ml) was added, and the mixture was stirred in an oil bath at 170 ℃ for 12 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20mL × 3). The combined organic phases were dried over anhydrous Na 2 SO 4 Dried, 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 the reduced pressure to give tert-butyl (6- (methyl-5- (anilino) pyridin-2-yl) carbamate I-22 (25mg, 84%) as a liquid phase mass spectrum m/z =300.2 +H +]+
Step 2: 6-methyl-N 5 -phenylpyridine-2, 5-diamine:
to tert-butyl (6- (methyl-5- (anilino) pyridin-2-yl) carbamate (25mg, 0.084 mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3h, ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, concentrated under reduced pressure, and the solvent was removed by silica gel column chromatography (ethyl acetate: hexane = 3), distillation under reduced pressure was performed to remove the solvent, to give 6-methyl-N 5 Liquid phase mass spectrum m/z =200.1[ 2[ M ] +H ] of-phenylpyridine-2, 5-diamine A-8 (12mg, 72%)]+
Example 19: (E) -6-methyl-5-styrylpyridin-2-amine A-9
Figure BDA0003124934430000221
The synthesis circuit is as follows:
Figure BDA0003124934430000222
step 1: tert-butyl (E) - (6-methyl-5-styrylpyridin-2-yl) carbamate I-24
(5-amino-6-methylpyridin-2-yl) carbamic acid tert-butyl ester (22mg, 0.1mmol) was mixed with styrene (13mg, 0.12mmol), bis (dibenzylideneacetone) -palladium (0) (5mg, 0.0087mmol),tert-butyl nitroso ester (0.5 ml), chloroacetic acid (0.5 ml), and acetic acid (3 ml) were added and stirred in an oil bath at 50 ℃ for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20mL × 3). The combined organic phases were in anhydrous Na 2 SO 4 Dried, 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 the reduced pressure to give tert-butyl (E) - (6-methyl-5-styrylpyridin-2-yl) carbamate I-24 (22mg, 71%) as a liquid phase mass spectrum m/z =311.2 ++ H +]+
And 2, step: (E) -6-methyl-5-styrylpyridin-2-amine:
to tert-butyl (E) - (6-methyl-5-styrylpyridin-2-yl) carbamate (22mg, 0.071mmol) was added a solution of HCl in dioxane (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3h, 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure to give (E) -6-methyl-5-styrylpyridin-2-amine A-9 (13mg, 88%) as a liquid phase mass spectrum m/z =211.1[ M ] +H ] +
Example 20: n- (6-amino-2-methylpyridin-3-yl) thiophene-2-sulfonamide A-10
Figure BDA0003124934430000231
The synthesis circuit is as follows:
Figure BDA0003124934430000232
step 1: (6-methyl-5- (thiophene-2-sulfanylamino) pyridin-2-yl) carbamic acid tert-butyl ester I-26
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (22mg, 0.1mmol) and thiophene-2-sulfonyl chloride (22mg, 0.12mmol) were mixed, triethylamine (0.5 ml), dichloromethane (3 ml) were added, and the mixture was stirred at room temperature for 24 hours under nitrogen atmosphere. To the reaction mixture were added ethyl acetate and water, 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) and the solvent was removed by distillation under the reduced pressure to give (6-methyl-5- (thiophene-2-sulfonamido) pyridin-2-yl) carbamic acid tert-butyl ester I-26 (19mg, 52%) as a liquid phase mass spectrum m/z =370.1[ M + H ] +
Step 2: n- (6-amino-2-methylpyridin-3-yl) thiophene-2-sulfonamide
To tert-butyl (6-methyl-5- (thiophene-2-sulfonamido) pyridin-2-yl) carbamate (19mg, 0.052mmol) was added a dioxane solution of HCl (5%, 5 mL), the mixture was heated and stirred at 70 ℃ for 3 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 subjected to silica gel column chromatography (ethyl acetate: hexane =3, 7), and the solvent was removed by distillation under the reduced pressure, to give N- (6-amino-2-methylpyridin-3-yl) thiophene-2-sulfonamide A-10 (10mg, 72%) as a liquid phase mass spectrum m/z =270.0[ 2 ], [ M ] +H ] +
Example 21: n- (2- ((6-amino-2-methylpyridin-3-yl) amino) ethyl) methanesulfonamide A-11
Figure BDA0003124934430000233
The synthesis circuit is as follows:
Figure BDA0003124934430000234
step 1: (5- ((2-aminoethyl) amino) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-28
Tert-butyl (5-amino-6-methylpyridin-2-yl) carbamate (44mg, 0.2mmol) and 2-bromoethane-1-amine (29mg, 0.24mmol) were mixed, water (5 ml) was added and the mixture was stirred in an oil bath at 95 ℃ 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 is on silica gel (CH) 2 Cl 2 Methanol-ammonia) to afford the desired product. The solvent was removed by distillation under the reduced pressure to give (5- ((2-aminoethyl) amino) -6-methylpyridin-2-yl) carbamic acid tert-butyl ester I-28 (38mg, 72%) liquid phase mass spectrum m/z =267..2[ M +. H ]]+
Step 2: (6-methyl-5- ((2- (methylsulfonylamino) ethyl) amino) pyridin-2-yl) carbamic acid tert-butyl ester I-30
Tert-butyl (5- ((2-aminoethyl) amino) -6-methylpyridin-2-yl) carbamate (38mg, 0.144mmol) was mixed with methanesulfonyl chloride (28mg, 0.16mmol), dichloromethane (5 ml) was added, and the mixture was stirred in an ice-water bath for 24 hours. To the reaction mixture were added ethyl acetate and water, 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 the reduced pressure to give (6-methyl-5- ((2- (methylsulfonamido) ethyl) amino) pyridin-2-yl) carbamic acid tert-butyl ester i-30 (20mg, 40%), liquid phase mass spectrum m/z =255.1[ m + h ] +
And step 3: n- (2- ((6-amino-2-methylpyridin-3-yl) amino) ethyl) methanesulfonamide
To tert-butyl (6-methyl-5- ((2- (methylsulfonylamino) ethyl) amino) pyridin-2-yl) carbamate (20mg, 0.057mmol) was added a dioxane solution of HCl (5%, 5 mL), heated and stirred at 70 ℃ for 3h, to the reaction mixture was added ethyl acetate and water, 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 the reduced pressure to give N- (2- ((6-amino-2-methylpyridin-3-yl) amino) ethyl) methanesulfonamide A-11 (11mg, 78%) as a liquid phase mass spectrum m/z =245.1[ M + H ] +
Example 22:3- ((1H-benzo [ d ] imidazol-6-yl) oxymethyl) benzonitrile B-1
Figure BDA0003124934430000241
The method comprises the following steps: 1H-benzo [ d ]]Imidazol-6-ol II-3 (9 mg, 0.07mmol), 3- (bromomethyl) benzonitrile (20mg, 0.1mmol) and K 2 CO3 (138mg, 1mmol) was mixed, acetonitrile (2 ml) was added, and the mixture was heated to 40 ℃ and stirred for 10 minutes. To the reaction mixture were added ethyl acetate and water, 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: methanol = 40)]Imidazol-6-yl) oxymethyl) benzonitrile B-1-1 (10 mg)57%) liquid phase mass spectrum m/z =249.3[ m + H ]]+
Example 23: 6-phenoxy-1H-benzo [ d ] imidazole B-1-2
Figure BDA0003124934430000242
The method comprises the following steps: 1H-benzo [ d ]]Imidazol-6-ol II-3 (9mg, 0.07mmol) and (2-bromoethyl) benzene (22mg, 0.12mmol) with K 2 CO 3 (138mg, 1mmol), acetonitrile (2 ml) was added, and the mixture was heated to 40 ℃ and stirred for 10 minutes. To the reaction mixture were added ethyl acetate and water, 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: methanol = 40)]Imidazole B-1-2 (12mg, 70%) liquid phase mass spectrum m/z =238.1[ m ] +H]+
Example 24:2- (1H-benzo [ d ] imidazol-6-yl) isoindol-1-one B-2
Figure BDA0003124934430000243
The synthetic route is as follows:
Figure BDA0003124934430000251
the method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.12mmol) and 2-formylbenzoic acid (18mg, 0.12mmol) were mixed, formic acid (0.2 ml), triethylamine (1 ml) and ethanol (1 ml) were added, and the mixture was heated to 80 ℃ and stirred for 60 minutes. To the reaction mixture were added ethyl acetate and water, 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 give 2- (1H-benzo [ d ] imidazol-6-yl) isoindol-1-one B-2 (13mg, 54%) liquid phase mass spectrum m/z =249.1[ m + H ] +
Example 25: n- (1H-benzo [ d ] imidazol-6-yl) thiophene-3-carboxamide B-3-1
Figure BDA0003124934430000252
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol), thiophene-2-carboxylic acid (15mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml), and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to obtain N- (1H-benzo [ d ] imidazol-6-yl) thiophene-3-carboxamide B-3-1 (9 mg, 37%) liquid phase mass spectrum m/z =243.1[ m + H ] +
Example 26: (E) -N- (1H-benzo [ d ] imidazol-6-yl) -2-methyl-2-enamide B-3-2
Figure BDA0003124934430000253
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol) and (E) -2-methyl-2-enoic acid (12mg, 0.12mmol) were mixed with 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol), N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) to give (E) -N- (1H-benzo [ d ] imidazol-6-yl) -2-methyl-2-enamide B-3-2 (10mg, 46%) as a liquid phase mass spectrum m/z =215.1[ m ] +H ] +
Example 27: n- (1H-benzo [ d ] imidazol-6-yl) -2, 3-dihydrobenzofuran-2-carboxamide B-3
Figure BDA0003124934430000254
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol), benzofuran-2-carboxylic acid (19mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) 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. To the reaction mixture were added ethyl acetate and water, 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) to give N- (1H-benzo [ d ] imidazol-6-yl) -2, 3-dihydrobenzofuran-2-carboxamide B-3-3 (12mg, 41%) as a liquid phase mass spectrum m/z =279.1[ m ] +H ] +
Example 28: n- (1H-benzo [ d ] imidazol-6-yl) -3, 4-dichlorobenzamide B-3-4
Figure BDA0003124934430000261
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol), 3, 4-dichlorobenzoic acid (23mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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 N- (1H-benzo [ d ] imidazol-6-yl) -3, 4-dichlorobenzamide B-3-4 (12mg, 39%) as a liquid phase mass spectrum m/z =305.0[ 2 ], [ m + H ] +
Example 29: n- (1H-benzo [ d ] imidazol-6-yl) benzo [ B ] thiophene-2-carboxamide B-3-5
Figure BDA0003124934430000262
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol), benzothiophene-2-carboxylic acid (21mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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 N- (1H-benzo [ d ] imidazol-6-yl) benzo [ B ] thiophene-2-carboxamide B-3-5 (13mg, 43%) as a liquid phase mass spectrum m/z =293.1[ 2 ], + m + H ] +
Example 30: n- (1H-benzo [ d ] imidazol-6-yl) -2-phenylacetamide B-3-6
Figure BDA0003124934430000263
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol), 2-phenylacetic acid (116mg, 0.12mmol), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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)
Example 31:1- (1H-benzo [ d ] imidazol-6-yl) -3- (p-tolyl) urea B-4-1
Figure BDA0003124934430000271
Step 1: p-nitrophenyl-p-toluidino carbonate II-9-1
P-toluidine (21mg, 0.2mmol) and 4-nitrophenylcarbonyl chloride (48mg, 0.24mmol) were mixed, and triethanolamine (0.2 ml), methylene chloride (1 ml) and tetrahydrofuran (2 ml) were added thereto and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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: hexane =5
Step 2:1- (1H-benzo [ d ] imidazol-6-yl) -3- (p-tolyl) urea
P-nitrophenyl p-toluidine carbamate (27mg, 0.1mmol) was reacted with 1H-benzo [ d ]]Imidazol-6-amine (16mg, 0.12mmol) with K 2 CO3 (0.2g, 1.45mmol) was mixed, acetonitrile (3 ml) was added, and the mixture was heated to 40 ℃ and stirred for 4 hours. After completion of the reaction (monitored by TLC), the reaction mixture was isolated and concentrated in vacuo. The crude reaction mixture thus obtained was washed with dichloromethane, then EtOAc and finally MeOH (1 mL each). Finally, the reaction product was recrystallized using EtOAc (under warm conditions) to give 1- (1H-benzo [ d ] in pure form]Imidazol-6-yl) -3- (p-tolyl) urea B-4-1 (20mg, 75%) liquid phase mass spectrum m/z =266.1[ m ] +H]+
Example 32:1- (1H-benzo [ d ] imidazol-6-yl) -3- (3-bromophenyl) urea B-4-2
Figure BDA0003124934430000272
Step 1: 4-Nitrophenyl (3-bromophenyl) aminocarbonate II-9-2
3-bromoaniline (34mg, 0.2mmol) and 4-nitrophenylcarbonyl chloride (48mg, 0.24mmol) were mixed, and triethanolamine (0.2 ml), methylene chloride (1 ml) and tetrahydrofuran (2 ml) were added and stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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: hexane =5: 1) to give 4-nitrophenyl (3-bromophenyl) aminocarbonate II-9-2 (46mg, 68%) liquid phase mass spectrum m/z =338.1[ m ] +H ] +
Step 2:1- (1H-benzo [ d ] imidazol-6-yl) -3- (3-bromophenyl) urea
4-Nitrophenyl (3-bromophenyl) aminocarbonate (34mg, 0.1mmol) was reacted with 1H-benzo [ d ]]Imidazol-6-amine (16mg, 0.12mmol) with K 2 CO3 (0.2g, 1.45mmol) was mixed, acetonitrile (3 ml) was added, and the mixture was heated to 40 ℃ and stirred for 4 hours. After completion of the reaction (monitored by TLC), the reaction mixture was isolated and concentrated in vacuo. The crude reaction mixture thus obtained was washed with dichloromethane, then EtOAc and finally MeOH (1 mL each). Finally, the reaction product was recrystallized using EtOAc (under warm conditions) to give 1- (1H-benzo [ d ] in pure form]Imidazol-6-yl) -3- (3-bromophenyl) urea B-4-2 (22mg, 68%) liquid phase mass spectrum m/z =330.0[ m ] +H]+
Example 33: N-methyl-N-phenyl-1H-benzo [ d ] imidazole-6-carboxamide B-5
Figure BDA0003124934430000281
The synthetic route is as follows:
Figure BDA0003124934430000282
step 1: 6-nitro-1H-benzo [ d ] imidazole II-10
To a solution of H2O2 (240mg, 2.2mmol) in fuming sulfuric acid (500. Mu.L) was added dropwise 1H-benzo [ d ] n]A solution of imidazol-6-amine (40mg, 0.3 mmol) in concentrated sulfuric acid (100. Mu.L) was maintained at 0 ℃ for the reaction. After stirring for 3h at 10-25 ℃, the reaction mixture was brought to pH =11-12 by adding 40% aqueous naoh solution at 0-5 ℃. The resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution over Na 2 Dried over SO4 and filtered. Distilling under reduced pressure to remove the solvent to obtain the desired 6-nitro-1H-benzo [ d]Imidazole II-10 (35mg, 72%) LC-mS m/z =163.0[ m ] +H]+
Step 2: 1H-benzo [ d ] imidazole-6-carboxylic acid II-11
Reacting 6-nitro-1H-benzo [ d ]]Imidazole (39mg, 0.24mmol) and KCN (195mg, 3mmol) were mixed and 1-butylimidazole tetrafluoroborate (3mg, 0.014mmol), etOH (2 ml), and water (2 ml) were added, and the mixture was heated to 80 ℃ and stirred for 19 hours. Then 10ml of water are added, using CH 2 Cl 2 The mixture was extracted (3X 5 ml) and diethyl ether (3X 10 ml). Acidified to pH1-2 with hydrochloric acid and extracted with diethyl ether (3X 10 ml). Magnesium sulfate (3 g) and activated carbon (1 g) were added and stirred for 5h. The solid is filtered off, the filtrate is evaporated and the residue is crystallized from the corresponding solvent to give the desired 1H-benzo [ d ]]Imidazole-6-carboxylic acid II-11 (14mg, 40%) LC-mS m/z =162.0[ 2 ], [ m + H ]]+
And step 3: N-methyl-N-phenyl-1H-benzo [ d ] imidazole-6-carboxamide
1H-benzo [ d ] imidazole-6-carboxylic acid (16mg, 0.1mmol), N-methylaniline (13mg, 0.12mmol) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (38mg, 0.1mmol) were mixed, N, N-diisopropylethylamine (0.2 ml) and N, N-dimethylformamide (2 ml) were added, and the mixture was stirred at room temperature for 18 hours. To the reaction mixture were added ethyl acetate and water, 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) and the solvent was removed by distillation under the reduced pressure, to give N-methyl-N-phenyl-1H-benzo [ d ] imidazole-6-carboxamide B-5 (1695g, 65%) as a liquid phase mass spectrum m/z =251.1 2[ 2 ] m + H ] +
Example 34: n- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d ] imidazol-6-amine B-6-1
Figure BDA0003124934430000283
The method comprises the following steps: by reacting 1H-benzo [ d ]]Imidazol-6-amine (13mg, 0.1mmol) and tetrahydro-4H-pyran-4-one (12mg, 0.12mmol) were mixed, methanol (2 ml) was added thereto, and the mixture was stirred at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol) and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20 mLx 3). The combined organic phases were in anhydrous Na 2 Dried over SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5) and the solvent was removed by distillation under the reduced pressure to give N- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d]Imidazole-6-amine B-6-1 (1695g, 75%) liquid phase mass spectrum m/z =217.1[ 2 ], [ m ] +H]+
Example 35: n- (pyridin-4-ylmethyl) -1H-benzo [ d ] imidazol-6-amine B-6-2
Figure BDA0003124934430000291
The method comprises the following steps: by reacting 1H-benzo [ d ]]Imidazol-6-amine (13mg, 0.1mmol) and isonicotinal (13mg, 0.12mmol) were mixed, methanol (2 ml) was added, and stirring was carried out at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol), and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20 mLx 3). The combined organic phases were dried over anhydrous Na 2 Dried over SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5) and the solvent was removed by distillation under the reduced pressure to give N- (pyridine-4-methyl) -1H-benzo [ d]Imidazole-6-amine B-6-2 (14mg, 64%) liquid phase mass spectrum m/z =224.1[ m ] +H]+
Example 36: n-cyclobutyl-1H-benzo [ d ] imidazol-6-amine B-6-3
Figure BDA0003124934430000292
The method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.1mmol) and cyclobutanone (8mg, 0.12mmol) were mixed, methanol (2 ml) was added, and the mixture was stirred at room temperature for 2 hours, followed by addition of sodium cyanoborohydride (20mg, 0.3mmol), and stirring at room temperature for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DC m (20 mLx 3). The combined organic phases were 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 reduced pressure distillation to obtain n-cyclobutyl-1H-benzo [ d ] imidazole-6-amine B-6-3 (13mg, 72%) liquid phase mass spectrum m/z =187.1[ m ] +H ] +
Example 37:2- (1H-benzo [ d ] imidazol-6-yl) isoindoline-1, 3-dione B-7
Figure BDA0003124934430000293
The synthetic route is as follows:
Figure BDA0003124934430000294
the method comprises the following steps: by reacting 1H-benzo [ d ]]Imidazole-6-amine (13mg, 0.1mmol) and isobenzofuran-1, 3-dione (18mg, 0.12mmol) were mixed, and N, N-dimethylacetamide (2 ml) was added thereto, and the mixture was stirred at room temperature for 24 hours, followed by addition of xylene (1 ml), and then stirred at 140 ℃ in an oil bath for 48 hours. Upon completion (monitored by TLC), the insoluble catalyst was isolated by filtration, washed with acetone and dried. The organic layer was concentrated under reduced pressure toThe desired product is obtained, washed with water and recrystallized from ethanol and the crude product is chromatographed on a silica gel Column (CH) 2 Cl 2 N-hexane =1: 1) Purifying, and distilling under reduced pressure to remove solvent to obtain 2- (1H-benzo [ d ]]Imidazol-6-yl) isoindoline-1, 3-dione B-7 (18mg, 68%) liquid phase mass spectrum m/z =263.1[ m ] +H]+
Example 38: N-phenyl-1H-benzo [ d ] imidazol-6-amine B-8
Figure BDA0003124934430000301
The synthetic route is as follows:
Figure BDA0003124934430000302
the method comprises the following steps: by reacting 1H-benzo [ d ]]Imidazole-6-amine (13mg, 0.1mmol) was mixed with iodobenzene (24mg, 0.12mmol), (N, N-bipyridinylimidazolidene) copper dibromide (10mg, 0.023mmol), cesium carbonate (100mg, 0.3mmol), 1, 4-dioxane (5 ml) was added, and stirred in an oil bath at 170 ℃ for 12 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20 mLx 3). The combined organic phases were dried over anhydrous Na 2 Dried over SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5) and the solvent was removed by distillation under the reduced pressure to give N-phenyl-1H-benzo [ d]Imidazole-6-amine B-8 (17mg, 82%) liquid phase mass spectrum m/z =209.1[ m ] +H]+
Example 39: (E) -6-styryl-1H-benzo [ d ] imidazole B-9
Figure BDA0003124934430000303
The synthetic route is as follows:
Figure BDA0003124934430000304
the method comprises the following steps: 1H-benzo [ d ]]Imidazol-6-amine (13mg, 0.1mmol) with benzeneEthylene (13mg, 0.12mmol), bis (dibenzylideneacetone) -palladium (0) (5mg, 0.0087mmol) were mixed, and tert-butyl nitroso ester (0.5 ml), chloroacetic acid (0.5 ml) and acetic acid (3 ml) were added thereto, and stirred in an oil bath at 50 ℃ for 2 hours. Then, an aqueous NaOH solution (10mL, 0.3mol/L) was added to the mixture. The resulting mixture was extracted with DCM (20 mLx 3). The combined organic phases were in anhydrous Na 2 Dried over SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5)]Imidazole B-9 (13mg, 61%) liquid phase mass spectrum m/z =220.1[ m ] +H]+
Example 40: n- (1H-benzo [ d ] imidazol-6-yl) thiophene-2-sulfonamide B-10
Figure BDA0003124934430000305
The synthesis circuit comprises:
Figure BDA0003124934430000311
the method comprises the following steps: 1H-benzo [ d ] imidazol-6-amine (13mg, 0.12mmol) and thiophene-2-sulfonyl chloride (22mg, 0.12mmol) were mixed, triethylamine (0.5 ml), dichloromethane (3 ml) were added, and the mixture was stirred at room temperature under nitrogen for 24 hours. To the reaction mixture were added ethyl acetate and water, 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) and the solvent was removed by distillation under the reduced pressure to give N- (1H-benzo [ d ] imidazol-6-yl) thiophene-2-sulfonamide B-10 (13mg, 45%) as a liquid phase mass spectrum m/z =279.0[ m ] +H ] +
Example 41: n- (2- (1 h benzo [ d ] imidazol-6-yl) aminoethyl) methanesulfonamide B-11
Figure BDA0003124934430000312
The synthesis circuit comprises:
Figure BDA0003124934430000313
step 1: n1- (1H-benzo [ d ] imidazol-6-yl) ethane-1, 2-diamine II-19
1H-benzo [ d ]]Imidazole-6-amine (27mg, 0.2mmol) was mixed with 2-bromoethane-1-amine (29mg, 0.24mmol), water (5 ml) was added and the mixture was stirred in an oil bath at 95 ℃ 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 is on silica gel (CH) 2 Cl 2 Methanol-ammonia) to afford the desired product. Removing the solvent by distillation under reduced pressure to obtain N1- (1H-benzo [ d ]]Imidazol-6-yl) ethane-1, 2-diamine II-19 (22mg, 61%) liquid phase Mass Spectrometry m/z =176.1[ m ] +H]+
Step 2: n- (2- (1 h benzo [ d ] imidazol-6-yl) aminoethyl) methanesulfonamide
N1- (1H-benzo [ d ] imidazol-6-yl) ethane-1, 2-diamine (23mg, 0.13mmol) and methanesulfonyl chloride (28mg, 0.113mmol) were mixed, dichloromethane (5 ml) was added, and the mixture was stirred in an ice-water bath for 24 hours. To the reaction mixture were added ethyl acetate and water, 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) and the solvent was removed by distillation under the reduced pressure to give N- (2- (1H benzo [ d ] imidazol-6-yl) aminoethyl) methanesulfonamide B-11 (11mg, 32%), liquid phase mass spectrum m/z =254.1[ 2 ], [ m + H ] +
Small molecule binding energy prediction:
the aminopyridine derivative molecules of examples 1-21, the benzimidazole derivative molecules of examples 22-41, and the OCT4 target protein were each molecularly docked using AutoDock Vina and LeDock software to produce 10 docking conformations, respectively. Calculating the binding energy and ligand efficiency of the optimal docking result of each aminopyridine derivative molecule and each benzimidazole derivative molecule and the OCT4 target protein, and performing molecular screening on the aminopyridine derivative molecule and the benzimidazole derivative molecule by synthesizing the docking result, wherein the specific docking results are respectively shown in tables 1 and 2: the second and third columns of the table show the binding free energy (binding energy) calculated by autodock vina and Ledock molecular docking software respectively, and 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, with larger absolute values indicating greater potential for small molecule activity. According to the invention, the binding energy level of the compound related to the invention is predicted according to independent algorithms of two pieces of software, and the predicted value shows that the binding energy of the compound is far larger than a threshold value 3 set according to target characteristics.
TABLE 1 prediction of binding of aminopyridine derivative molecules used in the present invention to target proteins
Figure BDA0003124934430000321
TABLE 2 prediction of binding energy of benzimidazole derivative molecules used in the present invention to targeting sequences
Figure BDA0003124934430000322
Figure BDA0003124934430000331
And (3) verifying the difference of transcription expression caused by small molecules:
the invention aims to achieve the effect of promoting a target gene by using a high-selectivity activator, and the important function of the activator is to promote the expression of Oct4, so that the expression abundance of Oct4 to downstream genes is increased. Therefore, when the small molecule function of the invention is verified, in addition to verifying the increase of the Oct4 gene itself, the increase of the expression of the downstream gene Nanog of the Oct4 gene is also an index for functional verification of the compound of the invention.
Human mesenchymal cells cultured at T25, as 4x10 5 The cells were seeded and cultured in serum-free Duchen modified eagle's medium (DMEM-F12 medium) to which 50nM of the above aminopyridine derivative small molecule and benzimidazole derivative small molecule were added, respectively, under 37 ℃ and 5% carbon dioxide. On day 5, RNeasy Mini or Micro K was usedIt (QIAGEN) was used for total RNA extraction, and 1mg of RNA was used for cDNA Synthesis using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of Quantitative PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Ct method. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 3. As a result, as shown in fig. 1 and 2, the above aminopyridine derivative small molecules and benzimidazole derivative small molecules significantly increased the basal expression of OCT4 and the expression of the downstream gene Nanog, compared to the control group without small molecules.
TABLE 3 Compound Effector QPCR primer sequences
Figure BDA0003124934430000332
Figure BDA0003124934430000341
Early cell pluripotency validation by small molecules:
the Rex1 gene is highly expressed in undifferentiated embryonic stem cells, and most closely approximates the natural state of in vivo embryonic stem cells and pre-implantation blastocysts 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. Expression of the Rex1 gene indicates that the cell is in a natural pluripotent state resembling the blastocyst from which the embryonic stem cell is actively implanted. Therefore, the expression of Rex1 is used as a terminal detection index of the small molecule function.
Human mesenchymal cells cultured at T25, as 4x10 5 The cells were seeded and cultured in serum-free Duchen modified eagle's medium (DMEM-F12 medium) to which 50nM of the above aminopyridine derivative small molecule and benzimidazole derivative small molecule were added, respectively, under 37 ℃ and 5% carbon dioxide. Immunofluorescence on day 5Dyeing and identifying: fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then permeabilized with 0.1% Triton X-100 for 5 minutes and washed twice with DPBS buffer; then incubating the cells overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then 2% horse serum and 0.1 Triton X-100 in DPBS buffer 1. As shown in FIG. 3, the small chemical molecule can promote mesenchymal cells to express Rex1 gene and make the mesenchymal cells form pluripotent stem cell clone-like cell masses.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggccgaggac tttgattgca ca 22
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gggcacgaag gctcatcatt caa 23

Claims (11)

1. A compound having the structure of formula (III):
Figure FDA0003124934420000011
wherein:
m1 and m2 are each 0 or 1;
A 2 is C1-C6 alkylene, C2-C6 alkenylene, -O (CH) 2 )q-、-NR 1 -、-SO 2 -、-(CH 2 ) V NHS(O) 2 -or a bond, wherein q is 1 or 2 or 3 or 4, v is 0 or 1 or 2, R1 is selected from H or C1-C4 alkyl;
A 3 is C1-C6 alkyl; C2-C6 alkenyl; C4-C6 cycloalkyl, wherein one carbon atom may be substituted by a N, O, S heteroatom;
Figure FDA0003124934420000012
z and Z 1 Are each N or CR 2 ，R 2 Selected from H, halogen, C1-C4 alkyl or cyano;
Figure FDA0003124934420000013
Z 3 is N, O, S or C = O, when Z is 4 And Z 5 When the bond between is a single bond, Z 4 Is N or CH, Z 5 Is CH 2 Or C = O, when Z 4 And Z 5 When the bond between is a double bond, Z 4 Is C, Z 5 Is CH;
and pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, esters, optical isomers, prodrugs or combinations thereof.
2. The compound of claim 1, wherein:
A 2 is-CH 2 -、-CH＝CH-、-C(CH 3 )＝CH-、-O(CH 2 )-、-O(CH 2 ) 2 -、-NH-、-N(CH 3 )-、-SO 2 -、-NHS(O) 2 -、-(CH 2 ) 2 NHS(O) 2 -or a bond.
3. The compound of claim 2, wherein:
A 3 is-CH 3 Butenyl, butenyl,
Figure FDA0003124934420000014
Figure FDA0003124934420000021
4. A compound according to claim 3, wherein:
m1 is 0, m2 is 1;
A 2 is-N (CH) 3 )-；
A 3 Is that
Figure FDA0003124934420000022
5. A compound according to claim 3, wherein:
m1 is 1, m2 is 0;
A 2 is-CH 2 -、-SO 2 -、-(CH 2 ) 2 NHS(O) 2 -or a bond;
A 3 is-CH 3 、
Figure FDA0003124934420000023
6. A compound according to claim 3, wherein:
m1 is 1, m2 is 1;
A 2 is-CH 2 -、-NH-、-C(CH 3 ) = CH-or bond;
A 3 is-CH 3 、-C(CH 3 )＝CH-CH 3 、
Figure FDA0003124934420000024
Figure FDA0003124934420000031
7. A compound according to claim 3, wherein:
m1 is 0, m2 is 0;
A 2 is-CH 2 -、-CH＝CH-、-O(CH 2 )-、-O(CH 2 ) 2 -or a bond;
A 3 is that
Figure FDA0003124934420000032
8. The compound of claim 1, selected from the group consisting of:
Figure FDA0003124934420000033
9. a pharmaceutical composition comprising a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, ester, optical isomer, prodrug or combination thereof, and at least one pharmaceutically acceptable carrier or excipient.
10. Use of the compound of any one of claims 1 to 8 and/or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, ester, optical isomer, prodrug thereof or combination thereof for the manufacture of a medicament for the induction of an OCT4 highly selective activator of pluripotent stem cells.
11. The use of claim 10, wherein the diseases treated by OCT4 high selectivity activator-induced 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, edentulous teeth, wound healing, bone marrow transplantation, osteoarthritis, rheumatoid arthritis, hair loss, blindness, deafness, crohn's disease, and genetic diseases, and other similar diseases.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011131748A2 (en) * 2010-04-21 2011-10-27 Probiodrug Ag Novel inhibitors
US20140154805A1 (en) * 2012-12-03 2014-06-05 City Of Hope Enhancers of induced pluripotent stem cell reprogramming

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011131748A2 (en) * 2010-04-21 2011-10-27 Probiodrug Ag Novel inhibitors
US20140154805A1 (en) * 2012-12-03 2014-06-05 City Of Hope Enhancers of induced pluripotent stem cell reprogramming

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Title
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FREI, RETO, ET AL: "2-Aminobenzimidazole Derivatives Strongly Inhibit and Disperse Pseudomonas aeruginosa Biofilms", 《ANGEWANDTE CHEMIE, INTERNATIONAL EDITION》, vol. 51, no. 21, 5 April 2012 (2012-04-05), pages 5226 - 5229, XP072079570, DOI: 10.1002/anie.201109258 *

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