Inducer for inducing transformation and reprogramming of mesenchymal cells into epithelial cells

Abstract

The present invention relates to an inducer for transforming mesenchymal cells into epithelial cells, and the pyrrolopyridine derivative is a compound for inducing the transformation of mesenchymal cells into epithelial cells, and is represented by the following formula: JPEG2026500029000078.jpg52143 having formula (I) (wherein m1, m2, A2, A3 are described herein).

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, and in particular to a group of compounds and their application in inducing transformation of mesenchymal cells into epithelial cells and in reprogramming. [Background technology]

[0002] Interconversion between epithelial and mesenchymal cells is a highly conserved and reversible cellular process in which polarized, immobile epithelial cells extend filopodia from their basal surface to generate migrating mesenchymal cells. Epithelial-mesenchymal and mesenchymal-epithelial transformations are well-known biological phenomena that play important roles not only in normal tissue and organ development but also in disease pathogenesis. The change in cell phenotype between epithelial and mesenchymal states is classified as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). These cell morphological transitions are not only central to the complex remodeling of embryonic and organ structures during gastrulation and organogenesis, but are also recognized as key events in many cancer metastases (Thiery, JP, Nat Rev Cancer. 2002;2:442-54).

[0003] During early Drosophila development, newly formed epithelial germ layers undergo complex morphogenetic movements to enable embryonic development. For example, newly formed ectodermal cells retain their epithelial phenotype (Tepass, U. Bioessays 19, 1997, 673-682; Tepass, U. & Hartenstein, V. Dev. Biol. 161, 1994:563-596). The EMT-MET cycle is also observed during mesoderm development, where these cells undergo invagination of the ventral furrow to form the dorsal vascular germ plate and Malpighian tubules (Campbell, K. et al., Mech. Dev. 2010, 127, 345-357). Development in most other metazoans progresses through a series of divisions of the fertilized egg and the stepwise assembly of epithelial-like structures (Stern, CD (ed.), Gastrulation: From Cells to Embryos (Cold Spring Harbor Laboratory Press, New York, 2004)). Therefore, MET occurs during normal developmental processes, such as somatic cell development, kidney development, heart development, liver development, and coelomogenesis (Bin L et al., PLoS One. 2011;6(2):e17092; Nakajima Y et al., Anat Rec. 2000;258:119-127). These findings suggest that although the mechanisms of MET are similar during organ morphogenesis, with a unifying trend of epithelial-related genes being upregulated and mesenchymal genes being downregulated, each process has its own unique signaling pathways for inducing MET-related changes in gene expression.

[0004] Similarly, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) also undergo EMT and MET to differentiate into somatic cell types. Differentiated somatic cells can then be reprogrammed to a pluripotent state through the EMT-MET sequence, with MET being a critical step for achieving pluripotency (Shu, X. & Pei, D. Curr. Opin. Genet. Dev. 28, 2014, 32-37). Studies have shown that MET in reprogramming achieves cell fate changes through a synergistic effect with metabolic transformations and epigenetic modifications (Wu, J., Ocampo, A. & Belmonte, JCI Cell, 2016, 166, 1371-1385). Studies have shown that MET plays a crucial role in the early somatic cell reprogramming process of mouse embryonic and human fibroblasts (Hofding, M.K. and Hyttel, P. Stem Cell Res. 2015, 14, 39-53; Subramanyam, D. et al. Nat. Biotechnol. 2011, 29, 443-448; Li, R. et al. Cell Stem Cell. 2010, 7, 51-63). In this process, the BMP4 pathway plays a key role in initiating MET, enhancing its role by activating CDH1 (encoding E-cadherin) intron 2 and the promoter CLDN4 (encoding claudin-4). Currently, protein structure prediction tools can predict compounds that bind to target proteins, and functional screening can identify compounds that upregulate target proteins. At the same time, various microRNAs (mirRNAs) such as mir-134, mir-145, mir-470, and mir-200c are highly correlated with MET, all of which negatively regulate MET (Esther E Creemers 1, Anke J Tijsen, Yigal M Pinto, Circ Res, 2012 Feb 3;110(3):483-95; Li, R. et al. Cell Stem Cell. 2010,7,51-63).Therefore, by designing inhibitory compounds based on the structure of this type of regulatory microRNA (mirRNA), it is possible to achieve positive regulation of the MET phenomenon.

[0005] Therefore, the biological phenomenon of MET plays an important role in various developmental processes. Currently, various methods for in vitro developmental control are available to reconstruct aging or dysfunctional tissues and organs, and to treat related diseases through various medical approaches. This is an important research direction in regenerative medicine, focusing on the characteristics and functional mechanisms of normal tissues, exploring the biological basis of post-traumatic repair, the mechanisms of tissue and organ regeneration, and the differentiation mechanisms of various stem cells, ultimately leading to the development of effective biological therapies. Among these, embryonic stem cells (ESCs, also abbreviated as ES, EK, or ESC cells) and induced pluripotent stem cells (induced pluripotent stem cells) are attracting the most attention. However, most developmental control methods currently involve genome modification, and various approaches, including viral vector control, all carry the potential risk of tumorigenesis. Therefore, it is particularly important to use control methods that do not alter the genome sequence. The discovery of compounds that can regulate MET could play a major role in the fields of reprogramming, cell differentiation, and tissue reconstruction, achieving safer and more flexible control goals. Summary of the Invention [Problem to be solved by the invention]

[0006] For the above reasons, the present invention designs pyrrolopyridine derivatives that can simultaneously bind to the Oct4 protein structure and the negative regulatory miRNA structure, based on the Oct4 protein structure and the negative regulatory miRNA structure that binds to the Oct4 complex. The pyrrolopyridine derivatives can chemically activate Oct4 and regulate the expression of its downstream genes. This avoids the need for viruses or other vectors to regulate Oct4, and enables the enhancement of biological expression functions using safe and convenient small chemical molecules. [Means for solving the problem]

[0007] The present invention provides a pyrrolopyridine derivative compound that can realize the biological phenomenon of MET in various cells, induce cell transformation, and simultaneously achieve the expression of epithelial cell-related genes and reprogramming of early genes, thereby providing a powerful promoter compound for chemical induction of reprogramming.

[0008] Among these, the present invention relates to a compound having the structure of formula (I) which is a pyrrolopyridine derivative, or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer or prodrug thereof, including a pharmaceutical composition of the compound having the structure of formula (I) and a highly selective Oct4 activator for cell reprogramming.

[0009] The present invention relates to a compound having the structure of formula (I): [ka] Formula (I) [In the formula, m1 and m2 are each 0 or 1; A2 is C1-C6 alkylene, C2-C6 alkenylene, -O(CH2)q-, -NR1-, -SO2-, -(CH2) V NHS(O)2- or a bond (q is 1 or 2 or 3 or 4, V is 0 or 1 or 2, and R1 is selected from H or C1-C4 alkyl); A3 is C1-C6 alkyl, C2-C6 alkenyl, C4-C6 cycloalkyl (one of the carbon atoms may be substituted with an N, O, or S heteroatom); [ka] wherein Z and Z1 are each N or CR2 (R2 is selected from H, halogen, C1-C4 alkyl, or cyano); [ka] (Z3 is N, O, S or C=O, and when the bond between Z4 and Z5 is a single bond, Z4 is N or CH, and Z5 is CH2 or C=O, and when the bond between Z4 and Z5 is a double bond, Z4 is C and Z5 is CH). or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination thereof.

[0010] In some embodiments, A2 is -CH2-, -CH=CH-, -C(CH3)=CH-, -O(CH2)-, -O(CH2)2-, -NH-, -N(CH3)-, -SO2-, -NHS(O)2-, -(CH2)2NHS(O)2-, or a bond.

[0011] In some embodiments, A3 is -CH3, butenyl, [ka] or [ka] is.

[0012] In some embodiments, m1 is 0, m2 is 1, A2 is -N(CH3)-, and A3 is [ka] is.

[0013] In some embodiments, m1 is 1, m2 is 0, A2 is —CH2—, —SO2—, —(CH2)2NHS(O)2— or a bond, and A3 is —CH3, [ka] is.

[0014] In some embodiments, m1 is 1, m2 is 1, A2 is -CH2-, -NH-, -C(CH3)=CH- or a bond, and A3 is -CH3, -C(CH3)=CH-CH3, [ka] or [ka] is.

[0015] In some embodiments, m1 is 0, m2 is 0, A2 is —CH2—, —CH═CH—, —O(CH2)—, —O(CH2)2— or a bond, and A3 is [ka] or [ka] is.

[0016] In some embodiments, the compound is [ka] is.

[0017] The present invention relates to a pharmaceutical composition comprising at least one of the compounds described above, or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination of two or more thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0018] The present invention relates to use of any one of the compounds described above and / or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination thereof in the preparation for inducing transformation of mesenchymal cells into epithelial cells.

[0019] The present invention relates to a method for activating Oct4, which comprises contacting an Oct4 target protein with any one of the compounds described above and / or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination thereof. [Brief explanation of the drawings]

[0020] [Figure 1] The upper half of Figure 1 shows the heat flux of the cell. The red peak indicates the heat generated by the binding of miR-145 to A-3-6, and the blue peak indicates the heat generated by the binding of A-3-6 to a buffer solution containing no miR-145. The lower half of Figure 1 shows the peak areas fitted to the Multiple Sites model curve. The calculated nSite values ​​are 3 and 1.8, respectively, with corresponding Kd(M) values ​​of 7.017E-8 and 7.544E-9. The blank control Kd(M) is 1.000E-3. The ITC experimental results demonstrate that the small molecules obtained in this invention can specifically bind to their targets. [Figure 2] A comparison with a control group to which a pyrrolopyridine derivative was added is shown. After cells were treated with only the pyrrolopyridine derivative for 24 hours, the MET phenomenon was induced and the cells exhibited a typical epithelial-like morphology (CK is the control group). [Figure 3] The results of basal expression of genes such as Oct4 by pyrrolopyridine derivatives are shown (CK is the control group). DETAILED DESCRIPTION OF THE INVENTION

[0021] For the purposes of the present invention, the following definitions are applicable:

[0022] The term "alkyl," as used herein, refers to a straight or branched chain saturated hydrocarbon containing 1 to 12 carbon atoms. Examples of (C1-C6)alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

[0023] The term "alkenyl" refers to a straight- or branched-chain saturated hydrocarbon containing 2 to 12 carbon atoms and containing at least one C=C double bond in the chain. Examples of alkenyl include vinyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl.

[0024] The term "alkylene" refers to a divalent alkyl. Any monovalent alkyl can be converted to an alkylene by abstracting a second hydrogen atom from the alkyl. As defined herein, alkylene can be a C1-C6 alkylene. Alkylene can further be a C1-C4 alkylene. Typical alkylenes include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like.

[0025] The term "alkenylene" refers to a divalent alkenyl. Any monovalent alkenyl can be converted to an alkenylene by abstracting a second hydrogen atom from the alkenyl. As defined herein, alkenylene can be a C2-C6 alkenylene. Typical alkenylenes include, but are not limited to, -CH=CH-, -CH=C(CH3)-, -CH=CHCH2-, -CH=CHCH2CH2-, -CH=CHCH2CH2CH2-, -CH=CHCH2CH2CH2-, and the like.

[0026] The term "cycloalkyl" refers to a monocyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. Cycloalkyl may further be a C4-C6 cycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0027] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0028] The term "cyano" refers to a substituent having a carbon atom attached to a nitrogen atom by a triple bond (ie, C≡N).

[0029] As used herein, the term "substituted" refers to the replacement of any one or more hydrogen atoms of a specified atom or group with a group selected from a specified range, provided that the replacement does not exceed the normal valence of the specified atom.

[0030] In some embodiments described herein, the present invention provides a compound of formula (I), wherein when A2 is a "bond", the structure of the compound of formula (I) is: [ka] is.

[0031] The compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures. In these cases, individual enantiomers or diastereomers, i.e., optically active configurations, can be obtained by asymmetric synthesis or by resolution of racemates or diastereomeric mixtures. Resolution of racemates or diastereomeric mixtures can be achieved by conventional methods, such as crystallization in the presence of a resolving agent or chromatography using a chiral high performance LC-MS (HPLC) column. Furthermore, these compounds include compounds with chiral centers of R- and S-configuration. These compounds also include crystalline forms, including crystalline polymorphs and clathrates. Similarly, the term "salt" also includes all isomers, racemates, other mixtures, R- and S-configurations, tautomers, and crystalline forms of salts of the compounds.

[0032] "Pharmaceutically acceptable salt" refers to a salt of a free acid or free base of a compound of Formula (I), Formula (II), or Formula (III) that is non-toxic, biologically acceptable, or otherwise biologically suitable for administration to a therapeutic subject. 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, a pharmaceutically acceptable salt refers to a salt that is pharmacologically effective and suitable for contact with a patient's tissues without unusual toxicity, irritation, or allergic response. The compounds of formula (I), formula (II), or formula (III) have sufficient acidic groups, sufficient basic groups, or both types of functional groups, and accordingly react with a number of inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, hydrogen sulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, and butyne-1,4-dioate. , hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.

[0033] A "solvate," such as a "hydrate," is formed by the interaction of a solvent with a compound. The term "compound" includes solvates of a compound, including hydrates. Similarly, a "salt" includes solvates of salts, such as hydrates. Suitable solvates are pharmaceutically acceptable solvates, for example, hydrates, including monohydrates and hemihydrates.

[0034] A "prodrug" can refer to a precursor of a specified compound obtained by converting the prodrug to a compound of formula (I) in vivo through a chemical or physiological process (e.g., solvation, enzymatic degradation) or under physiological conditions (e.g., under physiological pH conditions) after administration to a subject. A "pharmaceutically acceptable prodrug" is a prodrug that is non-toxic, biologically acceptable, 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 Prodrugs," edited by H. Bundgaard (Elsevier, 1985).

[0035] "Active metabolite" refers to a pharmaceutically active product to which a compound of Formula (I), Formula (II), or Formula (III) or a salt thereof is metabolized in vivo. Prodrugs and active metabolites of a compound can be determined using conventional techniques known or available in the art. See, for example, Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86(7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

[0036] A "therapeutically effective amount" refers to the amount of a compound disclosed herein that, when administered to a mammal (preferably a human), is sufficient to effect treatment (as defined below) for a disease or disorder in the mammal (preferably a human). The amount of a disclosed compound that constitutes a "therapeutically effective amount" will vary depending on the compound, the disorder and its severity, and the age of the mammal being treated, but can be determined routinely by one of ordinary skill in the art based on their own knowledge and the disclosures herein.

[0037] The term "treatment" refers to the administration of at least one compound described herein and / or at least one pharmaceutically acceptable salt thereof to a subject to slow (alleviate) an undesirable physiological change or disease, such as inflammation or the development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the condition, and disease remission (partial or complete), regardless of whether the disease is detected or not. "Treatment" also means prolonging survival compared to expected survival in the absence of treatment. Subjects in need of treatment include subjects with symptoms of these diseases or subjects with these diseases.

[0038] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising one or more of the compounds described herein or pharmaceutically acceptable salts or esters thereof as an active ingredient, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solutions and organic solvents, permeation enhancers, solubilizers, and adjuvants. The pharmaceutical compositions can be administered alone or together with other therapeutic agents. Such compositions can be prepared by methods well known in the pharmaceutical arts.

[0039] The pharmaceutical compositions may be administered in single or multiple doses by any method acceptable for drugs having similar uses, including rectal, buccal, intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, as an inhalant, or as an implant or coated device such as a stent, e.g., a polymeric stent inserted into an artery, as described in the patents and patent applications incorporated herein by reference.

[0040] The present invention further 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.

[0041] "Pharmaceutically acceptable carrier or excipient" refers to any other substance that is non-toxic, biologically acceptable, and biologically suitable for administration to a subject, e.g., an inert substance that is added to a pharmacological composition or that is compatible with facilitating administration of an active ingredient as a vehicle, carrier, or diluent. Examples of excipients include calcium carbonate, calcium phosphate, various types of sugars or starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0042] "Combinations of two or more thereof": As will be understood, "combinations of two or more thereof" refers to combinations of two or more of a compound, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, a pharmaceutically acceptable active metabolite thereof, a pharmaceutically acceptable crystalline polymorph thereof, a pharmaceutically acceptable ester thereof, a pharmaceutically acceptable optical isomer thereof, and a pharmaceutically acceptable prodrug thereof.

[0043] Uses of the compounds and compositions thereof The present invention provides the use of compounds and compositions thereof, which are primarily used as highly selective Oct4 activators to activate the function of Oct4, control the expression of downstream genes by chemically controlling the Oct4 promoter, and further transform mesenchymal cells into epithelial cells.

[0044] Below is a list of abbreviations used in the examples and elsewhere in this specification. CH2Cl2: dichloromethane, Cs2CO3: cesium carbonate, Cu2SO4: copper(I) sulfate, DCM: dichloromethane, DMAC: N,N-dimethylacetamide, DMF: N,N-dimethylformamide, DIEA: N,N-diisopropylethylamine, DIOX: 1,4-dioxane, EA: acetic acid, Et3N: triethylamine, ETOH: ethanol, EtOAc: ethyl acetate, FA: formic acid, g: gram, h: hour, HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HBr: hydrogen bromide, (Hbim)BF4: 1-butylimidazolium tetrafluoroborate, H2O: water, HAc: acetic acid, H2O 2: hydrogen peroxide, H2SO4: oleum, KCN: potassium cyanide, K2CO3: potassium carbonate, MCA: chloroacetic acid, MeCN: acetonitrile, MeOH: methanol, mg: milligram, mL: milliliter, mmol: millimole, mol: mole, mol / L: mole per liter, m / z: mass to charge ratio, N2: nitrogen, NaBH3CN: sodium cyanoborohydride, NaNO2: sodium nitrite, NaOH: sodium hydroxide, Na2SO4: sodium sulfate, PH: hydrogen ion index, TBN: tert-butyl nitrite, TEA: triethanolamine, THF: tetrahydrofuran, TLC: thin layer chromatography, μL: microliter; Xylene: xylene.

[0045] The technical means employed by the present invention to achieve the specified object of the invention will be further explained below with reference to the drawings and examples of the present invention. Unless otherwise specified, all experimental methods in the following examples are conventional methods. Unless otherwise specified, all raw materials, reagents, etc. used in the following examples are commercially available products.

[0046] General Synthesis The general synthetic routes described herein can be modified to obtain different corresponding products by replacing the starting materials with other materials having similar structures. The synthetic route descriptions below provide several examples of how the starting materials can be varied to obtain the corresponding products. General Method A-1-n [ka] [Wherein R is [ka] wherein q is 1, 2, 3, or 4, and Z and Z1 are each N or CR2 (wherein R2 is selected from H, halogen, C1-C4 alkyl, or cyano). General Method A-3-n [ka] [Wherein R is C2-C6 alkenyl, [ka] or [ka] wherein q is 0 or 1 or 2 or 3 or 4; Z and Z1 are each N or CR2 (R2 is selected from H, halogen, C1-C4 alkyl, or cyano); R1 is selected from H or C1-C4 alkyl; and Z3 is N, O, S, or C=0. General Method A-4-n [ka] [Wherein R is [ka] wherein q is 0 or 1 or 2 or 3 or 4, and Z and Z1 are each N or CR2 (wherein R2 is selected from H, halogen, C1-C4 alkyl, or cyano). General Method A-6-n [ka] wherein R is a C4-C6 cycloalkyl (one of the carbon atoms of which may be replaced by an N, O, or S heteroatom), or R=O is [ka] wherein q is 0 or 1 or 2 or 3 or 4, and Z and Z1 are each N or CR2 (wherein R2 is selected from H, halogen, C1-C4 alkyl, or cyano).

[0047] Intermediate synthesis Intermediate I-3: 1H-pyrrolo[2,3-c]pyridin-5-ol Synthetic Route: [ka] Step 1: Intermediate 1H-pyrrolo[2,3-c]pyridine-5-diazo I-2 To 1H-pyrrolo[2,3-c]pyridin-5-amine I-1 (66.6 mg, 0.5 mmol) was added water (2 mL) and 20% aqueous H2SO4 (1 mL). Under ice-cooling, a solution of sodium nitrite (42 mg, 0.6 mmol) in water (0.5 mL) and acetonitrile (2 mL) were added, and the mixture was stirred for 30 min. To the resulting reaction mixture was added a solution of Cu2SO4 (67 mg, 0.3 mmol) stored in 20% aqueous HBr (0.5 mL) at room temperature, and the mixture was stirred at 80 °C for 30 min. 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 the hydrogen sulfate salt of 1H-pyrrolo[2,3-c]pyridine-5-diazo I-2 (94 mg, 78%). LC-MS: m / z=145.1 [M]+. Step 2: Intermediate 1H-pyrrolo[2,3-c]pyridin-5-ol I-3 A solution (1 mL) of the hydrogen sulfate salt of 1H-pyrrolo[2,3-c]pyridine-5-diazo I-2 (48 mg, 0.2 mmol) was added dropwise to 5 mL of 40% aqueous sulfuric acid at 100 °C and stirred for 10 min. NaOH was added to the resulting reaction mixture until the pH was adjusted to approximately 3, and ethyl acetate was added to the reaction mixture. The organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform:methanol = 50:1) to give 1H-pyrrolo[2,3-c]pyridin-5-ol I-3 (20 mg, 77%). LC-MS: m / z = 135.1 [M+H]+.

[0048] Example 1: ((1H-pyrrolo[2,3-c]pyridin-5-yl)oxy)methyl)benzonitrile A-1-1 [ka] Step 1: 1H-Pyrrolo[2,3-c]pyridin-5-ol I-3 (10 mg, 0.07 mmol), 3-(bromomethyl)benzonitrile (20 mg, 0.1 mmol), and K2CO3 (138 mg, 1 mmol) were mixed, and acetonitrile (2 mL) was added. The mixture was heated to 40 °C and stirred for 10 min. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 40:1) to give 3-((1H-pyrrolo[2,3-c]pyridin-5-yl)oxy)methyl)benzonitrile A-1-1 (11 mg, 63%). LC-MS: m / z = 250.1 [M+H]+.

[0049] Example 2: 5-Phenylethoxy-1H-pyrrolo[2,3-c]pyridine A-1-2 [ka] Step 1: 1H-Pyrrolo[2,3-c]pyridin-5-ol I-3 (10 mg, 0.07 mmol), (2-bromoethyl)benzene (22 mg, 0.12 mmol), and K2CO3 (138 mg, 1 mmol) were mixed, and acetonitrile (2 mL) was added. The mixture was heated to 40 °C and stirred for 10 min. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 40:1) to give 5-phenylethoxy-1H-pyrrolo[2,3-c]pyridine A-1-2 (13 mg, 78%). LC-MS: m / z = 239.1 [M+H]+.

[0050] Example 3: (1H-Pyrrolo[2,3-c]pyridin-5-yl)isoindol-1-one A-2 [ka] Synthetic Route: [ka] Step 1: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) and 2-formylbenzoic acid (18 mg, 0.12 mmol) were mixed, and formic acid (0.2 mL), triethylamine (1 mL), and ethanol (1 mL) were added. The mixture was heated to 80 °C and stirred for 60 min. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate = 20:1) to give (1H-pyrrolo[2,3-c]pyridin-5-yl)isoindol-1-one A-2 (15 mg, 60%). LC-MS: m / z = 250.3 [M+H]+.

[0051] Example 4: (1H-pyrrolo[2,3-c]pyridin-5-yl)thiophene-3-carboxamide A-3-1 [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), thiophene-2-carboxylic acid (15 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give N-(1H-pyrrolo[2,3-c]pyridin-5-yl)thiophene-3-carboxamide A-3-1 (10 mg, 41%). LC-MS: m / z=244.1 [M+H]+.

[0052] Example 5: (E)-2-methyl-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)but-2-enamide A-3-2 [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), (E)-2-methyl-2-enoic acid (12 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give (E)-2-methyl-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)but-2-enamide A-3-2 (11 mg, 51%). LC-MS: m / z=216.1 [M+H]+.

[0053] Example 6: N-(1H-pyrrolo[2,3-c]pyridin-5-yl)-2,3-dihydrobenzofuran-2-carboxamide A-3-3 [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), benzofuran-2-carboxylic acid (19 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give N-(1H-pyrrolo[2,3-c]pyridin-5-yl)-2,3-dihydrobenzofuran-2-carboxamide A-3-3 (13 mg, 46%). LC-MS: m / z=280.1 [M+H]+.

[0054] Example 7: 3,4-Dichloro-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)benzamide A-3-4 [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), 3,4-dichlorobenzoic acid (23 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give 3,4-dichloro-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)benzamide A-3-4 (13 mg, 43%). LC-MS: m / z=306.0 [M+H]+.

[0055] Example 8: N-(1H-pyrrolo[2,3-c]pyridin-5-yl)benzothiophene-2-carboxamide A-3-5 [ka] Step 1: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), benzothiophene-2-carboxylic acid (21 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give N-(1H-pyrrolo[2,3-c]pyridin-5-yl)benzothiophene-2-carboxamide A-3-5 (14 mg, 48%). LC-MS: m / z=294.1 [M+H]+.

[0056] Example 9: 2-phenyl-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)acetamide A-3-6 [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol), 2-phenylacetic acid (16 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to give 2-phenyl-N-(1H-pyrrolo[2,3-c]pyridin-5-yl)acetamide A-3-6 (12 mg, 48%). LC-MS: m / z=252.1 [M+H]+.

[0057] Example 10: 1-(1H-pyrrolo[2,3-c]pyridin-5-yl)-3-(p-tolyl)urea A-4-1 [ka] Step 1: p-Nitrophenyl p-tolueneaminocarbonate I-9-1 [ka] p-Toluidine (21 mg, 0.2 mmol) and 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol) were mixed, and triethanolamine (0.2 mL), dichloromethane (1 mL), and tetrahydrofuran (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:hexane = 5:1) to give p-nitrophenyl p-tolueneaminocarbonate I-9-1 (42 mg, 77%). LC-MS: m / z = 273.1 [M+H]+. Step 2: 1-(1H-pyrrolo[2,3-c]pyridin-5-yl)-3-(p-tolyl)urea A-4-1 The above p-nitrophenyl p-tolueneaminocarbonate I-9-1 (27 mg, 0.1 mmol), 1H-pyrrolo[2,3-c]pyridin-5-amine I-1 (16 mg, 0.12 mmol), and K2CO3 (0.2 g, 1.45 mmol) were mixed, and acetonitrile (3 mL) was added. The mixture was heated to 40 °C and stirred for 4 h. After completion of the reaction (monitored by TLC), the reaction mixture was separated 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 from EtOAc (at warming) to give pure 1-(1H-pyrrolo[2,3-c]pyridin-5-yl)-3-(p-tolyl)urea A-4-1 (22 mg, 83%). LC-MS: m / z = 267.1 [M+H]+.

[0058] Example 11: 1-(3-bromophenyl)-3-(1H-pyrrolo[2,3-c]pyridin-5-yl)urea A-4-2 [ka] Step 1: 4-Nitrophenyl(3-bromophenyl)aminocarbonate I-9-2 [ka] 3-Bromoaniline (34 mg, 0.2 mmol) and 4-nitrophenylcarbonyl chloride (48 mg, 0.24 mmol) were mixed, and triethanolamine (0.2 mL), dichloromethane (1 mL), and tetrahydrofuran (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:hexane = 5:1) to give 4-nitrophenyl(3-bromophenyl)aminocarbonate I-9-2 (46 mg, 68%). LC-MS: m / z = 338.1 [M+H]+. Step 2: 1-(3-bromophenyl)-3-(1H-pyrrolo[2,3-c]pyridin-5-yl)urea A-4-2 The above 4-nitrophenyl(3-bromophenyl)aminocarbonate I-9-2 (34 mg, 0.1 mmol), 1H-pyrrolo[2,3-c]pyridin-5-amine I-1 (16 mg, 0.12 mmol), and K2CO3 (0.2 g, 1.45 mmol) were mixed, and acetonitrile (3 mL) was added. The mixture was heated to 40 °C and stirred for 4 h. After completion of the reaction (monitored by TLC), the reaction mixture was separated 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 from EtOAc (at warming) to give pure 1-(3-bromophenyl)-3-(1H-pyrrolo[2,3-c]pyridin-5-yl)urea A-4-2 (25 mg, 75%). LC-MS: m / z = 331.2 [M+H]+.

[0059] Example 12: N-methyl-N-phenyl-1H-pyrrolo[2,3-c]pyridine-5-carboxamide A-5 [ka] Synthetic Route: [ka] Step 1: 5-Nitro-1H-pyrrolo[2,3-c]pyridine I-10 [ka] To a solution of HO (240 mg, 2.2 mmol) in fuming sulfuric acid (500 μL), a solution of 1H-pyrrolo[2,3-c]pyridin-5-amine I-1 (40 mg, 0.3 mmol) in concentrated sulfuric acid (100 μL) was added dropwise while maintaining the reaction temperature at 0 °C. After stirring at 10–25 °C for 3 h, the reaction mixture was adjusted to pH = 11–12 by adding 40% aqueous NaOH at 0–5 °C. The resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over NaSO, and filtered. The solvent was removed under reduced pressure to give the desired 5-nitro-1H-pyrrolo[2,3-c]pyridine I-10 (39 mg, 80%). LC-MS: m / z = 164.0 [M+H]. Step 2: 1H-Pyrrolo[2,3-c]pyridine-5-carboxylic acid I-11 [ka] The above 5-nitro-1H-pyrrolo[2,3-c]pyridine I-10 (39 mg, 0.24 mmol) was mixed with KCN (195 mg, 3 mmol), 1-butylimidazolium tetrafluoroborate (3 mg, 0.014 mmol), EtOH (2 mL), and water (2 mL) were added. The mixture was heated to 80 °C and stirred for 19 h. Next, 10 mL of water was added, and the mixture was extracted with CHCl (3 × 5 mL) and diethyl ether (3 × 10 mL). The mixture was acidified with hydrochloric acid to a pH of 1–2 and extracted with diethyl ether (3 × 10 mL). Magnesium sulfate (3 g) and activated carbon (1 g) were added, and the mixture was stirred for 5 h. The solid was filtered, the filtrate was evaporated, and the residue was crystallized from the corresponding solvent to give the desired 1H-pyrrolo[2,3-c]pyridine-5-carboxylic acid I-11 (16 mg, 41%). LC-MS: m / z=163.0 [M+H]+. Step 3: N-methyl-N-phenyl-1H-pyrrolo[2,3-c]pyridine-5-carboxamide A-5 The above 1H-pyrrolo[2,3-c]pyridine-5-carboxylic acid I-11 (16 mg, 0.1 mmol), N-methylaniline (13 mg, 0.12 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.1 mmol) were mixed, and N,N-diisopropylethylamine (0.2 mL) and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7). The solvent was evaporated under reduced pressure to give N-methyl-N-phenyl-1H-pyrrolo[2,3-c]pyridine-5-carboxamide A-5 (18 mg, 72%). LC-MS: m / z=252.1 [M+H]+.

[0060] Example 13: N-(tetrahydro-2H-pyran-4-yl)-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-1 [ka] 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) and tetrahydro-4H-pyran-4-one (12 mg, 0.12 mmol) were mixed, added with methanol (2 mL), and stirred at room temperature for 2 hours. Sodium cyanoborohydride (20 mg, 0.3 mmol) was then added and stirred at room temperature for 2 hours. Next, aqueous NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1), and the solvent was removed under reduced pressure to give N-(tetrahydro-2H-pyran-4-yl)-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-1 (18 mg, 83%). LC-MS: m / z=218.1 [M+H]+.

[0061] Example 14: N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-2 [ka] 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) and isonicotinaldehyde (13 mg, 0.12 mmol) were mixed, added with methanol (2 mL), and stirred at room temperature for 2 hours. Sodium cyanoborohydride (20 mg, 0.3 mmol) was then added and stirred at room temperature for 2 hours. Next, aqueous NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1), and the solvent was removed under reduced pressure to give N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-2 (16 mg, 71%). LC-MS: m / z=225.1 [M+H]+.

[0062] Example 15: N-Cyclobutyl-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-3 [ka] 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) and cyclobutanone (8 mg, 0.12 mmol) were mixed, added with methanol (2 mL), and stirred at room temperature for 2 hours. Then, sodium cyanoborohydride (20 mg, 0.3 mmol) was added and stirred at room temperature for 2 hours. Next, aqueous NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1), and the solvent was removed under reduced pressure to give N-cyclobutyl-1H-pyrrolo[2,3-c]pyridin-5-amine A-6-3 (15 mg, 80%). LC-MS: m / z = 188.1 [M+H]+.

[0063] Example 16: 2-(1H-pyrrolo[2,3-c]pyridin-5-yl)isoindole-1,3-dione A-7 [ka] Synthetic Route: [ka] Step 1: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) and isobenzofuran-1,3-dione (18 mg, 0.12 mmol) were mixed, N,N-dimethylacetamide (2 mL) was added, and the mixture was stirred at room temperature for 24 h. After that, xylene (1 mL) was added and the mixture was stirred in an oil bath at 140 °C for 48 h. After 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, which was washed with water and recrystallized in ethanol. The crude product was purified by silica gel column chromatography (CHCl / n-hexane = 1:1). The solvent was evaporated under reduced pressure to give 2-(1H-pyrrolo[2,3-c]pyridin-5-yl)isoindole-1,3-dione A-7 (20 mg, 76%). LC-MS: m / z=264.1 [M+H]+.

[0064] Example 17: N-phenyl-1H-pyrrolo[2,3-c]pyridin-5-amine A-8 [ka] Synthetic Route: [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) was mixed with iodobenzene (24 mg, 0.12 mmol), (N,N-bipyridylimidazolidene)copper dibromide (10 mg, 0.023 mmol), and cesium carbonate (100 mg, 0.3 mmol). 1,4-Dioxane (5 mL) was added and the mixture was stirred in an oil bath at 170 °C for 12 h. Next, aqueous NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL × 3). The combined organic phase was dried over anhydrous NaSO, 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 evaporated under reduced pressure to give N-phenyl-1H-pyrrolo[2,3-c]pyridin-5-amine A-8 (19 mg, 91%). LC-MS: m / z=210.1 [M+H]+.

[0065] Example 18: (E)-5-Phenylvinyl-1H-pyrrolo[2,3-c]pyridine A-9 [ka] Synthetic Route: [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) was mixed with styrene (13 mg, 0.12 mmol) and bis(dibenzylideneacetone)-palladium(0) (5 mg, 0.0087 mmol), and tert-butyl nitrite (0.5 mL), chloroacetic acid (0.5 mL), and acetic acid (3 mL) were added. The mixture was stirred in an oil bath at 50 °C for 2 h. Next, aqueous NaOH (10 mL, 0.3 mol / L) was added to the mixture. The resulting mixture was extracted with DCM (20 mL × 3). The combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1), and the solvent was evaporated under reduced pressure to give (£)-5-phenylvinyl-1H-pyrrolo[2,3-c]pyridine A-9 (15 mg, 68%). LC-MS: m / z=221.1 [M+H]+.

[0066] Example 19: N-(1H-pyrrolo[2,3-c]pyridin-5-yl)thiophene-2-sulfonamide A-10 [ka] Synthetic Route: [ka] Step: 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (13 mg, 0.1 mmol) was mixed with thiophene-2-sulfonyl chloride (22 mg, 0.12 mmol), triethylamine (0.5 mL), and dichloromethane (3 mL) were added, and the mixture was stirred at room temperature for 24 hours under nitrogen protection. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7), and the solvent was removed under reduced pressure to give N-(1H-pyrrolo[2,3-c]pyridin-5-yl)thiophene-2-sulfonamide A-10 (14 mg, 50%). LC-MS: m / z = 280.0 [M+H]+.

[0067] Example 20: N-(2-((1H-pyrrolo[2,3-c]pyridin-5-yl)amino)ethyl)methanesulfonamide A-11 [ka] Synthetic Route: [ka] Step 1: N1-(1H-pyrrolo[2,3-c]pyridin-5-yl)ethane-1,2-diamine I-19 [ka] 1H-Pyrrolo[2,3-c]pyridin-5-amine I-1 (26 mg, 0.2 mmol) was mixed with 2-bromoethan-1-amine I-18 (29 mg, 0.24 mmol), water (5 mL) was added, and the mixture was stirred in an oil bath at 95 °C for 18 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The mixture was purified by flash chromatography on silica gel (CHCl methanol-aqueous ammonia) to give the desired product. The solvent was removed under reduced pressure to give N-(1H-pyrrolo[2,3-c]pyridin-5-yl)ethane-1,2-diamine I-19 (24 mg, 68%). LC-MS: m / z = 177.1 [M+H]. Step 2: N-(2-((1H-pyrrolo[2,3-c]pyridin-5-yl)amino)ethyl)methanesulfonamide A-11 The above N1-(1H-pyrrolo[2,3-c]pyridin-5-yl)ethane-1,2-diamine I-19 (24 mg, 0.13 mmol) was mixed with methanesulfonyl chloride I-20 (28 mg, 0.16 mmol), and dichloromethane (5 mL) was added. The mixture was stirred in an ice-water bath for 24 hours. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7). The solvent was removed under reduced pressure to give N-(2-((1H-pyrrolo[2,3-c]pyridin-5-yl)amino)ethyl)methanesulfonamide A-11 (12 mg, 36%). LC-MS: m / z = 255.1 [M+H]+.

[0068] Example 21: The present invention employs two compound prediction methods to predict pyrrolopyridine derivatives, as follows: Method 1: Protein structure docking prediction Using AutoDock Vina and LeDock software, the pyrrolopyridine derivative molecules in Examples 1-20 were docked with the Oct4 target protein molecule, respectively, to generate 10 docking conformations. The binding energy and ligand efficiency of the optimal docking results for each pyrrolopyridine molecule with the Oct4 target protein were calculated, and pyrrolopyridine molecules were screened based on the docking results. Specific docking results are shown in Table 1. Columns 2 and 3 of the table show the binding free energies calculated by AutoDock Vina and LeDock molecular docking software, respectively. The more negative the value, the stronger the binding ability of the small molecule ligand with the target protein. Column 4 of the table shows the ligand efficiency calculated by LeDock. The larger the absolute value, the higher the potential of the small molecule. In this invention, the binding energy levels of the compounds of the present invention were predicted based on independent algorithms from the two software programs. The predicted values ​​indicate that the binding energies of the compounds of the present invention are all significantly greater than the threshold value 3, which was established based on the target properties of the present invention.

[0069] Table 1. Predicted binding energy between the pyrrolopyridine derivative molecules used in the present invention and the target sequence [Table 1]

[0070] Method 2: mir-RNA structure docking prediction: Affinity prediction between miR-145 and small molecules was performed using a new computational tool, RLDOCK software. RLDOCK is a ligand-RNA binding ability prediction model based on physical deformation. A new global multi-step binding site search algorithm was employed to optimize search efficiency and stability, enabling the algorithm to effectively predict unknown binding sites. RLDOCK's multi-step calculation first thoroughly scans all possible binding sites on the miR-145 structure and all possible binding poses of small molecules at those sites. All binding possibilities are scored and ranked using a minimum Lennard-Jones potential energy scoring function. The results are shown in Table 2. The scoring results are negative, and the higher the absolute value, the higher the ligand-RNA affinity. The final score by RLDock is based on the sum of the various energy functions employed. Currently, the number of validated and available RNA targets is limited, and no mature computational system exists that can distinguish between actual interaction thresholds. This method ranks molecules based on their predicted binding energy, and higher-ranked molecules are prioritized for validation.

[0071] Table 2: Predicted binding energies of pyrrolopyridine derivatives with target mir-RNAs [Table 2]

[0072] The Oct4 protein structure docking prediction and the mir-145 structure docking prediction showed that pyrrolopyridine derivatives showed large binding energies in both structure docking predictions. Therefore, it was inferred that pyrrolopyridine derivatives have the ability to enhance Oct4 expression. Therefore, the expression-enhancing effect of pyrrolopyridine derivatives on Oct4 was verified by isothermal titration calorimetry and cytological experiments, and therefore the effect of pyrrolopyridine derivatives as highly selective activators of Oct4 was verified.

[0073] Example 22: Validating the interaction of small molecule compounds with target nucleic acids using isothermal titration calorimetry Isothermal titration calorimetry (ITC) utilizes the principle of power compensation to accurately capture the enthalpy change of intermolecular interactions occurring within a wide concentration range within a system through a single concentration scan, measuring the heat change upon binding and providing a complete thermodynamic characterization of nucleic acid-ligand interactions. Quantifying the thermodynamic properties and energy of the complex provides a molecular basis for analyzing the interaction between the micronucleic acid miR-145 and compounds. This invention uses a Waters Nano-ITC titration calorimeter to examine nucleic acid-small molecule interactions in vitro. The experimental procedure is as follows: A 300 μL volume of single-stranded miR-145 solution at a concentration of 50 μM was placed in a temperature-controlled cell and coupled to an equal volume of deionized water in a reference cell via a thermocouple loop. For example, a 50 μL volume of the A-3-6 small molecule at a concentration of 500 μM was placed in a syringe as the ligand. The heat flux of the cell was measured and displayed as an endothermic or exothermic peak by equilibrium calculation with a reference cell. The software provided with the instrument fitted the interaction patterns of both reactants, allowing direct calculation of the reaction binding enthalpy (ΔH), constant-pressure heat capacity (ΔCp), number of binding sites (n), and binding equilibrium constant (Ka) between two or more molecules in solution. These data can then be used to obtain kinetic data. The upper half of Figure 1 shows the heat flux of the cell. The red peak represents the heat generated by the binding of miR-145 to A-3-6, while the blue peak represents the heat generated by the binding of A-3-6 to a buffer solution containing no miR-145. The lower half of Figure 1 shows the peak areas fitted to the Multiple Sites model curve. The calculated nSite values ​​are 3 and 1.8, respectively, with corresponding Kd (M) values ​​of 7.017E-8 and 7.544E-9, and the blank control Kd (M) is 1.000E-3. The ITC experiment results demonstrate that the small molecules obtained in the present invention can specifically bind to targets.

[0074] Example 23: Verification of morphological changes in MET cells by small molecule compounds Human mesenchymal cells were cultured in a T25 tube, and 4 × 10 cells were added. 5The cells were inoculated and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM-F12) supplemented with 20 μM of the pyrrolopyridine derivative small molecule at 37°C and 5% carbon dioxide. Figure 2 shows that after 24 hours of treatment with the pyrrolopyridine derivative alone, MET was induced, and the cells exhibited a typical epithelial morphology, compared to the control group without the pyrrolopyridine derivative. These results morphologically demonstrate that the pyrrolopyridine derivative can rapidly induce the transformation of mesenchymal cells into epithelial cells.

[0075] Example 24: Verification of differences in transcriptional expression caused by small molecules The objective of the present invention is to achieve the effect of MET transformation using specific compounds, and the important function of such activators is to enhance the expression of MET transformation. According to previous literature reports, when verifying the function of the small molecules of the present invention, the structural protein KRT family formed with MET and the regulatory gene MSX series were used as detection indicators, and the results showed that pyrrolopyridine and its derivatives can induce the occurrence of MET phenomenon. Increased expression of downstream genes of the Oct4 gene is also an indicator for verifying the function of the compounds of the present invention. The present invention further verifies the Oct4 activation function of pyrrolopyridine and its derivatives by reprogramming the expression of early genes using Nanog.

[0076] Human mesenchymal cells were cultured in a T25 tube, and 4 × 10 cells were added. 5The cells were inoculated and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM-F12 medium) supplemented with 50 nM of the pyrrolopyridine derivative small molecule at 37°C and 5% CO2. On day 3, total RNA was extracted using the RNeasy Mini or Micro Kit (QIAGEN), and cDNA was synthesized from 1 mg of RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed using SYBR Premix Ex Taq (TaKaRa) and the Thermal Cycler Dice Real Time System (TaKaRa), with beta-Actin used as an internal control. All data were analyzed using the delta-Ct method. Each experiment was repeated in three groups, and variance statistics were performed. The primer sequences for the coding genes used to identify different cell markers are listed in Table 3. The results are shown below. Figure 3 shows that the expression of genes such as Oct4 was significantly increased by the pyrrolopyridine derivative small molecule compared to the control group without the small molecule.

[0077] Table 3. Compound effect gene QPCR primer sequences [Table 3]

[0078] (Addendum) (Appendix 1) An inducer for transforming mesenchymal cells into epithelial cells, comprising: The inducer is a compound of the structure of formula (I): [ka] Formula (I) [In the formula, m1 and m2 are each 0 or 1; A2 is C1-C6 alkylene, C2-C6 alkenylene, -O(CH2)q-, -NR1-, -SO2-, -(CH2) VNHS(O)2- or a bond (q is 1 or 2 or 3 or 4, V is 0 or 1 or 2, and R1 is selected from H or C1-C4 alkyl); A3 is C1-C6 alkyl, C2-C6 alkenyl, C4-C6 cycloalkyl (one of the carbon atoms may be substituted with an N, O, or S heteroatom); [ka] wherein Z and Z1 are each N or CR2 (R2 is selected from H, halogen, C1-C4 alkyl, or cyano); [ka] (Z3 is N, O, S or C=O, and when the bond between Z4 and Z5 is a single bond, Z4 is N or CH, and Z5 is CH2 or C=O, and when the bond between Z4 and Z5 is a double bond, Z4 is C and Z5 is CH). or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, or prodrug thereof.

[0079] (Appendix 2) In the compound, An inducer described in Appendix 1, characterized in that A2 is -CH2-, -CH=CH-, -C(CH3)=CH-, -O(CH2)-, -O(CH2)2-, -NH-, -N(CH3)-, -SO2-, -NHS(O)2-, -(CH2)2NHS(O)2- or a bond.

[0080] (Appendix 3) In the compound, A3 is -CH3, butenyl, [ka] The inducer according to claim 2, wherein

[0081] (Appendix 4) In the compound, m1 is 0 and m2 is 1; A2 is -N(CH3)-, A3 is [ka] The inducer according to claim 3, wherein

[0082] (Appendix 5) In the compound, m1 is 1 and m2 is 0; A2 is —CH2—, —SO2—, —(CH2)2NHS(O)2— or a bond; A3 is -CH3, [ka] The inducer according to claim 3, wherein

[0083] (Appendix 6) In the compound, m1 is 1, m2 is 1, A2 is -CH2-, -NH-, -C(CH3)=CH- or a bond; A3 is -CH3, -C(CH3)=CH-CH3, [ka] The inducer according to claim 3, wherein

[0084] (Appendix 7) In the compound, m1 is 0, m2 is 0, A2 is -CH2-, -CH=CH-, -O(CH2)-, -O(CH2)2- or a bond; A3 is [ka] The inducer according to claim 3, wherein

[0085] (Appendix 8) The compound is [ka] The inducer according to claim 1, wherein the inducer is selected from the group consisting of:

[0086] (Appendix 9) A pharmaceutical composition comprising at least one of the inducer according to any one of Appendices 1 to 8, or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination of two or more thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0087] (Appendix 10) Use of the inducer according to any one of Appendices 1 to 8 and / or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination thereof in the preparation for inducing transformation of mesenchymal cells into epithelial cells.

Claims

1. An inducer for transforming mesenchymal cells into epithelial cells, comprising: The inducer is a compound of the structure of formula (I): 【Chemistry 1】 Formula (I) [In the formula, m1 and m2 each represent 0 or 1; A ２ is C １ ~C ６ Alkylene, C ２ ~C ６ Alkenylene, —O(CH ２ ) q-, -NR １ -, -SO ２ -, -(CH ２ ) Ｖ NHS (O) ２ - or a bond (q is 1 or 2 or 3 or 4, V is 0 or 1 or 2, R １ is H or C １ ~C ４ alkyl), A ３ is C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ４ ~C ６ cycloalkyl (one of whose carbon atoms may be replaced by an N, O, S heteroatom); 【Chemistry 2】 (Z and Z １ are N or CR respectively ２ (R ２ is H, halogen, C １ ~C ４ alkyl or cyano) 【Transformation 3】 (Z ３ is N, O, S or C=O, and Z ４ and Z ５ When the bond between Z ４ is N or CH, and Z ５ is CH ２ or C═O, and Z ４ and Z ５ When the bond between Z ４ is C and Z ５ is CH) or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, or prodrug thereof.

2. In the compound, 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. In the compound, A ３ Ha-CH ３ , butenyl, 【Chemistry 4】 The inducer according to claim 2, characterized in that:

4. In the compound, m1 is 0 and m2 is 1; A ２ HA-N(CH ３ ) - and A ３ teeth 【Transformation 5】 The inducer according to claim 3, wherein

5. In the compound, m1 is 1 and m2 is 0; A ２ Ha-CH ２ -, -SO ２ -, -(CH ２ ) ２ NHS (O) ２ - or a bond, A ３ Ha-CH ３ , 【Transformation 6】 The inducer according to claim 3, characterized in that:

6. In the compound, m1 is 1 and m2 is 1; A ２ Ha-CH ２ -, -NH-, -C(CH ３ )=CH— or a bond, A ３ は-CH ３ 、-C(CH ３ )=CH-CH ３ 、 【Transformation 7】 The inducer according to claim 3, wherein

7. In the compound, m1 is 0, m2 is 0, A ２ Ha-CH ２ -, -CH=CH-, -O(CH ２ ) -, -O(CH ２ ) ２ - or a bond, A ３ teeth 【Transformation 8】 The inducer according to claim 3, wherein

8. The compound is 【Chemistry 9】 The inducer according to claim 1, characterized in that it is selected from the group consisting of:

9. A pharmaceutical composition comprising at least one of the inducer according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination of two or more thereof, and at least one pharmaceutically acceptable carrier or excipient.

10. Use of the inducer according to any one of claims 1 to 8 and / or a pharmaceutically acceptable salt, solvate, active metabolite, crystalline polymorph, ester, optical isomer, prodrug, or combination thereof in preparation for inducing transformation of mesenchymal cells into epithelial cells.

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