Applications of BMPR1B inhibitors in growth induction, and induction media.

Abstract

This application relates to the biopharmaceutical technology, and more particularly to the applications, induction media, and induction methods for the growth induction of BMPR1B inhibitors. The induction media comprises a BMPR1B inhibitor having the structure represented by formula I, or a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula I. JPEG2026517509000028.jpg49170

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical technology, and in particular, to the use of BMPR1B inhibitors in growth induction and induction media.

Background Art

[0002] Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily and plays an extremely important role in regulating cell growth and differentiation. There are mainly two types of TGF-β cell membrane receptors, namely TGF-β type I receptor and TGF-β type II receptor. In vertebrates, there are seven types of type I receptors (Activin-receptor like kinases), namely ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), ALK6 (BMPR1B) and ALK7 (ACVR1C), and five types of type II receptors, namely TGFβRII, ACTRII, ACTRIIB, BMPRII, AMHRII.

[0003] Among these, ALK6 belonging to the type I receptor, that is, BMPR1B (bone morphogenetic protein receptor, type IB), is an important transmembrane receptor protein that plays an important role in the control of bone formation differentiation, cell proliferation and follicle development. Therefore, artificially controlling BMPR1B to control the expression of downstream genes and physiological processes has become a research hotspot in the field of biopharmaceuticals.

Summary of the Invention

Problems to be Solved by the Invention

[0004] In view of the above, this application provides an induction medium, an induction method and the use of a BMPR1B inhibitor. [Means for solving the problem]

[0005] A first aspect of this application provides an induction medium containing a BMPR1B inhibitor, The BMPR1B inhibitor is having the structure represented by formula I, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula I. [ka] (In the formula, ring A is selected from one of the following structures, [ka] Y is -(CH2) n - and n is 0, 1, 2 or 3, R 1 is -H, -D, unsubstituted or R 2 Substitutions of C1-C6 alkyl, C1-C6 alkoxy, and C6-C 10 Aryl or C3~C 10 Selected from heteroaryls, R 2 These are selected from -F, -Cl, -Br, -I, -OH, -COOH, C1-C6 alkyl, or C1-C6 alkoxy. The asterisk (*) indicates a connecting point.

[0006] A second aspect of this application provides a method for differentiating induced pluripotent stem cells into neural lineage cells. The method includes the step of adhering pluripotent stem cells to the induction medium to obtain neural lineage cells.

[0007] A third aspect of this application provides the use of the BMPR1B inhibitor in the differentiation of induced pluripotent stem cells into neural lineage cells. [Brief explanation of the drawing]

[0008] [Figure 1]This shows the specificity of target binding of pyrazolecarbonylpiperazinone compounds based on kinase activity screening analysis. Figure 1A shows the effect of compound II-1 on the activity of 330 kinases, and Figures 1B and 1C show the results of kinase activity tests on TGFBR1 and BMPR2 at different concentrations of compound II-1. [Figure 2] The PyMol software displays information on the interaction between the compound and BMPR1B in the pyrazolecarbonylpiperazinone compound-BMPR1B complex. Of these, the compound in Figure 2A is II-13, the compound in Figure 2B is II-11, the compound in Figure 2C is II-2, and the compound in Figure 2D is II-1. [Figure 3] The specific details of the Western blot experiment in Example 3 are shown, with "12hr" indicating 12 hours, "24hr" indicating 24 hours, and "36hr" indicating 36 hours. [Figure 4] The figures show the changes in mRNA expression levels of marker genes after treating H9 cells with different concentrations of compound II-1. Figure 4A shows the mRNA expression level of the NANOG gene 7 days after treatment, Figure 4B shows the mRNA expression level of the PAX6 gene 7 days after treatment, Figure 4C shows the mRNA expression level of the NANOG gene 14 days after treatment, and Figure 4D shows the mRNA expression level of the PAX6 gene 14 days after treatment. [Figure 5] The figures show the changes in mRNA expression levels of marker genes after treating H1 cells with different concentrations of compound II-13. Figure 5A shows the mRNA expression level of the MAP2 gene 7 days after treatment, Figure 5B shows the mRNA expression level of the SOX9 gene 7 days after treatment, Figure 5C shows the mRNA expression level of the MAP2 gene 14 days after treatment, and Figure 5D shows the mRNA expression level of the SOX9 gene 14 days after treatment. [Figure 6]Figure 6A shows the changes in mRNA expression levels of marker genes after treating induced pluripotent stem cells (iPSCs) with different concentrations of compound II-13. Figure 6B shows the mRNA expression level of the SOX5 gene 7 days after treatment, Figure 6C shows the mRNA expression level of the VIMENTIN (VIM) gene 7 days after treatment, Figure 6D shows the mRNA expression level of the SOX1 gene 14 days after treatment, Figure 6E shows the mRNA expression level of the SOX5 gene 14 days after treatment, and Figure 6F shows the mRNA expression level of the VIMENTIN (VIM) gene 14 days after treatment. [Modes for carrying out the invention]

[0009] To facilitate understanding of this application, a more comprehensive description of this application will be provided below with reference to the relevant drawings. While the drawings illustrate preferred embodiments of this application, it can be implemented in a variety of forms and is not limited to the embodiments described herein. Rather, the purpose of presenting these embodiments is to provide a more complete and comprehensive understanding of the disclosures of this application.

[0010] Unless otherwise specified, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art. The terms used herein are for illustrative purposes of specific embodiments and are not intended to limit this application. The terms “and / or” as used herein encompass any one or more of the listed items, or any combination thereof.

[0011] In this application, the disclosed technical features include confidential technical solutions consisting of the listed features, as well as open technical solutions that include the listed features.

[0012] In this application, unless otherwise specified, numerical ranges are deemed to be continuous within that range and include the minimum and maximum values ​​of that range, as well as all values ​​between the minimum and maximum values. Furthermore, if a range refers to an integer, it includes all integers between the minimum and maximum values ​​of that range. In addition, for features or characteristics to which multiple ranges are assigned, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein are understood to include any and all subranges contained therein.

[0013] In this application, unless otherwise specified, percentage content refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0014] In this application, unless otherwise specified, percentage concentrations refer to the final concentration. The final concentration, as used herein, means the proportion of the component in the system after its addition.

[0015] Unless otherwise specified, the temperature parameters in this application may be constant temperature processing or processing within a specific temperature range. Constant temperature processing, as used herein, means that temperature fluctuations are permitted within a precision range controlled by the equipment.

[0016] (Explanation of terms) The term "alkyl" refers to a monovalent residue produced by the loss of one hydrogen atom from a saturated hydrocarbon containing a primary (n) carbon atom, secondary carbon atom, tertiary carbon atom, quaternary carbon atom, or a combination thereof. Expressions containing this term, such as "C1-C6 alkyl," refer to alkyl groups containing 1 to 6 carbon atoms, and each instance may independently be one of C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. Suitable examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), and 2-butyl (s-Bu, s-butyl, -CH(CH3 )CH2CH3), 2-methyl-2-propyl(t-Bu, t-butyl, -C(CH3)3), 1-pentyl(n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl(-CH(CH3)CH2CH2CH3), 3-pentyl(-CH(CH2CH3)2), 2-methyl-2-butyl(-C(CH3)2CH2CH3), 3-methyl-2-butyl(-CH(CH3)CH(CH3)2), 3-methyl-1-butyl(-C H2CH2CH(CH3)2), 2-methyl-1-butyl(-CH2CH(CH3)CH2CH3), 1-hexyl(-CH2CH2CH2CH2CH2CH3), 2-hexyl(-CH(CH3)CH2CH2CH2CH3), 3-hexyl(-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl(-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl(-CH(CH3)CH(CH3)) Examples include, but are not limited to, CH2CH3, 4-methyl-2-pentyl(-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl(-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl(-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl(-C(CH3)2CH(CH3)2), and 3,3-dimethyl-2-butyl(-CH(CH3)C(CH3)3).

[0017] The term "alkoxy" refers to a group having an -O-alkyl structure, that is, a group in which the alkyl group defined above is bonded to an adjacent group through an oxygen atom. Expressions containing this term, such as "C1-C6 alkoxy", refer to an alkyl moiety containing 1 to 6 carbon atoms, and each time it appears, it may independently be any of C1 alkoxy, C4 alkoxy, C5 alkoxy, or C6 alkoxy. Suitable examples include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-O-CH2CH3 or -OEt), and tert-butoxy (-OC(CH3)3 or -OtBu).

[0018] "Aryl" refers to an aromatic hydrocarbon group derived by removing one hydrogen atom from an aromatic ring compound, and may be any of monocyclic aryl, fused-ring aryl, or polycyclic aryl. In the case of polycyclic aryl, at least one is an aromatic ring system. For example, "C6-C 10 aryl" refers to an aryl group containing 6 to 10 carbon atoms, and each time it appears, it may independently be any of a C6 aryl group, a C7 aryl group, a C8 aryl group, a C9 aryl group, or a C 10 aryl group. Suitable examples include, but are not limited to, benzene, biphenyl, naphthalene, and their derivatives.

[0019] "Heteroaryl" refers to a group in which at least one carbon atom in an aryl group or a cyclopentadienyl group is replaced by a non-carbon atom such as an N atom, an O atom, or an S atom. For example, "C3-C 10"Heteroaryl" refers to a heteroaryl group containing 3 to 10 carbon atoms, and each instance may independently be a C3 heteroaryl, C4 heteroaryl, C5 heteroaryl, C6 heteroaryl, C7 heteroaryl, or C8 heteroaryl. Suitable examples include, but are not limited to, furan, benzofuran, thiophene, benzothiophene, pyrrole, pyrazole, triazole, imidazole, oxazole, oxadiazole, thiazole, tetrazole, indole, carbazole, pyrrole-imidazole, pyrrole-pyrrole, thiophene-pyrrole, thiophene-thiophene, furan-pyrrole, furan-furan, thiophene-furan, benzoisoxazole, benzoisothiazole, benzimidazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, quinoline, isoquinoline, cinnoline, quinoxaline, phenanthidine, primidine, quinazoline, and quinazolinone.

[0020] In this specification, the term “prodrug” refers to any compound that, upon administration to an organism, produces a drug, i.e., an active ingredient, through spontaneous chemical reactions, enzymatic catalyzed chemical reactions, photolysis, and / or metabolic chemical reactions. Thus, a prodrug is a covalently modified analog or potential form of a therapeutically active compound. Suitable examples include, but are not limited to, carboxylic acid esters, carbonate esters, phosphate esters, nitrate esters, sulfate esters, sulfone esters, sulfinate esters, amino compounds, carbamates, azo compounds, phosphoramides, glucosides, ethers, and acetals.

[0021] "Medically acceptable" means a ligand, material, composition, and / or dosage form that is appropriate for administration to a patient within the bounds of reasonable medical judgment and that has a reasonable benefit-to-risk ratio.

[0022] "Medically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the term "medically acceptable carrier" includes buffers suitable for drug administration, sterile water for injection, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. Each carrier must be "medically acceptable" in the sense of compatibility with other components in the formulation and non-harmful to the patient. Appropriate examples include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; and (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. Examples of non-toxic, suitable substances used in pharmaceutical formulations include, but are not limited to, (10) glycols such as propylene glycol, (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethanol, (20) phosphate buffer, and (21) other non-toxic, suitable substances used in pharmaceutical formulations.

[0023] A "pharmaceutically acceptable salt" refers to a salt formed by any compound having the given structure with an acid or base suitable for use as a pharmaceutical. pharmaceutically acceptable salts include both inorganic and organic salts. An example of a salt is a salt formed by the compound of this application with an acid. Acids suitable for salt formation include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another example of a salt is a salt formed by the compound of this application with a base. Suitable bases for salt formation include, but are not limited to, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0024] "Medicinally acceptable esters and amides" refers to esters or amides suitable for medicinal use, formed by any of the compounds having the indicated structure with other compounds. Medicinally acceptable esters include organic and inorganic esters.

[0025] If the compound represented by formula I has a carboxyl group, the compound represented by formula I or its active derivative (e.g., acylchloride, mixed acid anhydride, etc.) is reacted with the corresponding alcohol (e.g., C1-C6 alcohols, etc.) or its active derivative (e.g., (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl body, (pivaloyloxy)methyl body, benzofuranone body, (isopropoxycarbonyl)oxymethyl body, etc.), or ammonia or the corresponding amine (mono-C1-C6 alkylamine or di-C1-C6 alkylamine, etc.) according to a normal method (condensation reaction of carboxylic acid with alcohol or amine), and the ester of the compound represented by formula I is reacted with the corresponding alcohol (e.g., C1-C6 alcohols, etc.) (transesterification reaction), or ammonia or the corresponding amine (mono-C1-C6 alkylamine or di-C1-C6 alkylamine, etc.) Medicinally acceptable esters or amides can be prepared by reacting (amidation reaction) or by reacting an alkali metal salt of the compound represented by formula I with the corresponding halide (e.g., C1-C6 alkyl chlorides or bromides, (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methyl chloride or bromide, (pivaloyloxy)methyl chloride or bromide, benzofuranone chloride or bromide, [(isopropoxycarbonyl)oxy]methyl chloride or bromide, etc.). Similarly, if the compound represented by formula I has a hydroxyl group, medicamentally acceptable esters can be obtained by condensing the compound represented by formula I with carboxylic acids, acyl chlorides, acid anhydrides, etc., according to conventional methods.

[0026] The aforementioned esters include, for example, C1-C6 alkyl esters such as methyl esters, ethyl esters, propyl esters, isopropyl esters, butyl esters, sec-butyl esters, tert-butyl esters, pentyl esters, and hexyl esters; C3-C6 cycloalkyl esters such as cyclopentyl esters and cyclohexyl esters; and C6-C6 alkyl esters such as phenyl esters and naphthyl esters. 10 Aryl esters; such as benzyl esters, phenethyl esters, α-methylbenzyl esters, 3-phenylpropyl esters, 4-phenylbutyl esters, 6-phenylhexyl esters, diphenylmethyl esters, triphenylmethyl esters, etc. C6-C 10 Examples include aryl C1-C6 alkyl esters. Alternatively, they may be esters that can be hydrolyzed in vivo, such as (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methyl ester, (pivaloyloxy)methyl ester, benzofuranone ester, [(isopropoxycarbonyl)oxy]methyl ester, [(cyclohexyloxycarbonyl)oxy]methyl ester, and 1-[(cyclohexyloxycarbonyl)oxy]ethyl ester.

[0027] Examples of the amides include mono-C1~C6 alkylamides or mono-C3~C6 cycloalkylamides such as amide (-CONH2), N-formamide, N-acetamide, N-propionamide, N-isopropionamide, N-butylamide, N-sec-butylamide, N-tert-butylamide, N-pentanamide, N-hexaneamide, N-cyclopropionamide, N-cyclopentanamide, and N-cyclohexaneamide. Alternatively, amides such as N,N-diformamide, N,N-diacetamide, N,N-dipropionamide, N,N-diisopropionamide, N-methyl-N-acetamide, N-methyl-N-propionamide, N-methyl-N-butylamide, N-ethyl-N-propionamide, N-ethyl-N-butylamide, N-butyl-N-cyclopentanamide, N-ethyl-N-cyclopropionamide, and N,N-dicyclohexaneamide are di-C1~C6 alkylamides, N-C1~C6 alkyl-N-C3~C6 cycloalkylamides, or di-C3~C6 cycloalkylamides.

[0028] A "solvate" refers to a complex formed when a compound represented by general formula (I) coordinates with a solvent molecule to form a specific ratio. A "hydrate" refers to a complex formed when the compound of this application coordinates with water.

[0029] An "active metabolite" refers to a derivative of a compound that possesses its activity, which is produced during the metabolism of that compound.

[0030] "Crystal polymorphism" refers to the compound of this application that exists in different crystal lattice configurations.

[0031] "Isotope labeling" refers to the compounds of this application that are labeled with isotopes. For example, the isotopes in the compounds of this application may be various isotopes of elements such as H, C, N, O, P, F, and S, for example, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P,32 P, 35 S, 18 F, 36 This includes S, etc.

[0032] An "isomer" refers to an isomer resulting from a different spatial arrangement of atoms in a molecule. Because the compounds in this application contain structures such as chiral or asymmetric centers and double bonds, they may include various isomeric forms such as optical isomers, geometric isomers, tautomers, and atropisomers. These isomers, their single isomers, and racemates are all within the scope of this application. For example, optical isomers, specifically the optically active (R)-, (S)- isomers and D, L isomers, can be prepared by chiral separation, chiral synthesis, chiral reagents, or other conventional techniques. For example, they can be converted to diastereomers by reacting with a suitable optically active substance (e.g., a chiral alcohol or moscher chloride), and then separated to convert them to the corresponding single isomers (e.g., by hydrolysis). Furthermore, separation can also be performed, for example, by chromatography.

[0033] Neurodegenerative diseases and nerve injuries have devastating effects on human health and are often difficult to treat. However, advances in stem cell biology, neuroscience, and reprogramming techniques have led to the development of several effective treatment options. For example, stem cell replacement therapy can be used to reconstruct damaged nerve tissue using neurons and other nerve cells for diseases and injuries of the central and peripheral nervous systems. One of the main strategies to achieve the aforementioned ultimate goal is to reconstruct normal nervous system development by activating the endogenous regenerative capacity of neural stem cells or by transplanting nerve cells or embryonic cells. However, while the use of stem cells in neurological diseases is thriving, several significant challenges remain. For instance, the problem of how to efficiently, stably, and directionally differentiate stem cells has been a long-standing challenge that the field of stem cell biology research has strived to solve.

[0034] Based on the background described above, a first aspect of this application provides an induction medium containing a BMPR1B inhibitor. The BMPR1B inhibitor has a structure represented by formula I, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula I.

[0035] [ka]

[0036] In the formula, ring A is selected from one of the following structures.

[0037] [ka]

[0038] Y is -(CH2) n - and n is 0, 1, 2 or 3, R 1 is -H, -D, unsubstituted or R 2 Substitutions of C1-C6 alkyl, C1-C6 alkoxy, and C6-C 10 Aryl or C3~C 10 Selected from heteroaryls, R 2 These are selected from -F, -Cl, -Br, -I, -OH, -COOH, C1-C6 alkyl, or C1-C6 alkoxy. The asterisk (*) indicates a connecting point.

[0039] The culture medium provided by this application is a pyrazolecarbonylpiperazinone compound with a specially designed structure that contains a BMPR1B inhibitor. These compounds can form stable complexes with BMPR1B through hydrogen bonding interactions and hydrophobic interactions, thereby regulating BMPR1B and consequently controlling the expression of downstream genes and physiological processes. This enables highly efficient and directional differentiation of pluripotent stem cells into neural lineage cells, providing a novel and possible approach for applying stem cell biology to the treatment of neurodegenerative diseases and nerve damage.

[0040] In some embodiments, Y is -(CH2) n - where n is 0, 1, or 2. Preferably, Y is -(CH2) n - where n is 0 or 1. More preferably, Y is -(CH2) n - and n is either 0 or 1.

[0041] In some embodiments, R 1 -H, -D, unsubstituted or R 2 Substitution of C1-C6 alkyl groups, C6-C 10 Aryl, or C3~C 10 Selected from heteroaryl groups. Preferably, R 1 -H, -D, unsubstituted or R 2 Selected from substituted C3-C6 heteroaryl groups. More preferably, R 1 is -H, -D, unsubstituted or R 2 A substitution is selected from furan, thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyran, thiam, pyridine, pyridazine, or pyrimidine. More preferably, R 1 is selected from -H, -D, or one of the following substituents.

[0042] [ka]

[0043] The asterisk (*) indicates a connecting point.

[0044] R 1 When the heteroaryl group is selected, particularly a heteroaryl group substituted with a methyl or hydroxyl group, the resulting pyrazolecarbonylpiperazinone compound has a high binding energy to the BMPR1B docking target and low physiological toxicity. This is beneficial for improving the efficacy and biocompatibility of pyrazolecarbonylpiperazinone compounds as BMPR1B inhibitors.

[0045] In some embodiments, R 2 is selected from -OH or C1-C6 alkyl. Preferably, R 2 is selected from -OH or C1-C4 alkyl. More preferably, R 2 The propyl group is selected from -OH, methyl, ethyl, n-propyl, or isopropyl.

[0046] In some embodiments, the BMPR1B inhibitor has one of the structures represented by formulas I-1 to I-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of one of the structures represented by formulas I-1 to I-13.

[0047] [ka]

[0048] In some embodiments, the BMPR1B inhibitor is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug having the structure shown in formula II.

[0049] [ka]

[0050] Among them, rings A, Y, and R 1 This is the same as the definition described in any of the embodiments above.

[0051] In some embodiments, the BMPR1B inhibitor has one of the structures represented by formulas II-1 to II-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of one of the structures represented by formulas II-1 to II-13.

[0052] [ka]

[0053] In some embodiments, the concentration of the BMPR1B inhibitor is 1 μM to 50 μM, preferably 1 μM to 20 μM, more preferably 1 μM to 10 μM. In any choice, the concentration of the BMPR1B inhibitor may be, for example, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, or 45 μM. An appropriate concentration of the BMPR1B inhibitor can more effectively promote directional differentiation from pluripotent stem cells to neural lineage cells without damaging it.

[0054] In some embodiments, the induction medium further comprises a basal medium, growth factors, inorganic salts, and one or more additives. The growth factors include one or more vitamins, progesterone, 1,4-butanediamine, and insulin. The additives include one or more piperidinol oxide, luteolin, D(+)-galactose, and recombinant human transferrin.

[0055] In some embodiments, the induction medium is (1) The basal medium is DMEM medium or DMEM-F12 medium, and MEM non-essential amino acids are added to the basal medium. (2) The vitamins include one or more of vitamin E, vitamin B12, and vitamin C. (3) The inorganic salt contains one or more of sodium chloride and sodium selenite, satisfying one or more of the conditions.

[0056] In some embodiments, the induction medium is (1) The growth factors include vitamins, progesterone 2.0 ng / mL to 15 ng / mL, 1,4-butanediamine 2 μg / mL to 50 μg / mL, and insulin 5 mg / L to 40 mg / L, and the vitamins include vitamin E 0.1 μg / mL to 15 μg / mL, vitamin B12 0.5 μM to 2.4 μM, and vitamin C 32 mg / L to 128 mg / L. (2) The inorganic salt contains 0.5 g / L of sodium chloride and 13.6 μg / L of sodium selenite. (3) The additives include piperidinol oxide 10 μM to 200 μM, luteolin 5 μM to 150 μM, D(+)-galactose 5 μg / mL to 25 μg / mL, and recombinant human transferrin 50 ng / mL to 200 ng / mL. One or more conditions must be met.

[0057] In some embodiments, the induction medium is (1) The growth factors include vitamins, progesterone 6.3 ng / mL, 1,4-butanediamine 23 μg / mL, and insulin 22 mg / L, and the vitamins include vitamin E 1 μg / mL, vitamin B12 1.2 μM, and vitamin C 64 mg / L. (2) The inorganic salt contains 0.5 g / L of sodium chloride and 13.6 μg / L of sodium selenite, (3) The additive contains piperidinol oxide 50 μM, luteolin 60 μM, D(+)-galactose 12.5 μg / mL, and recombinant human transferrin 100 ng / mL, and satisfies one or more of the following conditions.

[0058] A second aspect of this application provides a method for differentiating induced pluripotent stem cells into neural lineage cells, comprising the step of adhering pluripotent stem cells to an induction medium according to any of the above embodiments to obtain neural lineage cells.

[0059] In some embodiments, NANOG is used as a marker to identify pluripotent stem cells, and PAX6 is used as a marker to identify neural lineage cells. Since NANOG is expressed only in pluripotent stem cells and PAX6 is expressed only in neural lineage cells, by selecting these two as identification markers, differentiation from pluripotent stem cells to neural lineage cells can be quantitatively tracked and the induction results verified.

[0060] In some embodiments, adherent culture is carried out in the presence of a basement membrane preparation. The basement membrane preparation preferably contains one or more of Matrigel, laminin, and vitrin.

[0061] In some embodiments, the pluripotent stem cells are mammalian pluripotent stem cells. In any choice, the pluripotent stem cells are human pluripotent stem cells, which may be, for example, H9 cell lines or H1 cell lines, or human induced pluripotent stem cells (which can be obtained, for example, by reprogramming CD34+ cells by the method disclosed in CN109628383B).

[0062] In some embodiments, the neural lineage cells include one or more neural progenitor cells, neurons, astrocytes, and oligodendrocytes.

[0063] A third aspect of this application provides the use of the BMPR1B inhibitor described in the induction medium according to any of the above embodiments in the differentiation of induced pluripotent stem cells into neural lineage cells.

[0064] Synthesis of compounds (In the following cases, if there is a discrepancy between the compound name and structural formula, the structural formula takes precedence):

[0065] [ka]

[0066] Intermediate M1-n (0.12 mmol), (4aS,8aS)-octahydroquinoline-2(1H)-one (M2, 15 mg, 0.1 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 380 mg, 1 mmol) were mixed, and then N,N-diisopropylethylamine (DIEA, 2 mL) and N,N-dimethylformamide (DMF, 20 mL) were added, and the mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to obtain the target product II-n.

[0067] Compounds II-1 and II-13 are obtained by synthetic route A. II-1: (4aS,8aS)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (12 mg, 41%), LC-MS m / z = 289.2 [M + H] + II-13: (4aS,8aS)-4-(1H-indazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-4-(1H-indazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (17mg, 57%), LC-MS m / z=299.2[M+H] +

[0068] [ka]

[0069] Compound II-12 is obtained by synthetic route B. II-12: (4aS,8aS)-4-(4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-4-(4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carbonyl)octahydroquinoxaline-2(1H)-one

[0070] (1) At 0°C, 1.5 mL of 1,4-dioxane solution containing 0.25 g (1.1 mmol) of di-tert-butyl dicarbonate (Boc2O) was added dropwise to 2 mL of 1 mol / L NaOH containing 4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (M3-1, 0.167 g, 1 mmol) and 0.6 mL of 1,4-dioxane solution. The reaction mixture was stirred at 0°C for 0.5 hours, and then allowed to react overnight at room temperature while maintaining the pH at 8-9. After the reaction, the mixture was diluted with 5 mL of H2O and extracted with n-hexane. The aqueous phase was acidified to pH 2-3 with citric acid and extracted with ethyl acetate. The organic phase was washed with saturated saline solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain 5-(tert-butoxycarbonyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (M3-2, 200 mg, 75%). LC-MS m / z = 268.1 [M + H] + That was the case. (2) M3-2 (20 mg, 0.075 mmol) was mixed with (4aS,8aS)-octahydroquinoline-2(1H)-one (M2, 15 mg, 0.1 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 38 mg, 0.1 mmol), and N,N-diisopropylethylamine (DIEA, 0.2 mL) and N,N-dimethylformamide (DMF, 2 mL) were added, and the mixture was stirred at room temperature for 18 hours. Ethyl acetate and water were added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 3:7) to obtain tert-butyl 3-((4aS,8aS)-3-oxodeoxyquinoline-1-carbonyl)-1,4,6,7-tetrahydro-5H-pyrazole[4,3-c]pyridine-5-carboxylic acid ester (M3-3, 18 mg, 60%). LC-MS m / z = 404.2 [M+H] + That was the case. (3) M3-3 (18 mg, 0.04 mmol) and trifluoroacetic acid (TFA, 35 mg, 0.3 mmol) were added to 2 mL of dichloromethane (DCM), and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate and water were then added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. II-12 (13 mg, 96%) was obtained. LC-MS m / z = 304.2 [M + H] + That was the case.

[0071] [ka]

[0072] (1) Sodium hydride (60% suspension, 1.07 g, 26.9 mmol) dissolved in tetrahydrofuran (5 mL) was cooled to 0°C, and a solution of M1-n (21.5 mmol) in tetrahydrofuran (50 mL) was added dropwise over 15 minutes, and the mixture was stirred at 0°C for 1 hour. A solution of (2-(chloromethoxy)ethyl)trimethylsilane (M4, 4.76 mL, 26.9 mmol) in tetrahydrofuran (50 mL) was added dropwise to the mixture, and the mixture was stirred at room temperature for 48 hours. Water and ethyl acetate were slowly added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure to obtain M5-n. (2) In a 20 mL reactor, R 1 -YX(0.698 mmol, where X represents halogen, R 1 (where Y is the same as defined above), (4aS,8aS)-octahydroquinoxaline-2(1H)-one (M2, 108 mg, 0.698 mmol, 1.0 eq.), cesium carbonate (796 mg, 2.443 mmol, 2.5 eq.), 4,5-bisdiphenylphosphino-9,9-dimethylxanthene (20 mg, 0.035 mmol, 0.05 eq.), and palladium acetate (7.84 mg, 0.035 mmol, 0.05 eq.) were dissolved in tetrahydrofuran (3 mL) and blown with nitrogen for 10 minutes. The reaction was heated to 70°C and maintained for 90 minutes. The mixture was filtered, washed with dichloromethane, and then concentrated. The residue was purified by silica gel column chromatography (0-10% ethyl acetate / dichloromethane) to obtain M6-n. (3) M6-n (1.0 mmol, 1.0 eq.) was dissolved in dichloromethane (10 mL), and M5-n (1.2 mmol, 1.2 eq.), triethylamine (200 mg, 2.0 mmol, 2.0 eq.), and hexafluorophosphate azabenzotriazole tetramethylurea (570 mg, 1.5 mmol, 1.5 eq.) were added to the mixture and stirred overnight. The reaction was observed using thin-layer chromatography, and when the reaction products were no longer visible, the mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain M7-n. (4) At 5°C, triethylsilane (2.30 g, 19.8 mmol) was added to a solution of M7-n (3.95 mmol) in tetrahydrofuran (39.5 mL), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was then concentrated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 20-100%) to obtain the target product II-n.

[0073] Compounds II-2 to II-10 are obtained via synthetic route C. II-2: (4aS,8aS)-1-(3-methylthiophen-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(3-methylthiophen-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1504 mg, 99%), LC-MS m / z=384.2[M+H] + II-3: (4aS,8aS)-1-(2-methyloxazol-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(2-methyloxazol-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1445 mg, 99%), LC-MS m / z=370.2[M+H] + II-4: (4aS,8aS)-1-(3-methylisothiazol-5-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(3-methylisothiazol-5-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1507 mg, 99%), LC-MS m / z=386.2[M+H] + II-5: (4aS,8aS)-1-(2-methylpyridin-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(2-methylpyridine-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1469 mg, 98%), LC-MS m / z=380.2[M+H] + II-6: (4aS,8aS)-1-(6-methylpyridin-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(6-methylpyridine-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1484 mg, 99%), LC-MS m / z=380.2[M+H] + II-7: (4aS,8aS)-1-(6-hydroxypyridin-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(6-hydroxypyridine-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1491 mg, 99%), LC-MS m / z=382.2[M+H] + II-8: (4aS,8aS)-1-(4-hydroxypyridin-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(4-hydroxypyridine-2-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1491 mg, 99%), LC-MS m / z=382.2[M+H] + II-9: (4aS,8aS)-1-(2-methylpyrimidin-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(2-methylpyrimidine-4-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1488 mg, 99%), LC-MS m / z=381.2[M+H] + II-10: (4aS,8aS)-1-(6-methylpyridazin-3-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-(6-methylpyridazin-3-yl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1413 mg, 94%), LC-MS m / z=381.2[M+H] +

[0074] [ka]

[0075] Compound II-11 is obtained via synthetic route D. II-11: (4aS,8aS)-1-((5-methyl-1H-pyrazol-4-yl)methyl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxalin-2(1H)-one (4aS,8aS)-1-((5-methyl-1H-pyrazole-4-yl)methyl)-4-(1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (1) Sodium hydride (60% suspension, 1.07 g, 26.9 mmol) dissolved in tetrahydrofuran (5 mL) was cooled to 0°C, and a solution of 5-methyl-1H-pyrazole-4-carbaldehyde (M8-1, 2.37 g, 21.5 mmol) in tetrahydrofuran (50 mL) was added dropwise over 15 minutes, and the mixture was stirred at 0°C for 1 hour. A solution of (2-(chloromethoxy)ethyl)trimethylsilane (M4, 4.76 mL, 26.9 mmol) in tetrahydrofuran (50 mL) was added dropwise to the mixture, and the mixture was stirred at room temperature for 48 hours. Water and ethyl acetate were slowly added to the reaction mixture, the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure to obtain 5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-carbaldehyde (M8-2, 4.96 g, 96%). LC-MS m / z = 240.1[M+H] + That was the case. (2) In a 20 mL reactor, M8-2 (168 mg, 0.698 mmol), (4aS,8aS)-octahydroquinoxaline-2(1H)-one (M2, 108 mg, 0.698 mmol, 1.0 eq.), cesium carbonate (796 mg, 2.443 mmol, 2.5 eq.), 4,5-bisdiphenylphosphino-9,9-dimethylxanthene (20 mg, 0.035 mmol, 0.05 eq.), and palladium acetate (7.84 mg, 0.035 mmol, 0.05 eq.) were dissolved in tetrahydrofuran (3 mL) and blown in with nitrogen for 10 minutes. The reaction was heated to 70°C and maintained for 90 minutes. The mixture was filtered, washed with dichloromethane, and then concentrated. The residue was purified by silica gel column chromatography (0-10% ethyl acetate / dichloromethane) to obtain (4aS,8aS)-1-((5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-yl)methyl)octahydroquinoxaline-2(1H)-one (M8-3, 244 mg, 92.2%). LC-MS m / z = 378.3 [M+H] + That was the case. (3) M8-3 (379 mg, 1.0 mmol, 1.0 eq.) was dissolved in dichloromethane (10 mL), and 1-((2-(trimethylsilyl)ethoxy)methyl)-1,4,5,6-tetrahydrocyclopentapyrazole-3-carboxylic acid (M8-4, 339 mg, 1.2 mmol, 1.2 eq.), triethylamine (200 mg, 2.0 mmol, 2.0 eq.), and hexafluorophosphate azabenzotriazole tetramethylurea (570 mg, 1.5 mmol, 1.5 eq.) were added and the mixture was stirred overnight. The reaction was observed using thin-layer chromatography, and when the reactants were no longer visible, the mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain (4aS,8aS)-1-((5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-yl)methyl)-4-(1-((2-(trimethylsilyl)ethoxy)methyl)-1,4,5,6-tetrahydrocyclopentapyrazole-3-carbonyl)octahydroquinoxaline-2(1H)-one (M8-5, 424 mg, 66%). LC-MS m / z = 642.4 [M+H] + That was the case. (4) At 5°C, triethylsilane (2.30 g, 19.8 mmol) was added to a solution of M8-5 (2540 mg, 3.95 mmol) in tetrahydrofuran (39.5 mL), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was then concentrated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 20-100%) to obtain II-11 (1510 mg, 99%). LC-MS m / z = 383.2 [M + H] + That was the case.

[0076] Example 1 Using AutoDock Vina software, the pyrazolecarbonylpiperazinone compounds described in this application were molecularly docked with the BMPR1B target protein to create 13 docking structures. The binding energy and ligand efficiency of the optimal docking result between each pyrazolecarbonylpiperazinone compound and the BMPR1B target protein were calculated. The docking results were integrated, and pyrrolopyridine molecular screening was performed. The specific docking results are shown in Table 1. The second column of Table 1 shows the binding free energy calculated by the AutoDock Vina molecular docking software; a more negative value indicates a stronger binding energy of the compound to the target protein. The predicted values ​​showed that the absolute values ​​of the binding energies of all pyrazolecarbonylpiperazinone compounds significantly exceeded the threshold 3 set based on the target characteristics.

[0077] Cytotoxicity predictions were performed for the pyrazolecarbonylpiperazinone compounds related to this application using eToxPred software. The toxicity calculation results for each pyrazolecarbonylpiperazinone compound are shown in the third column of Table 1. The predicted values ​​indicate that the toxicity of all pyrazolecarbonylpiperazinone compounds is below the threshold of 0.58 (a value above 0.58 indicates the possibility of toxicity).

[0078] [Table 1]

[0079] Example 2 330 kinase candidates (15–50 nM, 2.5 μL) were mixed with compound II-1 (2.5 mM, 25 nL, DMSO prepared) and incubated at 25°C for 10 minutes. Subsequently, a mixture of kinase peptide substrate (0.2 mg / mL, supplier: GenScript) and ATP (10–60 μM, supplier: Promega, product number: V915B) (total 2.5 μL, test buffer prepared) was added to the mixture and reacted at 25°C for 60–120 minutes. Finally, HTRF was performed. 1 or ADP-Glo 2The reaction products were quantitatively analyzed using a specific method. In the kinase activity tests, the consumption of each substrate was less than 10%. Each kinase activity test consisted of two technical replicates.

[0080] As a result, II-1 significantly inhibited the kinase activity of BMPR1B (Figure 1A; Note: All results are shown, but for readability, one kinase name is displayed on the x-axis for every 10 kinases). On the other hand, II-1 at the same concentration did not inhibit the kinase activity of TGFBR1 and BMPR2, and showed no inhibitory effect on TGFBR1 and BMPR2 at different concentrations (maximum concentration 12.5 μM, 3-fold serial dilution) (Figures 1B and 1C). This result indicates that II-1 has the ability to specifically bind to its target site.

[0081] To further verify the above experiments from a structural perspective, structural analysis was performed on the complexes formed by compounds II-1, II-2, II-11, and II-13 with BMPR1B using PyMol software (supplier: Schrodinger). As shown in Figure 2, the structures of the complexes were obtained by molecular docking. The N- and C-terminal structural regions of BMPR1B are shown in cartoon models of different shapes. The loop structure of BMPR1B and the activation loop structure that stimulates catalytic activity are shown in cartoon models of different grayscale values. The pyrazolecarbonylpiperazinone compounds are shown in stick-ring models. The interacting amino acid backbone / side chains are shown in stick models. Hydrogen bonding interactions are shown by solid lines. Hydrophobic interactions are shown by dashed lines. This result also indicates that the pyrazolecarbonylpiperazinone compounds have structural properties that allow them to bind with BMPR1B.

[0082] Example 3 Western blot experiments were used to further investigate the inhibitory effect of pyrazolecarbonylpiperazinone compounds on the BMP signaling pathway. First, commercially available human pluripotent stem cell line H1 was cultured in a 6-well cell culture plate with 2 mL of medium per well and a cell volume of 1.5 × 10⁴.6 Cells were seeded. After 1 day of culture, the medium was changed and 6 μL of 30 μM pyrazolecarbonylpiperazinone compound II-I was added. After treating the cells for 12, 24, and 36 hours, the cells were detached and protein samples were prepared. Sample preparation followed the instructions of the commercially available kit (Beyotime Biotechnology, product number: P0013), and the Western blot analysis method referred to the literature (H. Zhang et al., 2022; N. Zhang et al., 2021). As a result, within 24 hours of treatment, pyrazolecarbonylpiperazinone compound II-1 did not inhibit the phosphorylation level of the downstream signaling proteins SMAD2 / 3 of the TGF signaling pathway, but showed a significant inhibitory effect on the phosphorylation level of the downstream signaling proteins SMAD1 / 5 / 8 of the BMP signaling pathway (Figure 3). These results further demonstrate that pyrazolecarbonylpiperazinone compound II-1 selectively inhibits the BMP signaling pathway, which is consistent with the results of kinase activity tests (Figure 1B, C).

[0083] [Table 2]

[0084] Example 4: Induction and identification of the nervous system (1) Culture medium composition Dulbecco's Modified Eagle Medium (DMEM medium), MEM Non-Essential Amino Acids (Minimum Essential Medium Non-Essential Amino Acids, Product Number: 11140076, Thermo The medium contains (Fisher), 4-hydroxy-TEMPO (50 μM), D(+)-galactose (12.5 μg / mL), vitamin E (1 μg / mL), vitamin B12 (1.2 μM), vitamin C (64 mg / L), progesterone (6.3 ng / mL), 1,4-butanediamine (23 μg / mL), luteolin (60 μM), sodium chloride (0.5 g / L), sodium selenite (13.6 μg / L), insulin (22 μg / mL), recombinant human transferrin (100 ng / mL), and the BMPR1B inhibitor of the present invention (induction medium A uses BMPR1B inhibitor II-1, and induction medium B uses BMPR1B inhibitor II-13). (2) Proliferation and passage of pluripotent stem cells Commercially available human pluripotent stem cell lines H1 and H9, as well as induced pluripotent stem cell (iPSC) lines, were used. Induced pluripotent stem cells were prepared by reprogramming with CD34+ according to "Culture method by culture medium reprogramming, i.e., reprogramming of induced pluripotent stem cells" (CN109628383A, Example 2). T25 cell culture flasks were coated with Matrigel (STEMCELL Technologies), and the plates were cultured in a 37°C incubator for more than 1 hour. 1 × 10 6 The cells were seeded in T25 cell culture flasks, and then grown and subcultured. (3) Neural guidance Six-well cell culture plates were coated with 50 μg / mL polylysine (SIGMA, product number: P6407) and cultured in a 37°C incubator for at least 3 hours. Next, they were further coated with 5 μg / mL laminin (SIGMAALDRICH, product number: I2020) and cultured in a 37°C incubator for at least 3 hours. When the pluripotent stem cell confluence reached 70%, the cells were detached using EDTA at 37°C for 5 minutes, and cell detachment was stopped using DMEM. After washing and centrifugation of the cells, 2 × 10⁶ cells were collected per flask. 5 The cells were re-seed into T25 cell culture plates according to their cell proportions. Induction was performed using the induction media described above. Specifically, pluripotent stem cells H9 were induced using induction medium A, while pluripotent stem cells H1 and induced pluripotent stem cells were induced using induction medium B, respectively. The media were changed daily until day 14. (4) Sequence detection and data analysis Samples were collected on days 7 and 14 of induction, processed using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-01 / 02), and sequence detection was performed using Illumina. After obtaining the original sequence detection data, the data was sequentially processed using FASTP, HISAT2, and FeatureCounts to obtain gene expression levels. GAPDH was used as the reference gene to correct for mRNA expression levels in the transcriptome between batches.

[0085] Analysis revealed that as the concentration of BMPR1B inhibitor II-1 increased, the expression of the marker gene NANOG in pluripotent stem cells H9 treated with the BMPR1B inhibitor decreased (P<0.05, t-test), while the expression of the marker gene PAX6 in neural progenitor cells increased (P<0.05, t-test). This indicates that II-1 exerts a dose-dependent effect during the induction process. From day 7 to day 14, as the induction time was prolonged, NANOG expression decreased and PAX6 expression increased (Figure 3, error bars indicate the standard deviation of three biological replicates). These results indicate that the BMPR1B inhibitor II-1 according to the present invention has the effect of promoting differentiation from pluripotent stem cells to neural progenitor cells (Figure 4B, D).

[0086] Furthermore, as the concentration of the BMPR1B inhibitor II-13 increased, the expression of the neural marker gene MAP2 (A. Caseres, Developmental Brain Research, Volume 13, Issue 2, April 1984, Pages 314-318) and the glial progenitor cell marker gene Sox9 (Neyrinck, K et al., Stem Cell Rev and Rep 17, 185521) in pluripotent stem cells H1 treated with the BMPR1B inhibitor showed a dose-dependent effect on II-13 (Figure 5).

[0087] Furthermore, compared to induced pluripotent stem cells in a control group not treated with the BMPR1B inhibitor, induced pluripotent stem cells treated with the BMPR1B inhibitor II-13 showed significantly increased expression of key genes, including the neural progenitor markers Sox1 and Sox5 (Elkouris M et al., StemCells. 2011;29(1):89~98; Tina Lai et al., Neuron V57(2):232~247, 2008) and the neural differentiation marker gene VIM (Sofia Duarte et al., Nature Communications volume 10, 4200 (2019)), demonstrating a dose-dependent effect on II-13 (Figure 6). These results indicate that the BMPR1B inhibitor described in this application not only promotes the differentiation of pluripotent stem cells into neural progenitor cells but also promotes the formation and differentiation of multiple types of nerve cells.

[0088] The technical features of the embodiments described above can be combined in any way, and for the sake of brevity, not all possible combinations of the technical features in the embodiments described above have been explained. However, as long as these combinations of technical features are inconsistent, they should all be considered to fall within the scope described herein.

[0089] The embodiments described above represent only a few embodiments of this application, and although the descriptions are relatively specific and detailed, they should not be understood as limiting the scope of the claims of the invention. Those skilled in the art can make several modifications and improvements without departing from the concept of this application, and all of these fall within the scope of protection of this application. Therefore, the scope of protection of the patent in this application should be determined based on the attached claims, and the specification and drawings are used to interpret the content of the claims.

[0090] (Note) (Note 1) Contains a BMPR1B inhibitor, The BMPR1B inhibitor is an induction medium having the structure represented by formula I, or a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula I. [ka] (In the formula, ring A is selected from one of the following structures, [ka] Y is -(CH2) n - and n is 0, 1, 2 or 3, R 1 is -H, -D, unsubstituted or R 2 Substitutions of C1-C6 alkyl, C1-C6 alkoxy, and C6-C 10 Aryl or C3~C 10 Selected from heteroaryls, R 2 These are selected from -F, -Cl, -Br, -I, -OH, -COOH, C1-C6 alkyl, or C1-C6 alkoxy. The asterisk (*) indicates a connecting point.

[0091] (Note 2) The aforementioned BMPR1B inhibitor is (1) Y is -(CH2) n - and n is 0, 1 or 2, (2)R 1 is -H, -D, unsubstituted or R 2 Substitution of C1-C6 alkyl groups, C6-C 10 Aryl or C3~C 10 Selected from heteroaryls, (3)R 2 is selected from -OH or C1-C6 alkyl groups. An induction medium as described in Appendix 1, characterized by satisfying one or more of the conditions.

[0092] (Note 3) The aforementioned BMPR1B inhibitor is (1) Y is -(CH2) n -, n is 0 or 1, (2) R 1 is -H, -D, unsubstituted or R 2 substituted C3-C6 heteroaryl selected from, (3) R 2 is selected from -OH or C1-C4 alkyl, The induction medium according to Appendix 2, characterized by satisfying one or more of the conditions.

[0093] (Appendix 4) The BMPR1B inhibitor is, (1) Y is -(CH2) n -, n is 0 or 1, (2) R 1 is, -H, -D, unsubstituted or R 2 substituted furan, thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyran, thiopyran, pyridine, pyridazine or pyrimidine selected from, (3) R 2 is selected from -OH, methyl, ethyl, n-propyl or isopropyl, The induction medium according to Appendix 3, characterized by satisfying one or more of the conditions.

[0094] (Appendix 5) R 1 is selected from -H, -D or any of the following substituents, the induction medium according to any one of Appendices 1-4.

Chemical formula

[0095] (Appendix 6) The induction medium according to any one of the appendices 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by any of formulas I-1 to I-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of a structure represented by any of formulas I-1 to I-13. [ka]

[0096] (Note 7) The induction medium according to any one of the appendices 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by formula II, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula II. [ka] (A ring, Y and R ring) 1 (This is the same as the definition described in any one of the appendices 1 to 4.)

[0097] (Note 8) The induction medium according to any one of the appendices 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by any of formulas II-1 to II-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of a structure represented by any of formulas II-1 to II-13. [ka]

[0098] (Note 9) The induction medium according to any one of the appendices 1 to 4, characterized in that the concentration of the BMPR1B inhibitor is 1 μM to 50 μM.

[0099] (Note 10) The induction medium further comprises one or more of a basal medium, a growth factor, an inorganic salt, and an additive. The aforementioned growth factor comprises one or more of the following: vitamins, progesterone, 1,4-butanediamine, and insulin. The induction medium according to any one of the appendices 1 to 4, characterized in that the additive comprises one or more of piperidinol oxide, luteolin, D(+)-galactose, and recombinant human transferrin.

[0100] (Note 11) The induction medium is (1) The basal medium is DMEM medium or DMEM-F12 medium, and MEM non-essential amino acids are added to the basal medium. (2) The vitamins include one or more of vitamin E, vitamin B12, and vitamin C. (3) The inorganic salt comprises one or more sodium chloride and sodium selenite, An induction medium as described in Appendix 10, characterized by satisfying one or more of the conditions.

[0101] (Note 12) The induction medium is (1) The growth factor comprises vitamins, progesterone 2.0 ng / mL to 15 ng / mL, 1,4-butanediamine 2 μg / mL to 50 μg / mL, and insulin 5 mg / L to 40 mg / L, and the vitamins comprise vitamin E 0.1 μg / mL to 15 μg / mL, vitamin B12 0.5 μM to 2.4 μM, and vitamin C 32 mg / L to 128 mg / L. (2) The inorganic salt contains 0.5 g / L of sodium chloride and 13.6 μg / L of sodium selenite, (3) The additive comprises piperidinol oxide 10 μM to 200 μM, luteolin 5 μM to 150 μM, D(+)-galactose 5 μg / mL to 25 μg / mL, and recombinant human transferrin 50 ng / mL to 200 ng / mL. The induction medium described in Appendix 11, characterized by satisfying one or more of the conditions.

[0102] (Note 13) A method for differentiating induced pluripotent stem cells into neural lineage cells, comprising the step of obtaining neural lineage cells by adhering pluripotent stem cells to an induction medium described in any one of the appendices 1 to 12.

[0103] (Note 14) Uses of BMPR1B inhibitors in induction media described in any one of Appendix 1 to 12 in the differentiation of induced pluripotent stem cells into neural lineage cells.

Claims

1. Contains a BMPR1B inhibitor, The BMPR1B inhibitor is an induction medium having the structure represented by formula I, or a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of the structure represented by formula I. 【Chemistry 1】 (In the formula, ring A is selected from one of the following structures, 【Chemistry 2】 Y is - (CH ２ ) ｎ - and n is 0, 1, 2 or 3, R １ is -H, -D, unsubstituted or R ２ substituted C １ to C ６ alkyl, C １ to C ６ alkoxy, C ６ to C １０ aryl or C ３ to C １０ selected from heteroaryl, R ２ -F, -Cl, -Br, -I, -OH, -COOH, C １ ~C ６ Alkyl or C １ ~C ６ Selected from alkoxy, (* indicates a connecting point.)

2. The BMPR1B inhibitor is (1) Y is - (CH ２ ) ｎ - and n is 0, 1 or 2, (2) R １ is -H, -D, unsubstituted or R ２ Substitution C １ ~C ６ Alkyl, C ６ ~C １０ Aryl or C ３ ~C １０ Selected from heteroaryls, (3) Caution ２ is -OH or C １ ~C ６ Selected from alkyl groups, The induction medium according to claim 1, characterized in that it satisfies one or more conditions.

3. The BMPR1B inhibitor is (1) Y is - (CH ２ ) ｎ - and n is 0 or 1, (2) R １ is -H, -D, unsubstituted or R ２ Substitution C ３ ~C ６ Selected from heteroaryls, (3) Caution ２ is -OH or C １ ~C ４ Selected from alkyl groups, The induction medium according to claim 2, characterized in that it satisfies one or more conditions.

4. The BMPR1B inhibitor is (1) Y is - (CH ２ ) ｎ - and n is 0 or 1, (2) R １ is -H, -D, unsubstituted or R ２ Substitutions are selected from furan, thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyran, thiopyran, pyridine, pyridazine, or pyrimidine. (3) Caution ２ is selected from -OH, methyl, ethyl, n-propyl or isopropyl. The induction medium according to claim 3, characterized in that it satisfies one or more conditions.

5. R １ The induction medium according to any one of claims 1 to 4, characterized in that -H, -D, or any of the following substituents are selected. 【Transformation 3】 (The asterisk "*" indicates a connecting point.)

6. The induction medium according to any one of claims 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by any one of formulas I-1 to I-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of a structure represented by any one of formulas I-1 to I-13. 【Chemistry 4】

7. The induction medium according to any one of claims 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by formula II, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of a structure represented by formula II. 【Transformation 5】 (A ring, Y and R １ (This is the same as the definition described in any one of claims 1 to 4.)

8. The induction medium according to any one of claims 1 to 4, characterized in that the BMPR1B inhibitor has a structure represented by any one of formulas II-1 to II-13, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, crystalline polymorph, isotope-labeled, isomer, or prodrug of a structure represented by any one of formulas II-1 to II-13. 【Transformation 6】

9. The induction medium according to any one of claims 1 to 4, characterized in that the concentration of the BMPR1B inhibitor is 1 μM to 50 μM.

10. The induction medium further comprises one or more of a basal medium, a growth factor, an inorganic salt, and an additive. The growth factor comprises one or more of the following: vitamins, progesterone, 1,4-butanediamine, and insulin. The induction medium according to any one of claims 1 to 4, characterized in that the additive comprises one or more of piperidinol oxide, luteolin, D(+)-galactose, and recombinant human transferrin.

11. The induction medium is (1) The basal medium is DMEM medium or DMEM-F12 medium, and MEM non-essential amino acids are added to the basal medium. (2) The vitamins include one or more of vitamin E, vitamin B12, and vitamin C. (3) The inorganic salt comprises one or more of sodium chloride and sodium selenite, The induction medium according to claim 10, characterized in that it satisfies one or more conditions.

12. The induction medium is (1) The growth factor comprises vitamins, progesterone 2.0 ng / mL to 15 ng / mL, 1,4-butanediamine 2 μg / mL to 50 μg / mL, and insulin 5 mg / L to 40 mg / L, and the vitamins comprise vitamin E 0.1 μg / mL to 15 μg / mL, vitamin B12 0.5 μM to 2.4 μM, and vitamin C 32 mg / L to 128 mg / L. (2) The inorganic salt contains 0.5 g / L of sodium chloride and 13.6 μg / L of sodium selenite, (3) The additive comprises piperidinol oxide 10 μM to 200 μM, luteolin 5 μM to 150 μM, D(+)-galactose 5 μg / mL to 25 μg / mL, and recombinant human transferrin 50 ng / mL to 200 ng / mL. The induction medium according to claim 11, characterized in that it satisfies one or more conditions.

13. A method for differentiating induced pluripotent stem cells into neural lineage cells, comprising the step of obtaining neural lineage cells by adhering and culturing pluripotent stem cells using an induction medium according to any one of claims 1 to 12.

14. Use of the BMPR1B inhibitor in the induction medium according to any one of claims 1 to 12 in the differentiation of induced pluripotent stem cells into neural lineage cells.

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