Alkbh8 small molecule inhibitors, methods of making and uses thereof

Abstract

The present application relates to ALKBH8 small molecule inhibitors and preparation methods and uses thereof. Specifically, the present application discloses an ALKBH8 small molecule inhibitor of a compound shown in formula I, the present application also provides a preparation method of the compound, and proves by experiments that ALKBH8 is a potential new target of aging inhibitor, and the use of the compound in preventing and / or treating aging-related diseases is also disclosed. (I).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicinal chemistry, in particular, to ALKBH8 small molecule inhibitors and the preparation method and use thereof. BACKGROUND

[0002] ALKBH8 (alkB homolog 8) is a key RNA modification enzyme belonging to the AlkB family of dioxygenases, which mainly maintains the accuracy of protein translation and cellular stress response by regulating tRNA modification in the human body. Its functional loss leads to neurodevelopmental disorders, cancer susceptibility and stress response defects, highlighting its importance as a basic biological hub and potential therapeutic target.

[0003] Senescence promotes the occurrence and development of various diseases, including tumors, fibrotic diseases and metabolic diseases. Senescence-associated secretory phenotype, i.e. the secretion of inflammatory factors, mainly mediates these biological effects of senescent cells. Inhibition of the secretion of inflammatory factors can effectively inhibit the progression of inflammatory senescence and related diseases, but there is still a lack of molecular targets and intervention strategies.

[0004] Although it has been reported that ALKBH8 inhibits the occurrence of senescence (May Y Lee et al, Redox Biology, 2020, PMID: 31765888), it is not yet clear whether it affects the expression of senescence-related inflammatory factors in cells that have already senesced.

[0005] Therefore, there is an urgent need in the art to design a class of novel ALKBH8 inhibitors and develop a new target for preventing and / or treating senescence-related diseases in order to overcome the shortcomings of the prior art. SUMMARY

[0006] The main purpose of the present application is to provide a novel ALKBH8 inhibitor, and for the first time, propose ALKBH8 as a therapeutic target for senescence inflammation and aging-related diseases. The present application also provides a preparation method of the compound and its use in preventing and / or treating senescence-related diseases.

[0007] In a first aspect, the present application provides a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof,

[0008] (I)

[0009] wherein,

[0010] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C2-C8alkenyl, substituted or unsubstituted C2-C8alkynyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl, substituted or unsubstituted C3-C8heterocycloalkyl containing from 1 to 3 heteroatoms selected from N, O, or S;

[0011] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C2-C8alkenyl, substituted or unsubstituted C2-C8alkynyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl, substituted or unsubstituted C3-C8heterocycloalkyl containing from 1 to 3 heteroatoms selected from N, O, or S;

[0012] substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted 5-7 membered heteroaryl containing from 1 to 3 heteroatoms selected from N, O, or S;

[0013] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C2-C8alkenyl, substituted or unsubstituted C2-C8alkynyl, substituted or unsubstituted C3-C8cycloalkyl.

[0014] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl.

[0015] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl. substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted 5-7 membered heteroaryl containing from 1 to 3 heteroatoms selected from N, O, or S;

[0016] R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl. substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted 5-7 membered heteroaryl containing from 1 to 3 heteroatoms selected from N, O, or S;

[0017] In another preferred embodiment, the heteroaryl is selected from the group consisting of pyridine, pyrimidine, pyrazine, thiophene, thiazole, pyrazole, pyrrole, imidazole, furan, oxazole, isoxazole; and the heteroaryl is optionally substituted with a substituent selected from the group consisting of halogen, -OH, -CN, -NH2, -NO2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl.

[0018] In another preferred embodiment, the substituent is selected from the group consisting of halogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl.

[0019] In another preferred embodiment, the compound is selected from the group consisting of:

[0020] .

[0021] In a second aspect of the present application, there is provided a method for preparing the compound of the first aspect of the present application, comprising the following steps:

[0022]

[0023] Step one: condensation reaction of compound I-1 and 4-bromo-2-hydrazinylpyridine (II-1) in methanol with hydrochloric acid as catalyst to obtain compound III-1;

[0024] Step two: Suzuki coupling reaction of compound III-1 and compound IV-1 in a mixed solvent of water and organic solvent 1 in the presence of a palladium catalyst to obtain compound I;

[0025] When R1 is H, the method further comprises step three: hydrolysis reaction of compound I in solvent 2 by a base to obtain compound I-A.

[0026] In a third aspect of the present application, there is provided a composition comprising:

[0027] (i) a compound of the first aspect of the present application, or a pharmaceutically acceptable salt or solvate thereof; and

[0028] (ii) a pharmaceutically acceptable carrier.

[0029] In a fourth aspect of the present application, there is provided use of a compound of the first aspect of the present application, the use comprising:

[0030] (i) as an ALKBH8 inhibitor;

[0031] (ii) use as a skin anti-aging agent;

[0032] (iii) use for the preparation of a medicament for preventing and / or treating an aging-related disease.

[0033] In another preferred embodiment, the aging-related disease is selected from the group consisting of tumor, liver fibrosis, lung fibrosis, cardiovascular disease, neurodegenerative disease, skeletal muscle system disease, respiratory system disease, chronic obstructive pulmonary disease.

[0034] In another preferred embodiment, the cardiovascular disease is atherosclerosis.

[0035] In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer, Parkinson.

[0036] In another preferred embodiment, the skeletal muscle system disease is selected from the group consisting of osteoporosis, sarcopenia.

[0037] In another preferred embodiment, the respiratory system disease is pneumonia.

[0038] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 ALKBH8 protein is specifically up-regulated with aging process is verified in cell and animal experiments. Among them, a-b, the expression level of p16, p21, the aging markers after etoposide treatment of cells for 48 hours (a), or 4-hydroxytamoxifen induced RAS expression (b). c-d, the expression level of p21, the aging marker in liver tissue of young mice (2 months old), old mice (18 months old), or mice treated with 4Gy radiation (d).

[0040] Figure 2 ALKBH8 promotes aging-related secretory characteristics is shown. Among them, a-c, analysis of gene expression changes (a), signal pathway analysis (b) and expression changes of specific inflammatory factor genes (c) in young cells, aging control cells, aging ALKBH8 knockdown cells. d, cytokine chip detection of inflammatory factor secretion in normal culture of young cells, aging control cells, aging ALKBH8 knockdown cells.

[0041] Figure 3The ALKBH8 regulates the biological function mediated by the senescence-associated secretory phenotype is shown. Among them, a-b, the tumor cells TOV-21G and young cells, senescent control cells or senescent cells with different sequences of ALKBH8 knockdown are mixedly transplanted subcutaneously in nude mice, and the tumor growth curves at different time points (a) and the tumor weights at the last time point (b) are monitored. c-e, the Alkbh8 Flox / Flox The conditional knockout mice are subjected to 4Gy radiation to induce systemic aging. After 7 days, they are randomly divided into two groups, 5 mice in each group. The tail vein is injected with AAV-Cre virus to achieve liver-specific knockdown of the Alkbh8 gene (gene knockout) or AAV-GFP control virus to retain the Alkbh8 gene (wild type). After 7 days, the liver tissue is collected to detect the mRNA expression level changes of inflammatory factors (d), and the SA- -gal staining is performed and counted (e).

[0042] Figure 4 It is shown that the compounds of the present application can reduce inflammation in senescent tissues. Among them, a, the level changes of mcm5U and mchm5U caused by compounds 10, 12 and 13 are analyzed by in vitro enzyme activity experiment. b, after the senescent IMR90 cells are treated with different concentrations of compound 1, the mRNA expression of senescence inflammatory factors IL1A and IL1B is detected. c-g, the solvent or compound 1 is injected intraperitoneally, the body weight change of the mouse is monitored (c), the liver weight ratio change is compared (d), and the activity changes of glutamic-pyruvic transaminase (e) and glutamic-oxaloacetic transaminase (f) are detected. After 7 days of administration, the liver tissue is collected to compare the expression changes of inflammatory factors (g). DETAILED DESCRIPTION

[0043] The present inventors have made extensive and in-depth research, and for the first time found that ALKBH8 is significantly highly expressed in senescent cells and tissues, proving that the protein can be used as a biomarker of senescence. The present inventors have developed a series of ALKBH8 inhibitors, and for the first time applied ALKBH8 inhibitors to the field of anti-senescence. The compounds have novel structures, and in vitro biochemical experiments, in vivo cell and mouse experiments prove that the compounds of the present application have wide application prospects for reducing senescence-related inflammation and inhibiting aging-related diseases. On this basis, the present application is completed.

[0044] The present inventors have for the first time found that ALKBH8 promotes the expression and secretion of senescence inflammatory factors, and mediates the biological effects of senescent cells, including tumor growth promotion by senescent cells, regulation of immune surveillance, etc. The present application designs and proves that small molecule compound 10 can inhibit the enzyme activity of ALKBH8 in vitro, and compound 1 can inhibit the expression of inflammatory factor genes of senescent cells and the tissue inflammation mediated thereby in vivo, thereby relieving senescence-related diseases.

[0045] TERMS

[0046] In the present application, the terms used have the general meanings known to those skilled in the art, unless specifically indicated otherwise.

[0047] In the present application, the term "halogen" means F, Cl, Br or I.

[0048] In the present application, "C1-C6 alkyl" means a straight or branched chain alkyl group including 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, neopentyl, tert-pentyl, or the like. The similar term "C1-C4 alkyl" has a similar definition.

[0049] In the present application, the term "C1-C6 alkoxy" means a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, and the like. The similar term "C1-C4 alkoxy" has a similar definition.

[0050] In the present application, the term "C2-C6 alkenyl" means a straight or branched chain alkenyl group having 2 to 6 carbon atoms containing at least one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl, and the like.

[0051] In the present application, the term "C2-C6 alkynyl" means a straight or branched chain alkynyl group having 2 to 6 carbon atoms containing at least one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like.

[0052] In the present application, the term "C3-C8 cycloalkyl" represents a cyclic aliphatic group consisting of 3 to 8 ring-forming carbon atoms, and the like; it is to be understood that the "cycloalkyl" groups described herein include not only monocyclic aliphatic groups, but also fused, spiro, and bridged ring systems consisting of multiple cyclic aliphatic groups; it is to be understood that the "cycloalkyl" groups described herein include not only aliphatic groups in which the carbon atoms are fully saturated, but also aliphatic groups in which some of the carbon atoms have unsaturated bonds; examples of "cycloalkyl" groups described herein include, but are not limited to: 、 、 、 、 、 、 、 and the like. When "cycloalkyl" is a substituent, the point of attachment to the molecular backbone can occur at any position on the "cycloalkyl" group that a chemical bond permits. The similar term "C3-C6 cycloalkyl" has a similar definition.

[0053] In the present application, the term "aryl" means monocyclic and bicyclic ring systems composed of a specific number of carbon atoms, which obey the Hückel rule; it is to be understood that when the "aryl" of the present application is a bicyclic ring system, it includes not only the case where all the rings are aromatic, but also the case where only one of the rings is aromatic and the other is a non-aromatic aliphatic ring.

[0054] In the present application, the term "C6-C10 aryl" means a cyclic system having 6 to 10 carbon atoms, and at least one of the rings is aromatic; examples of the "aryl" of the present application include, but are not limited to , etc.; when the "aryl" is a substituent, the connection point to the main body of the molecule is on the aromatic ring.

[0055] In the present application, the term "heterocycloalkyl" means a cyclic group composed of a specific number of ring-forming atoms, containing at least one ring-forming heteroatom (N, O or S), saturated or partially unsaturated, and non-aromatic; it is to be understood that the "heterocycloalkyl" of the present application includes not only monocyclic heterocyclic systems, but also polycyclic heterocyclic systems such as fused, spiro and bridged rings; when the "heterocycloalkyl" is a polycyclic system, at least one of the rings contains a ring-forming heteroatom, and the other rings can contain a ring-forming heteroatom or be a cycloalkyl; for example, the term "4-8 membered heterocycloalkyl" means a monocyclic or polycyclic system having 4 to 8 ring-forming atoms, at least one of which is a heteroatom, saturated or partially unsaturated; the definitions of other similar terms are analogous; more preferably, the number of heteroatoms is 1 to 3. Examples include (but are not limited to) the following groups: , etc.; it is to be understood that when the "heterocycloalkyl" is a substituent, the connection point to the main body of the molecule can be at any position allowed by the chemical bonds on the "heterocycloalkyl".

[0056] ​​​​​​​​​​​​​​In the present application, the term "heteroaryl" means a cyclic group having a specified number of ring-forming atoms, containing at least one ring-forming heteroatom (N, O or S), and having aromaticity; unless otherwise specified, "heteroaryl" as used herein encompasses not only monocyclic heteroaromatic systems, but also polycyclic heteroaromatic systems, such as bicyclic heteroaryl groups, tricyclic heteroaryl groups, tetracyclic heteroaryl groups; when the "heteroaryl" group is a polycyclic heteroaromatic system, at least one ring is aromatic, and the other rings can be aromatic or non-aromatic, and the heteroatoms can be in the aromatic rings or in the non-aromatic rings; polycyclic heteroaromatic systems include fused ring systems, as well as bridged and spirocyclic ring systems. The term "5-7 membered heteroaryl" means a cyclic group having 5 to 7 ring-forming atoms, at least one of which is a heteroatom, and having aromaticity. Definitions of other similar terms are analogous.

[0057] In the present application, the term "halo" means substituted with a halogen.

[0058] In the present application, the term "optionally" means that when there is a list of candidate groups to choose from, one can choose some, or none.

[0059] In the present application, "each independently" means that when several substituents are defined simultaneously, they are selected from the same list of candidate groups, and they can be the same or different.

[0060] In the present application, the term "substituted" means that one or more hydrogen atoms on a specified group are replaced with a specified substituent. The specified substituent is a substituent described in the foregoing, or a substituent appearing in each embodiment. Unless otherwise specified, a substituted group can have one substituent selected from a specified group at any substitutable position on the group, and the substituents can be the same or different at each position. Those skilled in the art will appreciate that the combinations of substituents contemplated by the present application are those that are stable or chemically feasible.

[0061] In the present application, the term "1-6" means having 1, 2, 3, 4, 5 or 6, and each of the other similar terms has an analogous independent meaning.

[0062] It is understood that when a group is present simultaneously at multiple different positions in a compound, the definition of the group at each position is independent of the definition at the other positions, and can be the same or different. That is, the term "selected from the group consisting of" has the same meaning as the term "each independently selected from the group consisting of".

[0063] Compounds of the present application of formula I

[0064] The present application provides a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof,

[0065] (I)

[0066] wherein each group is as defined above.

[0067] Preferably, R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C3-C8halocycloalkyl.

[0068] Preferably, is substituted or unsubstituted C6-C10aryl or substituted or unsubstituted 5-7 membered heteroaryl containing 1-3 heteroatoms selected from N, O, or S;

[0069] In another preferred embodiment, the compound is one wherein R1and each of which is independently the corresponding group in the particular compound of the application.

[0070] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of Formula (I) formed between a positively charged group on the compound and an anion, or a salt of a compound of Formula (I) formed between a negatively charged group on the compound and a cation. Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate, and the like. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, ammonium ion, and the like.

[0071] In another preferred embodiment, the pharmaceutically acceptable salt of the application refers to a salt of a compound of Formula (I) with an acid, such as, but not limited to, hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzoic acid, 2-acetoxybenzoic acid, and isethionic acid, and the like; or a salt of a compound of Formula (I) with an inorganic base, such as, but not limited to, a sodium salt, a potassium salt, a calcium salt, an aluminum salt, or an ammonium salt; or a salt of a compound of Formula (I) with an organic base, such as, but not limited to, a methylamine salt, an ethylamine salt, an ethanolamine salt, a TRIS ammonium salt, and the like.

[0072] Methods of making compounds of Formula I

[0073] The present application specifically describes the preparation of the compounds of formula (I) of the present application, but these specific methods do not limit the present application in any way. The compounds of the present application can also be readily prepared by combining various synthetic methods described in the present specification or known in the art, such combinations being readily made by one skilled in the art to which the present application pertains.

[0074] Typically, the preparation of the compounds of the present application follows the procedure as shown below, wherein the starting materials and reagents used, if not specifically mentioned, are commercially available.

[0075] Specifically, the present application provides a chemical synthesis method for preparing the compounds, and the specific steps are as follows:

[0076]

[0077] Step one: compound I-1 is condensed with 4-bromo-2-hydrazinylpyridine (II-1) in methanol, with hydrochloric acid as a catalyst, to obtain a compound of formula III-1;

[0078] Step two: the compound of formula III-1 is subjected to Suzuki coupling reaction with a compound of formula IV-1 in a mixed solvent of water and an organic solvent 1 in the presence of a palladium-containing catalyst, to obtain a compound of formula I;

[0079] When R1 is H, the method further comprises step three: the compound of formula I is subjected to hydrolysis reaction by a base in a solvent 2, to obtain a compound of formula I-A.

[0080] The palladium-containing catalyst in step two is selected from the group consisting of tetrakis(triphenylphosphine)palladium(0), bis(triphenylphosphine)palladium(II) dichloride, [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, or a combination thereof.

[0081] Preferably, the organic solvent 1 is selected from the group consisting of tetrahydrofuran, 1,4-dioxane, acetonitrile, N , N dimethylformamide, N , N dimethylacetamide, or a combination thereof.

[0082] Preferably, the base in step three is selected from the group consisting of lithium hydroxide (including monohydrate thereof), sodium hydroxide, potassium hydroxide, or a combination thereof.

[0083] Preferably, the solvent 2 is selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran, 1,4-dioxane, water, or a combination thereof.

[0084] Pharmaceutical compositions and methods of administration

[0085] The pharmaceutical composition of the present application comprises a safe and effective amount of the compound of the present application or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably, 5-1000 mg of the compound of the present application per dose. Preferably, the "dose" is one capsule or tablet.

[0086] The "pharmacologically acceptable carrier" means one or more compatible solid or liquid filler or gel materials which are suitable for human use and which are sufficiently nontoxic in the amounts used. "Compatible" means that the components of the composition are capable of being commingled with the compound of the present application and with each other in the composition, without any component tending to impair the effectiveness of another component. Examples of suitable pharmacologically acceptable carriers are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0087] The pharmaceutical composition is in the form of an injection, a capsule, a tablet, a pill, a powder or a granule.

[0088] The mode of administration of the compound or the pharmaceutical composition of the present application is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0089] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate or with such other ingredients as binders, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, and corn starch; fillers, e.g., lactose, calcium sulfate, calcium carbonate, and sodium chloride; humectants, e.g., glycerol; disintegrating agents, e.g., agar agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; absorption accelerators, e.g., quaternary ammonium compounds; and lubricants, e.g., magnesium stearate, stearic acid, and colloidal silica. Solid compositions of a similar type are also employed as fillers in soft and hard filled gelatin capsules. Tablets can additionally be prepared, if desired, with coatings and shells, such as enteric coatings and other materials well known in the art. They can optionally contain opacifying agents and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0090] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials well known in the art. They can optionally contain opacifying agents and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds are also present as a mixture with excipients, if desired.

[0091] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and the like, as well as mixtures thereof.

[0092] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0093] Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and sodium carbomate, among others.

[0094] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0095] Dosage forms for topical administration of a compound of this application include ointments, powders, sprays, and inhalers. The active component is admixed with a carrier, which can be a sterile, physiologically acceptable carrier, and any buffers, preservatives, or propellants as can be required.

[0096] A compound of this application can be administered alone or in combination with other pharmaceutically acceptable compounds (e.g. anti-aging drugs).

[0097] The therapeutic methods of this application can be administered alone or in combination with other therapeutic procedures or therapeutic agents.

[0098] Pharmaceutical compositions are used in the form of a safe and effective amount of a compound of this application in admixture with a pharmaceutical carrier or vehicle that is suitable for the intended application. A safe and effective amount of a compound of this application is an amount that is sufficient to significantly induce the desired effect, but low enough to avoid serious side effects. The specific safe and effective amount will depend on the particular compound of this application, the particular composition, the mode of delivery, and the mammal involved. In general, a safe and effective amount will be in the range of 1-2000 mg, preferably 5-1000 mg, per day for a 60 kg mammal, administered in a suitable number of doses. The specific dose will vary depending on the route of administration, the health and weight of the patient, and the disease being treated.

[0099] The advantages of the present application compared to the prior art are mainly:

[0100] 1. The present application first discovers ALKBH8 as a new target for the treatment of inflammatory aging.

[0101] 2. The present application first designs a series of compounds to achieve the effect of treating inflammatory aging by targeting ALKBH8 receptors.

[0102] 3. The compounds described in the present application can effectively inhibit the secretion of inflammatory factors and the ability of senescent cells to promote tumor growth.

[0103] 4. The compounds described in the present application specifically inhibit the catalytic activity of ALKBH8, blocking the downstream senescence-related inflammatory pathway.

[0104] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are presented by way of example only and are not intended to limit the scope of the application.

[0106] Unless otherwise specified, in the following examples, the NMR measurement is carried out using a Varian Mercury Plus 400 (400 MHz room temperature probe), and the measurement solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCI3), and the internal standard is tetramethylsilane (TMS). The chemical shift is expressed as δ in ppm, and the peak shape is expressed as s (singlet), d (doublet), t (triplet), dd (doublet of doublets), q (quartet), dt (doublet of triplets), and m (multiplet). The coupling constant (J) is expressed in Hz. The results are analyzed using MestReNova 14. The MS measurement is carried out using an Agilent 6120 liquid chromatograph-mass spectrometer (ESI); and the column chromatography is carried out using a SANTAI SepaBean machine T rapid preparation instrument. d 6 ), deuterated chloroform (CDCI3), and the internal standard is tetramethylsilane (TMS). The chemical shift is expressed as δ in ppm, and the peak shape is expressed as s (singlet), d (doublet), t (triplet), dd (doublet of doublets), q (quartet), dt (doublet of triplets), and m (multiplet). The coupling constant (J) is expressed in Hz. The results are analyzed using MestReNova 14. The MS measurement is carried out using an Agilent 6120 liquid chromatograph-mass spectrometer (ESI); and the column chromatography is carried out using a SANTAI SepaBean machine T rapid preparation instrument. J

[0107] Preparation of compounds

[0108] Example 1: 1-(4-phenylpyridin-2-yl)-1 H - pyrazole-4-carboxylate (Compound 1)

[0109]

[0110] Step 1: 1-(4-bromopyridin-2-yl)-1 H - pyrazole-4-carboxylate

[0111] ​Ethyl 2-formyl-3-oxopropanoate (317.1 mg, 2.2 mmol, 1.1 equiv.) was dissolved in 5 mL of methanol with 4-bromo-2-hydrazinylpyridine (376.5 mg, 2.0 mmol, 1.0 equiv.) and 0.7 mL of 6 mol / L hydrochloric acid was added. The mixture was stirred at 25 °C for 8 h, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was filtered under reduced pressure, and the filter cake was washed with 5 mL of water and dried under reduced pressure to obtain 1-(4-bromopyridin-2-yl)-1 H Ethyl pyrazole-4-carboxylate was a light yellow solid, 510 mg, yield 85%. 1 H NMR (400MHz, CDCl3) δ 8.93 (s, 1H), 8.19 (d, J = 5.2 Hz, 1H), 8.16 (s, 1H), 8.04 (s,1H), 7.35 (dd, J = 5.2, 1.4 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz,3H).

[0112] Step 2: 1-(4-phenylpyridin-2-yl)-1 H Ethyl pyrazole-4-carboxylate (compound 1)

[0113] Ethyl 1-(4-bromopyridin-2-yl)-1 H - pyrazole-4-carboxylate (100 mg, 0.34 mmol, 1.0 equiv.), phenylboronic acid (45.1, 0.37 mmol, 1.1 equiv.), anhydrous potassium carbonate (116.6 mg, 0.844 mmol, 2.5 equiv.) and tetrakis(triphenylphosphine)palladium (34.7 mg, 0.03 mmol, 0.1 equiv.) were placed in a two-necked flask, 5 mL of 1,4-dioxane / water (v / v = 4:1) mixed solvent was added, and the mixture was reacted at 100 °C for 3 h under N2atmosphere. After the mixture was cooled to room temperature, the solvent was removed under reduced pressure, and column chromatography was used for purification to obtain ethyl 1-(4-phenylpyridin-2-yl)-1 H Ethyl 1-(4-phenylpyridin-2-yl)-1 1 H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.26 (d, J= 1.0 Hz, 1H), 8.14 (s, 1H), 7.77 – 7.70 (m, 2H), 7.54 – 7.47 (m, 4H), 4.35(q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

[0114] Example 2: 1-(4-(4-methoxyphenyl)pyridin-2-yl)-1 H - pyrazole-4-carboxylate (Compound 2)

[0115]

[0116] Preparation method according to Example 1, replacing phenylboronic acid in Step 2 with 4-methoxyphenylboronic acid. 1 H NMR (400MHz, CDCl3) δ 9.11 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.24 (d, J = 1.0 Hz, 1H), 8.14 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 5.1, 1.3 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H).

[0117] Example 3: 1-(4-(4-fluorophenyl)pyridin-2-yl)-1 H - pyrazole-4-carboxylate (Compound 3)

[0118]

[0119] Preparation method according to Example 1, replacing phenylboronic acid in Step 2 with 4-fluorophenylboronic acid. 1 H NMR (400MHz, CDCl3) δ 9.08 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.20 (s, 1H), 8.13 (s,1H), 7.71 (dd, J= 8.7, 5.2 Hz, 2H), 7.49 – 7.42 (m, 1H), 7.25 – 7.06 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).

[0120] Example 4: 1-(4-(3-fluorophenyl)pyridin-2-yl)-1 H 4-Pyrazole-4-carboxylic acid ethyl ester (compound 4)

[0121]

[0122] The preparation method is the same as in Example 1, except that the phenylboronic acid in step 2 is replaced with 3-fluorophenylboronic acid. 1 H NMR (400MHz, CDCl3) δ 9.01 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.16 (d, J = 0.8 Hz, 1H),8.07 (s, 1H), 7.48 – 7.32 (m, 4H), 7.12 (d, J = 7.4 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H).

[0123] Example 5: 1-(4-(4-trifluoromethylphenyl)pyridin-2-yl)-1 H 4-Pyrazole-4-carboxylic acid ethyl ester (compound 5)

[0124]

[0125] The preparation method is the same as in Example 1, except that the phenylboronic acid in step 2 is replaced with 4-trifluoromethylphenylboronic acid. 1 H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.26 (s, 1H), 8.14(s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.48 (dd, J = 5.1, 1.4Hz, 1H), 4.35 (q, J= 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

[0126] Example 6: 1 -(4-(4-trifluoromethoxyphenyl)pyridin-2-yl)-1 H - pyrazole-4-carboxylate (Compound 6)

[0127]

[0128] Preparation method according to Example 1, replacing phenylboronic acid in Step 2 with 4-trifluoromethoxyphenylboronic acid. 1 H NMR (400 MHz, CDC13) δ 9.09 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.22 (s, 1H), 8.14(s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.45 (dd, J = 5.1, 1.4 Hz, 1H), 7.36 (d, J = 8.5Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

[0129] Example 7: 1 -(4-(3,5-dimethoxyphenyl)pyridin-2-yl)-1 H - pyrazole-4-carboxylate (Compound 7)

[0130]

[0131] Preparation method according to Example 1, replacing phenylboronic acid in Step 2 with 3,5-dimethoxyphenylboronic acid. 1 H NMR (400 MHz, CDC13) δ 9.08 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.25 - 8.19 (m, 1H), 8.14 (s, 1H), 7.45 (dd, J = 5.2, 1.6 Hz, 1H), 6.83 (d, J = 2.2 Hz, 2H), 6.57 (t, J = 2.2 Hz, 1H), 3.87 (s, 6H).

[0132] Example 8: 1-([4,4'-Bipyridyl]-2-yl)-1 H -ethyl pyrazole-4-carboxylate (Compound 8)

[0133]

[0134] The procedure of Example 1 was followed, replacing the phenylboronic acid in Step 2 with pyridin-4-ylboronic acid. 1 H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.80 (d, J = 6.0 Hz, 2H), 8.58 (d, J = 5.1 Hz, 1H),8.30 (s, 1H), 8.15 (s, 1H), 7.69 (d, J = 5.6 Hz, 2H), 7.51 (dd, J = 5.2, 1.5 Hz,1H), 4.36 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

[0135] Example 9: 1-(4-(4-methylthiazol-5-yl)pyridin-2-yl)-1 H -ethyl pyrazole-4-carboxylate (Compound 9)

[0136]

[0137] The procedure of Example 1 was followed, replacing the phenylboronic acid in Step 2 with (4-methylthiazol-5-yl)boronic acid pinacol ester. 1 H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 8.82 (s, 1H), 8.48 (d, J = 5.1 Hz,1H), 8.12 (d, J = 5.9 Hz, 2H), 7.33 (dd, J = 5.1, 1.1 Hz, 1H), 4.35 (q, J = 7.1 Hz,2H), 2.68 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H).

[0138] Example 10: 1-(4-phenylpyridin-2-yl)-1 H -pyrazole-4-carboxylic acid (Compound 10)

[0139]

[0140] Compound 1 (60 mg, 0.20 mmol) was dissolved in 2 mL of methanol / tetrahydrofuran (v / v / v = 4:1) mixed solvent, 2 mol / L NaOH solution (1 mL, 2 mmol, 10.0 equiv.) was added, and the reaction was carried out at 50 °C for 8 h. After being cooled to room temperature, 5 mL of water was added, 6 mol / L hydrochloric acid solution was added to adjust the pH to 4, most of the organic solvent was removed under reduced pressure, and the filter cake was washed with 2 mL of water, dried under reduced pressure, and Compound 10 was obtained as a white solid, 40.7 mg, yield 75%. 1 H NMR (400 MHz, DMSO) δ 12.82 (s, 1H), 9.00 (s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 8.25 – 8.19 (m, 2H),7.91 (dd, J = 8.0, 1.4 Hz, 2H), 7.80 (dd, J = 5.2, 1.6 Hz, 1H), 7.63 – 7.53 (m,3H).

[0141] Example 11: 1-(4-(4-methoxyphenyl)pyridin-2-yl)-1 H pyrazole-4-carboxylic acid (Compound 11)

[0142]

[0143] The preparation method is referred to Example 10, and Compound 1 is replaced with Compound 2. 1 H NMR (400 MHz, DMSO) δ 12.82 (s, 1H), 9.00 (s, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.21 (d, J = 2.6 Hz, 2H),7.87 (d, J = 8.5 Hz, 1H), 7.78 (dd, J = 5.3, 1.6 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H),2.56 (s, 3H).

[0144] Example 12: 1-(4-(4-fluorophenyl)pyridin-2-yl)-1 H pyrazole-4-carboxylic acid (Compound 12)

[0145]

[0146] The preparation method refers to Example 10, and compound 1 is replaced with compound 3. 1 H NMR (400 MHz, DMSO) δ 12.83 (s, 1H), 8.97 (s, 1H), 8.58 (s, 1H), 8.19 (s, 2H), 7.96 (s, 2H), 7.77(s, 1H), 7.40 (s, 2H).

[0147] Example 13: 1-(4-(4-trifluoromethylphenyl)pyridin-2-yl)-1 H -pyrazole-4-carboxylic acid (compound 13)

[0148]

[0149] The preparation method refers to Example 10, and compound 1 is replaced with compound 5. 1 H NMR (400 MHz, DMSO) δ 12.83 (s, 1H), 9.02 (s, 1H), 8.67 (s, 1H), 8.26 (d, J = 21.7 Hz, 2H), 8.13 (s,2H), 7.94 (d, J = 6.9 Hz, 2H), 7.87 (s, 1H).

[0150] Example 14: 1-([4,4'-bipyridinyl]-2-yl)-1 H -pyrazole-4-carboxylic acid (compound 14)

[0151]

[0152] The preparation method refers to Example 10, and compound 1 is replaced with compound 8. ESI-MS: m / z 267.2 [M+H] + C 14 H 10 N4O2.

[0153] Example 15: 1-(4-(4-methylthiazol-5-yl)pyridin-2-yl)-1 H -pyrazole-4-carboxylic acid (compound 15)

[0154]

[0155] The preparation method refers to Example 10, and compound 1 is replaced with compound 9. 1H NMR (400 MHz, DMSO) δ 12.81 (s, 1H), 9.21 (s, 1H), 8.98 (s, 1H), 8.62 (d, J = 5.2 Hz, 1H), 8.22 (s,1H), 8.04 (s, 1H), 7.62 (dd, J = 5.2, 1.4 Hz, 1H), 2.63 (s, 3H).

[0156] Biological experimental test example

[0157] Test Example 1 verifies that specific up-regulation of ALKBH8 protein is a biomarker of aging or senescence

[0158] Principle of experiment:

[0159] Using chemotherapy drug Etoposide to treat or 4-hydroxytamoxifen (4-OHT) to induce proto-oncogene RAS expression to promote senescence and other models, using immunoblotting method to analyze the relationship between ALKBH8 protein level and classical senescence markers p16 and p21. Using 4Gy X-ray irradiation of mice to induce systemic aging, and comparing the protein level of ALKBH8 in young tissues and aged tissues of naturally aging (18-month-old) mice.

[0160] Experimental materials and instruments:

[0161]

[0162] Experimental method:

[0163] Immunoblotting: After RIPA lysis of cell or tissue samples, the protein concentration was determined using BCA quantitative kit. After loading the same amount of protein, protein gel electrophoresis was performed.

[0164] 1. Running gel conditions: constant voltage 110 V, electrophoresis 80 min.

[0165] 2. Transmembrane (wet transfer) transfer conditions: constant voltage 110 V, transfer 80 min.

[0166] 3. Blocking. Shake bed at room temperature for 1 h with 5% skim milk.

[0167] 4. Primary antibody incubation. Add the corresponding primary antibody and incubate overnight at 4°C on a shaking bed.

[0168] 5. Secondary antibody incubation. After recovering the primary antibody, wash the membrane three times with 1 x TBST on a shaker at room temperature, add the corresponding rabbit or mouse antibody, and incubate on a shaker at room temperature for 1 h. After incubation, wash the membrane three times with 1 x TBST on a shaker at room temperature.

[0169] 6. ECL protein band development. Data is collected using a protein gel imaging system.

[0170] The structure of etoposide is as follows:

[0171]

[0172] The structure of 4-OHT is as follows:

[0173]

[0174] Experimental conclusion:

[0175] like Figure 1 a and Figure 1 As shown in Figure b, in senescent cells, the ALKBH8 protein level is specifically upregulated, consistent with classic aging markers p16 and p21. This is consistent with cell experiments, as... Figure 1 c and Figure 1 As shown in Figure d, ALKBH8 is also specifically upregulated in aging tissues. This together demonstrates that ALKBH8 is a biomarker of aging or senescence.

[0176] Test Example 2 verifies that ALKBH8 promotes the mRNA expression and protein secretion of aging inflammatory factors.

[0177] Experimental materials:

[0178]

[0179] Experimental methods:

[0180] Three experimental groups were used: young cells, control cells knocked down from senescent cells, and ALKBH8 knocked-down senescent cells. Transcriptome changes in the three groups were compared using a transcriptome sequencing system. Changes in the secretion levels of inflammatory factors in the three groups were detected using a cytokine microarray.

[0181] Experimental results:

[0182] from Figure 2 As shown in a, the deletion of ALKBH8 causes changes in the expression of a large number of genes. Among them, such as Figure 2 As shown in Figure b, multiple signaling pathways related to inflammatory factors were significantly altered in the ALKBH8 knockdown senescent cell group, such as... Figure 2As shown in Fig. 2c, the expression levels of some important senescence inflammatory factors were significantly reduced. Cytokine array is a high-throughput secreted protein detection technology, which is used to analyze the expression levels of various cytokines (such as interleukins, chemokines, growth factors, etc.), such as Figure 2 As shown in Fig. 2d, it is proved that the deletion of ALKBH8 significantly reduces the secretion of senescence-related inflammatory factors. Therefore, the transcriptome data combined with the cytokine array data indicate that ALKBH8 promotes the expression and secretion of senescence inflammatory factors.

[0183] Test Example 3 Biological effects of ALKBH8-mediated senescence inflammatory factors in promoting tumor growth and immune surveillance

[0184] Experimental materials:

[0185]

[0186] Experimental methods:

[0187] The ability of ALKBH8 to regulate senescent cells to promote tumor cell proliferation was detected by using immunodeficient mouse animal experiments. TOV-21G tumor cells and cells were mixed in PBS / matrix glue at a ratio of 1:2 (5x105 tumor cells, 1x106 senescent cells) and inoculated subcutaneously on the dorsal side of nude mice, and the cells were grouped into young cells, senescent control cells, and senescent cells with different sequences of ALKBH8 knockdown. After one week, the body weight and tumor volume of nude mice were monitored every 3 days, and the tumor growth curves were compared.

[0188] Using a sublethal dose of 4Gy radiation, Alkbh8 flox / flox Genetically engineered mice were systemically aged. After 7 days, AAV-TBG-Cre or AAV-TBG-GFP control virus was injected into the tail vein to reach the liver, and after the Alkbh8 gene was specifically knocked out in the liver, the liver tissue was collected after 7 days, and the mRNA expression of inflammatory factors and SA- -gal staining and statistical analysis.

[0189] SA- -gal staining:

[0190] The staining solution is prepared with reagents at pH 5.5.

[0191]

[0192] (1) The liver tissue was embedded in OCT and cut on ice.

[0193] (2) 0.5% glutaraldehyde fixation, room temperature for 15 min.

[0194] (3) Add SA-β-gal staining solution, place in a 37 ℃ CO2-free incubator, and after 6-8 hours, observe the tissue staining under a bright field. After the color develops appropriately, terminate the reaction after washing with PBS, and use eosin staining and perform mounting, take pictures under a microscope, and perform statistics on the positive proportion.

[0195] RNA extraction and fluorescent quantitative PCR:

[0196] Use the RNA extraction kit of Shengong to extract RNA, take the same amount of RNA, use the reverse transcription kit of Nuozhan to perform reverse transcription, obtain cDNA, and dilute the template into a 1:25 ratio. Prepare the quantitative PCR system according to the following component allocation:

[0197]

[0198] Quantitative PCR procedure:

[0199]

[0200] 2-3 steps are repeated for 40 cycles. According to the Ct value, the expression of the target gene mRNA is analyzed by the 2-ΔΔCt method.

[0201] Experimental results:

[0202] As shown in a and b of the present application, the experimental results show that the growth of tumor cells in the senescent cell group with mixed knockdown of ALKBH8 is slowed down. In the radiation-aged animal model, from c and d of the present application, compared with the wild type control, after knocking out Alkbh8 by using Cre enzyme expressed in the liver, the expression of inflammatory factors in the liver is analyzed, and it is found that the knockout Alkbh8 mouse group expresses a lower level of inflammatory factors, indicating that the degree of tissue inflammation is at a lower level compared with the wild type control group. As shown in e of the present application, further SA-β-gal staining analysis of the liver is performed, and it is found that the staining signal after gene knockout is significantly reduced, also indicating that the degree of tissue aging is lower. Figure 3 Figure 3 Figure 3 Figure 3 Figure 3

[0203] Test Example 4: The compound of the present application specifically inhibits the enzyme activity of ALKBH8 and its biological function of promoting the expression of senescence inflammatory factors

[0204] Experimental method:

[0205] Use the recombinant protein ALKBH8 to perform in vitro enzyme activity experiment and combine LC-MS characterization to detect the influence of the compound of the present application on the level of 5-carboxymethoxyuridine (mchm5U). ​​​​​

[0206] The enzyme activation reaction system was prepared at pH 7.5.

[0207]

[0208] After treating senescent cells with different doses of compound 1 for 96 hours, the mRNA expression levels of senescence-related inflammatory factors were analyzed. To assess the toxicity of the small molecule, mice were administered 25 mg / kg intraperitoneally daily for one month. The biosafety of the compound was verified by measuring liver weight ratio and serum enzyme activity levels, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST). One week after irradiation, mice were treated with the same concentration of compound 1 or a solvent (10% Solutol HS15) for one week, and the mRNA expression levels of senescence-related inflammatory factors were observed.

[0209] Experimental results:

[0210] like Figure 4 As shown in Figure a, in vitro enzyme activity assays revealed that compounds 10, 12, and 13 specifically reduced the production of the ALKBH8 product mchm5U. From... Figure 4 It was learned from study b that compound 1 can significantly reduce the mRNA expression of aging-related inflammatory factors. Through... Figure 4 From middle C to Figure 4 The results of the study showed that the body weight of normal mice did not change significantly, and indicators such as liver weight ratio and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity levels remained unchanged, demonstrating the biocompatibility of the small molecule compound. Figure 4 As shown in the study, compound 1 significantly reduced the expression of aging-related inflammatory factors (Cxcl1, Oas1, and Mmp3).

[0211] This invention marks the first discovery of ALKBH8 as a novel therapeutic target for inflammatory aging, and the successful development of a series of small-molecule inhibitory compounds. These compounds effectively inhibit the secretion of inflammatory factors, the tumor-promoting ability of senescent cells, and inflammation in aging tissues. Experiments at the enzyme, cellular, and animal levels validated the specific inhibition of ALKBH8 catalytic activity by these compounds, blocking downstream aging-related inflammatory pathways. This invention develops a novel target for the prevention and / or treatment of aging-related diseases and can also be used as a skin anti-aging agent, showing broad application prospects.

[0212] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition targeting ALKBH8, characterized in that, Comprising: (i) a compound of Formula I, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier; (I) wherein, R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl; selected from the group consisting of substituted or unsubstituted C6-C10aryl, substituted or unsubstituted 5-10 membered heteroaryl containing from 1-3 heteroatoms selected from N, O, or S; said substitution means substituted with one or more substituents selected from the group consisting of halogen, -OH, -CN, azido, -NH2, -NO2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl.

2. The pharmaceutical composition of claim 1, wherein R1is H or substituted or unsubstituted C1-C8alkyl.

3. The pharmaceutical composition of claim 1, wherein R1is substituted or unsubstituted C1-C8alkyl.

4. The pharmaceutical composition of claim 1, wherein selected from the group consisting of substituted or unsubstituted C6-C10aryl, substituted or unsubstituted 5-7 membered heteroaryl containing from 1-3 heteroatoms selected from N, O, or S.

5. The pharmaceutical composition of claim 1, wherein selected from the group consisting of pyridine, pyrimidine, pyrazine, thiophene, thiazole, pyrazole, pyrrole, imidazole, furan, oxazole, isoxazole; said heteroaryl is optionally substituted with a substituent selected from the group consisting of halogen, -OH, -CN, -NH2, -NO2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl.

6. The pharmaceutical composition of claim 1, wherein said compound of Formula I is selected from the group consisting of: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 7. A compound of Formula I: ###0002### or a pharmaceutically acceptable salt thereof. having the structure: (I) wherein, R1is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl, substituted or unsubstituted C1-C8haloalkyl; selected from the group consisting of substituted or unsubstituted C6-C10aryl, substituted or unsubstituted 5-10 membered heteroaryl containing from 1-3 heteroatoms selected from N, O, or S; said substitution means substituted with one or more substituents selected from the group consisting of halogen, -OH, -CN, azido, -NH2, -NO2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl; The additional condition is that: is not phenyl.

8. The compound of claim 7, wherein R1is H or substituted or unsubstituted C1-C8alkyl.

9. The compound of claim 7, wherein R1is substituted or unsubstituted C1-C8alkyl.

10. The compound of claim 7, wherein said heteroaryl is selected from the group consisting of pyridine, pyrimidine, pyrazine, thiophene, thiazole, pyrazole, pyrrole, imidazole, furan, oxazole, isoxazole; and said heteroaryl is optionally substituted with a substituent selected from the group consisting of halogen, -OH, -CN, -NH2, -NO2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl.

11. A compound, characterized in that, said compound is selected from the group consisting of: 、 、 、 、 、 、 、 、 、 、 、 、 、 。 12. The pharmaceutical composition of claim 1, the use of the compound of claim 7 or 11, wherein, said use comprises: (i) use as an ALKBH8 inhibitor; (ii) use as a skin anti-aging agent; (iii) use for the manufacture of a medicament for the prevention and / or treatment of an aging-related disease.

13. The use according to claim 12, characterized in that, said aging-related disease is selected from the group consisting of tumor, liver fibrosis, lung fibrosis, cardiovascular disease, neurodegenerative disease, skeletal muscle system disease, respiratory system disease, chronic obstructive pulmonary disease.

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