Use of a class of compounds containing a 2,4-thiazole ring in anti-aging

Abstract

This invention relates to the field of biomedicine and discloses the application of a compound containing a 2,4-thiazole ring in anti-aging. The compound containing a 2,4-thiazole ring is a compound of formula X or a pharmaceutically acceptable salt thereof, wherein the compound of formula X includes class I compounds with a pyrazolopyrimidine skeleton and class II compounds with an indole skeleton. The inventors have for the first time discovered that IL4I1 is a key molecule regulating cellular senescence and elucidated the mechanism by which the compound of formula X maintains mitochondrial homeostasis and delays cellular and tissue senescence by targeting IL4I1, relieving IL4I1's ubiquitination and degradation of EIF4B, and upregulating EIF4B-PGAM5 axis expression. Experimental results show that the compound containing a 2,4-thiazole ring described in this invention can effectively prevent radiotherapy and chemotherapy-induced cellular senescence, reduce radiotherapy-induced overall aging in mice, improve motor function, reduce the secretion of aging-related secretory phenotypic factors, and alleviate organ fibrosis. This invention provides a new chemical molecular basis and target for the development of novel anti-aging drugs.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of a class of compounds containing a 2,4-thiazole ring in anti-aging. Background Technology

[0002] Any discussion of prior art throughout the specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0003] Aging can be divided into natural aging and exogenously induced aging. Among the many exogenous factors, chemical drugs and radiation are important inducers of accelerated aging. Studies have shown that radiotherapy and chemotherapy can lead to the accumulation of senescent cells in various tissues and organs, thereby causing a variety of age-related diseases such as metabolic dysfunction, multi-organ fibrosis, and degenerative changes.

[0004] Aging is characterized by a variety of hallmarks, including: (1) fundamental markers: genomic instability, telomere depletion, epigenetic alterations, protein homeostasis imbalance, and autophagy disorders; (2) antagonistic markers: malnutrition sensing disorder, mitochondrial dysfunction, and cellular senescence; and (3) integrative markers: stem cell depletion, abnormal intercellular communication, chronic inflammation, and dysbiosis. Current anti-aging interventions include: senolytics (such as dasatinib / quercetin) to clear senescent cells; senomorphics (such as rapamycin and metformin) to regulate aging phenotypes; and lifestyle interventions such as calorie restriction and regular exercise.

[0005] Mitochondrial dysfunction plays a central role in the aging process, manifesting as functional decline, redox imbalance, and genomic instability. Senescent cells undergo dynamic changes in mitochondria, particularly PGAM5-mediated DRP1 dephosphorylation, which inhibits mitochondrial division and activates the mTOR signaling pathway, thereby promoting aging. Notably, apoptosis-induced mini mitochondrial outer membrane potential (miMOMP) triggers the mtDNA-cGAS-STING signaling pathway, driving the formation of age-associated secretory phenotypes (SASPs) and exacerbating the aging burden. These mechanisms suggest that maintaining mitochondrial homeostasis is a key therapeutic point for anti-aging interventions.

[0006] IL4I1 was initially discovered to be upregulated upon stimulation by IL-4. The protein possesses L-amino acid oxidase activity, with aromatic amino acids, primarily tryptophan, as its substrate. IL4I1 activates the AHR pathway and promotes the production of pro-inflammatory factors by catalyzing the conversion of tryptophan to indole-3-propionic acid, thus playing a role in tumor immunosuppression and inflammatory responses. Summary of the Invention

[0007] To address the lack of safe, effective, and clearly targeted anti-aging drugs in existing technologies, this invention aims to provide a novel application of compounds containing a 2,4-thiazole ring in the preparation of anti-aging drugs. This invention demonstrates for the first time that these compounds can specifically target IL4I1, maintaining mitochondrial homeostasis by regulating the IL4I1-EIF4B-PGAM5 signaling axis, and effectively reducing aging-related indicators in in vitro and in vivo experiments, providing new candidate molecules and targets for the development of anti-aging drugs.

[0008] The inventor made the following discovery for the first time:

[0009] (1) IL4I1 is a key molecule regulating cellular senescence; (2) IL4I1 regulates aging by promoting the K48-linked ubiquitination degradation of EIF4B; (3) The EIF4B-PGAM5 axis maintains mitochondrial homeostasis; (4) The present invention demonstrates through in vitro and in vivo experiments that the general formula compound shown in Formula X has anti-aging activity; among which, the representative compound IA 3 can target IL4I1 and regulate IL4I1 EIF4B The PGAM5 signaling axis maintains mitochondrial homeostasis, thereby playing an anti-aging role.

[0010] The discovery of the above mechanism provides new targets and theoretical basis for the development of anti-aging drugs.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of a compound of formula X or a pharmaceutically acceptable salt thereof in the preparation of an anti-aging medicament:

[0012] Wherein, structure A is pyrazolopyrimidine or indole, and the compound conforms to the structure shown in formula X1 or formula X2:

[0013] Z is absent, carbonyl, or –C(O)NH–; X is either O or S; Y is -O-, -NH-, or ; R1 is hydrogen or C. 1-6 alkyl; R2 is selected from C1-C3 alkyl groups, C5-C6 alkyl groups, and C2 is selected from C1-C3 alkyl groups. 15 Alkenyl, alkynyl, 5-10 membered heterocyclic groups, C6-C 12Aryl, 5-12 membered heteroaryl, sterol, and 5-10 membered cycloalkyl; Y is directly attached to R2, or Y is attached to R2 to form a ring; R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, 3-10 membered heterocyclic group, C6-C. 12 Aryl, 5-12 membered heteroaryl, 3-10 membered cycloalkyl, ester group, carboxyl group, trihalomethyl and adamantyl; R2 or R3 is either unsubstituted or substituted by one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl. When R2 is a C1-C3 alkyl group, R3 is not hydrogen.

[0014] In this invention, the term "C1-C6 alkyl" refers to a straight-chain saturated hydrocarbon group having 1 to 6 carbon atoms on the chain, including, without limitation, methyl, ethyl, propyl, etc.

[0015] The term "C5-C" 15 "Alkenyl" refers to a straight or branched hydrocarbon group with 5 to 15 carbon atoms and containing one or more double bonds.

[0016] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group with 2 to 15 carbon atoms on the chain and containing one or more carbon-carbon triple bonds, such as ethynyl, propynyl, butynyl, etc.

[0017] The term "3-10 membered heterocyclic group" refers to a heterocyclic group containing one or more saturated and / or partially saturated rings, comprising 3-10 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen or S(0)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon; for example, propylene oxide, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.

[0018] The term "C6-C" 12 "Aryl" refers to an aromatic cyclic group containing 6-12 ring atoms, but without heteroatoms, such as phenyl, naphthyl, biphenyl, etc.

[0019] The term "5-12 membered heteroaryl" refers to an aromatic cyclic group containing 5-12 ring atoms, with 1-4 heteroatoms as ring members. The heteroatoms can be selected from nitrogen, oxygen, or sulfur. The heteroaryl can be a monocyclic heteroaryl with 5-7 ring atoms or a bicyclic heteroaryl with 7-12 ring atoms. In the bicyclic heteroaryl, only one ring needs to be a heteroaryl; the other can be an aromatic or non-aromatic ring, containing or not containing heteroatoms. Furthermore, the bicyclic heteroaryl can be a fused ring structure or two heterocycles directly linked. Examples of heteroaryl groups include, but are not limited to, pyrrole, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyridinyl, pyrimidinyl, furanyl, thiophene, isoxazolyl, and indole.

[0020] The term "sterol group" refers to a cyclopentane polyhydrophenanthrene derivative group formed by the fusion of three hexane rings and one cyclopentane ring, such as sitosterol group, cholesterol group, ergosterol group, solanamine group, diosgenin group, etc.

[0021] The term "3-10 membered cycloalkyl" refers to a group containing one or more saturated and / or partially saturated rings, all of which are carbon atoms, including 3 to 10 carbon atoms; for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptenyl, cyclohepttrienyl, adamantyl, etc.

[0022] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0023] The term "halomethyl" refers to a methyl group that has been substituted with a halogen. It can be monosubstituted, disubstituted, or trisubstituted, including trifluoromethyl, bromomethyl, etc.

[0024] The term "trihalomethyl" refers to -CX3, where each X is independently selected from F, Cl, Br, or I, such as trifluoromethyl, trichloromethyl, or tribromomethyl.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable salt may be a hydrochloride, sulfate, phosphate, maleate, fumarate, citrate, methanesulfonate, p-toluenesulfonate, or tartrate of the compound, etc.

[0026] In some embodiments of the present invention, R2 is selected from C1-C3 alkyl groups and C5-C6 alkyl groups. 15 Alkenyl, C5-C 15 Dieneyl, C5-C 15 Trienyl, alkynyl, 5-6 membered cycloalkyl, phenyl, 5-6 membered heterocyclic, 5-6 membered heteroaryl, sterol; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R2 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

[0027] Furthermore, in some embodiments of the present invention, R2 is selected from methyl, ethyl, propyl, C5-alkenyl, C6-ethyl, C7-ethyl ... 10 Dieneyl, C 15 Trienyl, alkynyl, cyclopentyl, cyclohexyl, triazolyl, phenyl, piperidinyl, piperazine, pyrrolyl, pyridyl, pyrimidinyl, sterolyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R2 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl. Wherein, the sterol group is selected from , , .

[0028] In some embodiments of the present invention, R3 is selected from hydrogen, halogen, amino, acetyl, 5-6 membered heterocyclic group, phenyl, biphenyl, naphthyl, 5-6 membered heteroaryl, 5-6 membered cycloalkyl, ester group, carboxyl group, amide group, trihalomethyl, adamantyl. R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

[0029] Furthermore, in some embodiments of the present invention, R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, phenyl, biphenyl, naphthyl, cyclopentyl, cyclohexyl, piperidinyl, piperazine, pyrrolyl, pyridinyl, pyrimidinyl, ester, carboxyl, amide, trihalomethyl, and adamantyl. R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

[0030] In some embodiments of the present invention, the compound has the structure described in Formula I or Formula II:

[0031] Among them, X, Y, R1, R2, and R3 are the same as defined above.

[0032] In these embodiments, in the compound of formula I, X is O or S; Y is -O-, -NH-, or ; R1 is hydrogen or a C1-C2 alkyl group; R2 is selected from methyl, ethyl, C5-enyl, C6-ethyl, C7-ethyl, C6-ethyl 10 dienyl, cyclohexyl, phenyl, pyridyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R3 is selected from hydrogen, phenyl, pyridyl, pyrimidinyl, ester, and trihalomethyl; R2 or R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, and carboxyl.

[0033] In these embodiments, in compounds of formula II, X is O or S; Y is -O-, -NH-, or ; R2 is selected from methyl, ethyl, propyl, C5-enyl, C6-ethyl, C7-ethyl, C6 ... 10 Dieneyl, C 15 Trienyl, alkynyl, cyclopentyl, cyclohexyl, phenyl, triazolyl, pyridyl, sterolyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; The sterol group is selected from... , , ; R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, phenyl, biphenyl, naphthyl, cyclopentyl, cyclohexyl, pyrrolyl, pyridyl, pyrimidinyl, ester, carboxyl, amide, trihalomethyl, and adamantyl. R2 or R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

[0034] In some embodiments of the present invention, when X is O, the compound has the structure shown in Formula IA or Formula IIA:

[0035] Among them, Y, R1, R2, and R3 are defined as above, Y is directly connected to R2, or Y is connected to R2 to form a loop.

[0036] In some embodiments of the present invention, when X is S, the compound has I B The structure shown is as follows:

[0037] Where Y is -NH-, and R1, R2, and R3 are as defined above; Furthermore, in some embodiments, in the IB compound, R2 is selected from methyl, ethyl, and pyridyl; R3 is selected from hydrogen, pyridyl, and pyrimidinyl.

[0038] In some embodiments of the present invention, Y is -O-, -NH-, or When X is 0; When Y is -O- or -NH-, Y and R2 can be directly connected; Or, Y is When Y connects to R2 to form a ring, it forms the R2' structure, and the N atom acts as a ring-forming atom in the R2' structure. The compound has the structure shown in IA1, IA2, IA3, IIA1, IIA2 or IIA3:

[0039]

[0040] Among them, R1, R2, and R3 are as defined above; R2' is selected from... , or .

[0041] In some embodiments of the present invention, R2' is selected from... , or .

[0042] Furthermore, in some embodiments of the present invention, in formula IIA2, R2 is selected from... , , In this case, R3 is selected from halogens, hydroxyl groups, phenyl groups, naphthyl groups, and adamantyl groups.

[0043] As an example, the present invention provides a series of compounds selected from the following structures: .

[0044] In some embodiments of the present invention, the pharmaceutically acceptable salt is a hydrochloride, sulfate, phosphate, maleate, fumarate, citrate, methanesulfonate, p-toluenesulfonate, or tartrate. Preferably, the pharmaceutically acceptable salt is a hydrochloride.

[0045] In some embodiments of the present invention, the anti-aging drug exerts its effect by targeting IL4I1. The inventors, through techniques such as chemical probe enrichment and surface plasmon resonance, have for the first time discovered and confirmed that IL4I1 is the direct target of the compound shown in Formula X. The dissociation equilibrium constant (Ka) between the representative compound IA-3 and the IL4I1 protein is shown. D The value is 14.8 µM.

[0046] In some embodiments of the present invention, the anti-aging drug exerts its effect by inhibiting IL4I1-mediated EIF4B ubiquitination degradation. The inventors have discovered that IL4I1 mediates the proteasome degradation of EIF4B via a K48-linked ubiquitination pathway, and that the compound shown in Formula X can stabilize EIF4B protein levels by targeting IL4I1.

[0047] In some embodiments of the present invention, the anti-aging drug maintains mitochondrial homeostasis by regulating the IL4I1-EIF4B-PGAM5 signaling axis. The inventors have discovered that EIF4B is an upstream regulator of PGAM5, and the absence of either EIF4B or PGAM5 leads to impaired mitochondrial division and reduced mitochondrial turnover, thereby promoting cellular senescence. The compound shown in Formula X maintains PGAM5 expression and mitochondrial homeostasis by inhibiting IL4I1 activity and reducing EIF4B degradation, thus exerting an anti-aging effect.

[0048] In some embodiments of the present invention, the anti-aging drug is used to prevent or alleviate aging induced by radiotherapy and chemotherapy. The aging induced by radiotherapy and chemotherapy includes cellular aging induced by radiotherapy and chemotherapy and / or tissue aging induced by radiotherapy and chemotherapy.

[0049] Furthermore, the cellular senescence induced by radiotherapy and chemotherapy includes, but is not limited to, doxorubicin-induced endothelial cell senescence and X-ray-induced endothelial cell senescence.

[0050] Furthermore, the radiotherapy-induced tissue aging includes, but is not limited to, radiotherapy-induced cardiac aging, lung aging, kidney aging, liver aging, or adipose tissue aging.

[0051] In some embodiments of the present invention, the anti-aging drug is used to prevent or alleviate replicative aging.

[0052] In some embodiments of the present invention, the anti-aging drug is used for: (a) Reduce the proportion of aging-associated β-galactosidase (SA-β-gal) positive cells; and / or (b) Downregulating the expression of aging markers p21 and / or p16; and / or (c) Inhibit the expression of the DNA damage marker γ-H2AX; and / or (d) Reduce the expression or secretion of aging-associated secretory phenotype (SASP) factors.

[0053] Furthermore, the aging-related secretory phenotypic factor is selected from one or more of IL-1α, IL-1β, IL-6, IL-8, IL-23, IL-32, TNF-α, CCL5, CXCL8, CXCL11, MMP1, MMP3, and MMP10.

[0054] A second aspect of the present invention provides a pharmaceutical composition comprising a compound of formula X or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient or carrier, said pharmaceutical composition for anti-aging purposes.

[0055] In some embodiments of the present invention, the active ingredient is IA-3 or its hydrochloride salt.

[0056] In some embodiments of the present invention, the pharmaceutical composition is an oral formulation or an injectable formulation. The oral formulation includes, but is not limited to, tablets, capsules, granules, and oral liquids; the injectable formulation includes, but is not limited to, injection solutions and lyophilized powder injections.

[0057] Pharmaceutically acceptable excipients refer to components in the pharmaceutical composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, solvents, solubilizers, fillers, lubricants, disintegrants, buffers, stabilizers, or preservatives. The drug carrier can be a pharmaceutically acceptable solvent, suspending agent, or carrier for delivering the compound to animals or humans.

[0058] In a third aspect, the present invention also provides the application of IL4I1 as a target in screening anti-aging drug candidates. The present invention provides a method for screening anti-aging drug candidates, the method comprising: contacting a candidate substance with IL4I1 protein or cells expressing IL4I1, and detecting at least one of IL4I1 activity, the interaction between IL4I1 and EIF4B, or the ubiquitination level of EIF4B; and screening, based on the detection results, candidate substances capable of inhibiting IL4I1 activity, blocking the interaction between IL4I1 and EIF4B, reducing the ubiquitination level of EIF4B, or upregulating the expression level of EIF4B as anti-aging drug candidates.

[0059] Furthermore, the use of IL4I1 inhibitors in the preparation of medicaments for the prevention, treatment, or alleviation of age-related pathological conditions also falls within the scope of protection of this invention. In some embodiments, the IL4I1 inhibitor is a compound of formula X above or a pharmaceutically acceptable salt thereof; in other embodiments, the IL4I1 inhibitor may also be selected from antibodies targeting IL4I1, siRNA, shRNA, antisense nucleic acids, nucleic acid aptamers, or CRISPR / Cas system components.

[0060] In a fourth aspect, the present invention also provides a kit for screening anti-aging drug candidates. The kit comprises at least one of the following components: IL4I1 protein or a functional fragment thereof, a cell line expressing IL4I1, a substrate and / or indicator for detecting IL4I1 enzyme activity, EIF4B protein or a functional fragment thereof, and a reagent for detecting protein-protein interactions. In some embodiments, the kit further comprises an anti-IL4I1 antibody, an anti-EIF4B antibody, and / or an anti-ubiquitin antibody. In some preferred embodiments, the kit further comprises a compound of formula X of the present invention as a positive control.

[0061] This invention also provides the use of IL4I1 detection reagents in the preparation of detection products for assessing the aging state of cells or tissues. The IL4I1 detection reagents include, but are not limited to, anti-IL4I1 antibodies, IL4I1 nucleic acid probes, IL4I1 PCR primer pairs, or IL4I1 enzyme activity detection substrates.

[0062] The present invention also provides a kit for assessing aging status or predicting the effect of anti-aging treatment, the kit comprising reagents for detecting IL4I1 protein expression level or enzyme activity, EIF4B protein expression level or ubiquitination level, and / or PGAM5 protein expression level.

[0063] The beneficial effects of this invention are as follows: (1) This invention is the first to discover that compounds containing a 2,4-thiazole ring have significant anti-aging activity, providing new candidate compounds for the development of anti-aging drugs. The compounds described in this invention have different mechanisms of action from existing anti-aging drugs (such as senolytics drugs dasatinib / quercetin and senomorphics drugs rapamycin), enriching the research and development strategies for anti-aging drugs.

[0064] (2) This invention reveals for the first time that IL4I1 is a new target for anti-aging. Existing research mainly focuses on the role of IL4I1 in tumor immunity and other fields, while its role in the regulation of aging related to radiotherapy and chemotherapy stress still lacks systematic research. This invention demonstrates through in vitro and in vivo experiments that inhibiting or knocking out IL4I1 can significantly reduce radiotherapy and chemotherapy-induced aging, providing a new direction for the screening of anti-aging drug targets.

[0065] (3) This invention elucidates for the first time the mechanism of the IL4I1-EIF4B-PGAM5 signaling axis in aging regulation. This invention discovers that IL4I1 mediates EIF4B degradation through K48-linked ubiquitination, and EIF4B in turn regulates PGAM5 to maintain mitochondrial division and turnover, revealing a new aging regulatory pathway and providing a new theoretical basis for understanding the molecular mechanisms of aging.

[0066] (4) The compounds described in this invention can effectively prevent cell senescence induced by radiotherapy and chemotherapy in vitro. The representative compound IA-3 can reduce the accumulation of senescent cells, alleviate cell cycle arrest, inhibit DNA damage, and downregulate SASP factor expression in a dose-dependent manner, and has no obvious toxicity to normal cells.

[0067] (5) The compounds described in this invention can significantly reduce the radiotherapy-induced aging phenotype in mice in vivo. IA-3 treatment can reduce hair graying, improve motor function, reduce serum inflammatory factor levels, and alleviate pathological changes and fibrosis in multiple organs such as the heart, lungs, and kidneys.

[0068] (6) Representative compound IA-3 has good pharmacokinetic properties and safety. IA-3 has good oral bioavailability (about 22%), no obvious toxicity was observed at the tested concentration, and has good drug-like properties, and is expected to be developed into a new anti-aging drug. Attached Figure Description

[0069] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. The embodiments of this application will be described in detail below with reference to the accompanying drawings, wherein: Figure 1This study illustrates the prevention of radiotherapy- and chemotherapy-induced cellular senescence by compound IA-3, including: (a) the chemical structure of compound IA-3; and (b) representative images of senescence-associated β-galactosidase (SA-β-gal) staining. The top row shows the doxorubicin (Dox)-induced senescence model of human umbilical vein endothelial cells (HUVECs), in order: control group, Dox group, Dox+IA-3 4 μM group, Dox+IA-3 2 μM group, and Dox+IA-3 1 μM group. The bottom row shows the X-ray-induced HUVEC cell senescence model, in order: control group, X-ray group, X-ray+IA-3 40 μM group, X-ray+IA-3 20 μM group, and X-ray+IA-3 10 μM group. (c) Percentage of SA-β-gal positive cells in chemotherapy-induced senescence model (left) and radiotherapy-induced senescence model (right); (d) Cell morphology of mouse primary lung cell replication senescence model (cultured for 21 days), the upper figure is the dimethyl sulfoxide (DMSO) solvent control group, and the lower figure is the IA-3 treatment group; (e) Percentage of SA-β-gal positive cells in mouse primary lung cell replication senescence model; (f) Flow cytometry analysis of cell cycle distribution; (g) Cell proliferation curve detected by real-time cell analyzer (xCELLigence); (h) Cell index statistics at different time points; (i) γ-H2AX immunofluorescence staining results, including γ-H2AX, DAPI and Merge three-channel images; (j) Western spectroscopy. blot detection of p21 protein expression results, with Actin as an internal control; (k) RNA sequencing heatmap analysis of aging-related gene expression profiles; (l) real-time quantitative PCR (qRT-PCR) verification of mRNA expression changes of aging biomarkers and aging-related secretory phenotype (SASP) factors; (m) Reactome pathway enrichment analysis results, with the left figure comparing the Dox group and the control group, and the right figure comparing the IA-3 pretreatment group and the Dox group.

[0070] Figure 2The study demonstrates how compound IA-3 alleviates radiotherapy-induced aging in mice, including: (a) a flowchart of the animal experiment design, where 8-week-old C57BL / 6J mice were divided into a blank control group (0 Gy TBI), a model group (5 Gy TBI), a low-dose IA-3 hydrochloride group (5 Gy TBI + 20 mg / kg), and a high-dose IA-3 hydrochloride group (5 Gy TBI + 30 mg / kg). One week after pre-administration, mice underwent total body irradiation (TBI), followed by gavage administration every other day. Phenotypic assessment was performed at week 10, and samples were collected at week 11; (b) photographs of the mice in each group; (c) statistics of the drop latency in the rotating bar test; (d) statistics of the drop velocity in the rotating bar test; (e) detection of serum SASP factor levels in each group using enzyme-linked immunosorbent assay (ELISA); (f) SA-β-gal tissue staining results for adipose tissue, kidney, and liver; and (g) Western chromatographic analysis of the mice. blot analysis of p21 protein expression in kidney, liver and heart, with Actin as internal control; (h) qPCR analysis of cdkn1a and cdkn2a mRNA levels in heart, lung and spleen; (i) hematoxylin-eosin (HE) staining results of heart, lung and kidney; (j) Masson trichrome staining results of heart, lung and kidney; (k) immunohistochemical staining results of p16, p21 and γ-H2AX in heart; (l) immunohistochemical staining results of p16, p21 and γ-H2AX in lung; (m) immunohistochemical staining results of p16, p21 and γ-H2AX in kidney.

[0071] Figure 3This study demonstrates that compound IA-3 targets IL4I1 and exerts anti-aging effects in vitro, including: (a) silver staining analysis of the target protein enriched by the IA-3 probe, with Western blot verification on the right confirming IL4I1 as the target protein enriched by the IA-3 probe; (b) immunoprecipitation assay verifying IL4I1 as the binding target of IA-3; (c) surface plasmon resonance (SPR) assay detecting the binding affinity of IA-3 and the IA-3 probe to IL4I1 protein; (d) cell thermal displacement assay (CETSA) detecting the effect of IA-3 treatment on the thermal stability of IL4I1 protein; (e) CETSA quantitative analysis results; (f) immunoprecipitation assay of purified IL4I1 protein with the IA-3 probe in vitro; and (g) Western blot analysis. (h) Representative images of SA-β-gal staining in wild-type (WT) and IL4I1-KO cells in a radiotherapy-induced aging model; (i) Statistical analysis of the percentage of SA-β-gal positive cells; (j) Western blot analysis of p21 protein expression in a radiotherapy-induced aging model; (k) Western blot analysis of p21 protein expression in a chemotherapy-induced aging model; (l) Immunofluorescence staining results of γ-H2AX in a radiotherapy-induced aging model; (m) Immunofluorescence staining results of p21 in a radiotherapy-induced aging model; (n) qRT-PCR analysis of SASP factor mRNA expression; (o) Representative images of SA-β-gal staining in WT and IL4I1-KO cells in a mouse primary kidney cell replication aging model (cultured for 21 days); (p) Statistical analysis of the percentage of SA-β-gal positive cells in a mouse primary kidney cell replication aging model.

[0072] Figure 4 The diagram illustrates how Il4i1 knockout mice are protected from radiation-induced aging, including: (a) a flowchart of the animal experimental design, showing 8-week-old wild-type (WT) and Il4i1 knockout mice. Il4i1 - / - C57BL / 6J mice were irradiated with 0 or 5 Gy whole body, respectively, to form WT-0 Gy. Il4i1 - / - -0 Gy, WT-5 Gy and Il4i1 - / --5 Gy Four groups, phenotypic assessment at week 10, and sampling at week 11; (b) Appearance photographs of mice in each group; (c) Statistics of drop latency in the rotating bar test; (d) Statistics of drop velocity in the rotating bar test; (e) Serum SASP factor level detected by ELISA; (f) SA-β-gal tissue staining results of adipose tissue, kidney and liver; (g) Western blot detection of p21 protein expression in kidney, liver and heart, with Actin as internal control; (h) Immunohistochemical staining results of p16, p21 and γ-H2AX in heart; (i) Immunohistochemical staining results of p16, p21 and γ-H2AX in lung; (j) Immunohistochemical staining results of p16, p21 and γ-H2AX in kidney; (k) HE staining results of heart, lung and kidney; (l) Masson staining results of heart, lung and kidney.

[0073] Figure 5 This study illustrates the ubiquitination and degradation of eukaryotic translation initiation factor 4B (EIF4B) mediated by IL4I1, including: (a) KEGG pathway enrichment analysis of the IL4I1 interacting proteome; (b) silver staining identification of Flag-IL4I1 immunoprecipitation products, with EIF4B identified as the interacting protein; (c) co-immunoprecipitation verification of the interaction between IL4I1 and EIF4B; (d) detection of changes in EIF4B protein levels after gradient transfection of Myc-IL4I1 in HEK293T cells; (e) cyclohexylimide (CHX) tracking assay to detect the dynamics of EIF4B protein degradation; (f) quantitative analysis results of the CHX tracking assay; (g) ubiquitination detection assay showing the total ubiquitination level of EIF4B; (h) K48-linked ubiquitin chain analysis results; and (i) Western blot analysis. (j) Validation of CRISPR / Cas9-constructed EIF4B knockout HUVEC cells; (k) Representative images of SA-β-gal staining in WT and EIF4B-KO cells in a radiotherapy-induced aging model; (l) Statistical analysis of the percentage of SA-β-gal positive cells; (m) Western blot analysis of EIF4B and p21 protein expression under control and X-Ray conditions; (n) qRT-PCR analysis of the relative expression level of p21 mRNA under control and X-Ray conditions; (d) qRT-PCR analysis of the relative expression level of SASP factor mRNA under control and X-Ray conditions.

[0074] Figure 6This study demonstrates how the EIF4B-PGAM5 axis maintains mitochondrial homeostasis and mitigates aging-related secretory phenotypes, including: (a) silver staining to identify EIF4B interacting proteins; (b) Western blot analysis of changes in EIF4B and PGAM5 protein expression after EIF4B knockout; (c) qPCR analysis of changes in EIF4B and PGAM5 mRNA expression after EIF4B knockout; (d) co-immunoprecipitation to verify the interaction between EIF4B and PGAM5; (e) Western blot analysis of changes in EIF4B and PGAM5 protein expression during radiation-induced aging; (f) Western blot analysis of CRISPR / Cas9-constructed PGAM5 knockout cells; (g) representative images of SA-β-gal staining in WT, PGAM5-KO1, and PGAM5-KO2 cells under control and Dox conditions; (h) statistical analysis of the percentage of SA-β-gal positive cells; (i) Western blot analysis of PGAM5 and p21 protein expression in WT and PGAM5-KO cells; and (j) [followed by a specific method / method]. (k) qRT-PCR detection of SASP factor mRNA expression in WT and PGAM5-KO cells; (l) qRT-PCR detection of mitochondrial-related protein expression in PGAM5 knockout cells; (m) qRT-PCR detection of related gene mRNA expression in PGAM5 knockout cells; (n) qRT-PCR detection of related gene mRNA expression in EIF4B knockout cells; (o) MitoTimer fluorescence detection of mitochondrial turnover in PGAM5-KO cells; (p) MitoTimer fluorescence detection of mitochondrial turnover in EIF4B-KO cells; (q) Western blot detection of the effect of IA-3 pretreatment on Dox-induced changes in EIF4B and PGAM5 protein expression. Detailed Implementation

[0075] This application is further illustrated with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope thereof. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0076] Unless otherwise defined, all technical and scientific terms used in this application shall have meanings familiar to those skilled in the art. Unless otherwise specified, all reagents or raw materials used in this application are obtainable through conventional means and used in accordance with conventional methods or product instructions in the art. Furthermore, any methods or materials similar to or equivalent to those described may be applied to the methods of this application. The preferred embodiments and materials described in this application are for illustrative purposes only.

[0077] Unless otherwise stated, all cells used in the following examples were cultured under standard culture conditions in the art, and all reagents, antibodies, kits, and instruments used were from conventional sources in the art. Experimental procedures not specifically defined were performed according to standard methods in the art or the instructions for use of the corresponding kits / instruments; the solvent volume fraction used in the control group and model group was kept consistent. Each detection index could be replicated in parallel and statistically analyzed. Statistical methods could be selected according to the experimental design, such as t-tests or one-way ANOVA.

[0078] Animal experiments were conducted in accordance with laboratory animal management regulations and ethical requirements. Animals were randomly grouped and housed under the same conditions.

[0079] The preparation methods of compounds IA-1~IA-31, IB-1~IB-4, IIA-1~IIA-30 and IIB-1~IIB-5 of the present invention are as follows: they are prepared according to the method disclosed in the applicant's prior patent CN113292570A, the entire contents of which are incorporated herein by reference and constitute a part of the present invention.

[0080] Example 1: Screening of in vitro anti-aging activity of compounds of general formula This embodiment aims to systematically evaluate the in vitro anti-aging activity of compounds of the general formula having the structure shown in Formula X.

[0081] 1. Test compounds: The test compounds listed in Table 1 2. Experimental methods: Dox-induced cell senescence model and treatment with test compounds: Human umbilical vein endothelial cells (HUVECs) were seeded into culture plates and cultured until adherent. Control, model, and test compound groups were established. The control group received an equal volume of solvent but no Dox. The model group received Dox to induce senescence and received the same volume of solvent as the test compound groups. The test compound groups were pretreated with 10 μM of the corresponding test compound for 6 h, followed by incubation with 60 nM Dox for 48 h. After Dox treatment, the culture medium was discarded and replaced with medium containing the corresponding test compound for another 48 h.

[0082] SA-β-galactosidase staining and statistics: Cell senescence staining was performed using an SA-β-galactosidase staining kit. The staining working solution was prepared according to the kit instructions, and the cells were fixed, washed, stained, and imaged. Cells were observed and photographed under a regular optical microscope, and the percentage of SA-β-gal positive cells [i.e., (number of SA-β-gal positive cells / total number of cells) × 100%] was counted to evaluate the inhibitory effect of the test compound on the Dox-induced senescence phenotype.

[0083] 3. Experimental results: See Table 1 Table 1. In vitro anti-aging activity of compounds represented by formula X

[0084] The results showed that, compared with the model group, all tested compounds reduced the proportion of Dox-induced SA-β-gal positive cells, indicating that the compounds of formula X as a whole have an inhibitory effect on cell senescence. Among them, IA-3 showed a better inhibitory effect, therefore IA-3 was selected as a representative compound for subsequent in vitro and in vivo efficacy and mechanism of action studies.

[0085] Example 2: Compound IA-3 prevents radiotherapy and chemotherapy-induced cellular senescence Based on the activity screening results of Example 1, this example selects IA-3, a representative compound with the best activity, to further verify its in vitro anti-aging activity and dose-response relationship. The chemical structural formula of compound IA-3 is as follows: Figure 1 As shown in (a), it belongs to Formula I compounds and has a pyrazolopyrimidine skeleton.

[0086] The anti-aging effects of IA-3 were evaluated using the chemotherapy drug doxorubicin (Dox) and an X-ray-induced aging model of human umbilical vein endothelial cells (HUVEC).

[0087] Experimental methods: Dox-induced cell senescence model: HUVEC cells were pretreated with 0, 1, 2 or 4 μM IA-3 for 6 h, then treated with 60 nM Dox for 48 h. The Dox was discarded, and the culture medium containing IA-3 was replaced, and the cells were cultured for another 48 h.

[0088] X-ray-induced cell senescence model: HUVEC cells were pretreated with 0, 10, 20 or 40 μM IA-3 for 6 h and then subjected to 5 Gy X-rays for 10 days.

[0089] Replicative senescence model: Primary mouse lung cells were cultured continuously for 21 days to induce replicative senescence; during the induction process, compound IA was added according to the experimental groups. 3. Perform treatment; after culture, observe cell morphology and SA. β gal staining statistics (SA) β The proportion of gal-positive cells was used to assess the degree of replication senescence.

[0090] Cell senescence staining: Aspirate the cell culture medium, wash once with PBS, and fix with fixative at room temperature for 15 minutes. Aspirate the cell fixative, wash the cells three times with PBS for 3 minutes each time. Aspirate the PBS, add staining working solution (Solution A:Solution B:Solution C:X-Gal solution = 1:1:93:5), and incubate overnight at 37°C. Observe the staining under a regular optical microscope, take pictures, and count the proportion of senescent cells.

[0091] Cell cycle analysis: Collect cells, centrifuge, and discard the supernatant. Resuspend in pre-chilled PBS, centrifuge again, discard the supernatant, add 1 mL of pre-chilled 70% ethanol for fixation, and incubate overnight at -20 ℃. The next day, centrifuge, discard the supernatant, and add 0.5 mL of pyridine iodide staining solution (0.5 mL buffer + 25 μL 20×pyridine iodide staining solution + 10 μL 50×RNase A) to each sample, incubate at 37 ℃ for 20 min. After cell filtration, transfer to flow cytometry tubes, analyze samples using a flow cytometer in the FL2 channel, and analyze the data using FlowJo V10.

[0092] xCELLigence Real-Time Cell Analysis: The xCELLigence real-time cell analyzer was placed in a cell culture incubator, 50 μL of culture medium was added to detect background impedance, and cells were seeded in E-Plates. After culturing at 37 ℃ for 18 h, the cells were pretreated with compounds, and then Dox was added for induction after 6 h. The cells were cultured for another 48 h, and the experimental data were exported and analyzed. Immunofluorescence: Cells were seeded in confocal microscopy dishes and treated as described above. Cell culture medium was aspirated, and the cells were washed once with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were then incubated overnight at 4 °C with γ-H2AX working solution. Cells were washed three times with PBST for 5 min each time. Cells were then incubated with TRITC-labeled secondary antibody (1:200) for 2 h. Cells were washed three times with PBST for 5 min each time. Cells were then incubated with DAPI (1:1000) for 10 min. Cells were washed twice with PBS for 5 min each time. Imaging was performed under a confocal microscope.

[0093] Western blotting: Proteins are extracted from cells by adding lysis buffer. The prepared protein samples are then loaded, electrophoresed, transferred to a membrane, blocked, incubated with primary and secondary antibodies, and then developed.

[0094] RNA-seq: Cells were treated with Trizol, flash-frozen in liquid nitrogen, and then sent for analysis. Transcriptome data were analyzed, and heatmaps and bubble charts were generated.

[0095] Real-time quantitative PCR: RNA was extracted, reverse transcribed into cDNA, a qPCR reaction system was prepared, and the gene expression level was calculated and analyzed using the 2-ΔΔCt formula.

[0096] Experimental results: such as Figure 1 As shown.

[0097] The results of SA-β-galactosidase staining are as follows: Figure 1 As shown in (b), pretreatment with compound IA-3 reduced the accumulation of SA-β-gal positive cells in chemotherapy- and radiotherapy-induced aging models in a dose-dependent manner. Figure 1 (c) shows the statistical results of the percentage of SA-β-gal positive cells in the chemotherapy-induced senescence model. Given the heterogeneity of senescent cell subtypes, a replicative senescent cell model was constructed by culturing primary mouse lung cells in vitro for 21 days, as shown below. Figure 1 As shown in (de), the IA-3 treatment group significantly reduced the number of SA-β-gal positive cells and improved cell morphology compared with the control group.

[0098] Flow cytometry analysis results as follows Figure 1 As shown in (f), 4 μM IA-3 pretreatment alleviated doxorubicin-induced G2 / M phase cell cycle arrest. Real-time cell analyzer results are shown below. Figure 1 As shown in (gh), the cell proliferation rate in the doxorubicin treatment group was lower than that in the control group, while IA-3 pretreatment significantly increased the cell proliferation rate.

[0099] Radiotherapy-induced cellular senescence leads to an increase in the DNA damage marker γ-H2AX. For example... Figure 1 As shown in (i), immunofluorescence experiments revealed that IA-3 pretreatment inhibited the formation of γ-H2AX foci. Figure 1 As shown in (j), Western blot analysis revealed that IA-3 pretreatment also downregulated the expression level of another important marker of cellular senescence, p21.

[0100] To further investigate the effects of IA-3 on aging, RNA sequencing analysis was performed on HUVEC cells. For example... Figure 1 As shown in (k), the heatmap reveals that the expression of senescence-related genes is downregulated after IA-3 pretreatment. Figure 1 As shown in (l), RNA sequencing results were verified by qRT-PCR. The results showed that, compared with the model control group, aging-related markers CDKN1A, CDKN2AIP, and SERPINE1 were downregulated in cells pretreated with compound IA-3, while LAMNB1 expression was upregulated. In addition, the expression levels of aging-related secretory phenotype (SASP) factors such as IL-1β, CCL5, CXCL8, CXCL11, IL-32, MMP1, MMP3, and MMP10 were also significantly reduced.

[0101] like Figure 1 As shown in (m), Reactome functional enrichment pathway analysis revealed synergistic alterations in aging-related signaling nodes, including pathways related to cellular stress response, telomere maintenance, oxidative stress-induced aging, cellular senescence, telomere end packaging, DNA damage / telomere stress-induced aging, and aging-associated secretory phenotypes (SASP). These results indicate that compound IA-3 can effectively prevent radiotherapy- and chemotherapy-induced cellular senescence, and its mechanism of action involves inhibiting the expression of aging markers and reducing SASP secretion.

[0102] The above results indicate that the representative compound IA-3 can reduce the positive proportion of SA-β-gal in Dox or X-ray induced HUVEC aging models and replication aging models, accompanied by relief of G2 / M blockade, improved proliferation capacity, decrease of γ-H2AX and p21, and regression of aging / SASP-related gene expression profiles.

[0103] Example 3: Compound IA-3 alleviates radiotherapy-induced aging in mice This embodiment aims to verify the anti-aging effect of compound IA-3 in vivo.

[0104] Experimental methods: Animal experimental protocol: Eight-week-old C57BL / 6J mice were used as experimental animals and divided into a blank control group, a model group, a low-dose IA-3 hydrochloride group (20 mg / kg), and a high-dose IA-3 hydrochloride group (30 mg / kg), with five mice in each group. The mice were administered the drugs via gavage. Mice were pre-administered the blank solvent, a low-dose (20 mg / kg) or high-dose (30 mg / kg) IA-3 hydrochloride for one week, followed by whole-body irradiation with 5 Gy. While this radiation dose was not lethal, it caused the continuous accumulation of senescent cells. The drugs were administered every two days during the experiment. Phenotypic assessment was performed at week 10 post-irradiation, and tissue samples were collected at week 11.

[0105] Mouse Rotary Cyclic Experiment: An accelerated rotating cylinder system was used to assess the fall latency and velocity in mice. Mice were trained on the rotating cylinder at 10 rpm and 20 rpm for 10 minutes each time for two consecutive days. In the formal experiment, mice were placed on a rotating cylinder, which accelerated from 5 rpm to 40 rpm over 200 seconds. The fall velocity and fall latency were recorded.

[0106] ELISA: Blood was collected from mouse eyeballs, centrifuged, and the supernatant was collected as serum. The levels of IL-1α, CXCL11, and CCL5 in the serum were detected using an ELISA kit.

[0107] SA-β-galactosidase staining of tissues: Adipose tissue, kidney and liver tissue samples were cut into appropriate sizes and stained, imaged and analyzed using the SA-β-galactosidase staining kit according to the instructions to evaluate positive signals of tissue aging.

[0108] Western blot and qPCR: Proteins or total RNA were extracted from tissue samples such as kidney, liver, heart, lung or spleen. Western blot was used to detect the expression of proteins such as p21, and qPCR was used to detect the mRNA levels of cdkn1a, cdkn2a, etc. The specific operations were performed according to the conventional methods in this field.

[0109] Histological staining (HE and Masson staining): Tissues such as heart, lung, and kidney were fixed, embedded, and sectioned for HE and Masson staining to evaluate histopathological changes and the degree of fibrosis.

[0110] Immunohistochemical staining: Tissue sections were routinely dewaxed, hydrated, antigen-retrievaled, and blocked. Immunohistochemical staining and imaging analysis were performed using p16, p21, and / or γ-H2AX antibodies, respectively.

[0111] Experimental results: The results are as follows Figure 2 As shown.

[0112] like Figure 2 As shown in (b), visual observation revealed significant phenotypic differences among the groups. The model control group exhibited significant hair whitening, while the IA-3 treatment group showed a significant reduction in the degree of hair whitening. Figure 2 As shown in (cd), the motor function was assessed by the rotating bar test. The results showed that the fall latency and fall speed were increased in the high-dose IA-3 group, indicating that the motor coordination was improved.

[0113] like Figure 2 As shown in (e), serum inflammatory markers in mice were detected by ELISA. Compared with the model group, IL-1α and CXCL11 levels were significantly reduced after IA-3 treatment; CCL5 showed a decreasing trend. Figure 2 As shown in (f), the SA-β-gal staining results of adipose tissue, kidney and liver of mice in each group also confirmed that IA-3 can alleviate radiotherapy-induced aging in mice.

[0114] like Figure 2 As shown in (g), Western blot results indicate that IA-3 treatment reduces the protein expression level of the aging marker p21 in the kidneys, liver, and heart. Figure 2 As shown in (h), qPCR detection indicated that IA-3 treatment reduced the mRNA levels of cdkn1a and cdkn2a in the heart, lungs, and spleen. Figure 2As shown in (ij), HE staining and Masson staining revealed that IA-3 administration alleviated age-related pathological changes in the heart, lungs, and kidneys. Compared with the model group, IA-3 treatment reduced inflammatory infiltration of cardiomyocytes, resulting in more regular cardiomyocyte arrangement; reduced inflammatory cell infiltration and fibrosis in the lungs, maintaining alveolar integrity; and alleviated pathological changes such as decreased glomerular numbers and renal tubular epithelial cell edema.

[0115] like Figure 2 As shown in the image (km), immunohistochemical staining of the heart, lungs, and kidneys revealed that, compared to the model group, IA-3 administration downregulated the expression of p16, p21, and γ-H2AX. In summary, these results indicate that IA-3 treatment effectively alleviates radiotherapy-induced overall aging in mice, improves motor function, and reduces SASP secretion and histopathological changes.

[0116] The in vivo experimental results above indicate that the representative compound IA-3 can effectively alleviate radiotherapy-induced overall aging in mice, improve motor function, and reduce SASP secretion and histopathological changes. The in vivo anti-aging effect of IA-3 further confirms the anti-aging activity of compounds of the general formula shown in Formula X, providing in vivo pharmacodynamic evidence for the clinical development of this type of compound.

[0117] Example 4: Compound IA-3 exerts anti-aging effects in vitro by targeting IL4I1. This embodiment aims to identify the target of compound IA-3 and verify the role of IL4I1 in anti-aging. The experimental results are as follows: Figure 3 As shown. To explore the mechanism of action of IA-3, an IA-3 probe was designed to identify its potential targets and elucidate the pathway by which it exerts its anti-aging effects.

[0118] Experimental methods: Silver staining: Place the gel in fixative and fix overnight on a shaker at 4°C. Discard the fixative, add 30% ethanol, wash at room temperature for 10 minutes, then wash with double-distilled water for another 10 minutes. Add silver staining solution and shake at room temperature for 10 minutes, then wash with water for 1 minute. Add silver staining development solution and shake for 3-10 minutes. When the expected protein band appears, add stop solution to stop the development. Finally, rinse with double-distilled water, cut differential bands from the gel, and analyze the sample using liquid chromatography-mass spectrometry (LC-MS / MS).

[0119] Surface plasmon resonance: The S-series CM5 sensor chip was inserted into the sensor chip port, and the ligand was immobilized by amine coupling. Subsequently, analyte dilutions were prepared in flow buffer, and 200 μL of different concentrations of analyte were used for surface testing. Finally, kinetic and affinity data were analyzed.

[0120] Cellular thermal displacement assay (CETSA): HUVEC cells were treated with DMSO or 10 μM IA-3 for 48 h, respectively. Cells were lysed with lysis buffer, centrifuged, and the supernatant was subjected to gradient heating. The resulting samples were denatured and then subjected to Western blotting to evaluate the effect of IA-3 treatment on the thermostability of IL4I1 protein.

[0121] Other experiments: affinity enrichment of IA-3 probe, immunoprecipitation / co-immunoprecipitation verification, LC-MS / MS identification, in vitro purified protein binding verification, CRISPR / Cas9 construction of IL4i1 knockout cells, SA-β-gal staining, immunofluorescence, Western blot and qRT-PCR detection, etc., were all performed according to conventional methods in the field or the corresponding kit / instrument operation instructions; the mouse primary kidney cell replication senescence model was performed according to the method for replicating the senescence model in Example 2, with the cell source replaced by wild-type (WT) and IL4i1 knockout (IL4i1 - / - Primary kidney cells from mice were cultured for 21 days, and the degree of replication and senescence was evaluated by SA-β-gal staining.

[0122] Experimental results: such as Figure 3 As shown.

[0123] like Figure 3 As shown in (a), the proteins enriched by the IA-3 probe were analyzed using silver staining, and IL4I1 was identified as a target of IA-3. Figure 3 As shown in (b), immunoprecipitation experiments further validated that IL4I1 is a target of IA-3. IL4I1 exerts a negative regulatory effect on antitumor immunity by activating the indolepyruvate pathway in the transcription factor AHR, through enzymatic degradation of tryptophan. Figure 3 As shown in (c), after in vitro purification of IL4I1 protein, the dissociation equilibrium constant (KD value) between IA-3 and IL4I1 protein was determined to be 14.8 µM using surface plasmon resonance (SPR) experiments, while the KD value between the IA-3 probe and IL4I1 protein was 50.52 µM. Figure 3 As shown in (d), CETSA Western blot results indicate that treatment with compound IA-3 enhances the thermal stability of IL4I1 protein under gradient temperatures. Figure 3 As shown in (e), CETSA quantitative analysis further confirmed that the retention of IL4I1 protein in the IA-3 treatment group at high temperature was significantly higher than that in the DMSO control group. Figure 3 As shown in (f), the purified IL4I1 protein and the IA-3 probe were subjected to an immunoprecipitation experiment, which further confirmed that IL4I1 is the direct target of IA-3.

[0124] To assess the effect of IL4I1 on cellular senescence, IL4I1 knockout HUVEC cells were established using CRISPR / Cas9 technology, such as... Figure 3 As shown in (g). After irradiation with 6 Gy and incubation for 7 days, as... Figure 3 As shown in (hi), SA-β-galactosidase staining revealed that the proportion of senescent cells in IL4I1 knockout HUVEC cells was significantly lower than that in the wild-type control group. Figure 3 As shown in (jk), in chemotherapy- and radiotherapy-induced aging models, Western blot analysis revealed that p21 expression in IL4I1 knockout HUVEC cells was significantly downregulated compared to the wild-type control group. Figure 3 As shown in (lm), in a radiotherapy-induced aging model, immunofluorescence analysis of γ-H2AX and p21 verified the anti-aging effect of IL4I1 knockout cells compared to the wild-type control group.

[0125] like Figure 3 As shown in (n), qRT-PCR experiments indicated that after doxorubicin-induced senescence, the expression of multiple SASP factor genes in IL4I1 knockout cells was downregulated, including IL-1β, IL-6, IL-8, IL-23, IL-32, TNF-α, and MMP3. Figure 3 As shown in (op), in wild-type and mouse IL4I1 knockout primary kidney cells passaged in vitro for 21 days, SA-β-gal staining was significantly reduced in the knockout group, confirming that IL4I1 deficiency can alleviate replication senescence. These results indicate that IL4I1 is a direct target of IA-3, and IL4I1 knockout can alleviate radiotherapy- and chemotherapy-induced cell senescence and replication senescence in vitro.

[0126] The above results indicate that IL4I1 is a representative compound IA IL4I1 is a direct target of 3; and the inhibition or absence of IL4I1 can alleviate radiotherapy and chemotherapy-induced cellular senescence and replication senescence, supporting IL4I1 as a key target for screening anti-aging drugs and studying its mechanism of action.

[0127] Example 5: Il4i1 deficiency protects mice from radiation-induced aging This embodiment aims to verify the role of IL4I1 in in vivo anti-aging through an animal knockout model.

[0128] Experimental methods: 8-week-old WT and Il4i1 were selected. - / -Five C57BL / 6J mice were used as experimental animals, receiving either 0 or 5 Gy irradiation. Behavioral assessments and overall appearance observations were performed at week 10 post-irradiation, and serum and tissue samples were collected at week 11. A rotating rod test was used to assess motor coordination; an ELISA kit was used to detect the levels of IL-1α, TNF-α, IL-1β, and CXCL11 in serum; and SA samples were collected from adipose tissue, kidneys, and livers. β Gal staining is used to evaluate tissue senescence; Western blot is used to detect p21 expression in tissues such as the kidney, liver, and heart; immunohistochemistry is used to detect p16, p21, and / or γ in the heart, lung, and kidney. H2AX expression; HE and Masson staining were used to evaluate histopathological changes and the degree of fibrosis. All the above detection steps were performed according to standard methods in the field or the instructions of the corresponding kits.

[0129] Experimental results: such as Figure 4 As shown.

[0130] like Figure 4 As shown in (a), 8-week-old wild-type (WT) and Il4i1 knockout (Il4i1) cells were selected. - / - Mice were used as experimental animals and received whole-body irradiation of 0 or 5 Gy. Behavioral assessment and overall appearance monitoring were performed at week 10 post-irradiation, and tissue samples were collected at week 11.

[0131] like Figure 4 As shown in (b), visual observation revealed that, compared to WT mice, Il4i1 - / - The whitening of the mice's fur was significantly reduced after irradiation. Figure 4 As shown in (cd), the test results of the rotating rod indicate that Il4i1 - / - The mice exhibited enhanced motor coordination, with prolonged fall time and increased fall speed. For example... Figure 4 As shown in (e), SASP analysis revealed that, compared to the WT group, the irradiated Il4i1 - / - Serum levels of IL-1α, IL-1β, and TNF-α were significantly reduced in mice, and CXCL11 showed a decreasing trend, all of which indicate that age-related inflammation in mice was weakened.

[0132] like Figure 4 As shown in (f), SA-β-galactosidase staining results of adipose tissue, kidney, and liver indicate that, compared with WT mice, irradiated Il4i1 - / - The number of senescent positive cells in mice was significantly reduced. For example... Figure 4 As shown in (g), Western blot analysis revealed that, compared with WT mice, irradiated Il4i1 - / -p21 expression was significantly downregulated in the kidneys, liver, and heart of mice. Figure 4 As shown in (hj), immunohistochemical analysis confirmed the irradiation of Il4i1. - / - The expression of aging markers p21, p16 and γ-H2AX was decreased in the heart, lungs and kidneys of mice.

[0133] like Figure 4 As shown in (k), HE staining reveals the irradiated Il4i1 - / - Mice exhibited lower levels of inflammatory cell infiltration in various tissues, more normalized cardiomyocyte arrangement, more intact alveolar structure, and reduced vacuolation of renal tubular epithelial cells. For example... Figure 4 As shown in (l), Masson staining reveals the irradiated Il4i1 - / - The degree of fibrosis in the heart, lungs, and kidneys of mice was lower than that in WT mice. These results indicate that Il4i1 deficiency maintains tissue homeostasis under radiotherapy stress by regulating SASP secretion and the expression of aging-related biomarkers, thereby embodying the body's ability to resist radiotherapy-induced aging.

[0134] Il4i1 gene knockout mice exhibited an anti-aging phenotype after radiotherapy, providing in vivo evidence from a reverse genetics perspective to support IL4i1 as an anti-aging intervention target.

[0135] Example 6: IL4I1-mediated ubiquitination and degradation of EIF4B. This example aims to elucidate the molecular mechanism by which IL4I1 regulates cellular senescence.

[0136] Experimental methods: Immunoprecipitation: 293T cells were transfected as required, and lysed after 48 hours to extract protein. Protein concentration was determined using a BCA kit to ensure consistency across groups. Magnetic beads and protein lysis buffer were incubated overnight at 4°C in a rotary shaker. After incubation, the magnetic beads were washed at least five times with PBST buffer. The protein bound to the magnetic beads was then boiled with SDS-PAGE loading buffer to elute the protein, followed by Western blotting.

[0137] Other experiments: immunoprecipitation and silver staining identification of IL4I1 interacting proteins, LC-MS / MS analysis and KEGG pathway enrichment analysis; Myc-IL4I1 gradient transfection; CHX tracking experiment and proteasome inhibitor MG132 intervention; detection of EIF4B ubiquitination level and K48-linked ubiquitin chain analysis; construction and verification of EIF4B knockout cells by CRISPR / Cas9; SA-β-gal staining, Western blot, qPCR and qRT-PCR detection, etc., all performed according to conventional methods in this field or corresponding kits / instructions.

[0138] Experimental results: such as Figure 5 As shown. To investigate the regulatory mechanism of IL4I1 in cellular senescence, an immunoprecipitation-mass spectrometry analysis was used to construct its protein-protein interaction map. Figure 5 As shown in (a), KEGG pathway enrichment analysis of the IL4I1 interactome reveals its association with multiple aging-related signaling pathways, including antigen processing and presentation, lifespan regulation pathways, Parkinson's disease and Alzheimer's disease pathways.

[0139] like Figure 5 As shown in (b), eukaryotic translation initiation factor 4B (EIF4B) was identified as a novel interacting protein of IL4I1 by silver staining analysis of the Flag-IL4I1 immunoprecipitate. Figure 5 As shown in (c), the interaction between IL4I1 and EIF4B was further verified using co-immunoprecipitation in HEK293T cells. Figure 5 As shown in (d), in order to investigate the IL4I1-mediated EIF4B stability regulation mechanism, Myc-IL4I1 was gradient-transfected into HEK293T cells. Western blot results showed that the EIF4B protein level decreased in a dose-dependent manner, suggesting that IL4I1 may regulate EIF4B homeostasis by affecting the protein stability of EIF4B (including proteasome-related degradation pathway).

[0140] like Figure 5 As shown in (ef), treatment of human umbilical vein endothelial cells with cycloheximide (CHX) accelerates EIF4B degradation, while the proteasome inhibitor MG132 attenuates this effect, indicating the existence of a proteasome-dependent degradation pathway. Figure 5 As shown in (g), to determine whether ubiquitination mediates this process, transfection of Flag-IL4I1 into HEK293T cells increased EIF4B ubiquitination levels in the immunoprecipitation assay. Figure 5 As shown in (h), further investigation determined the type of ubiquitination linkage involved in the IL4I1-mediated EIF4B degradation process. The results showed that IL4I1 can specifically enhance the formation of K48-linked (proteasome-targeted) ubiquitin chains on EIF4B.

[0141] In EIF4B functional studies, CRISPR / Cas9 technology was used to construct EIF4B knockout HUVEC cells, such as... Figure 5 As shown in (i), the results showed that EIF4B knockout exacerbated the cellular senescence phenotype. Figure 5 As shown in (jk), compared with the wild-type control group, the number of SA-β-gal positive cells increased in irradiated EIF4B knockout cells. Figure 5As shown in (lm), Western blot and qPCR analyses indicated that p21 expression was upregulated in EIF4B knockout cells. Figure 5 As shown in (n), qRT-PCR analysis revealed increased secretion of SASP factors (IL-1β, IL-8, IL-23, IL-32, CXCL11, and MMP3) in EIF4B knockout cells. In summary, these findings confirm that IL4I1 plays a dual regulatory role in EIF4B stability through the K48-linked ubiquitination pathway, while EIF4B depletion activates aging-related pathways.

[0142] IL4I1 mediates EIF4B degradation through a K48-linked ubiquitination pathway, elucidating the molecular mechanism by which IL4I1 regulates aging and providing a mechanistic basis for the anti-aging effects of compounds of the general formula shown in Formula X.

[0143] Example 7: The EIF4B-PGAM5 axis maintains mitochondrial homeostasis and reduces age-related secretory phenotypes This embodiment aims to illustrate how EIF4B regulates mitochondrial homeostasis and cellular senescence through PGAM5.

[0144] Experimental Methods: CRISPR-Cas9 Gene Knockout: Cells were exposed to a virus derived from the CRISPR / Cas9 KO plasmid, and the medium was changed after 12 hours. After selection with puromycin, the knockout effect of polyclonal cells was verified by Western blot. After successful polyclonal construction, single cells were sorted into 96-well plates, and clones containing only a single cell or derived from a single cell were selected from each well. Single-clonal cells were expanded, and the gene knockout effect was verified by Western blot.

[0145] Other experiments: Immunoprecipitation, silver staining identification, and mass spectrometry analysis of EIF4B interacting proteins; co-immunoprecipitation verification of the interaction between EIF4B and PGAM5; Dox- or radiation-induced cell senescence model treatment; SA β gal staining, Western blot, qPCR / qRT PCR detection; MitoTimer fluorescence detection of mitochondrial turnover: MitoTimer plasmid was transfected into target cells, and after appropriate culture time, green (newborn mitochondria) and red (aging mitochondria) fluorescence signals were detected using fluorescence microscopy. The red / green fluorescence ratio was used to evaluate the mitochondrial turnover rate; and IA 3. The effects of treatment on EIF4B and PGAM5 expression (refer to the Dox-induced cell senescence model treatment protocol in Example 2) were all performed according to conventional methods in the field or the corresponding kit / instrument operation instructions.

[0146] Experimental results: such as Figure 6 As shown, EIF4B, as a eukaryotic translation initiation factor, can regulate the cell cycle, but its regulation of aging-related secretory phenotypes has not been reported in the literature. Immunoprecipitation was used to investigate the interacting proteins of EIF4B to elucidate its regulatory mechanism on SASP.

[0147] like Figure 6 As shown in (a), several differentially expressed proteins were identified based on silver staining results, including SPTAN1, MYO1C, and PGAM5. To investigate the potential interaction between EIF4B and these proteins, EIF4B knockout human umbilical vein endothelial cells were constructed using CRISPR / Cas9 gene editing technology. Figure 6 As shown in (bc), subsequent Western blot and qPCR analyses revealed a significant downregulation of PGAM5 expression after EIF4B knockout, while MYO1C and SPTAN1 expression levels remained unchanged. PGAM5, as a mitochondrial serine / threonine phosphatase, can dephosphorylate various substrates and participate in biological processes such as cell senescence and mitophagy. Figure 6 As shown in (d), the interaction between EIF4B and PGAM5 was verified by co-immunoprecipitation assay. Figure 6 As shown in (e), Western blot analysis revealed that the expression of both EIF4B and PGAM5 proteins was downregulated during radiation-induced cell senescence, indicating that EIF4B plays a role as a novel upstream regulator of PGAM5.

[0148] like Figure 6 As shown in (f), PGAM5 knockout human umbilical vein endothelial cells were established using CRISPR-Cas9 technology to validate their aging phenotype in vitro. Figure 6 As shown in (gh), when PGAM5 knockout cells were induced with doxorubicin, their SA-β-galactosidase activity was significantly increased compared to the control group. Figure 6 As shown in (i), p21 protein expression was upregulated in PGAM5 knockout cells compared to wild-type cells. Figure 6 As shown in (j), the levels of multiple SASP markers (IL-1α, IL-1β, IL-6, IL-8, IL-23, IL-32, CXCL11, MMP3) were significantly increased in PGAM5 knockout cells.

[0149] Studies have shown that PGAM5 deficiency leads to increased mitochondrial fusion and reduced cell turnover, and enhances the mTOR and IRF / IFN-β signaling pathways, thereby inducing cellular senescence both in vivo and in vitro. Figure 6As shown in (kl), Western blot and qPCR results revealed that PGC1α, a key transcriptional co-regulator of mitochondrial biogenesis, was significantly downregulated in PGAM5 knockout cells, while the mitochondrial matrix protein cyclic peptidin D (PPID) and the mitochondrial outer membrane protein Tom20 were significantly upregulated by 2-4 times, indicating that the increased mitochondrial mass does not originate from mitochondrial biogenesis. When DRP1 is phosphorylated at Ser637, mitochondrial division is inhibited while mitochondrial fusion is enhanced. Consistent with PGAM5 regulating mitochondrial division through dephosphorylation of DRP1, the phosphorylation level of DRP1 at Ser637 is increased in PGAM5 knockout cells. Figure 6 As shown in (mn), similar changes in mitochondrial protein expression were also observed in EIF4B-deficient cells.

[0150] like Figure 6 As shown in (op), mitochondrial turnover was directly detected using the MitoTimer fluorescent protein. After translation, its color gradually changes from green to red. Consistent with the Western blot results, both PGAM5 and EIF4B deletions led to the accumulation of more red mitochondria than in control cells, indicating reduced mitochondrial turnover. In conclusion, PGAM5 deficiency in cells leads to impaired mitochondrial division and reduced mitochondrial turnover.

[0151] like Figure 6 As shown in (q), the study found that IA-3 pretreatment can alleviate the reduction in EIF4B and PGAM5 expression caused by doxorubicin-induced senescence, indicating that IA-3 regulates cellular senescence through the IL4I1-EIF4B-PGAM5 axis. In summary, this invention reveals the molecular mechanism by which compound IA-3 targets IL4I1 and maintains mitochondrial homeostasis by regulating the IL4I1-EIF4B-PGAM5 signaling axis, thereby delaying cellular and tissue senescence. This provides a new chemical molecular basis and mechanism of action for the development of novel anti-aging drugs.

[0152] EIF4B maintains mitochondrial division and turnover by regulating PGAM5, and IA-3 pretreatment can alleviate the decline in EIF4B and PGAM5 expression during aging. These results indicate that the representative compound IA... 3 can be transmitted via IL4I1 EIF4B The PGAM5 signaling axis affects mitochondrial homeostasis and mitigates aging-related phenotypes; this mechanism provides a pathway-level explanation and support for the anti-aging applications of the compound represented by Formula X.

[0153] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of this application. Although this application has been described in detail with reference to the above embodiments, those skilled in the art can still make various modifications or equivalent substitutions of some technical features after reading this description. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be considered to fall within the protection scope of this application.

Claims

1. The use of a compound of general formula X or a pharmaceutically acceptable salt thereof in the preparation of an anti-aging drug: in, Structure A is pyrazolopyrimidine or indole, and the compound conforms to the structure shown in formula X1 or X2: Z is absent, carbonyl, or –C(O)NH–; X is either O or S; Y is -O-, -NH-, or ; R1 is hydrogen or C. 1-6 alkyl; R2 is selected from C1-C3 alkyl groups, C5-C6 alkyl groups, and C2 is selected from C1-C3 alkyl groups. 15 Alkenyl, alkynyl, 5-10 membered heterocyclic groups, C6-C 12 Aryl, 5-12 membered heteroaryl, sterol, and 5-10 membered cycloalkyl; Y is directly attached to R2, or Y is attached to R2 to form a ring; R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, 3-10 membered heterocyclic group, C6-C. 12 Aryl, 5-12 membered heteroaryl, 3-10 membered cycloalkyl, ester group, carboxyl group, trihalomethyl and adamantyl; R2 or R3 is either unsubstituted or substituted by one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl. When R2 is a C1-C3 alkyl group, R3 is not hydrogen.

2. The application according to claim 1, characterized in that, R2 is selected from C1-C3 alkyl groups, C5-C6 alkyl groups, and C2 is selected from C1-C3 alkyl groups. 15 Alkenyl, C5-C 15 Dieneyl, C5-C 15 Trienyl, alkynyl, 5-6 membered cycloalkyl, phenyl, 5-6 membered heterocyclic, 5-6 membered heteroaryl, sterol; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R2 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl. Preferably, R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, 5-6 membered heterocyclic group, phenyl, biphenyl, naphthyl, 5-6 membered heteroaryl, 5-6 membered cycloalkyl, ester group, carboxyl group, amide group, trihalomethyl, adamantyl. R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

3. The application according to claim 1, characterized in that, R2 is selected from methyl, ethyl, propyl, C5-enyl, C6-ethyl, C7-ethyl, C6 ... 10 Dieneyl, C 15 Trienyl, alkynyl, cyclopentyl, cyclohexyl, triazolyl, phenyl, piperidinyl, piperazine, pyrrolyl, pyridyl, pyrimidinyl, sterolyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R2 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, and carboxyl. Wherein, the sterol group is selected from , , ; Preferably, R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, phenyl, biphenyl, naphthyl, cyclopentyl, cyclohexyl, piperidinyl, piperazine, pyrrolyl, pyridinyl, pyrimidinyl, ester, carboxyl, amide, trihalomethyl, and adamantyl. R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, carboxyl, and phenyl.

4. The application according to any one of claims 1 to 3, characterized in that, The compound has the structure described in Formula I or Formula II: Wherein, X, Y, R1, R2, and R3 are as described in any one of claims 1 to 3; Preferably, in compound I, X is either O or S; Y is -O-, -NH-, or ; R1 is hydrogen or a C1-C2 alkyl group; R2 is selected from methyl, ethyl, C5-enyl, C6-ethyl, C7-ethyl, C6-ethyl 10 dienyl, cyclohexyl, phenyl, pyridyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; R3 is selected from hydrogen, phenyl, pyridyl, pyrimidinyl, ester, and trihalomethyl; R2 or R3 is either unsubstituted or substituted by one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, and carboxyl. Preferably, in the compound of formula II, X is either O or S; Y is -O-, -NH-, or ; R2 is selected from methyl, ethyl, propyl, C5-enyl, C6-ethyl, C7-ethyl, C6 ... 10 Dieneyl, C 15 Trienyl, alkynyl, cyclopentyl, cyclohexyl, phenyl, triazolyl, pyridyl, sterolyl; wherein Y is directly connected to R2, or Y is connected to R2 to form a ring; Wherein, the sterol group is selected from , , ; R3 is selected from hydrogen, halogen, amino, hydroxyl, acetyl, phenyl, biphenyl, naphthyl, cyclopentyl, cyclohexyl, pyrrolyl, pyridyl, pyrimidinyl, ester, carboxyl, amide, trihalomethyl, and adamantyl. R2 or R3 is either unsubstituted or substituted with one or more of the following groups: C1-C6 alkyl, hydroxyl, halogen, trihalomethyl, and carboxyl.

5. The application according to any one of claims 1 to 4, characterized in that, When X is O, the compound has the structure shown in formula IA or formula IIA: Wherein, Y, R1, R2, and R3 are as described in any one of claims 1 to 3, Y and R2 are directly connected, or Y and R2 are connected to form a ring; Preferably, when X is S, the compound has I B The structure shown is as follows: Wherein, Y is -NH-, and R1, R2, and R3 are as described in any one of claims 1 to 4; Preferably, in the IB compound, R2 is selected from methyl, ethyl, and pyridyl; R3 is selected from hydrogen, pyridyl, and pyrimidinyl.

6. The application according to any one of claims 1 to 5, characterized in that, Y is -O-, -NH-, or When X is 0, When Y is -O- or -NH-, Y and R2 can be directly connected; Or, Y is When Y connects to R2 to form a ring, it forms the R2' structure, and the N atom acts as a ring-forming atom in the R2' structure. The compound has the structure shown in IA1, IA2, IA3, IIA1, IIA2 or IIA3: Wherein, R1, R2, and R3 are as described in any one of claims 1 to 5; R2' is selected from... , or ; Preferably, in formula IIA2, R2 is selected from... , , In this case, R3 is selected from halogens, hydroxyl groups, phenyl groups, naphthyl groups, and adamantyl groups.

7. The application according to any one of claims 1 to 6, characterized in that, The compound is selected from one or more of the following structures: ； Preferably, the pharmaceutically acceptable salt is a hydrochloride, sulfate, phosphate, maleate, fumarate, citrate, methanesulfonate, p-toluenesulfonate, or tartrate.

8. The application according to any one of claims 1 to 7, characterized in that, The anti-aging drug works by targeting IL4I1; Preferably, the anti-aging drug works by inhibiting IL4I1-mediated EIF4B ubiquitination degradation; Preferably, the anti-aging drug maintains mitochondrial homeostasis by regulating the IL4I1-EIF4B-PGAM5 signaling axis; Preferably, the anti-aging drug is used to prevent or alleviate aging induced by radiotherapy and chemotherapy; Preferably, the senescence induced by radiotherapy and chemotherapy includes cellular senescence induced by radiotherapy and chemotherapy and / or tissue senescence induced by radiotherapy and chemotherapy. Preferably, the cellular senescence induced by radiotherapy and chemotherapy is endothelial cell senescence induced by doxorubicin or X-rays; Preferably, the radiotherapy-induced tissue aging is radiotherapy-induced aging of the heart, lungs, kidneys, liver, or adipose tissue; Preferably, the anti-aging drug is used to prevent or alleviate recurrent aging; Preferably, the anti-aging drug is used for: (a) Reduce the proportion of aging-associated β-galactosidase-positive cells; and / or (b) Downregulating the expression of aging markers p21 and / or p16; and / or (c) Inhibit the expression of the DNA damage marker γ-H2AX; and / or (d) Reduce the expression or secretion of aging-related secretory phenotypic factors; Preferably, the aging-related secretory phenotypic factor is selected from one or more of IL-1α, IL-1β, IL-6, IL-8, IL-23, IL-32, TNF-α, CCL5, CXCL8, CXCL11, MMP1, MMP3, and MMP10.

9. A method for screening anti-aging drug candidates, characterized in that, Includes the following steps: (1) Contact the candidate substance with IL4I1 protein or cells expressing IL4I1; (2) Test at least one of the following indicators: a) Enzymatic activity of IL4I1; b) The interaction between IL4I1 and EIF4B; c) The ubiquitination level of EIF4B; d) Protein expression level of EIF4B; (3) Candidate substances that can inhibit IL4I1 activity, block the interaction between IL4I1 and EIF4B, reduce EIF4B ubiquitination level or upregulate EIF4B expression level are identified as anti-aging candidate drugs. Preferably, the method for detecting the interaction between IL4I1 and EIF4B is selected from immunoprecipitation, surface plasmon resonance, fluorescence resonance energy transfer, or bimolecular fluorescence complementarity. Preferably, the detection of the ubiquitination level of EIF4B includes the detection of K48-linked ubiquitination.

10. A kit for screening anti-aging drug candidates, characterized in that, The kit contains at least two of the following components: (1) IL4I1 protein or its functional fragments; (2) EIF4B protein or its functional fragment; (3) Reagents for detecting the interaction between IL4I1 and EIF4B; (4) Anti-ubiquitin antibodies used to detect EIF4B ubiquitination levels; Preferably, the kit further comprises a positive control compound, which is the compound of formula X according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

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