Use of RIPK1 inhibitor in prevention and treatment of vascular injury and diabetes

Abstract

A use of an RIPK1 inhibitor in the prevention and treatment of vascular injury and diabetes. Specifically provided are a method for regulating the levels of spermidine and acetylated spermidine and a use of the RIPK1 inhibitor in the prevention and treatment of related diseases caused by decreased levels of spermidine and acetylated spermidine due to arylamine N-acetyltransferase 2 deficiency or other reasons, including aging, diabetes, and other diseases with vascular injury. It has been demonstrated by means of a related animal disease model that the inhibition of RIPK1 kinase activity can treat vascular injury in the animal model, and related diseases and symptoms caused by vascular injury, such as weight gain, inflammation, insulin resistance, impaired glucose tolerance, lipid and amino acid metabolism disorders, and pancreatitis, thereby providing a novel and effective intervention for the treatment of human vascular injury, aging, and diabetes-related diseases and symptoms.

Description

Use of RIPK1 inhibitors in the prevention and treatment of vascular damage and diabetes Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to methods for regulating spermidine and acetylated spermidine levels. The RIPK1 inhibitor and the method for regulating spermidine and acetylated spermidine levels are used in the prevention and treatment of diseases caused by decreased spermidine and acetylated spermidine levels due to aryl N-acetyltransferase 2 deficiency or other reasons, vascular injury and its resulting obesity, insulin resistance, impaired glucose tolerance, lipid and amino acid metabolism abnormalities, pancreatic inflammation, diabetes and its complications such as cardiovascular and cerebrovascular diseases, diabetic retinopathy, diabetic nephropathy, diabetic foot, as well as neurodegenerative diseases and cerebral small vessel damage caused by treatment. This invention also provides a novel small molecule drug that can be used to treat vascular injury and diabetes. Background Technology

[0002] Over the past few decades, the global incidence of diabetes has been steadily increasing. According to statistics from the International Diabetes Federation, as of 2021, the number of people with diabetes worldwide had reached 537 million, accounting for 10.5% of the global population, and is projected to grow to 700 million by 2045. Currently, China has the largest diabetic population in the world. Between 2011 and 2021, the number of people with diabetes in my country increased from 90 million to 140 million, accounting for one-tenth of the national population, on par with the global incidence rate. From the invention of artificial insulin synthesis in my country in the 1960s to the use of metformin in the 1970s, and now to the availability of various drugs developed to enhance insulin sensitivity, diabetes remains incurable and irreversible. Complications of diabetes, such as cardiovascular disease, neuropathy, kidney disease, and retinopathy, impose a huge medical and economic burden on society. Therefore, developing novel and effective treatments for diabetes is an urgent problem to be solved.

[0003] Recent studies using genome-wide association studies (GWAS) have revealed a significant association between non-synonymous polymorphisms (SNPs) of arylamine N-acetyltransferase 2 (Nat2, homologous to Nat1 in mice) and insulin resistance in humans. Nat1-deficient mice also exhibit elevated fasting blood glucose, insulin, and triglyceride levels, as well as decreased insulin sensitivity. However, the relationship between Nat1 and vascular damage in diabetes remains unclear.

[0004] TNFα is a classic pleiotropic pro-inflammatory cytokine and the first to be described as an "adipokine" produced by adipose tissue. Elevated TNFα levels in adipose tissue are closely associated with obesity and diabetes. Other studies have found that anti-TNFα therapy in arthritis patients can increase insulin sensitivity. However, in clinical trials, blocking TNFα did not improve insulin resistance in non-insulin-dependent diabetes mellitus. TNFα deficiency also did not rescue insulin resistance in rodent obesity models. Although chronic inflammation in adipose tissue is considered a key risk factor for insulin resistance and type 2 diabetes in obese individuals, how inflammation modulates insulin sensitivity remains unclear.

[0005] [Corrected according to Rule 91, 07.08.2025] Receptor Interacting Serine / Threonine Protein Kinase 1 (RIPK1) is a key protein mediating the Tumor Necrosis Factor Alpha (TNFα) pathway. RIPK1's scaffolding and kinase functions jointly regulate the activation of NF-κB downstream of TNFα, as well as RIPK1-dependent apoptosis (RDA) and programmed cell death (necroptosis), promoting the occurrence of autonomous cellular inflammation and the development of chronic inflammation, including promoting the sustained production of inflammatory factors such as TNFα, CXCL8, and CCL2. RIPK1 is involved in various human diseases such as Inflammatory Bowel Disease (IBD), stroke, Alzheimer's Disease (AD), and Amyotrophic Lateral Sclerosis (ALS). However, the role and mechanism of RIPK1 kinase activity and RIPK1-dependent cell death in insulin resistance are unclear.

[0006] Spermidine is an aliphatic polyamine that naturally exists in cells and organisms. Studies have found that spermidine concentrations in various human organs, including the brain, mice, and fruit flies, decrease with age. Spermidine supplementation is considered an intervention for prolonging health and delaying aging. Existing research has shown that dietary spermidine can inhibit various age-related pathological phenotypes in animal models and human epidemiological studies, including those affecting the cardiovascular system, nervous system, intestinal barrier integrity, and promoting lifespan extension; however, the underlying molecular mechanisms remain unclear.

[0007] In summary, there is an urgent need in the field to develop novel pharmaceutical compositions for the treatment of diabetes and the resulting vascular damage. Summary of the Invention

[0008] The purpose of this invention is to provide a method for regulating the levels of spermidine and acetylated spermidine.

[0009] The purpose of this invention is to provide a novel pharmaceutical composition for the prevention and treatment of diseases caused by decreased levels of spermidine and acetylated spermidine.

[0010] The purpose of this invention is to provide a novel pharmaceutical composition for the prevention and treatment of diabetes, and the vascular damage it causes, as well as neurodegenerative diseases and the treatment of small cerebral vessel damage.

[0011] One object of the present invention is to provide a method for preventing and treating diabetes or vascular damage caused by diabetes, or mitochondrial dysfunction, as well as neurodegenerative diseases and treating small cerebral vessel damage caused by diabetes.

[0012] One object of the present invention is to provide a method for preventing and treating diabetes or vascular damage caused by diabetes as a RIPK1 inhibitor, with dietary spermidine and its modifications.

[0013] Another object of the present invention is to provide a novel small molecule compound for the prevention and treatment of diabetes or vascular damage caused by diabetes.

[0014] A first aspect of the present invention provides the use of an RIPK1 inhibitor, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in the preparation of a pharmaceutical composition, characterized in that the pharmaceutical composition is used to prevent or treat diseases or conditions selected from the group consisting of: symptoms of vascular injury, obesity, insulin resistance, diseases related to impaired glucose tolerance, diseases related to lipid and amino acid metabolism disorders, pancreatitis, diabetes, or complications of diabetes.

[0015] Preferably, the disease is caused by a deficiency of aryl N-acetyltransferase 2 or other reasons leading to a decrease in spermidine and acetylated spermidine levels; more preferably, the symptoms are selected from the group consisting of: mitochondrial dysfunction, insulin resistance, glucose intolerance, lipid and amino acid metabolism abnormalities, pancreatic inflammation and other related diseases; vascular damage complications caused by diabetes, including cardiovascular and cerebrovascular damage, diabetic retinopathy, diabetic nephropathy, diabetic foot, etc.; and diabetes-related symptoms such as insulin resistance, glucose intolerance, lipid and amino acid metabolism abnormalities, pancreatic inflammation, etc. caused by vascular damage; and other diseases with pathological features of vascular damage, including cerebrovascular damage caused by neurodegenerative diseases and treatment side effects, neurodegenerative diseases, and cerebral small vessel damage caused by treatment.

[0016] In another preferred embodiment, the lipid metabolism disorder is a disease related to overall lipid metabolism dysregulation.

[0017] In another preferred embodiment, the lipid metabolism disorder is selected from the group consisting of: metabolic dysfunction (including lysophospholipids, phospholipids, glycerides, phospholipid precursors choline, phosphorylcholine and sn-glycerol-3-phosphate ethanolamine, or amino acids such as arginine, proline, L-aspartic acid, L-arginine, sarcosine, ornithine, L-proline and L-glutamate), and mitochondrial dysfunction.

[0018] In another preferred embodiment, the treatment is selected from the group consisting of: improving, reducing, alleviating, curing, delaying the corresponding disease or symptom, or combinations thereof.

[0019] In another preferred embodiment, the diabetes is type II diabetes.

[0020] In another preferred embodiment, the diabetes is diabetes caused by vascular damage.

[0021] In another preferred embodiment, the diabetes is inflammation caused by vascular damage.

[0022] In another preferred embodiment, the RIPK1 inhibitor is selected from the group consisting of: small molecules, siRNA, shRNA, microRNA, antibodies, aptamers, DNA enzymes, enzymes, gene editing systems, hormones, inorganic compounds, oligonucleotides, organic compounds, polynucleotides, peptides, ribozymes, or synthetic compounds.

[0023] In another preferred embodiment, the RIPK1 inhibitor is selected from the group consisting of: RIPK1-i1, RIPK1-i2, spermidine, acetylated spermidine, or small molecule RIPK1 inhibitors selected from the group consisting of:

[0024] Or compounds as shown in Formula I or Formula II:

[0025] In the formula:

[0026] for

[0027] M is selected from the following group: chemical bond, O, S, NR3, CHR3 or C(R3)2;

[0028] X1 is selected from the following group: CR2, NR, O, S, CR, N;

[0029] X2 is selected from the following group: CR, N;

[0030] R is selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl;

[0031] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1 and R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups.

[0032] Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0033] Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0034] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O;

[0035] in,

[0036] Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;

[0037] n = 0, 1, or 2;

[0038] R 4Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SR b -N(R) b )2、-C(O)-NR 6 -R b -C(O)-NR 6 -C 1-4 Alkylene-N(R) b )2、-NR 6 -C(O)-R b ;

[0039] Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms;

[0040] R 6 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0041] Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups;

[0042] L 1 and L 2 Each is independently a divalent group selected from the following group:

[0043] none,

[0044] And L 1 and L 2 Not simultaneously equal to none;

[0045] R 1 and R 2 Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl;

[0046] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0047] Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl;

[0048] Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0049] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0050] Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0051] Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups;

[0052] Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R3-8 Cycloalkyl, 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O, optionally substituted with R, -C 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O;

[0053] R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl.

[0054] In another preferred embodiment, the small molecule RIPK1 inhibitor is selected from any compound from QY-15-59 to QY-17-67.

[0055] In another preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, sulfate, phosphate, nitrate, lactate, hydrobromide, hydroiodide, maleate, fumarate, citrate, formate, benzoate, acetate, trifluoroacetate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, succinate, citrate, salicylate, and ascorbate.

[0056] In another preferred embodiment, when the RIPK1 inhibitor is spermidine, the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, phosphate, lactate, fumarate, and citrate; preferably, the pharmaceutically acceptable salt of spermidine is a phosphate.

[0057] In another preferred embodiment, the vascular injury is vascular injury caused by RIPK1 activation, including peripheral vascular injury.

[0058] In another preferred embodiment, the diabetes is type II diabetes caused by vascular damage.

[0059] In another preferred embodiment, the symptoms include one or a combination of the following: vascular damage caused by the disease and related symptoms, including weight gain, insulin resistance, decreased insulin sensitivity, impaired glucose tolerance, hyperinsulinemia, lipid and amino acid metabolism disorders, and pancreatic inflammation.

[0060] In another preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or a second therapeutically active ingredient.

[0061] In another preferred embodiment, the second therapeutically active ingredient is selected from the group consisting of: diabetes treatment drugs, vascular injury treatment drugs, lipid metabolism promoting drugs, pancreatitis treatment drugs, mitochondrial function supplements, or combinations thereof.

[0062] In another preferred embodiment, the diabetes treatment drug is selected from the group consisting of: metformin (biguanide), meglitinides, sulfonylureas, DPP-4 inhibitors, thiazolidinediones, α-glucosidase inhibitors, amylin mimics, incretin mimics, and insulin.

[0063] In another preferred embodiment, the vascular injury treatment drug is selected from the group consisting of: antihypertensive agents, agents that reduce sympathetic tension, perfusion enhancers and / or agents with antithrombotic effects, and antioxidants, aldosterone- and mineralocorticoid-receptor antagonists, vasopressin receptor antagonists, organic nitrates and NO donors, IP receptor agonists, active substances that enhance contractility, calcium sensitizers, ACE inhibitors, compounds that regulate cGMP and cAMP, natriuretic peptides, NO-independent guanylate cyclase stimulators, NO-independent guanylate cyclase activators, inhibitors of human neutrophil elastase, compounds that inhibit signal transduction cascades, chemokine receptor antagonists, p38 kinase inhibitors, NPY agonists, and orexin agonists.

[0064] In another preferred embodiment, the lipid metabolism-promoting drug is selected from the group consisting of: CETP inhibitors, thyroid receptor agonists, cholesterol synthesis inhibitors (such as HMG-CoA reductase inhibitors or squalene synthesis inhibitors), ACAT inhibitors, MTP inhibitors, PPAR-α, PPAR-γ and / or PPAR-δ agonists, cholesterol absorption inhibitors, polymerized bile acid adsorbents, bile acid reabsorption inhibitors, lipase inhibitors and lipoprotein(a) antagonists, and desialyl glycoprotein inhibitors.

[0065] In another preferred embodiment, the second therapeutically active ingredient is selected from the group consisting of: β-adrenergic receptor agonists, antimuscarinic substances, corticosteroids, antithrombotic agents, platelet aggregation inhibitors, thrombin inhibitors, GPIIb / IIIa antagonists, factor Xa inhibitors, heparin or low molecular weight (LMW) heparin derivatives, vitamin K antagonists, calcium antagonists, α-1 receptor blockers, β-receptor blockers, angiotensin AII antagonists, ACE inhibitors, endothelin antagonists, renin inhibitors, mineralocorticoid receptor antagonists, diuretics, lipid metabolism regulators, and CE. TP inhibitors, thyroid receptor agonists, statin HMG-CoA reductase inhibitors, squalene synthesis inhibitors, ACAT inhibitors, MTP inhibitors, PPAR-γ agonists, PPAR-δ agonists, cholesterol absorption inhibitors, lipase inhibitors, polymerized bile acid adsorbents, bile acid reabsorption inhibitors, respiratory stimulants, psychostimulants, serotonin reuptake inhibitors, norepinephrine, serotonergic and tricyclic antidepressants, sGC stimulants, mineralocorticoid receptor antagonists, anti-inflammatory drugs, immunomodulators, immunosuppressants and cytotoxic drugs, or combinations thereof.

[0066] In another preferred embodiment, the mitochondrial functional supplement is selected from the group consisting of coenzyme Q10 (CoQ10), alpha-lipoic acid (ALA), nicotinamide adenine dinucleotide (NAD+), carnitine, glutathione, or combinations thereof.

[0067] A second aspect of the present invention provides a compound selected from the group consisting of:

[0068] A third aspect of the invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound as described in the second aspect of the invention, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable carrier.

[0069] A fourth aspect of the invention provides the use of the compound as described in the second aspect of the invention, characterized in that it is for the preparation of pharmaceutical compositions for treating or preventing diseases or conditions associated with programmed cell death and / or the activity or expression level of human receptor-interacting protein 1 kinase (RIPK1).

[0070] In a fifth aspect, the present invention provides the use of a spermidine level regulator or an acetylated spermidine level regulator, characterized in that it is used to prepare a pharmaceutical composition for the prevention and treatment of diseases or conditions selected from the group consisting of: diseases related to dysregulation of spermidine and acetylated spermidine levels; preferably, the diseases are selected from the group consisting of: symptoms caused by vascular injury, obesity, insulin resistance, diseases related to abnormal glucose tolerance, diseases related to abnormal lipid and amino acid metabolism, pancreatic inflammation, diabetes or complications of diabetes including but not limited to cardiovascular and cerebrovascular diseases, diabetic retinopathy, diabetic nephropathy, diabetic foot, etc., or mitochondrial dysfunction, as well as neurodegenerative diseases and cerebral small vessel damage caused by treatment.

[0071] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0072] Figure 1: Construction of Nat1-inducible Nat1 knockout and RIPK1 kinase inactivation hybrid mice. fl / fl Ubc creERT2+ Nat1 fl / fl Ubc creERT2 / + Ripk1D138N / D138N;

[0073] Figure 2: Construction of induced Nat1 vascular endothelial cell conditional knockout mice and RIPK1 kinase inactivated hybrid mice Nat1 fl / fl ;Cdh5 creERT2 / + Nat fl / fl Ripk1D138N / D138N; Cdh5 creERT2 ;

[0074] Figure 3: Ripk1D138N / D138N inhibits Nat1 fl / fl Ubc creERT2 / + Mice fed a normal diet (A) and a high-fat diet (B) gained weight to the level of wild-type mice;

[0075] Figure 4: Ripk1D138N / D138N inhibits Nat1 fl / fl ;Cdh5 creERT2 / + Mice fed a normal diet (A) and a high-fat diet (B) gained weight to the level of wild-type mice;

[0076] Figure 5: Ripk1D138N / D138N rescues Nat1 fl / fl Ubc creERT2 / + (A) and Nat1 fl / fl ;Cdh5creERT2 / + (B) Glucose tolerance in mice was reduced to wild-type mouse levels;

[0077] Figure 6: Ripk1D138N / D138N rescues Nat1 fl / fl Ubc creERT2 / + (A) and Nat1 fl / fl ;Cdh5 creERT2 / + (B) Insulin sensitivity in mice was reduced to wild-type mouse levels;

[0078] Figure 7: Ripk1D138N / D138N inhibits Nat1 fl / fl ;Cdh5 creERT2 / + The white adipose tissue (A) and pancreatic tissue (B) of mice were enlarged;

[0079] Figure 8: Ripk1D138N / D138N rescues Nat1 fl / fl Ubc creERT2 / + and Nat1 fl / fl ;Cdh5 creERT2 / + Abnormal lipid and amino acid metabolism levels in mice;

[0080] Figure 9: Evan Blue perfusion assay demonstrates that RIPK1 kinase inactivation inhibits Nat1. fl / fl ;Cdh5 creERT2 / + Increased vascular permeability in mouse brain tissue;

[0081] Figure 10: Evan Blue perfusion assay demonstrates that RIPK1 kinase inactivation inhibits Nat1. fl / fl ;Cdh5 creERT2 / + Increased vascular permeability in the mouse pancreas;

[0082] Figure 11: CD31 immunofluorescence staining demonstrates that RIPK1 kinase inactivation inhibits Nat1. fl / fl ;Cdh5 creERT2 / + Loss of vascular endothelial cells in mouse pancreatic tissue;

[0083] Figure 12: Ripk1D138N / D138N inhibits Nat1 fl / fl ;Cdh5 creERT2 / + Immune cell infiltration in mouse pancreatic tissue;

[0084] Figure 13: Ripk1D138N / D138N inhibits Nat1 fl / fl ;Cdh5 creERT2 / + Inflammation of pancreatic vascular endothelial cells in mice;

[0085] Figure 14: Evan Blue perfusion assay demonstrates that RIPK1 kinase inactivation inhibits Nat1. fl / fl ;Cdh5creERT2 / + Increased vascular permeability in mouse adipose tissue;

[0086] Figure 15: Evan Blue perfusion assay demonstrates that RIPK1 kinase inactivation inhibits Nat1. fl / fl ;Cdh5 creERT2 / + Increased vascular permeability in mouse kidney tissue;

[0087] Figure 16: Nat1 fl / fl Ubc creERT2 / + (A) and Nat1 fl / fl ;Cdh5 creERT2 / + (B) The level of spermine in the pancreas of mice was lower than that in wild-type mice;

[0088] Figure 17: Nat1 knockout leads to a decrease in spermidine and acetylated spermidine levels in mouse embryonic fibroblasts (MEFs), while spermidine supplementation can increase intracellular spermidine and acetylated spermidine levels.

[0089] Figure 18: Spermine can inhibit TNFα-induced RIPK1-dependent apoptosis (A) and cell necrosis (B);

[0090] Figure 19: Spermine can inhibit the activation of RIPK1 and Caspase-3 in apoptosis (A) and the activation of RIPK1 and its downstream RIPK3 and MLKL in cell necrosis (B);

[0091] Figure 20: Spermine can inhibit the activation of RIPK1 overexpression in 293T cells, increase spermine-mediated RIPK1K140 acetylated putrefaction modification, and the effect is dose-dependent.

[0092] Figure 21: RIPK1-i1 saves Nat1 fl / fl Ubc creERT2 / + The glucose tolerance (A) and insulin sensitivity (B) of mice were brought down to wild-type mouse levels;

[0093] Figure 22: RIPK1-i2 saves Nat1 fl / fl Ubc creERT2 / + The glucose tolerance (A) and insulin sensitivity (B) of mice were brought down to wild-type mouse levels;

[0094] Figure 23: Spermine rescues Nat1 fl / fl Ubc creERT2 / + The glucose tolerance (A) and insulin sensitivity (B) of mice were brought down to wild-type mouse levels. Detailed Implementation

[0095] Through long-term and in-depth research, the inventors discovered that administering drugs or treatments that inhibit RIPK1 kinase activity or expression to patients in need can effectively improve diabetic symptoms, especially those of type II diabetes caused by vascular damage. Therefore, RIPK1 inhibitors have potential applications in the treatment of diabetes. Based on these findings, the inventors completed this invention.

[0096] the term

[0097] In this document, unless otherwise specified, the term "substitution" refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of: halogen, amino, hydroxyl, nitro, cyano, trifluoromethyl, C1-C 12 Alkyl or cycloalkyl, C1-C 12 Alkoxy group, oxygen atom (i.e., =O), unsubstituted or C-substituted 1-4 Alkylamine-substituted C1-C 12 Alkylamine, C2-C6 ester, C2-C6 acyl, C2-C6 amide, thioC1-C 12 Alkyl, carboxyl, C5-C 12 Aryl or heteroaryl, C5-C 12 Heterocyclic group (containing 1-5, preferably 1-3, heteroatoms selected from N, O or S).

[0098] The term "C1-C" 12 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0099] The term "C1-C" 12 "Cycloalkyl" refers to a compound having 1-12 alkyl groups, preferably 3-12 (i.e., C12-12 alkyl groups). 3-12 ) A cycloalkyl group with a carbon atom, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or similar groups.

[0100] The term "C1-C" 12 "Alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 12 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups.

[0101] The term "halogen" refers to F, Cl, Br, and I.

[0102] The term "C1-C" 12 "Alkylamine group (or alkylamine group)" refers to a C1-C group that has been substituted with an amino group. 12 Alkyl groups, for example, those having "C1-C 12 Alkyl-NH- or (alkyl)2-N- (total number of carbon atoms is 1-12) or -C1-C12 Groups with the structures "alkylene-NH2", "alkyl-N-alkylene-(total number of carbon atoms 1-12)", or "(alkyl)2-N-alkylene-(total number of carbon atoms 1-12)", such as CH3NH-, C2H5NH-, C3H7NH-, (CH3)2N-, -CH2NH2, -C2H5NH2, -C3H7NH2, -C2H4N(CH3)2, or similar groups. Where C... 1-12 The definition of alkyl groups is as described above.

[0103] The term "C2-C6 ester group" refers to a substituent with a structure of "straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-oxy-" having 1-5 carbon atoms, such as ethyl ester, propyl ester, butyl ester, or similar groups.

[0104] The term "C1-C6 amide group" refers to a substituent with a structure of "a straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-amine-" having 0-5 carbon atoms, such as acetamido, propionamido, butyramido, or similar groups.

[0105] The term "C6-C" 10 "Aryl" refers to a group having 1-12 (preferably 6-10, i.e., C) groups. 6-10 The aryl group of the carbon atom, such as phenyl, naphthyl, etc., may be substituted or unsubstituted.

[0106] The term "C1-C" 12 "Heteroaryl" refers to a heteroaryl group having 1-12 carbon atoms and one or more (preferably 1-3) heteroatoms selected from O, S and / or N, preferably C5-C8 heteroaryl. The heteroaryl group may be substituted or unsubstituted.

[0107] The term "5-7 membered heterocycle" refers to a cyclic saturated, partially unsaturated or aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0108] The term "5-7 membered heteroaryl" refers to a cyclic aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0109] Specifically, expressions in the form "C1-Cn" indicate that the group has 1 to n carbon atoms. For example, expressions in the form "C1-C12" indicate that the group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms; "C6~C10" indicates that the group has 6, 7, 8, 9 or 10 carbon atoms.

[0110] In this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0111] In this invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is useless to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0112] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0113] As used herein, the term "compound of the invention" refers to a compound of Formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of compounds of Formula I.

[0114] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0115] RIPK1 inhibitors

[0116] The inventors have discovered that compounds with RIPK1 inhibitory effects exhibit excellent therapeutic effects on diabetes or vascular damage caused by diabetes. Therefore, patients with diabetes can achieve disease or symptom improvement (e.g., improvement of diabetes symptoms or reversal of symptoms of vascular damage caused by diabetes) by administering RIPK1 inhibitors. This treatment can be achieved by administering any therapeutic ingredient or method known or unknown in the art that targets the RIPK1 expression level, such as small molecule RIPK1 inhibitors, antibody drugs targeting RIPK1 activity or expression levels, conjugates, or genetic engineering methods. Typical RIPK1 inhibitors can be commercially available clinical small molecule drugs, such as representative RIPK1 inhibitors selected from the following group:

[0117] Other small molecule RIPK1 inhibitors in this field can also be used as RIPK1 inhibitors here.

[0118] In another preferred embodiment, the RIPK1 inhibitor may be the compound described in US2022 / 0213077A1, having the general formula structure shown in Formula I:

[0119] In the formula:

[0120] for

[0121] M is selected from the following group: chemical bond, O, S, NR3, CHR3 or C(R3)2;

[0122] X1 is selected from the following group: CR2, NR, O, S, CR, N;

[0123] X2 is selected from the following group: CR, N;

[0124] R is selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl;

[0125] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1 and R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups.

[0126] Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0127] Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl;

[0128] Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O.

[0129] Preferred molecules are shown in US2022 / 0213077A1, the entire contents of which are incorporated herein by reference.

[0130] In another preferred embodiment, the RIPK1 inhibitor may be the compound described in WO2022 / 057787A1, having the general formula structure shown in Formula III:

[0131] in,

[0132] Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;

[0133] n = 0, 1, or 2;

[0134] R 4 Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SR b -N(R) b )2、-C(O)-NR 6 -R b -C(O)-NR 6 -C 1-4 Alkylene-N(R) b)2、-NR 6 -C(O)-R b ;

[0135] Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms;

[0136] R 6 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0137] Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups;

[0138] L 1 and L 2 Each is independently a divalent group selected from the following group:

[0139] none,

[0140] And L 1 and L 2 Not simultaneously equal to none;

[0141] R 1 and R 2 Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl;

[0142] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0143] Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl;

[0144] Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0145] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0146] Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0147] Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups;

[0148] Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3-8 Cycloalkyl, 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O, optionally substituted with R, -C 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O;

[0149] R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl.

[0150] Preferred molecules are shown in WO2022 / 057787A1, the entire contents of which are incorporated herein by reference. In addition, another class of preferred compounds is shown in the table below:

[0151] In addition, other known or unknown RIPK1 small molecule inhibitors in the art can also be used for the purposes of this invention.

[0152] combination therapy

[0153] The compounds of the present invention, due to their excellent therapeutic effects on type 2 diabetes and the vascular damage caused by type 2 diabetes, can be used in combination with conventional diabetes treatment drugs to achieve improved therapeutic effects compared to single-component use. Furthermore, because the compounds of the present invention have therapeutic effects on diabetes, they can be used in combination with drugs known in the art that readily cause diabetic side effects. In preferred embodiments, pharmaceutically active agents that can be used in combination with the compositions of the present invention include, but are not limited to:

[0154] (i) Acetylcholinesterase inhibitors, such as donepezil hydrochloride (ARICEPT, MEMAC), physostigmine salicylate (ANTILIRIUM), physostigmine sulfate (ESERINE), metrifolate, neostigmine, ganstigmine, pyridostigmine (MESTINON), mytELASE, demarcarium, Debio 9902 (also known as ZT-1; Debiopharm), rivastigmine (EXELON), radoteg, NP-0361, galantamine hydrobromide (RAZADYNE, RIMINYL, NIVALIN), tacrine (COGNEX), tolserine, retinoic acid maleate, memoquin, huperzine A (HUP-A; NeuroHitech), filserin, tensilon (ENLON, TENSILON), and INM-176;

[0155] (ii) Amyloid-β (or fragments thereof), such as Aβ1-15, ACC-001 (Elan / Wyeth), ACI-01, ACI-24, AN-1792, Affitope AD-01, CAD106 and V-950 conjugated to the pan-HLA DR binding epitope (PADRE);

[0156] (iii) Antibodies against amyloid-β (or fragments thereof), such as ponezumab, sorapizumab, bapilizumab (also known as AAB-001), AAB-002 (Wyeth / Elan), ACI-01-Ab7, BAN-2401, intravenous Ig (GAMMAGARD), LY2062430 (humanized m266; Lilly), R1450 (Roche), ACU-5A5, huC091, aducanumab, lecanemab (B iogen), and those disclosed in International Patent Publications WO04 / 032868, WO05 / 025616, WO06 / 036291, WO06 / 069081, WO06 / 118959, US Patent Publications US2003 / 0073655, US2004 / 0192898, US2005 / 0048049, US2005 / 0019328, European Patent Publications EP0994728 and 1257584, and US Patent No. 5,750,349;

[0157] (iv) Amyloid-lowering or inhibitory agents (including active ingredients that reduce amyloid production, accumulation, and fibrosis), such as dimebon, davunetide, irodexacin, leuprorelin, SK-PC-B70M, celecoxib, lovastatin, anapesos, piracetam, plasracetam, varenicline, nicergoline, colostrinin, bisnorcymserine (also known as BNC), NIC5-15 (Humanetics), E-2012 (Eisai), pioglitazone, cloiodine hydroxyquine (also known as PBT1), PBT2 (Prana Biotechnology), flurbiprofen (ANSAID, FROBEN) and its R-enantiomer tarenflurbil (FLURIZAN), nitroflurbiprofen, fenofofen (FENOPRON, NALFON), ibuprofen (ADVIL, MOTRIN, NUROFEN), ibuprofen lysine salt, meclofenac, meclofenac sodium (MECLOMEN), indomethacin (INDOCIN), diclofenac sodium (VOLTAREN), diclofenac potassium, sulindac (CLINORIL), sulindac sulfide, diflunisal (DOLOBID), naproxen (NAPROSYN), naproxen sodium (ANAPROX, ALEVE), ARC031 (Archer) Pharmaceuticals), CAD-106 (Cytos), LY450139 (Lilly), Insulin Degrading Enzyme (also known as Insulin Lysozyme), Ginkgo Biloba Extract EGb-761 (ROKAN, TEBONIN), High Taurine (CEREBRIL, ALZHEMED), Irodex (FIBRILLEX, KIACTA), Compound W [3,5-bis(4-nitrophenoxy)benzoic acid], NGX-96992, Enkephalinase (also known as Neutral Endopeptidase (NEP)), Squalene (also known as Squalene), Atorvastatin (LIPITOR), Simvastatin (ZOCOR), KLVFF- ( EEX)3, SKF-74652, ibuprofen mesylate, BACE inhibitors such as ASP-1702, SCH-745966, JNJ-715754, AMG-0683, AZ-12304146, BMS-782450, GSK-188909, NB-533, E2609 and TTP-854; γ-secretase modulators such as ELND-007; and RAGE (receptor for advanced glycation end products) inhibitors such as TTP488 (Transtech) and TTP4000 (Transtech), as well as those disclosed in U.S. Patent No. 7,285,293, including PTI-777;

[0158] (v) Alpha-adrenergic receptor agonists, such as guanifacin (INTUNIV, TENEX), clonidine (CATAPRES), metaraminol (ARAMINE), methyldopa (ALDOMET, DOPAMET, NOVOMEDOPA), tizanidine (ZANAFLEX), phenylephrine (also known as neo-ephrine), methoxyamine, cilerazoline, guanifacin (INTUNIV), lofezepine, toluenethiazide, modafinil (PROVIGIL), adrolidine, and armodafinil (NUVIGIL);

[0159] (vi) β-adrenergic receptor blockers (β-blockers), such as carteolol, esmolol (BREVIBLOC), labetalol (NORMODYNE, TRANDATE), oxenolol (LARACOR, TRASACOR), indololol (VISKEN), propranolol (INDERAL), sotalolol (BETAPACE, SOTALEX, SOTACOR), timolol (BLOCADREN, TIMOPTIC), acebutolol (SECTRAL, PRENT), nadololol (CORGARD), metoprolol tartrate (LOPRESSOR), metoprolol succinate (TOPROL-XL), atenolol (TENORMIN), butorxamin, and SR 59230A (Sanofi);

[0160] (vii) Anticholinergic drugs, such as amitriptyline (ELAVIL, ENDEP), butatriptyline, bentropin mesylate (COGENTIN), trihexyphenidyl (ARTANE), diphenhydramine (BENADRYL), oxifenesin (NORFLEX), hyoscyamine, atropine (ATROPEN), scopolamine (TRANSDERM-SCOP), methylscopolamine bromide (PARMINE), bicyclovir (BENTYL, BYCLOMINE, DIBENT, DILOMINE), tolterodine (DETROL), and oxybutynin (DITROPAN, LYRINEL). XL, OXYTROL), pentylammonium bromide, pro-BANTHINE, cycladine, imipramine hydrochloride, imipramine maleate, lofepramine, desipramine, doxepin, trimethoprim, and glycopyrronium bromide.

[0161] When used as a regulator of mitochondrial dysfunction, the compounds of the present invention can also be used in combination with any mitochondrial function supplement, typically exemplary (but not limited to) mitochondrial function supplements selected from the group consisting of: coenzyme Q10 (CoQ10), alpha-lipoic acid (ALA), nicotinamide adenine dinucleotide (NAD+), carnitine, glutathione, or combinations thereof.

[0162] Pharmaceutical Compositions and Administration

[0163] Because the compounds of the present invention have excellent lipid-lowering activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredients can be used to treat, prevent and alleviate diabetes, or related diseases caused by diabetes such as vascular damage.

[0164] In this invention, by administering RIPK1 inhibitors to patients, the levels of spermidine and acetylated spermidine are regulated. This method can be used for diabetes, including related diseases such as insulin resistance, glucose intolerance, lipid and amino acid metabolism abnormalities, and pancreatic inflammation; vascular complications caused by diabetes, including cardiovascular and cerebrovascular damage, diabetic retinopathy, diabetic nephropathy, and diabetic foot; prevention and treatment of diabetes-related symptoms such as insulin resistance, glucose intolerance, lipid and amino acid metabolism abnormalities, and pancreatic inflammation caused by vascular damage; and prevention and treatment of other diseases with pathological features of vascular damage, including cerebrovascular damage caused by neurodegenerative diseases and treatment side effects.

[0165] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 5-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0166] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0167] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0168] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0169] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0170] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0171] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0172] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0173] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0174] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0175] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0176] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0177] Compared with the prior art, the main advantages of the present invention include:

[0178] (1) A novel treatment mechanism for diabetes (especially type 2 diabetes) has been discovered, which involves symptom improvement in patients with type 2 diabetes by downregulating the expression or activity of RIPK1 kinase.

[0179] (2) A class of novel compounds for the treatment of diabetes or its related vascular damage are provided, which have good reversal activity for the symptoms of type II diabetes and its vascular damage.

[0180] The present invention will be further illustrated below 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 of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0181] Example 1. Model Establishment

[0182] We designed and inserted LoxP sequences at both ends of the only exon of the mouse Nat1 gene using the CRISPR-Cas9 method. After verifying successful insertion, we crossed the mice with Ubc-creERT2 and Cdh5-creERT2 transgenic mice, induced knockout with tamoxifen, and then collected tissues and extracted vascular endothelial cells from the offspring for testing, proving that Nat1 was effectively knocked out (Figures 1 and 2). We then crossed these mice with Ripk1D138N / D138N mice to obtain Ripk1D138N / D138N genotype mice, thus obtaining Nat1... fl / fl Ubc creERT2 / + Nat1 fl / fl ;Cdh5 creERT2 / + Nat1 fl / fl Ubc creERT2 / + ;Ripk1D138N / D138N,Nat1 fl / fl ;Cdh5 creERT2 / + Ripk1D138N / D138N. The husbandry and management of laboratory animals were carried out strictly in accordance with the standards stipulated by the National Protein Center for Laboratory Animal Management. Mice were housed in specific pathogen-free (SPF) cages with free access to food and water. The living conditions in the SPF-grade animal housing were as follows: circadian rhythm (6:00 AM to 6:00 PM, with light), room temperature 22±2℃, and relative humidity 50±10%. Mice aged 6-12 weeks were fed normal and high-fat diets to establish control and experimental mouse models of obesity, insulin resistance, and hyperglycemia.

[0183] By measuring Nat1 fl / fl Ubc creERT2 / + Nat1 fl / fl Ubc creERT2 / + ;Ripk1D138N / D138N,Nat1 fl / fl ;Cdh5 creERT2 / + Nat1 fl / fl ;Cdh5 creERT2 / +Ripk1D138N / D138N mice underwent changes in food intake, body weight, white fat, pancreas, glucose tolerance, and insulin tolerance under normal and high-fat diets. Nat1 was found to be... fl / fl Ubc creERT2 / + and Nat1 fl / fl ;Cdh5 creERT2 / + There was no significant difference in food intake among the mice, but their body weight was significantly increased compared to wild-type mice under both normal and high-fat diets. Furthermore, Ripk1D138N / D138N significantly inhibited the weight gain of these mice to the level of wild-type mice (Figs. 3A, B; Figs. 4A, B). Nat1 fl / fl ;Cdh5 creERT2 / + The white adipose tissue and pancreas of mice were significantly enlarged, while Ripk1D138N / D138N significantly suppressed this phenotype (Figure 5A, B). Glucose and insulin tolerance experiments showed that Nat1... fl / fl Ubc creER2 / + and Nat1 fl / fl ;Cdh5 creERT2 / + In mice, glucose (Fig. 6A, B) and insulin sensitivity were significantly reduced (Fig. 7A, B), and Ripk1D138N / D138N significantly rescued this phenotype to wild-type mouse levels. This result indicates that RIPK1 kinase inactivation effectively improves insulin resistance induced by NAT1 deficiency.

[0184] Mass spectrometry analysis of small molecule metabolites extracted from mouse plasma revealed that Ripk1D138N / D138N rescued Nat1. fl / fl Ubc creER2 / + and Nat1 fl / fl ;Cdh5 creERT2 / + The mice exhibited abnormal amino acid and lipid metabolism (Figure 8).

[0185] After perfusion of mice with Evans blue dye, spectrophotometric analysis revealed that the dye remained effective in Nat1 mice for 3 months. fl / fl ;Cdh5 creERT2 / + The phenotype diffused in mouse brain tissue and pancreatic tissue at 3, 5, and 7 months of age, while it was observed in Nat1. fl / fl ;Cdh5 creERT2 / + In Ripk1D138N / D138N mice, the levels were significantly reduced to control levels (Figures 9 and 10), suggesting that RIPK1 kinase inactivation can inhibit the increase in vascular permeability caused by conditional knockout of Nat1 in vascular endothelial cells. Nat1 was used... fl / fl ;Cdh5 creERT2 / + Immunofluorescence assays of mouse pancreatic tissue, focusing on the endothelial cell marker CD31, revealed Na1... fl / fl ;Cdh5 creERT2 / +Endothelial cells were reduced in mouse pancreatic tissue (Fig. 11), and Ripk1D138N / D138N was able to rescue this phenotype (Fig. 11), suggesting that RIPK1 kinase inactivation can inhibit endothelial cell loss caused by Nat1 endothelial cell conditional knockout.

[0186] Immunofluorescence staining of macrophage marker F4 / 80 was performed on mouse pancreatic tissue, and Na1 was found. fl / fl ;Cdh5 creERT2 / + Macrophage infiltration was significantly increased in mouse pancreatic tissue, while Ripk1D138N / D138N significantly inhibited this phenotype (Figure 12), suggesting that conditional knockout of Nat1 vascular endothelial cells promotes RIPK1-dependent macrophage infiltration in the pancreas. qPCR analysis of inflammatory factors such as TNFα, IL-6, and CRP in vascular endothelial cells extracted from mouse pancreatic tissue revealed that conditional knockout of Nat1 vascular endothelial cells significantly promoted the expression of these inflammatory factors, while Ripk1D138N / D138N significantly inhibited this phenotype (Figure 13), suggesting that RIPK1 kinase inactivation can suppress pancreatic inflammation induced by conditional knockout of Nat1 vascular endothelial cells.

[0187] In addition, Evans' blue dye infusion experiment also showed that Nat1 fl / fl ;Cdh5 creERT2 / + After 5 months, blue dye diffused into the adipose and kidney tissues of mice, a phenotype observed in Nat1. fl / fl ;Cdh5 creERT2 / + The level was significantly reduced to the control level in Ripk1D138N / D138N mice (Figure 14, Figure 15), suggesting that RIPK1 kinase inactivation can inhibit progressive vascular damage induced by conditional knockout of Nat1 in vascular endothelial cells.

[0188] Mass spectrometry quantification of polyamine metabolite levels in mouse pancreatic tissue and mouse embryonic fibroblasts (MEFs) revealed that Nat1 knockout decreased spermidine and acetylated spermidine levels (Fig. 16A-B, Fig. 17). Speridine supplementation in Nat1 knockout MEFs increased spermidine and acetylated spermidine levels (Fig. 16). Compared with wild-type MEFs, Nat1 knockout further promoted RIPK1-dependent apoptosis or necrosis induced by tumor necrosis factor TNFα+SM164 or TNFα+SM164+zVAD, and spermidine treatment partially inhibited the increased cell death sensitivity caused by Nat1 knockout (Fig. 18A-B). Western blotting experiments showed that spermidine treatment inhibited RIPK1 activation characterized by S166 phosphorylation, inhibited Caspase-3 activation, and inhibited the activation of downstream RIPK3 and MLKL (Fig. 19A-B). Overexpression of RIPK1 in 293T cells and spermidine treatment increased spermidine-mediated acetylation and hydroxycorrosive modification of RIPK1 K140, inhibiting RIPK1 activation characterized by S166 phosphorylation in a dose-dependent manner (Figure 20).

[0189] Example 2: Administration of the RIPK1 inhibitor RIPK1-i1

[0190] To further verify the effect of RIPK1 kinase inhibitors on glucose tolerance reduction caused by arylamine N-acetyltransferase 2 knockout, Nat1 knockout mice were given small molecule RIPK1 inhibitors and their effects were observed. The groups were set as follows:

[0191] RIPK1-i1 group: 40 mg of RIPK1-i1 was dissolved in 100 μL of dimethyl sulfoxide (DMSO). This solution was then added to 10 mL of 35% polyethylene glycol (PEG) and 10 mL of 20% sucrose solution, mixed well, filtered for sterilization, and then added to 80 mL of drinking water for mice. This treatment was administered for 14 consecutive days. Mice were fed a normal diet.

[0192] The RIPK1-i1 has the structure shown in the following formula:

[0193] (Necrostatin-1s enantiomer, CAS number: 852391-20-9)

[0194] Control group: The control group did not receive RIPK1-i1, and was otherwise the same as the treatment group.

[0195] Test Example 1. RIPK1-i1 completely saves Nat1 fl / fl Ubc creERT2 / + Glucose tolerance in mice was reduced to wild-type mouse levels.

[0196] After RIPK1-i1 treatment, the glucose tolerance of mice was detected using the glucose tolerance test (GTT).

[0197] In the glucose tolerance test for mice, the glucose dosage is generally 1.5 g / kg body weight. The required glucose solution is prepared beforehand by dissolving a certain amount of glucose in phosphate-buffered saline (PBS) at a concentration of 0.25 g / ml. The injection volume is determined based on the mouse's body weight, with 6 μL of glucose solution injected per gram of body weight.

[0198] 1. Mouse preparation: At 11 p.m. the night before the experiment, the mice were placed in clean cages and fasted for 10 hours until 9 a.m. the next morning; during the fasting period, the mice were allowed to drink water normally.

[0199] 2. At 9:00 a.m. the following morning, the glucose tolerance test began; the weight of each mouse was measured and a serial number was marked at the base of the mouse's tail so that the mice being tested could be quickly identified during the experiment;

[0200] 3. Measurement of fasting basal blood glucose: Remove the mouse from its cage and gently place it on a wire mesh. Cut off about 1-2 mm from the end of the mouse's tail, gently squeeze the tail to collect a drop of blood, and measure the fasting blood glucose using a blood glucose meter. The measured value is recognized as the blood glucose level at 0 minutes. The procedure should be gentle to avoid excessively startling the mouse.

[0201] 4. Gently pick up the mouse and inject it with a 1 ml syringe. The injection volume depends on the mouse's weight, at 6 μL of glucose solution per gram of body weight. Start timing immediately after injection.

[0202] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0203] 6. Experiment completed.

[0204] The results showed that RIPK1-i1 completely saved Nat1. fl / fl Ubc creERT2 / + Mice's glucose tolerance was brought down to wild-type mouse levels (Figure 21A).

[0205] Test Example 2. RIPK1-i1 completely saves Nat1 fl / fl Ubc creERT2 / + Insulin sensitivity in mice was reduced to wild-type mouse levels.

[0206] After RIPK1-i1 treatment, insulin sensitivity in mice was detected using an insulin sensitivity test (ITT).

[0207] Dilute the insulin to the appropriate concentration according to the specific experimental requirements. For example, if the insulin dosage is 0.75 units / kg, prepare an insulin solution of 0.125 units / mL using phosphate-buffered saline (PBS). The injection volume is determined based on the mouse's body weight, with 6 μL injected per gram of body weight.

[0208] 1. Mice preparation: At 9:00 AM, transfer the mice to clean cages and fast them for 6 hours until 3:00 PM. During the fasting period, ensure the mice have access to normal water.

[0209] 2. At 1 p.m., the insulin tolerance test will begin. Each mouse will be weighed, and a serial number will be marked at the base of its tail to facilitate quick identification during the experiment.

[0210] 3. Remove the mouse from its cage and gently place it on the wire mesh. Cut off about 1-2 millimeters from the tip of the mouse's tail with scissors. Gently squeeze the tail to collect a drop of blood. Measure the blood glucose level using a blood glucose meter. The measured value is considered the blood glucose level at 0 minutes. Handle the mouse as gently as possible to avoid excessively startling it.

[0211] 4. Gently pick up the mouse and administer insulin solution using a 1 ml syringe, following standard intraperitoneal injection procedures. The injection volume is determined by the mouse's weight, at 6 μL per gram of body weight. Start timing immediately after injection.

[0212] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0213] 6. Experiment completed.

[0214] The results showed that RIPK1-i1 completely saved Nat1. fl / fl Ubc creERT2 / + The insulin sensitivity of mice was reduced to the level of wild-type mice (Figure 21B).

[0215] Example 3. Administration of the RIPK1 inhibitor RIPK1-i2

[0216] To further verify the effect of RIPK1 kinase inhibitors on glucose tolerance reduction caused by arylamine N-acetyltransferase 2 knockout, Nat1 knockout mice were given small molecule RIPK1 inhibitors and their effects were observed. The groups were set as follows:

[0217] RIPK1-i2 group: 30 mg of RIPK1-i2 was dissolved in 5 mL of 0.5% sodium carboxymethyl cellulose solution, mixed well, filtered for sterilization, and then this mixture was administered to mice by gavage at doses of 10 mg / kg and 30 mg / kg of body weight for 14 consecutive days. Mice were fed a normal diet.

[0218] The RIPK1-i2 has the structure shown in the following formula:

[0219] (QY-13-33, recorded in WO2022057787A1)

[0220] Control group: The control group did not receive RIPK1-i2, and was otherwise the same as the treatment group.

[0221] Test Example 1. RIPK1-i2 completely saves Nat1 fl / fl Ubc creERT2 / + Glucose tolerance in mice was reduced to wild-type mouse levels.

[0222] After RIPK1-i2 treatment, the glucose tolerance of mice was detected using the glucose tolerance test (GTT).

[0223] In the glucose tolerance test for mice, the glucose dosage is generally 1.5 g / kg body weight. The required glucose solution is prepared beforehand by dissolving a certain amount of glucose in phosphate-buffered saline (PBS) at a concentration of 0.25 g / ml. The injection volume is determined based on the mouse's body weight, with 6 μL of glucose solution injected per gram of body weight.

[0224] 1. Mouse preparation: At 11 p.m. the night before the experiment, the mice were placed in clean cages and fasted for 10 hours until 9 a.m. the next morning; during the fasting period, the mice were allowed to drink water normally.

[0225] 2. At 9:00 a.m. the following morning, the glucose tolerance test began; the weight of each mouse was measured and a serial number was marked at the base of the mouse's tail so that the mice being tested could be quickly identified during the experiment;

[0226] 3. Measurement of fasting basal blood glucose: Remove the mouse from its cage and gently place it on a wire mesh. Cut off about 1-2 mm from the end of the mouse's tail, gently squeeze the tail to collect a drop of blood, and measure the fasting blood glucose using a blood glucose meter. The measured value is recognized as the blood glucose level at 0 minutes. The procedure should be gentle to avoid excessively startling the mouse.

[0227] 4. Gently pick up the mouse and inject it with a 1 ml syringe. The injection volume depends on the mouse's weight, at 6 μL of glucose solution per gram of body weight. Start timing immediately after injection.

[0228] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0229] 6. Experiment completed.

[0230] The results showed that RIPK1-i2 completely saved Nat1.fl / fl Ubc creERT2 / + The glucose tolerance of mice was brought down to the level of wild-type mice (Figure 22A).

[0231] Test Example 2. RIPK1-i2 completely saves Nat1 fl / fl Ubc creERT2 / + Insulin sensitivity in mice was reduced to wild-type mouse levels.

[0232] After RIPK1-i2 treatment, insulin sensitivity in mice was detected using an insulin sensitivity test (ITT).

[0233] Dilute the insulin to the appropriate concentration according to the specific experimental requirements. For example, if the insulin dosage is 0.75 units / kg, prepare an insulin solution of 0.125 units / mL using phosphate-buffered saline (PBS). The injection volume is determined based on the mouse's body weight, with 6 μL injected per gram of body weight.

[0234] 1. Mice preparation: At 9:00 AM, transfer the mice to clean cages and fast them for 6 hours until 3:00 PM. During the fasting period, ensure the mice have access to normal water.

[0235] 2. At 1 p.m., the insulin tolerance test will begin. Each mouse will be weighed, and a serial number will be marked at the base of its tail to facilitate quick identification during the experiment.

[0236] 3. Remove the mouse from its cage and gently place it on the wire mesh. Cut off about 1-2 millimeters from the tip of the mouse's tail with scissors. Gently squeeze the tail to collect a drop of blood. Measure the blood glucose level using a blood glucose meter. The measured value is considered the blood glucose level at 0 minutes. Handle the mouse as gently as possible to avoid excessively startling it.

[0237] 4. Gently pick up the mouse and administer insulin solution using a 1 ml syringe, following standard intraperitoneal injection procedures. The injection volume is determined by the mouse's weight, at 6 μL per gram of body weight. Start timing immediately after injection.

[0238] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0239] 6. Experiment completed.

[0240] The results showed that RIPK1-i2 completely saved Nat1. fl / fl Ubc creERT2 / + The insulin sensitivity of mice was reduced to that of wild-type mice (Figure 22B).

[0241] Example 4. Application of spermidine

[0242] To further verify the effect of spermidine on glucose tolerance reduction caused by aryl N-acetyltransferase 2 knockout, spermidine was administered to Nat1 knockout mice and its effects were observed. The groups were set as follows:

[0243] Spermine group: Spermine was added to the drinking water of mice to prepare a spermine concentration of 800 uM, filtered for sterilization, and administered continuously for 14 days. Mice were fed a normal diet.

[0244] Control group: No spermidine was added to the drinking water of the control group.

[0245] Test Example 1. Spermine completely rescues Nat1 fl / fl Ubc creERT2 / + Glucose tolerance in mice was reduced to wild-type mouse levels.

[0246] After spermidine treatment, the glucose tolerance of mice was tested using the glucose tolerance test (GTT).

[0247] In the glucose tolerance test for mice, the glucose dosage is generally 1.5 g / kg body weight. The required glucose solution is prepared beforehand by dissolving a certain amount of glucose in phosphate-buffered saline (PBS) at a concentration of 0.25 g / ml. The injection volume is determined based on the mouse's body weight, with 6 μL of glucose solution injected per gram of body weight.

[0248] 1. Mouse preparation: At 11 p.m. the night before the experiment, the mice were placed in clean cages and fasted for 10 hours until 9 a.m. the next morning; during the fasting period, the mice were allowed to drink water normally.

[0249] 2. At 9:00 a.m. the following morning, the glucose tolerance test began; the weight of each mouse was measured and a serial number was marked at the base of the mouse's tail so that the mice being tested could be quickly identified during the experiment;

[0250] 3. Measurement of fasting basal blood glucose: Remove the mouse from its cage and gently place it on a wire mesh. Cut off about 1-2 mm from the end of the mouse's tail, gently squeeze the tail to collect a drop of blood, and measure the fasting blood glucose using a blood glucose meter. The measured value is recognized as the blood glucose level at 0 minutes. The procedure should be gentle to avoid excessively startling the mouse.

[0251] 4. Gently pick up the mouse and inject it with a 1 ml syringe. The injection volume depends on the mouse's weight, at 6 μL of glucose solution per gram of body weight. Start timing immediately after injection.

[0252] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0253] 6. Experiment completed.

[0254] The results showed that spermidine completely rescued Nat1. fl / fl Ubc creERT2 / + Mice's glucose tolerance was brought down to wild-type mouse levels (Figure 23A).

[0255] Test Example 2. Spermine completely rescues Nat1 fl / fl Ubc creERT2 / + The insulin sensitivity of mice was reduced to the level of wild-type mice.

[0256] After spermidine treatment, insulin sensitivity in mice was detected using an insulin sensitivity test (ITT).

[0257] Dilute the insulin to the appropriate concentration according to the specific experimental requirements. For example, if the insulin dosage is 0.75 units / kg, prepare an insulin solution of 0.125 units / mL using phosphate-buffered saline (PBS). The injection volume is determined based on the mouse's body weight, with 6 μL injected per gram of body weight.

[0258] 1. Mice preparation: At 9:00 AM, transfer the mice to clean cages and fast them for 6 hours until 3:00 PM. During the fasting period, ensure the mice have access to normal water.

[0259] 2. At 1 p.m., the insulin tolerance test will begin. Each mouse will be weighed, and a serial number will be marked at the base of its tail to facilitate quick identification during the experiment.

[0260] 3. Remove the mouse from its cage and gently place it on the wire mesh. Cut off about 1-2 millimeters from the tip of the mouse's tail with scissors. Gently squeeze the tail to collect a drop of blood. Measure the blood glucose level using a blood glucose meter. The measured value is considered the blood glucose level at 0 minutes. Handle the mouse as gently as possible to avoid excessively startling it.

[0261] 4. Gently pick up the mouse and administer insulin solution using a 1 ml syringe, following standard intraperitoneal injection procedures. The injection volume is determined by the mouse's weight, at 6 μL per gram of body weight. Start timing immediately after injection.

[0262] 5. Measure the blood glucose levels of each mouse at 30 minutes, 60 minutes, and 120 minutes, following the procedure in step 3;

[0263] 6. Experiment completed.

[0264] The results showed that spermidine completely rescued Nat1. fl / fl Ubc creERT2 / + The insulin sensitivity of mice was reduced to that of wild-type mice (Figure 23B).

[0265] 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. The use of a RIPK1 inhibitor, or a pharmaceutically acceptable salt, hydrate, or solvation thereof, in the preparation of a pharmaceutical composition, characterized in that, The pharmaceutical composition is used to prevent or treat diseases or conditions selected from the group consisting of decreased spermidine and acetylated spermidine levels caused by aryl N-acetyltransferase 2 deficiency or other reasons: symptoms of vascular damage, obesity, inflammation, aging, insulin resistance, glucose intolerance-related diseases, lipid and amino acid metabolism-related diseases, pancreatic inflammation, diabetes or complications of diabetes. Preferably, the symptoms are selected from the following group: inflammation, mitochondrial dysfunction, insulin resistance, glucose intolerance, lipid and amino acid metabolism disorders, pancreatic inflammation and other related diseases; vascular damage complications caused by diabetes, including cardiovascular and cerebrovascular damage, diabetic retinopathy, diabetic nephropathy, diabetic foot, etc.; and diabetes-related symptoms such as insulin resistance, glucose intolerance, lipid and amino acid metabolism disorders, pancreatic inflammation, etc. caused by vascular damage; and other diseases with pathological features of vascular damage, including cerebrovascular damage caused by neurodegenerative diseases and treatment side effects, neurodegenerative diseases, and small cerebral vessel damage caused by treatment.

2. The use as described in claim 1, characterized in that, The RIPK1 inhibitor is selected from the group consisting of: small molecules, siRNA, shRNA, microRNA, antibodies, aptamers, DNA enzymes, enzymes, gene editing systems, hormones, inorganic compounds, oligonucleotides, organic compounds, polynucleotides, peptides, ribozymes, or synthetic compounds.

3. The use as described in claim 1, characterized in that, The RIPK1 inhibitor is selected from the group consisting of: RIPK1-i1, RIPK1-i2, spermidine, acetylated spermidine, or small molecule RIPK1 inhibitors selected from the group consisting of: Or compounds as shown in Formula I or Formula II: In the formula: for M is selected from the following group: chemical bond, O, S, NR3, CHR3 or C(R3)2; X1 is selected from the following group: CR2, NR, O, S, CR, N; X2 is selected from the following group: CR, N; R is selected from the following group: H, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 deuterated alkyl; or R1 and R2 together with the carbon atom attached to them to form substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 4-6 membered heterocyclic groups. Ring A is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl; Ring B is a group selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 heteroaryl; Wherein, the substitution refers to the hydrogen atom on the substituent group being replaced by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkoxy, halogenated C1-C6 alkoxy, methyl sulfone, -S(=O)2NH2, oxo(=O), -CN, hydroxyl, -NH2, carboxyl, C2-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, where Rc is H or a C1-C5 alkyl), C1-C6 alkyl-(C2-C6 amide), or substituted or unsubstituted groups selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amino, C6-C10 aryl, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH2)-C6-C10 aryl, -(CH2)-(5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O), and the substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH2, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amide, 5-10 heteroaryl having 1-3 heteroatoms selected from N, S and O; in, Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms; n = 0, 1, or 2; R 4 Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, -OR b -SR b -N(R) b )2、-C(O)-NR 6 -R b -C(O)-NR 6 -C 1-4 Alkylene-N(R) b )2、-NR 6 -C(O)-R b ; Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms; R 6 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups; L 1 and L 2 Each is independently a divalent group selected from the following group: none, And L 1 and L 2 Not simultaneously equal to none; R 1 and R 2 Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-4 alkyl; R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 alkyl; Or, when ring C is L 1 for And L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being substituted with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms; R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy; Or when ring C is nonexistent and L 2 for At that time, R 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms; Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups; Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen, nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3- 8-cyclic alkyl groups, 5-12-membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S, and O, optionally substituted with R, and -C optionally substituted with R. 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O; R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl.

4. The use as described in claim 1, characterized in that, The small molecule RIPK1 inhibitors mentioned are selected from the following group:

5. The use as described in claim 1, characterized in that, The vascular damage mentioned refers to vascular damage caused by RIPK1 activation, including peripheral vascular damage.

6. The use as described in claim 1, characterized in that, The diabetes mentioned is type 2 diabetes caused by vascular damage.

7. The use as described in claim 1, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or a second therapeutically active ingredient.

8. The use as described in claim 1, characterized in that, The second therapeutic active ingredient is selected from the group consisting of: drugs for treating diabetes, drugs for treating vascular damage, drugs for promoting lipid metabolism, drugs for treating pancreatitis, mitochondrial function supplements, or combinations thereof.

9. A compound selected from the group consisting of:

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises (a) a therapeutically effective amount of the compound as described in claim 9, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable carrier.

11. The use of the compound as claimed in claim 9, characterized in that, Pharmaceutical compositions for the treatment or prevention of diseases or conditions associated with inflammation, programmed cell death and / or human receptor-interacting protein 1 kinase (RIPK1) activity or expression levels.

12. The use of a spermidine level regulator or an acetylated spermidine level regulator, characterized in that, Pharmaceutical compositions for the prevention and treatment of diseases or conditions selected from the group consisting of: diseases related to dysregulation of spermidine and acetylated spermidine levels; preferably, the diseases are selected from the group consisting of: symptoms of vascular injury, obesity, insulin resistance, diseases related to glucose intolerance, diseases related to lipid and amino acid metabolism disorders, pancreatitis, diabetes or complications of diabetes including but not limited to cardiovascular and cerebrovascular diseases, diabetic retinopathy, diabetic nephropathy, diabetic foot, etc., or mitochondrial dysfunction, neurodegenerative diseases, and treatment-induced damage to small blood vessels in the brain.

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