Ferroptosis-inhibiting phosphatide-like material and application thereof

Abstract

The present application relates to the technical field of biological medicine, and in particular to a ferroptosis inhibition type phospholipid-like material and application thereof.The ferroptosis inhibition type phospholipid-like material is one of the following structural general formulae (1)-(5): The biomimetic phospholipid ferroptosis inhibitor has a long retention characteristic at the main position of cell ferroptosis (cell membrane and endoplasmic reticulum organelle membrane), thus significantly improving the ferroptosis inhibition efficiency.This new type of phospholipid material can be used as an active drug molecule and as a pharmaceutical excipient for constructing liposomes, micelles and other drug delivery carriers and implant coating, and is suitable for various drug delivery routes such as oral administration, injection and local administration.The new biomimetic ferroptosis inhibitor can efficiently relieve cell ferroptosis and has a wide application prospect in the field of treating or alleviating ferroptosis related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a ferroptosis-inhibiting phospholipid material and its application. Background Technology

[0002] Ferroplasmosis is a ferrous ion-dependent regulated cell death pathway that differs from traditional apoptosis, necrosis, and autophagy in terms of genes, biochemistry, and morphology. The three key elements of feroplasmosis are the presence of ferrous ions, the accumulation of specific lipid peroxides, and the loss or impairment of lipid peroxide repair function. Three major feroplasmosis-inhibiting systems exist within cells: (1) using GPX4 to reduce lipid peroxides to fatty alcohols; (2) using reducing coenzyme Q10 as an endogenous free radical scavenger to remove lipid peroxide free radicals; and (3) using tetrahydrobiopterin as another type of endogenous free radical scavenger to remove lipid peroxide free radicals. Previous studies have shown that the endoplasmic reticulum membrane and cell membrane are key sites for lipid peroxidation leading to feroplasmosis.

[0003] Ferroprelation is closely associated with a variety of diseases, including neurodegenerative diseases, uncontrolled inflammation, multiple organ dysfunction syndrome, and ischemia-reperfusion injury. Therefore, the use of ferroprelation inhibitors to treat these diseases has attracted widespread attention. Existing Chinese patents have reported the application of various ferroprelation inhibitors in related diseases, such as free radical scavengers Fer-1 and Liproxstatin-1, selenium-enriched Cordyceps militaris activating GPX4, as well as selenocarbon dots, tetrahydroquinolinol, and 2-amino-6-methylphenol, which are used to improve myocardial injury (e.g., doxorubicin cardiotoxicity, myocardial ischemia-reperfusion injury), ophthalmic diseases (e.g., ocular hypertension, age-related macular degeneration), liver and kidney injuries (e.g., non-alcoholic steatohepatitis, acute liver injury, kidney injury), lung diseases (e.g., acute lung injury, pneumonia), bone diseases (e.g., osteoarthritis, osteoporosis), as well as iron overload, biliary tract diseases, ischemia-reperfusion injury, and chemotherapy-related pain. These patents all demonstrate the important role of ferroprelation inhibitors in the treatment of these diseases.

[0004] Given the therapeutic potential of ferroptosis inhibitor therapy for various related diseases, the development of novel ferroptosis inhibitors has gradually become a hot topic in the biomedical field. Ferroptosis inhibitors mainly exert their effects by reducing free iron, scavenging free radicals, and inhibiting lipid peroxidation. Although cell membranes and organelle membranes are key sites of ferroptosis, the retention time of currently reported ferroptosis inhibitors in these membranes is very short, severely limiting their efficacy.

[0005] The main component of biofilms is phospholipids, characterized by a hydrophilic group consisting of a phosphate-linked substituent and a hydrophobic group consisting of two fatty acid chains. Phospholipid molecules possess excellent biocompatibility, amphiphilicity, and the ability to self-assemble in aqueous phases. Therefore, integrating ferroptosis-inhibiting active groups into phospholipids or their skeletal structure can construct novel amphiphilic biomimetic phospholipid ferroptosis inhibitors. Specifically, the ferroptosis-inhibiting active group can replace one or two fatty acid chains in the phospholipid structure, or the active group can be covalently linked to a polar group at the phospholipid head. These biomimetic ferroptosis inhibitors are amphiphilic, thus easily accumulating at the interface between two phases. Simultaneously, due to their phospholipid-like properties, they have a high affinity for cell membranes and organelle membranes, enabling targeted accumulation at these membranes and subcellular organelle membranes, thereby efficiently inhibiting ferroptosis. Furthermore, due to the amphiphilicity and surface activity of these biomimetic phospholipid ferroptosis inhibitors, they can be used as pharmaceutical excipients to construct various drug delivery systems. Summary of the Invention

[0006] This invention covalently couples ferroptosis-inhibiting active groups with phospholipids (or their backbones) to construct a biomimetic inhibitor with a phospholipid-like structure, which is expected to solve the problem of insufficient membrane retention of existing inhibitors, while also possessing drug activity and delivery carrier functions.

[0007] The purpose of this invention is to provide a ferroptosis-inhibiting phospholipid material and its application, in order to solve the problems of short retention time and limited inhibition efficiency of existing ferroptosis inhibitors at key sites of ferroptosis (cell membranes and organelle membranes such as endoplasmic reticulum). At the same time, this material can both act directly as an active drug molecule and be used as a pharmaceutical excipient to construct a drug delivery system, thereby effectively alleviating ferroptosis and providing a new solution for the improvement or treatment of ferroptosis-related pathological conditions.

[0008] To achieve the above objectives, a ferroptosis-inhibiting phospholipid material is one of the following general structural formulas (1)-(5):

[0009]

[0010] In the general structural formulas (1)-(5), R is selected from free radical scavengers and their derivatives, small molecule organoselenic compounds and their derivatives that mimic the function of glutathione peroxidase 4 (GPX4), iron chelating agents and their derivatives, monounsaturated fatty acids and their derivatives, 7-dehydrocholesterol and its derivatives, royal jelly acid (10-hydroxy-2-decenoic acid) and its derivatives, α-lipoic acid and its derivatives.

[0011] In the general structural formulas (1)-(4), X is a spacer arm selected from saturated or unsaturated aliphatic chains with 0-23 carbon atoms that do not contain heteroatoms; R is connected to X by an amide bond, an ester bond or other covalent bonds.

[0012] In the general structural formulas (1)-(4), L is selected from the hydrophilic head group of phospholipids, including one of phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS, phosphatidylglycerol PG, phosphatidylinositol PI, and phosphatidic acid PA.

[0013] In the general structural formulas (3)-(4), Y is a saturated or unsaturated aliphatic chain, and the length of the Y carbon chain is 5-25.

[0014] In the general structural formulas (3)-(4), R is covalently linked to the sn-1 or sn-2 position of the glycerophospholipid;

[0015] In the general formula (3), the ferroptosis inhibitor releases an active group R or a conjugate of R with spacer arm X under the action of phospholipase in the cell membrane and organelle membrane. The released active group or its conjugate with spacer arm is retained and diffused in the cell membrane and organelle membrane, thereby enhancing the ferroptosis inhibitory effect.

[0016] In the general formula (5), the phospholipid module is distearate phosphatidylethanolamine-polyethylene glycol DSPE-PEG, wherein the molecular weight of polyethylene glycol PEG is 400-3000 Da; the PEG end group is carboxyl, amino, mercapto, maleimide, succinimide; the PEG end group is covalently linked to the ferroptosis inhibitory active group R.

[0017] In the general formula (5), n is 8-75.

[0018] Further, in the general structural formulas (1)-(5), when R is selected from free radical scavengers and their derivatives, it is Ferrostatin-1 (Fer-1) and its derivatives, SRS11-92 (AA9) and its derivatives, SRS16-86 and its derivatives, Liproxstatin-1 and its derivatives, phenoxazine (Pnx) and its derivatives, phenothiazine and its derivatives; when R is selected from small molecule organoselenium compounds that mimic the function of glutathione peroxidase 4 and their derivatives, it is ebuselenline (Ebs); when R is selected from iron chelating agents and their derivatives, it is deferoxone, deferoxamine, deferasirox, ciclopirox, catechol or dextropropane; when R is selected from monounsaturated fatty acids and their derivatives, it is oleic acid.

[0019] In the general structural formulas (1)-(4), X is a saturated aliphatic chain and a monounsaturated aliphatic chain, with 0-10 carbon atoms;

[0020] In the general structural formulas (1)-(4), L is the hydrophilic head group of the phospholipid, and the hydrophilic head group is PC and PE;

[0021] In the general structural formulas (3)-(4), Y is a saturated aliphatic chain and a monounsaturated aliphatic chain, and the carbon chain length of Y is 13-21.

[0022] In the general structural formulas (3)-(4), R is covalently attached to the sn-2 position of the glycerophospholipid;

[0023] In the general structural formula (5), the molecular weight of polyethylene glycol (PEG) is 750-2000 Da, and the PEG end groups are carboxyl and amino groups.

[0024] Furthermore, in the general structural formulas (1)-(5), R is selected from Ferrostatin-1 and its derivatives, SRS11-92 and its derivatives, ebuselenline and its derivatives, phenoxazine and its derivatives;

[0025] In the general structural formulas (1)-(4), the number of carbon atoms in X is 0-8;

[0026] In the general structural formulas (1)-(4), L is selected as a hydrophilic head group of phospholipid, and the hydrophilic head group is PC;

[0027] In the general structural formulas (3)-(4), the length of the Y carbon chain is 15-17;

[0028] In the general formula (5), the molecular weight of polyethylene glycol (PEG) is 2000 Da.

[0029] Furthermore, in the general structural formulas (1)-(5), R is selected from ebuselenline and its derivatives, phenoxazine and its derivatives;

[0030] In the general structural formulas (1)-(4), the number of carbon atoms in X is 4-8, preferably 6;

[0031] In the general structural formulas (3)-(4), the length of the Y carbon chain is 17.

[0032] Furthermore, the partial chemical structural formula of general formula (3) is as follows:

[0033]

[0034] Among them, 18:0-(Fer-C x )PC and 18:0-(Pnx-C x The carbon chain lengths of the PC spacer arms are X = 2, 4, 6, 8, 10, which correspond to the repeating units n = 1, 2, 3, 4, 5 in the structural formula, respectively.

[0035] Furthermore, when R is a small-molecule organoselenium compound that mimics the function of glutathione peroxidase 4, the partial chemical structural formula of the ferroptosis-inhibiting phospholipid material is as follows:

[0036]

[0037] Furthermore, when R is an iron chelating agent, the partial chemical structure of the ferroptosis-inhibiting phospholipid material is as follows:

[0038]

[0039] This invention also provides the application of a ferroptosis-inhibiting phospholipid material in the preparation of a drug for improving ferroptosis-related diseases, including ophthalmic diseases, orthopedic diseases, cardiovascular diseases, kidney diseases, liver diseases, nervous system diseases, lung diseases, organ damage caused by ischemia-reperfusion, multiple organ dysfunction, sepsis, uncontrollable inflammation, chemotherapy-induced neuropathic pain, radiotherapy-induced tissue and organ damage, antiviral infections, cerebral infarction, iron overload diseases, and biliary tract diseases.

[0040] Furthermore, ophthalmic diseases include: age-related macular degeneration / AMD, dry eye syndrome, guttate keratosis / Fuchs' endothelial dystrophy, and neurotrophic keratitis;

[0041] Orthopedic diseases include: osteoarthritis, rheumatoid arthritis, and osteoporosis;

[0042] Cardiovascular diseases include: anthracycline-induced cardiotoxicity, ischemic heart disease, atherosclerosis, and myocardial infarction;

[0043] Kidney diseases include: platinum-based drug-induced kidney injury, acute kidney injury, chronic kidney disease, polycystic kidney disease, and diabetic nephropathy;

[0044] Liver diseases include: drug-induced liver injury, acute liver failure, autoimmune liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, and liver fibrosis.

[0045] Neurological disorders include: Parkinson's disease, Alzheimer's disease, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis (ALS), and Huntington's disease;

[0046] Lung diseases include: chronic obstructive pulmonary disease, asthma, acute lung injury, and pulmonary fibrosis.

[0047] This invention also provides the application of ferroptosis-inhibiting phospholipid materials in constructing ferroptosis-inhibiting drug delivery systems, wherein the ferroptosis inhibitor can be used alone or as an excipient; the drug delivery system includes liposomes, micelles, lipid nanoparticles, composite hybrid nanoparticles, scaffolds, or implant coatings; it can encapsulate active drug molecules, including anthracycline drugs, platinum-based drugs, and active molecules for treating neurodegenerative diseases, ophthalmic diseases, and orthopedic diseases; the routes of administration include oral administration, intravenous administration, nasal administration, pulmonary administration, intra-articular injection, transdermal administration, ocular administration, intraperitoneal administration, and mucosal administration.

[0048] Therefore, the advantages and positive effects of the ferroptosis-inhibiting phospholipid material and its application provided by this invention are as follows:

[0049] Biomimetic phospholipid-based ferroptosis inhibitors significantly improve ferroptosis inhibition efficiency due to their long retention characteristics at the main sites of ferroptosis (cell membranes and organelle membranes such as the endoplasmic reticulum). This novel phospholipid-like material can be used both as an active drug molecule and as a pharmaceutical excipient for constructing drug delivery carriers such as liposomes and micelles, as well as implant coatings, suitable for various routes of administration including oral, injection, and topical administration. These novel biomimetic ferroptosis inhibitors can effectively alleviate ferroptosis and have broad application prospects in the treatment or mitigation of ferroptosis-related diseases.

[0050] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0051] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 In diagram A, the synthetic route of the free radical scavenger small molecule Fer-COOH is shown; in diagram B, the synthetic route of GPC-Fer and GPC-(Fer)2 is shown; in diagram C, the synthetic route of 18:0-Fer PC is shown; and in diagram D, the synthetic route of Fer-18:0 PC is shown.

[0053] Figure 2 In A, (1) is the 1H NMR spectrum of the free radical scavenger small molecule Fer-COOH and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of GPC-Fer and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of GPC-(Fer)2 and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of 18:0-Fer PC and (2) is the mass spectrum; in E, (1) is the 1H NMR spectrum of Fer-18:0PC and (2) is the mass spectrum.

[0054] Figure 3 In Example 2, A represents the CNA-C series derivatives based on the free radical scavenger small molecule CNA. X The synthetic route diagram is shown in Figure 1, and B is the synthetic route diagram of the corresponding sn-2 position coupled biomimetic ferroptosis inhibitor.

[0055] Figure 4In A, (1) is the 1H NMR spectrum of CNA-C2 and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of CNA-C4 and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of CNA-C6 and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of CNA-C8 and (2) is the mass spectrum; in E, (1) is the 1H NMR spectrum of CNA-C6 and (2) is the mass spectrum. 10 (1) is the proton NMR spectrum, and (2) is the mass spectrum.

[0056] Figure 5 In A, (1) is the 1H NMR spectrum of 18:0-(Fer-C2)PC and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of 18:0-(Fer-C4)PC and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of 18:0-(Fer-C6)PC and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of 18:0-(Fer-C8)PC and (2) is the mass spectrum; and in E, (1) is the 1H NMR spectrum of 18:0-(Fer-C8)PC. 10 (1) is the proton NMR spectrum of PC, and (2) is the mass spectrum;

[0057] Figure 6 In Figure A, the synthetic route of the free radical scavenger small molecule AA9-COOH in Example 3 is shown; in Figure B, the synthetic route of the biomimetic ferroptosis inhibitors GPC-AA9 and GPC-(AA9)2 is shown; and in Figure C, the synthetic route of the biomimetic ferroptosis inhibitor 18:0-AA9 PC is shown.

[0058] Figure 7 In A, (1) is the 1H NMR spectrum of GPC-AA9 and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of GPC-(AA9)2 and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of 18:0-AA9 PC and (2) is the mass spectrum.

[0059] Figure 8 In Figure A, the synthetic route of the free radical scavenger small molecule Pnx-COOH in Example 4 is shown; in Figure B, the synthetic route of the biomimetic ferroptosis inhibitors GPC-Pnx and GPC-(Pnx)2 is shown; and in Figure C, the synthetic route of the biomimetic ferroptosis inhibitor 18:0-Pnx PC is shown.

[0060] Figure 9 In A, (1) is the 1H NMR spectrum of Pnx-COOH and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of GPC-Pnx and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of GPC-(Pnx)2 and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of 18:0-Pnx PC and (2) is the mass spectrum.

[0061] Figure 10 In Example 4, A is the synthetic route of Pnx-CX, a derivative based on the free radical scavenger small molecule Pnx-COOH; and B is the synthetic route of the corresponding sn-2 position coupled biomimetic ferroptosis inhibitor.

[0062] Figure 11 In A, (1) is the 1H NMR spectrum of CNA-Pnx2 and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of CNA-Pnx4 and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of CNA-Pnx6 and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of CNA-Pnx8 and (2) is the mass spectrum; and in E, (1) is the CNA-Pnx... 10 (1) is the proton NMR spectrum, and (2) is the mass spectrum.

[0063] Figure 12 In A, (1) is the 1H NMR spectrum of 18:0-(Pnx-C2)PC and (2) is the mass spectrum; in B, (1) is the 1H NMR spectrum of 18:0-(Pnx-C4)PC and (2) is the mass spectrum; in C, (1) is the 1H NMR spectrum of 18:0-(Pnx-C6)PC and (2) is the mass spectrum; in D, (1) is the 1H NMR spectrum of 18:0-(Pnx-C8)PC and (2) is the mass spectrum; and in E, (1) is the 1H NMR spectrum of 18:0-(Pnx-C8)PC. 10 (1) is the proton NMR spectrum of PC, and (2) is the mass spectrum;

[0064] Figure 13 In Example 5, A is the synthetic route of GPX4 simulating the small molecule Ebs-COOH; B is the synthetic route of the biomimetic ferroptosis inhibitors GPC-Ebs and GPC-(Ebs)2; C is the synthetic route of the biomimetic ferroptosis inhibitor 18:0-Ebs PC; and D is the synthetic route of the biomimetic ferroptosis inhibitor Ebs-18:0PC.

[0065] Figure 14 In A, (1) is the 1H NMR spectrum of the simulated small molecule Ebs-COOH of GPX4 in Example 5, and (2) is the mass spectrum. In B, (1) is the 1H NMR spectrum of GPC-Ebs, and (2) is the mass spectrum. In C, (1) is the 1H NMR spectrum of GPC-(Ebs)2, and (2) is the mass spectrum. In D, (1) is the 1H NMR spectrum of 18:0-Ebs PC, and (2) is the mass spectrum. In E, (1) is the 1H NMR spectrum of Ebs-18:0PC, and (2) is the mass spectrum.

[0066] Figure 15This is a synthetic route diagram of GPC-Dfx, a representative biomimetic ferroptosis inhibitor corresponding to the iron chelating molecule deirarosi (Dfx) in Example 6;

[0067] Figure 16 In Figure A, the 1H NMR spectrum of GPC-Dfx, a representative biomimetic ferroptosis inhibitor corresponding to the iron chelating molecule derarosipros Dfx in Example 6, is shown; and in Figure B, the mass spectrum is shown.

[0068] Figure 17 The representative biomimetic ferroptosis inhibitor DSPE-PEG corresponds to the iron chelating molecule deferoxamine Dfo in Example 7. 2000 -Dfo synthesis route map;

[0069] Figure 18 The representative biomimetic ferroptosis inhibitor DSPE-PEG corresponds to the iron chelating molecule deferoxamine Dfo in Example 7. 2000 -Dfo's proton NMR spectrum;

[0070] Figure 19 The representative biomimetic ferroptosis inhibitor DSPE-PEG corresponds to the iron chelating molecule deferoxamine Dfo in Example 7. 2000 -Dfo mass spectrometry;

[0071] Figure 20 The cytotoxicity of representative free radical-scavenging biomimetic ferroptosis inhibitors in rat cardiomyocytes H9c2 (n=3) is shown, where A represents the cytotoxicity of GPC-Fer, B represents the cytotoxicity of GPC-(Fer)2, C represents the cytotoxicity of 18:0-Fer PC, D represents the cytotoxicity of 18:0-(Fer-C2)PC, E represents the cytotoxicity of 18:0-(Fer-C4)PC, F represents the cytotoxicity of 18:0-(Fer-C6)PC, G represents the cytotoxicity of 18:0-(Fer-C8)PC, and H represents the cytotoxicity of 18:0-(Fer-C8)PC. 10 The cytotoxicity of GPC is represented by I, J, K, L, and L.

[0072] Figure 21 The cytotoxicity (n=3) of representative biomimetic ferroptosis inhibitors in rat H9c2 cardiomyocytes (based on cell viability) is shown in the table. A represents the cytotoxicity of GPC-(Pnx)2, B represents the cytotoxicity of 18:0-Pnx PC, C represents the cytotoxicity of 18:0-(Pnx-C2)PC, D represents the cytotoxicity of 18:0-(Pnx-C4)PC, E represents the cytotoxicity of 18:0-(Pnx-C6)PC, F represents the cytotoxicity of 18:0-(Pnx-C8)PC, and G represents the cytotoxicity of 18:0-(Pnx-C8)PC.10 The cytotoxicity of GPC is represented by H, GPC-Ebs, I, GPC-(Ebs)2, J, 18:0-Ebs PC, K, GPC-Dfx, and L, DSPE-PEG2000-Dfo.

[0073] Figure 22 The inhibitory effects of representative biomimetic ferroptosis inhibitors on RSL3 (0.5 μM)-induced H9c2 ferroptosis in cardiomyocytes (as an indicator of cell viability) (n=3) were shown, where A represents the inhibitory effect of GPC-Fer, B represents the inhibitory effect of GPC-(Fer)2, C represents the inhibitory effect of 18:0-FerPC, D represents the inhibitory effect of GPC-AA9, and E represents the inhibitory effect of GPC-(AA9). )2 The inhibitory effects of GPC-(Pnx)2, GPC-(Pnx)2, 18:0-Pnx PC, GPC-Ebs, GPC-(Ebs)2, LPC-(Pnx)2, Dfx, and DSPE-PEG2000-Dfo were defined as follows: F = 18:0-AA9 PC, G = 18:0-AA9 PC, H = 18:0-AA9 PC, I = 18:0-AA9 PC, J = 18:0-AA9 PC, K = 18:0-AA9 PC, L = 18:0-Ebs PC, M = 18:0-AA9 PC, N = 18:0-Pnx PC, NS = 18:0-AA9 PC, * = 18:0-AA9 PC, M = 18:0-Pnx ... J = 18:0-AA9 PC, K = 18:0-Pnx PC, L = 18:0-Pnx PC, M = 18:0-Pnx PC, N = 18:0-Pnx PC,

[0074] Figure 23 The inhibitory effects of biomimetic ferroptosis inhibitors Fer-18:0PC and 18:0-FerPC, and the phospholipase A2 (PLA2) inhibitor Darapladib on RSL3 (0.5 μM)-induced H9c2 ferroptosis in cardiomyocytes (as measured by cell viability) were evaluated (n=3). In this study, A represents the inhibitory effect of Fer-18:0PC and 18:0-FerPC without Darapladib, and B represents the inhibitory effect of Fer-18:0PC and 18:0-FerPC with 1.0 μM Darapladib. ns indicates no statistically significant difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0075] Figure 24The effect of the coupling positions (sn-1 and sn-2) of the active molecule Ebs on the efficacy of biomimetic ferroptosis inhibitors (n=3) is shown in Figure A, where A is a schematic diagram of the structures of three ferroptosis inhibitors, B is the rescue effect of the ferroptosis inhibitor Ebs on the degeneration of human retinal pigment epithelial cells induced by sodium iodate (SI, 20mM) in the presence / absence of the phospholipase A2 (PLA2) inhibitor Darapladib (as an indicator of cell viability), C is the rescue effect of Ebs-18:0PC, and D is the rescue effect of 18:0-Ebs PC. ns indicates no statistical difference, * indicates p<0.05, and *** indicates p<0.001.

[0076] Figure 25 The effect of the adipose chain length at the sn-2 position on the efficacy of Fer-based biomimetic ferroptosis inhibitors (n=3) was investigated, where A represents the inhibitory effect of 18:0-Fer PC on RSL3 (0.5 μM)-induced H9c2 ferroptosis in cardiomyocytes (as an indicator of cell viability), B represents the inhibitory effect of 18:0-(Fer-C2) PC, C represents the inhibitory effect of 18:0-(Fer-C4) PC, D represents the inhibitory effect of 18:0-(Fer-C6) PC, E represents the inhibitory effect of 18:0-(Fer-C8) PC, and F represents the inhibitory effect of 18:0-(Fer-C8) PC. 10 The inhibitory effect of PC;

[0077] Figure 26 The effect of the length of the fatty acid chain at the sn-2 position on the efficacy of Pnx-based biomimetic ferroptosis inhibitors (n=3) was investigated, where A represents the inhibitory effect of 18:0-Pnx PC on RSL3 (0.5 μM)-induced H9c2 ferroptosis in cardiomyocytes (as an indicator of cell viability), B represents the inhibitory effect of 18:0-(Pnx-C2) PC, C represents the inhibitory effect of 18:0-(Pnx-C4) PC, D represents the inhibitory effect of 18:0-(Pnx-C6) PC, E represents the inhibitory effect of 18:0-(Pnx-C8) PC, and F represents the inhibitory effect of 18:0-(Pnx-C8) PC. 10 The inhibitory effect of PC is shown in ***, where p < 0.001.

[0078] Figure 27 The inhibitory effects of different biomimetic phospholipid ferroptosis inhibitors on H9c2 ferroptosis (as an indicator of cell viability) induced by the combined use of doxorubicin hydrochloride (2 μM) and ferric ammonium citrate (1 mM) in cardiomyocytes were evaluated (n=3). A represents the inhibitory effect of GPC-Fer, B represents the inhibitory effect of GPC-(Fer)2, C represents the inhibitory effect of 18:0-Fer PC, D represents the inhibitory effect of GPC-AA9, E represents the inhibitory effect of GPC-(AA9)2, F represents the inhibitory effect of 18:0-AA9 PC, G represents the inhibitory effect of GPC-Ebs, and H represents the inhibitory effect of GPC-(Ebs).)2 The inhibitory effects of 18:0-Ebs PC, J, and K are given. I represents the inhibitory effect of GPC-Dfx, J represents the inhibitory effect of DSPE-PEG2000-Dfo, ns indicates no statistical difference, * indicates p<0.05, and ** indicates p<0.01.

[0079] Figure 28 The rescue effect of the biomimetic ferroptosis inhibitor 18:0-PnxPC on H9c2 cardiomyocyte ferroptosis induced by different types of anthracycline chemotherapy drugs (as an indicator of cell viability) (n=3) was studied. Among them, A represents the rescue effect on ferroptosis induced by doxorubicin / THP, B represents the rescue effect on ferroptosis induced by desmethyldaunorubicin / IDA, C represents the rescue effect on ferroptosis induced by epirubicin / EPI, D represents the rescue effect on ferroptosis induced by daunorubicin / DNR, E represents the rescue effect on ferroptosis induced by mitoxantrone / MTX, and F represents the rescue effect on ferroptosis induced by aclarubicin / ACM. ns indicates no statistical difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0080] Figure 29 The inhibitory effects of different biomimetic phospholipid ferroptosis inhibitors on cisplatin (15 μM)-induced ferroptosis in HK2 renal tubular epithelial cells (as an indicator of cell viability) were evaluated (n=3). A represents the inhibitory effect of GPC-Fer, B represents the inhibitory effect of GPC-(Fer)2, C represents the inhibitory effect of 18:0-Fer PC, D represents the inhibitory effect of GPC-AA9, E represents the inhibitory effect of GPC-(AA9)2, F represents the inhibitory effect of 18:0-AA9 PC, G represents the inhibitory effect of GPC-Ebs, H represents the inhibitory effect of GPC-(Ebs)2, I represents the inhibitory effect of 18:0-Ebs PC, J represents the inhibitory effect of GPC-Dfx, and K represents the inhibitory effect of DSPE-PEG2000-Dfo. ns indicates no statistically significant difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0081] Figure 30The efficacy of different biomimetic ferroptosis inhibitors in inhibiting lipid peroxidation in the RSL3 (0.5 μM)-induced H9c2 cardiomyocyte model (n=3) was evaluated. A represents the qualitative analysis of intracellular lipid peroxide content in H9c2 cells treated with the biomimetic inhibitor using the specific fluorescent probe Liperfluo (excitation / emission wavelength: 532 nm / 535–650 nm, scale bar: 25 μm). B represents the quantitative fluorescence analysis of lipid peroxides. C represents the content of malondialdehyde (MDA), a ferroptosis marker, in H9c2 cells after treatment with the biomimetic inhibitor. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. All statistical comparisons were based on the RSL3 group. Detailed Implementation

[0082] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art to which this application pertains. The instruments, equipment, reagents, and materials used in the embodiments are all commercially available; the method steps not described in detail in the embodiments are all conventional technical means in the art.

[0083] This invention provides a novel class of highly efficient biomimetic ferroptosis inhibitors with phospholipid-like structures. These inhibitors can be used alone or in combination as active molecules to treat ferroptosis-related diseases, and can also be used as pharmaceutical excipients in the construction of ferroptosis-inhibiting drug delivery systems. The amphiphilic nature of the (phospholipid-like) materials facilitates their accumulation on cell membranes and organelle membranes, enhancing the efficacy of the ferroptosis inhibitors. Simultaneously, phospholipases on cell and organelle membranes enable the sustained-release and efficient diffusion of the active groups in the biomimetic phospholipid ferroptosis inhibitors within the membranes, further improving their efficacy. This invention also provides the application effects of the biomimetic phospholipid ferroptosis inhibitors in three ferroptosis-related cell models (RSL3-induced ferroptosis, doxorubicin-induced cardiomyocyte injury, and cisplatin-induced renal cell injury), and evaluates the ferroptosis-inhibiting effect of the biomimetic phospholipid inhibitors using cell activity and ferroptosis markers, as well as the structure-activity relationship of the biomimetic phospholipid inhibitors.

[0084] In this invention: Ferrostatin-1 is abbreviated as Fer-1, SRS11-92 is abbreviated as AA9, phenoxazine is abbreviated as Pnx, glutathione peroxidase 4 is abbreviated as GPX4, and ebuseline is abbreviated as Ebs.

[0085] Example 1

[0086] This embodiment selects Fer-1 as the active group to illustrate how to prepare a free radical scavenging biomimetic phospholipid ferroptosis inhibitor. The specific synthetic route is as follows: Figure 1 As shown:

[0087] (1) Weigh 1.0 g (5 mmol) of 4-chloro-3-nitrobenzoic acid and dissolve it in 5 mL of anhydrous DMSO. After dissolution, add 1.4 g (10 mmol) of potassium carbonate and stir until homogeneous. Then add 686 μL (6 mmol) of cyclohexylamine and stir the mixture at 60 °C for 30 h under argon protection. Subsequently, transfer the reaction solution to ice water to precipitate the precipitate. Filter and collect the filter cake. Add a mixed solvent of ethanol and water (7:3, v / v), filter and remove the residue. Recrystallize the filtrate at -20 °C to obtain a golden yellow solid, 4-(cyclohexylamino)-3-nitrobenzoic acid, abbreviated as CNA, with a yield of 69%.

[0088] (2) Weigh 50.2 mg (0.19 mmol) CNA and dissolve it in 5 mL of anhydrous ethanol. Add palladium on carbon (10% w / w, 10 mg), evacuate the solution, purge with hydrogen gas, and stir at room temperature for 17 h. After the reaction is complete, filter with diatomaceous earth, collect the filtrate and rotary evaporate to obtain 30.2 mg of the product. This product is a derivative of the free radical scavenger small molecule (Fer-1), abbreviated as Fer-COOH.

[0089] (3) Weigh 187.4 mg (0.8 mmol) Fer-COOH into a 10 mL round-bottom flask and add 2 mL of anhydrous DMSO to dissolve it. Weigh 194.5 mg (1.2 mmol) N,N'-carbonyldiimidazole (CDI), dissolve it in 1 mL of anhydrous DMSO, and add it dropwise to the above flask. Under nitrogen protection, activate at 35 °C for 3 h. Then add 51.4 mg (0.2 mmol) glycerophosphorylcholine (GPC) and 30 μL (0.2 mmol) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to the system in sequence. Under nitrogen protection, stir at 40 °C for 24 h. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol:water = 3:1:0 → 65:25:0 → 65:25:2 → 65:25:4, v / v / v) to elute the phospholipid compounds GPC-(Fer)2 and GPC-Fer corresponding to Fer-COOH. Finally, the product was obtained by freeze drying.

[0090] (4) Weigh 234.3 mg (1 mmol) of Fer-COOH into a 10 mL round-bottom flask and add 5 mL of anhydrous chloroform to disperse it evenly. Weigh 243.2 mg (1.5 mmol) of CDI, dissolve it in 2 mL of anhydrous chloroform, and add it dropwise to the above flask. At this time, the solution becomes clear. Activate at 35 °C for 3 h, and then add 149.4 μL (1 mmol) of DBU and 261.8 mg (0.5 mmol) of 1-stearoyl-sn-glycerol-3-phosphocholine (18:0 Lyso PC) to the system in sequence. Under nitrogen protection, stir at 40 °C for 24 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v), the organic phase is removed by rotary evaporation, and the corresponding phospholipid product 18:0-Fer PC is obtained by freeze drying.

[0091] (5) Weigh 427 mg (1.5 mmol) stearic acid into a 10 mL round-bottom flask and add 2 mL of anhydrous dichloromethane to disperse it evenly. Weigh 325 mg (2.0 mmol) CDI, dissolve it in 2 mL of anhydrous DMSO, and add it dropwise to the above flask. The solution becomes clear at this point. Activate at 35 °C for 3 h, and then add 224 μL (1.5 mmol) DBU and 237 mg (0.5 mmol) GPC-Fer to the system sequentially. Under nitrogen protection, stir at 35 °C for 24 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v), the organic phase is removed by rotary evaporation, and finally the pure product Fer-18:0PC is obtained by lyophilization.

[0092] The 1H NMR and mass spectra of Fer-1 derivative Fer-COOH and its corresponding representative ferroptosis-inhibiting phospholipid compounds GPC-Fer, GPC-(Fer)2, 18:0-FerPC, and Fer-18:0PC are shown below. Figure 2 :

[0093] (a) Characterization results of Fer-COOH are as follows Figure 2 China A: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.25–7.05 (m, 2H), 6.43 (d, J = 8.4Hz, 1H), 4.82 (d, J = 7.4Hz, 1H), 4.69 (s, 2H), 3.51 (s, 1H), 2.02–1.14 (m, 10H). HRMS (ESI): m / z calculation results: C 13 H 18 N₂O₂[M+H] + 235.14; Analysis of measured results: 235.05.

[0094] (b) The characterization results of GPC-Fer are as follows Figure 2 B: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.31–7.08 (m, 2H), 6.46 (d, J = 8.4Hz, 1H), 5.01–4.69 (m, 2H), 4.49 (s, 1H), 4.28–4.01 (m, 4H), 3.93–3.68 (m, 3H), 3.52 (dq, J = 8.2, 2.9Hz, 2H), 3.39 (t, J = 2.5Hz, 1H), 3.12 (d, J = 4.4Hz, 9H), 2.01–1.12 (m, 10H). HRMS (ESI): m / z calculation results: C 21 H 36 N3O7P[M+H] + 474.51; Analytical measurement result: 474.12.

[0095] (c) Characterization results of GPC-(Fer)2 are as follows Figure 2 C: 1 H NMR (400MHz, DMSO-d6) δ7.69–7.58(m,1H),7.58–7.50(m,1H),7.36(t,J=7.4Hz,1H),7.24–7.11(m,2H),6.48–6 .38(m,1H),5.55–5.22(m,1H),5.02–4.65(m,2H),4.47(ddtd,J=50.9,18.5,12.2,5.1Hz,2H),4.24–3.86(m,4H ), 3.51(tt, J=8.3, 4.7Hz, 2H), 3.12(q, J=6.3, 4.5Hz, 9H), 2.08(d, J=12.8Hz, 3H), 1.94(d, J=12.1Hz, 2H), 1.82(d, J=12.2Hz, 3H), 1.76–1.57(m, 8H), 1.43(s, 2H), 1.41–1.38(m, 1H), 1.38–1.02(m, 3H). HRMS(ESI): m / z calculation results: C 34 H 52 N5O8P[M+H] + 690.36; Analytical measurement result: 690.24.

[0096] (d) The characterization results of 18:0-FerPC are as follows Figure 2 D: 1¹H NMR (600MHz, DMSO-d⁶) δ 7.71–7.10 (m, 2H), 6.43 (dd, J = 8.4, 6.2Hz, 1H), 4.98 (dd, J = 7.6, 3.2Hz, 1H), 4.53–3.65 (m, 6H), 3.51 (ddq, J = 19.2, 9.9, 5.0, 3.9Hz, 2H), 3.12 (dd, J = 15.2, 5.2Hz, 9H), 2.30–1.57 (m, 8H), 1.50–1.01 (m, 35H), 0.85 (t, J = 6.9Hz, 3H). HRMS (ESI): m / z calculation results: C 39 H 70 N3O8[M+H] + 740.49; Analytical measurement result: 740.48.

[0097] (e)Characteristics results of Fer-18:0PC are as follows Figure 2 E: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.69–7.05 (m, 2H), 6.43 (dd, J = 8.5, 4.0Hz, 1H), 4.99 (d, J = 7.2Hz, 1H), 4.36–3.69 (m, 6H), 3.50 (tt, J = 9.8, 4.8Hz, 2H), 3.18–3.04 (m, 9H), 2.28–1.54 (m, 9H), 1.55–0.97 (m, 30H), 0.85 (t, J = 6.7Hz, 3H). HRMS (ESI): m / z calculation results: C 39 H 70 N3O8P[M+H] + 740.49; Analytical measurement result: 740.32.

[0098] Example 2

[0099] This embodiment selects Fer-1 as the active group and, based on Example 1, illustrates how to prepare a radical-scavenging biomimetic phospholipid death inhibitor with different linker arm lengths. The specific synthetic route is as follows: Figure 3 As shown:

[0100] (1) Weigh 264.3 mg (1.0 mmol) CNA, add 5 mL of anhydrous chloroform, add 243.2 mg (1.5 mmol) CDI while stirring, stir at 35 °C for 2 h, and then add 1.25 mmol H2N (C2H4) of bifunctional linker. nCOOH (n = 1-5) and 150 μL (1.0 mmol) DBU were added, and the mixture was stirred at 40 °C for 6 h. After the reaction, the product was purified by column chromatography (petroleum ether / ethyl acetate / glacial acetic acid = 2:1:0.005 → 1:1:0.005, v / v / v). The product was dissolved in ethyl acetate, and glacial acetic acid was washed away with ultrapure water. The product was then rotary evaporated and dried at 40 °C for 4 h to obtain a yellow powder product, abbreviated as CNA-C. x (x = 2, 4, 6, 8, 10).

[0101] (2) Weigh 1.00 mmol CNA-C x (x=2,4,6,8,10), add 5 mL of anhydrous chloroform, then add 243.2 mg (1.5 mmol) CDI while stirring. After stirring at 35 °C for 1 h, add 261.9 mg (0.5 mmol) 18:0 Lyso PC and 150 μL (1.0 mmol) DBU. Continue stirring at room temperature for 6 h. Concentrate to obtain a yellow residue, which is purified by silica gel chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v) to obtain 18:0-(CNA-C x PC (x = 2, 4, 6, 8, 10). The organic phase was removed by rotary evaporation, and finally the product was freeze-dried to obtain the pure product.

[0102] Conjugates of CNA with molecules of different carbon chain lengths: CNA-C2, CNA-C4, CNA-C6, CNA-C8, CNA-C 10 See 1H NMR and mass spectra. Figure 4 :

[0103] (a) Characterization results of CNA-C2 are as follows Figure 4 China A: 1 ¹H NMR (400MHz, D₂O) δ 8.44 (s, J = 2.2Hz, 1H, Ar-H), 7.71 (d, 1H, Ar-H), 6.96 (d, J = 9.2Hz, 1H, Ar-H), 3.56 (d, 2H, CH₂; s, 1H, CH), 2.49 (d, 2H, CH₂), 2.08–1.88 (m, 2H, CH₂), 1.73 (dd, J = 5.2Hz, 2H, CH₂), 1.63 (d, J = 7.4Hz, 2H, CH₂), 1.48–1.13 (m, 4H, CH₂). HRMS (ESI): m / z calculation results: C 16 H 21 N3O5[M+H] + 336.15; Analysis of measured results: 336.10.

[0104] (b) Characterization results of CNA-C4 are as follows Figure 4 B:1 H NMR(400MHz,D2O)δ8.46(s,1H,Ar-H),7.74(d,J=9.7Hz,1H,Ar-H),6.98(d,J=9.5 Hz,1H,Ar-H),3.57(s,1H,CH),3.36(s,2H,CH2),2.24(m,2H,CH2),1.65(s,1H,one of CH2),1.54(s,4H,CH2;s,1H,the other H of CH2),1.40(m,2H,CH2),1.38-1.09(m,6H,CH2).HRMS(ESI):m / z calculation result:C 18 H 25 N3O5[M+H] + 364.18; Analysis of measured results: 364.08.

[0105] (c) Characterization results of CNA-C6 are as follows Figure 4 C: 1 H NMR(400MHz,D2O)δ8.41(s,1H,Ar-H),7.72(s,1H,Ar-H),6.82(s,1H,Ar-H),3.4 3(s,1H,CH),3.28(s,2H,CH2),2.26-2.05(m,2H,CH2),1.88(s,4H,CH2,s,1H,one H of CH2),1.62(s,1H,the other H of CH2),1.52(s,6H,CH2),1.23(d,6H,CH2).HRMS(ESI):m / z calculation result:C 20 H 29 N3O5[M+H] + 392.21; Analysis of measured results: 392.09.

[0106] (d) The characterization results of CNA-C8 are as follows: Figure 4 D: 1H NMR(600MHz,DMSO-d6)δ8.16(d,J=7.8Hz,1H),7.98(ddd,J=11.3,8.9,2.3Hz, 1H),7.17(dd,J=9.3,2.9Hz,1H),3.71(dt,J=9.3,4.4Hz,1H),3.27–3.18(m,2 H),2.18(t,J=7.4Hz,2H),2.00–1.92(m,2H),1.71(dq,J=13.5,4.1Hz,2H),1. 60(dt,J=13.3,4.0Hz,1H),1.53–1.34(m,7H),1.26(dt,J=16.1,7.3Hz,10H). HRMS(ESI): m / z calculation result: C 22 H 33 N3O5[M+H] + 420.24; Analysis of measured results: 420.24.

[0107] (e)CNA-C 10 The characterization results are as follows Figure 4 E: 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.16 (d, J = 7.8Hz, 1H), 7.97 (dd, J = 9.1, 2.2Hz, 1H), 7.17 (d, J = 9.2Hz, 1H), 3.71 (d, J = 8.7Hz, 1H), 3.22 (q, J = 6.7Hz, 2H), 2.18 (t, J = 7.4Hz, 2H), 1.96 (d, J = 12.6Hz, 2H), 1.75–1.67 (m, 2H), 1.63–1.57 (m, 1H), 1.53–1.34 (m, 8H), 1.32–1.21 (m, 13H). HRMS (ESI): m / z calculation results: C 24 H 37 N3O5[M+H] + 448.27; Analysis of measured results: 448.27.

[0108] The corresponding representative biomimetic phospholipid iron death inhibitors are 18:0-(Fer-C2)PC, 18:0-(Fer-C4)PC, 18:0-(Fer-C6)PC, 18:0-(Fer-C8)PC, and 18:0-(Fer-C... 10 The 1H NMR and mass spectra of PC are shown in [reference needed]. Figure 5 .

[0109] (a) The characterization results of 18:0-(Fer-C2)PC are as follows Figure 5 China A: 1H NMR(600MHz,DMSO-d6)δ9.17(t,J=5.5Hz,1H),8.17(d,J=1.7Hz,1H),7.79–7.64(m,2H),5.06(dd d,J=9.1,7.3,4.0Hz,1H),4.33–4.21(m,2H),4.15–4.04(m,2H),3.92–3.79(m,2H),3.60–3.44(m ,4H), 3.14(d,J=10.6Hz,9H), 2.58(d,J=5.1Hz,3H), 2.31–2.10(m,4H), 1.91–1.81(m,3H), 1.70(d,J=12.6Hz,1H), 1.54–1.35(m,5H), 1.21(dd,J=19.6,7.8Hz,28H), 0.85(td,J=7.0,3.1Hz,3H). HRMS(ESI): m / z calculation results: C 42 H 75 N4O9P[M+H] + 811.53; Analysis of measured results: 811.53.

[0110] (b) The characterization results of 18:0-(Fer-C4)PC are as follows Figure 5 B: 1 H NMR(600MHz,DMSO-d6)δ8.63(dt,J=76.0,5.8Hz,1H),8.10(d,J=19.8Hz,1H),7.76–7.66(m,2H),5 .10(ddd,J=20.6,10.1,5.5Hz,1H),4.33–4.22(m,2H),4.15–4.02(m,3H),3.78(q,J=8.4,7.0Hz,2 3.54 (t, J = 4.7 Hz, 3H), 3.31–3.22 (m, 2H), 3.14 (s, 9H), 2.58 (s, 2H), 2.39–2.09 (m, 5H), 1.90–1.82 (m, 3H), 1.70 (d, J = 12.3 Hz, 1H), 1.62–1.42 (m, 8H), 1.30–1.13 (m, 28H), 0.84 (t, J = 6.6 Hz, 3H). HRMS(ESI): m / z calculation results: C 44 H 79 N4O9P[M+H] + 839.56; Analysis of measured results: 839.56.

[0111] The characterization results of (c)18:0-(Fer-C6)PC are as follows Figure 5 C: 1H NMR(600MHz,DMSO-d6)δ8.50(dt,J=31.5,5.6Hz,1H),8.08(d,J=7.1Hz,1H),7.69(d,J=3.3Hz,2H),5.10–5.05(m,1H) ,4.29(td,J=12.4,3.6Hz,2H),4.14–4.00(m,3H),3.75(t,J=5.9Hz,2H),3.53(t,J=4.9Hz,2H),3.25(q,J=6.6Hz,2H), 3.14(s,9H), 2.58(s,3H), 2.28(dq,J=14.6,7.4Hz,4H), 2.15(qd,J=12.8,12.4,4.1Hz,2H), 1.92–1.81(m,4H), 1.70(d,J=12.9Hz,1H), 1.51(dq,J=31.7,8.5,7.9Hz,8H), 1.34–1.29(m,4H), 1.21(t,J=10.2Hz,28H), 0.84(t,J=6.8Hz,3H). HRMS(ESI): m / z calculation results: C 46 H 83 N4O9P[M+H] + 867.59; Analysis of measured results: 867.46.

[0112] The characterization results of (d)18:0-(Fer-C8)PC are as follows: Figure 5 D: 1 H NMR (600MHz, DMSO-d6) δ8.52(s,1H),8.06(d,J=10.1Hz,1H),7.69(q,J=7.6,6.6Hz,2H),5.06(qd,J=5.4,2.8H z,1H),4.29(dt,J=12.2,4.6Hz,1H),4.11–3.98(m,2H),3.73(h,J=5.4Hz,2H),3.51(dd,J=6.0,3.6Hz,2H),3.2 4 (q, J = 6.8 Hz, 2H), 3.13 (s, 9H), 2.58 (s, 2H), 2.29–2.24 (m, 3H), 2.03 (q, J = 8.0, 7.5 Hz, 6H), 1.86 (t, J = 13.1 Hz, 2H), 1.45 (tt, J = 12.6, 7.3 Hz, 7H), 1.35 (q, J = 7.0, 6.2 Hz, 6H), 1.27–1.19 (m, 33H), 0.85 (dt, J = 9.6, 6.8 Hz, 3H). HRMS(ESI): m / z calculation results: C 48 H 87 N4O9P[M+Na]+ 917.62; Analytical measurement result: 917.57.

[0113] (e)18:0-(Fer-C 10 The characterization results of PC are as follows: Figure 5 E: 1 H NMR(600MHz,DMSO-d6)δ8.46(dt,J=14.0,5.6Hz,1H),8.07(s,1H),7.69(s,2H),5.07(dp,J=8.1,2.6Hz,1H),4.2 9(ddq,J=8.5,6.1,3.7Hz,2H),4.13–3.98(m,3H),3.74(h,J=5.5Hz,2H),3.51(t,J=4.9Hz,2H),3.25(q,J=6.7Hz, 2H), 3.14(d, J = 41.2 Hz, 9H), 2.58(s, 2H), 2.31–2.22(m, 4H), 2.15(qd, J = 12.8, 12.3, 4.1 Hz, 1H), 1.91–1.81(m, 3H), 1.74–1.67(m, 1H), 1.62(dq, J = 13.6, 4.4 Hz, 1H), 1.56–1.45(m, 7H), 1.33–1.14(m, 40H), 0.84(q, J = 7.2 Hz, 3H). HRMS(ESI): m / z calculation results: C 50 H 91 N4O9P[M+H] + 923.65; Analysis of measured results: 923.65.

[0114] Example 3

[0115] This embodiment selects AA9 as the active group to illustrate how to prepare a biomimetic phospholipid ferroptosis inhibitor with free radical scavenging properties. The specific synthetic route is as follows: Figure 6 As shown:

[0116] (1) Weigh 281.1 mg (1.2 mmol) of Fer-COOH into a 25 mL round-bottom flask, and add 3 mL of 1,2-dichloroethane / methanol (2:1, v / v) to dissolve it. Measure 101.6 μL (1.0 mmol) of benzaldehyde and add it to the above solution, then sieve the solution through a molecular sieve. The mixture was stirred at room temperature for 1 hour under nitrogen protection. The reaction mixture was treated with 10 mL of saturated NaHCO3 aqueous solution, followed by extraction with ethyl acetate (5 × 6 mL). The ethyl acetate extract was dried over anhydrous MgSO4 and filtered. The organic phase was removed by rotary evaporation to obtain a solid product. 6 mL of ethyl acetate / methanol (2:1, v / v) was added to this product to dissolve it, and 10% palladium on carbon (reactant:palladium on carbon = 5:1, w / w) was added to the system. Hydrogen was introduced, and the mixture was stirred at room temperature for 8 hours. After the reaction was complete, the palladium on carbon was removed by diatomaceous earth filtration. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1 → 5:1 → 3:1, v / v), and the organic phase was removed by rotary evaporation to obtain the pure product, abbreviated as AA9-COOH.

[0117] (2) Weigh 324.1 mg (1 mmol) of AA9-COOH into a 25 mL round-bottom flask and dissolve it in 2 mL of anhydrous DMSO. Weigh 243.2 mg (1.5 mmol) of CDI, dissolve it in 1 mL of anhydrous DMSO, and add it dropwise to the above flask. Under nitrogen protection, activate at 35 °C for 3 h. Then, add 128.7 mg (0.5 mmol) of GPC and 149.4 μL (1 mmol) of DBU to the system sequentially. Under nitrogen protection, stir at 40 °C for 24 h. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol:water = 3:1:0 → 65:25:0 → 65:25:2 → 65:25:4, v / v / v) to elute the phospholipid compounds GPC-(AA9)2 and GPC-AA9 corresponding to AA9-COOH. Finally, the product was obtained by freeze-drying.

[0118] (3) Weigh 486.3 mg (1.5 mmol) of AA9-COOH into a 25 mL round-bottom flask and add 3 mL of anhydrous chloroform to disperse it evenly. Weigh 364.8 mg (2.25 mmol) of CDI, dissolve it in 2 mL of anhydrous chloroform, and add it dropwise to the above flask. The solution becomes clear at this point. Activate at 35 °C for 3 h, and then add 224.1 μL (1.5 mmol) of DBU and 261.8 mg (0.5 mmol) of 18:0 Lyso PC to the system. Under nitrogen protection, stir at 40 °C for 24 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v) and freeze-dried to obtain the corresponding phospholipid product 18:0-AA9 PC.

[0119] The 1H NMR and mass spectra of AA9-COOH and its corresponding biomimetic phospholipid ferroptosis inhibitors GPC-AA9, GPC-(AA9)2, and 18:0-AA9 PC are shown in [reference needed]. Figure 7 :

[0120] (a) Characterization results of AA9-COOH are as follows Figure 7 China A: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.47–7.17 (m, 7H), 6.93 (d, J = 1.9Hz, 1H), 6.49 (d, J = 8.5Hz, 1H), 5.44 (t, J = 5.7Hz, 1H), 5.13 (d, J = 7.3Hz, 1H), 4.31 (d, J = 5.4Hz, 2H), 3.71 (s, 1H), 2.11–1.29 (m, 11H). HRMS (ESI): m / z calculation results: C 20 H 24 N₂O₂[M+H] + 325.18; Analysis of measured results: 325.18.

[0121] (b) Characterization results of GPC-AA9 are as follows Figure 7 B: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.41–7.20 (m, 6H), 6.98–6.92 (m, 1H), 6.48 (dd, J = 16.2, 8.6Hz, 1H), 5.22 (s, 1H), 4.30 (s, 2H), 4.12–3.99 (m, 4H), 3.82–3.68 (m, 3H), 3.54–3.49 (m, 2H), 3.15–3.02 (m, 9H), 2.03–1.93 (m, 3H), 1.74 (d, J = 13.3Hz, 3H), 1.62 (s, 2H), 1.34 (s, 2H), 1.30 (s, 1H). HRMS (ESI): m / z calculation results: C 28 H 42 N3O7P[M+H] + 564.28; Analytical measurement result: 564.16.

[0122] (c) The characterization results of GPC-(AA9)2 are as follows Figure 7 C: 1H NMR (600MHz, DMSO-d6) δ7.39–7.15(m,12H),6.96(dd,J=28.2,2.0Hz,2H),6.46(d,J=8.5Hz,2H),5.46–5. 43(m,1H),4.40(dd,J=11.9,3.3Hz,1H),4.23(ddd,J=22.6,11.0,6.1Hz,5H),3.99(s,2H),3.84(t,J=6.0H z, 2H), 3.44(s, 2H), 3.12–3.06(m, 9H), 2.03(t, J = 5.9Hz, 4H), 1.97(d, J = 12.7Hz, 4H), 1.73(d, J = 13.3Hz, 4H), 1.64(dd, J = 17.1, 10.3Hz, 3H), 1.49(dd, J = 45.0, 4.2Hz, 7H), 1.40–1.31(m, 7H). HRMS(ESI): m / z calculation results: C 48 H 64 N5O8P[M+H] + 870.45; Analytical measurement result: 870.31.

[0123] (d) The characterization results of 18:0-AA9PC are as follows Figure 7 D: 1 H NMR (400MHz, DMSO-d6) δ7.46–7.13(m,6H),6.95(dd,J=9.0,1.9Hz,1H),6.47(d,J=8.6Hz,1H),5. 51(s,1H),4.28(d,J=3.2Hz,2H),4.01(s,3H),3.78(d,J=5.5Hz,2H),3.47(dt,J=13.1,5.7Hz,3H) ,3.17–3.01(m,9H),2.23(dt,J=14.2,7.3Hz,3H),2.05–1.87(m,4H),1.74(d,J=12.2Hz,2H),1.63(d,J=14.0Hz,4H),1.34(s,2H),1.31–1.06(m,28H),0.85(t,J=6.7Hz,3H).HRMS(ESI):m / z calculation results:C 46 H 76 N3O8P[M+H] + 830.54; Analytical measurement result: 830.40.

[0124] Example 4

[0125] This embodiment selects Pnx as the active group to illustrate how to prepare a biomimetic phospholipid ferroptosis inhibitor with free radical scavenging properties. The specific synthetic route is as follows: Figure 8 As shown:

[0126] (1) Weigh 1.09 g (10 mmol) of o-aminophenol and 3.0 g of potassium carbonate into a 100 mL round-bottom flask, add 12 mL of toluene and 20 mL of anhydrous DMF (20 mL), heat under nitrogen protection and reflux for 0.5 h, and remove water using a water separator. After adding 1.39 g (10 mmol) of 3,4-difluorocarbonitrile, cool the reaction solution to about 80 °C, and then heat under reflux for 4 h. Distill the toluene and replace it with anhydrous DMF. Heat the mixture at 150 °C for 4.5 h, remove the organic phase by rotary evaporation, and the crude product is subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v / v) to obtain the solid product 10H-phenoxazine-3-carboxynitrile.

[0127] (2) Weigh 50 mg (0.24 mmol) of 10H-phenoxazine-3-carboxylon into a 25 mL round-bottom flask, add 2 mL of ethylene glycol and 2 mL of 10% KOH, and heat at 170 °C for 15 min. After acid treatment, a dark brown solid is obtained, which is then separated by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1, v / v) to give a light yellow solid product: 10H-phenoxazine-3-carboxylic acid (Pnx).

[0128] (3) Weigh 19.33 mg (0.085 mmol) of Pnx and dissolve it in a 25 mL round-bottom flask. Add 1 mL of dry DMF. Separately weigh 20.71 mg (0.13 mmol) of CDI and dissolve it in 0.5 mL of DMF. Add the dissolved CDI to the flask with stirring and activate at 35 °C for 3 h. Weigh 11.06 mg (0.043 mmol) of GPC and dissolve it in 1 mL of chloroform. Add the dissolved GPC to the flask with stirring using a syringe. Separately disperse 12.7 μL (0.085 mmol) of DBU in 0.5 mL of chloroform and add the dissolved GPC to the flask with stirring using a syringe. Stir at 45 °C for 8 h. The crude product was purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v), eluting out the phospholipid compounds GPC-(Pnx)2 and GPC-Pnx corresponding to Pnx. The organic phase was removed by rotary evaporation, and finally the pure product was obtained by lyophilization.

[0129] (4) Weigh 19.33 mg (0.085 mmol) of Pnx and dissolve it in a 25 mL round-bottom flask. Add 1 mL of dry DMF. Separately weigh 20.71 mg (0.13 mmol) of CDI and dissolve it in 0.5 mL of DMF. Add the CDI to the flask with stirring and activate at 35 °C for 3 h. Weigh 22.30 mg (0.043 mmol) of 18:0 Lyso PC and dissolve it in 1 mL of chloroform. Transfer the solution to the flask with stirring using a syringe. Separately disperse 12.7 μL (0.085 mmol) of DBU in 0.5 mL of chloroform. Transfer the solution to the flask with stirring using a syringe and stir at 45 °C for 8 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v). Remove the organic phase by rotary evaporation and finally lyophilize to obtain pure 18:0-Pnx PC.

[0130] The 1H NMR and mass spectra of Pnx and its corresponding representative ferroptosis-inhibiting phospholipid compounds GPC-Pnx, GPC-(Pnx)2, and 18:0-Pnx PC are shown in [reference needed]. Figure 9 :

[0131] (a) The characterization results of Pnx are as follows Figure 9 China A: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 12.44 (s, 1H), 8.76 (s, 1H), 7.34 (dd, J = 8.2, 1.8Hz, 1H), 7.02 (d, J = 1.8Hz, 1H), 6.75 (ddd, J = 7.8, 5.4, 3.5Hz, 1H), 6.66–6.59 (m, 2H), 6.47 (dd, J = 8.3, 6.7Hz, 2H). HRMS (ESI): m / z calculation results: C 13 H9NO3[MH] － 226.06; Analytical measured result: 225.96.

[0132] (b) Characterization results of GPC-Pnx are as follows Figure 9 B: 1¹H NMR (600MHz, DMSO-d⁶) δ 7.38–7.35 (m, 1H), 7.04 (d, J = 1.8Hz, 1H), 6.75 (dt, J = 8.2, 3.7Hz, 1H), 6.62 (d, J = 4.2Hz, 2H), 6.49 (dd, J = 8.0, 5.0Hz, 2H), 5.57 (s, 1H), 4.06 (s, 2H), 3.83 (s, 1H), 3.75 (s, 2H), 3.71–3.65 (m, 2H), 3.52 (t, J = 4.9Hz, 2H), 3.18–3.09 (m, 9H). HRMS (ESI): m / z calculation results: C 21 H 27 N₂O₈P[M+H] + 467.15; Analytical measurement result: 467.04.

[0133] (c) The characterization results of GPC-(Pnx)2 are as follows Figure 9 C: 1 H NMR (400MHz, DMSO-d6) δ8.89(s,2H),7.35(ddd,J=16.9,8.2,1.9Hz,2H),7.05(d,J=1.9Hz,2H ),6.98(d,J=1.9Hz,1H),6.74(td,J=6.8,5.4,2.9Hz,2H),6.62(d,J=4.6Hz,4H),6.47(dd,J= 8.1, 4.5Hz, 4H), 5.32(t, J = 4.8Hz, 1H), 4.49(dd, J = 12.0, 3.5Hz, 2H), 4.36(dd, J = 11.7, 6.7Hz, 2H), 3.90(t, J = 5.9Hz, 2H), 3.52–3.48(m, 2H), 3.11(d, J = 10.8Hz, 9H). HRMS(ESI): m / z calculation results: C 35 H 38 N3O 10 P[M+H] + 676.20; Analytical measurement result: 676.06.

[0134] (d) The characterization results of 18:0-PnxPC are as follows Figure 9 D: 1H NMR(400MHz, DMSO-d6)δ8.94(s,1H),7.35(tt,J=7.1,3.5Hz,1H),7.22(s,1H),7.10–6.97( m,1H),6.69–6.57(m,2H),6.55–6.42(m,2H),5.33(t,J=4.9Hz,1H),4.30(d,J=6.9Hz,2H),4 .02(s,2H), 3.89–3.73(m,2H), 3.51(s,2H), 3.13(d,J=6.4Hz,9H), 2.27(dt,J=13.1,7.2Hz,3H), 1.99(p,J=6.9,6.4Hz,3H), 1.24(s,28H), 0.85(d,J=7.0Hz,3H). HRMS(ESI): m / z calculation results: C 39 H 61 N₂O₉P[M+H] + 733.41; Analytical measurement result: 733.32.

[0135] Example 5

[0136] This embodiment selects Pnx as the active group and, based on Example 4, illustrates how to prepare free radical scavenging biomimetic phospholipid death inhibitors with different linker arm lengths. The specific synthetic route is as follows: Figure 10 As shown:

[0137] (1) Weigh 227.1 mg (1.0 mmol) Pnx-COOH, add 5 mL of anhydrous chloroform, add 243.2 mg (1.5 mmol) CDI while stirring, stir at 35 °C for 2 h, and then add 1.25 mmol H2N (C2H4) of bifunctional linker. n COOH (n = 1-5) and 150 μL (1.0 mmol) DBU were added, and the mixture was stirred at 40 °C for 6 h. After the reaction, the product was purified by column chromatography (petroleum ether / ethyl acetate / glacial acetic acid = 2:1:0.005 → 1:1:0.005, v / v / v). The product was dissolved in ethyl acetate, and glacial acetic acid was washed away with ultrapure water. The product was then rotary evaporated and dried at 40 °C for 4 h to obtain a yellow powder product, abbreviated as Pnx-C. x (x = 2, 4, 6, 8, 10).

[0138] (2) Weigh 1.00 mmol Pnx-C x(x=2,4,6,8,10), add 5 mL of anhydrous chloroform, then add 243.2 mg (1.5 mmol) CDI while stirring. After stirring at 35 °C for 1 h, add 261.9 mg (0.5 mmol) 18:0 Lyso PC and 150 μL (1.0 mmol) DBU. Continue stirring at room temperature for 6 h. Concentrate to obtain a yellow residue, which is purified by silica gel chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v) to obtain 18:0-(Pnx-C x PC (x = 2, 4, 6, 8, 10). The organic phase was removed by rotary evaporation, and finally the product was freeze-dried to obtain the pure product.

[0139] Couplings of Pnx-COOH with molecules of different carbon chain lengths: Pnx-C2, Pnx-C4, Pnx-C6, Pnx-C8, Pnx-C 10 The 1H NMR spectrum and mass spectrum are shown below. Figure 11 :

[0140] (a) The characterization results of Pnx-C2 are as follows Figure 11 China A: 1 ¹H NMR (600MHz, D₂O) δ 6.97 (d, J = 8.0Hz, 1H), 6.71–6.65 (m, 2H), 6.57 (t, J = 7.8Hz, 1H), 6.46 (d, J = 7.9Hz, 1H), 6.29 (d, J = 7.7Hz, 1H), 6.24 (d, J = 8.1Hz, 1H), 3.40 (t, J = 7.2Hz, 2H), 2.39 (t, J = 7.1Hz, 2H). HRMS (ESI): m / z calculation results: C 16 H 14 N2O4[MH] － 297.10; Analytical measurement result: 297.04.

[0141] (b) The characterization results of Pnx-C4 are as follows Figure 11 B: 1 ¹H NMR (600MHz, D₂O) δ 6.99 (s, 1H), 6.70 (s, 1H), 6.67 (s, 1H), 6.58 (s, 1H), 6.47 (d, J = 8.0Hz, 1H), 6.30 (s, 1H), 6.25 (d, J = 8.3Hz, 1H), 3.20 (d, J = 7.5Hz, 2H), 2.16 (t, J = 7.0Hz, 2H), 1.54 (p, J = 7.1Hz, 2H), 1.49 (d, J = 8.0Hz, 2H). HRMS (ESI): m / z calculation results: C 18 H 18 N2O4[MH]－ 325.13; Analytical measurement result: 325.04.

[0142] (c) The characterization results of Pnx-C6 are as follows Figure 11 C: 1 ¹H NMR (600MHz, D₂O) δ 6.99(s, ¹H), 6.72(s, ¹H), 6.67(s, ¹H), 6.58(s, ¹H), 6.49(s, ¹H), 6.32(s, ¹H), 6.28(s, ¹H), 3.17(s, 2H), 2.10(d, J = 8.3Hz, 2H), 1.48(s, 4H), 1.27(s, 4H). HRMS (ESI): m / z calculation results: C 20 H 22 N2O4[MH] － 353.16; Analytical measurement result: 323.05.

[0143] (d) The characterization results of Pnx-C8 are as follows Figure 11 D: 1 H NMR(600MHz,DMSO-d6)δ8.63(s,1H),7.80–7.68(m,1H),7.30(dd,J=8.1,2.0Hz ,1H),7.12(d,J=1.9Hz,1H),6.75(ddd,J=8.3,6.4,2.6Hz,1H),6.64–6.58(m,2H ),6.51–6.47(m,1H),6.45(d,J=8.1Hz,1H),3.01(q,J=6.0Hz,2H),2.04(q,J=8 .0,6.9Hz,2H),1.47–1.44(m,2H),1.35(d,J=10.2Hz,2H),1.28–1.22(m,8H).HR MS(ESI): m / z calculation result: C 22 H 26 N2O4[MH] － 381.19; Analytical measurement result: 381.14.

[0144] (e)Pnx-C 10 The characterization results are as follows Figure 11 E: 1H NMR (600MHz, DMSO-d6) δ8.52(s,1H),8.07(t,J=5.6Hz,1H),7.27(dd,J=8.1,1.9Hz,1H),7.10(d,J=1.9Hz,1H),6 .77–6.68(m,1H),6.64–6.57(m,2H),6.46(dd,J=18.8,7.7Hz,2H),3.33(s,2H),3.23–3.15(m,2H),2.18(t,J=7.4

[0145] Hz, 2H), 1.48 (q, J = 7.2 Hz, 4H), 1.26 (d, J = 11.2 Hz, 10H). HRMS(ESI): m / z calculation results: C 24 H

[0146] 30 N2O4[MH] － 409.32; Analytical measurement result: 409.14.

[0147] The corresponding representative biomimetic ferroptosis inhibitors are 18:0-(Pnx-C2)PC, 18:0-(Pnx-C4)PC, 18:0-(Pnx-C6)PC, 18:0-(Pnx-C8)PC, and 18:0-(Pnx-C... 10 The 1H NMR spectrum and mass spectrum of PC are shown below. Figure 12 :

[0148] (a) The characterization results of 18:0-(Pnx-C2)PC are as follows Figure 12 China A: 1H NMR (600MHz, DMSO-d6) δ8.86(t,J=5.5Hz,1H),8.65(s,1H),7.36(dd,J=8.1,1.9Hz,1H),7.16(d,J=1.8Hz,1H),6.72(ddd,J=8 .1,6.0,2.8Hz,1H),6.62–6.55(m,2H),6.49–6.40(m,2H),5.06(dt,J=7.6,4.1Hz,1H),4.31–4.22(m,1H),4.13(ddd,J=10.7, 7.2, 3.6Hz, 1H), 4.08–4.05(m, 2H), 3.91–3.77(m, 2H), 3.59–3.50(m, 2H), 3.50–3.38(m, 2H), 3.14(d, J = 5.9Hz, 9H), 2.53(q, J = 6.8Hz, 2H), 2.23(t, J = 7.4Hz, 2H), 1.49–1.44(m, 2H), 1.27–1.18(m, 28H), 0.85(td, J = 7.0, 3.1Hz, 3H). HRMS(ESI): m / z calculation results: C 42 H 66 N3O 10 P[M+H] + 804.45; Analytical measurement result: 804.23.

[0149] (b) The characterization results of 18:0-(Pnx-C4)PC are as follows Figure 12 B: 1H NMR (600MHz, DMSO-d6) δ8.66(s,1H),8.22(t,J=5.6Hz,1H),7.28(ddd,J=6.5,4.7,1.9Hz,1H),7.10(dd,J=6.7,1.9Hz,1H) ,6.73(ddd,J=8.2,5.8,3.1Hz,1H),6.59(q,J=4.0,3.0Hz,2H),6.50–6.42(m,2H),5.07(dtd,J=8.2,5.3,3.1Hz,1H),4.28 (dd, J = 12.0, 3.2 Hz, 1H), 4.10 (dd, J = 12.0, 7.1 Hz, 1H), 4.03 (s, 2H), 3.79–3.72 (m, 2H), 3.51 (t, J = 4.9 Hz, 2H), 3.22–3.14 (m, 2H), 3.13 (s, 9H), 2.34–2.23 (m, 4H), 1.50 (dq, J = 27.6, 7.0 Hz, 4H), 1.21 (d, J = 7.1 Hz, 30H), 0.85 (q, J = 7.1, 6.5 Hz, 3H). HR MS(ESI): m / z calculation results: C 44 H 70 N3O 10 P[M+H] + 832.48; Analysis of measured results: 832.48.

[0150] The characterization results of (c)18:0-(Pnx-C6)PC are as follows Figure 12 C: 1 H NMR (600MHz, DMSO-d6) δ8.57(s,1H),8.21(t,J=5.6Hz,1H),7.28(ddd,J=8.2,4.7,1.9Hz,1H),7.10(dd,J=7.2,1.9Hz,1H ),6.73(ddd,J=7.8,5.8,3.0Hz,1H),6.62–6.57(m,2H),6.49–6.41(m,2H),5.07(dt,J=10.5,4.0Hz,1H),4.28(dd,J=12.0 ,3.2Hz,1H), 4.09(dd,J=12.0,7.1Hz,1H), 4.03(s,2H), 3.74(t,J=6.1Hz,2H), 3.51(dd,J=6.0,3.6Hz,2H), 3.17(q,J=6.6Hz,2H), 3.13(s,9H), 2.34–2.25(m,4H), 1.53–1.47(m,4H), 1.32–1.17(m,34H), 0.87–0.81(m,3H).HRMS(ESI):m / z calculation results: C46 H 74 N3O 10 P[M+H] + 860.51; Analytical measurement result: 860.50.

[0151] The characterization results of (d)18:0-(Pnx-C8)PC are as follows: Figure 12 D: 1 H NMR (600MHz, DMSO-d6) δ8.65(s,1H),8.14(dt,J=15.5,5.6Hz,1H),7.27(dt,J=8.2,2.1Hz,1H),7.10(d,J=1.9Hz,1H),6.75– 6.70(m,1H),6.62–6.56(m,2H),6.54–6.42(m,2H),5.09–5.04(m,1H),4.31–4.26(m,1H),4.09(ddd,J=12.0,7.1,1.7Hz,1H), 4.02 (d, J = 8.1 Hz, 2H), 3.73 (hept, J = 5.6 Hz, 2H), 3.53–3.48 (m, 2H), 3.17 (q, J = 6.6 Hz, 2H), 3.12 (d, J = 2.3 Hz, 9H), 2.26 (tdd, J = 7.4, 5.3, 2.4 Hz, 4H), 1.49 (dt, J = 13.5, 6.7 Hz, 4H), 1.32–1.15 (m, 38H), 0.84 (td, J = 7.0, 4.4 Hz, 3H). HRMS(ESI): m / z calculation results: C 48 H 78 N3O 10 P[M+H] + 888.54; Analytical measurement result: 888.53.

[0152] (e)18:0-(Pnx-C 10 The characterization results of PC are as follows: Figure 12 E: 1H NMR(600MHz,DMSO-d6)δ8.64(s,1H),8.10(d,J=5.6Hz,1H),7.33–7.19(m,1H),7.09(d,J=1.9Hz,1H) ,6.76–6.66(m,1H),6.65–6.53(m,2H),6.46(dd,J=20.5,8.0Hz,2H),5.06(s,1H),4.31–4.26(m,1H) ,4.09(dd,J=12.0,7.3Hz,1H),4.02(s,2H),3.73(q,J=5.7Hz,2H),3.17(d,J=5.6Hz,2H),3.12(s,9H),2.25(td,J=7.3,6.2,2.8Hz,4H),1.50–1.45(m,4H),1.25–1.20(m,42H),0.85(q,J=6.9Hz,3H).HRM S(ESI):m / z Calculation results:C 50 H 82 N3O 10 P[M+H] + 916.57; Analytical measurement result: 916.56.

[0153] Example 6

[0154] In this embodiment, Ebs, a small-molecule organoselenium compound with GPX4 function, is selected as the active group to prepare a biomimetic phospholipid ferroptosis inhibitor. The specific synthetic route is as follows: Figure 13 As shown:

[0155] (1) Weigh 2.0 g (5 mmol) of 2,2'-diselenobenzoic acid into a 25 mL round-bottom flask, add 10 mL of thionyl chloride (SOCl2) to dissolve it, and add 3 drops of DMF. Under N2 protection, heat to 85 °C and reflux for 3 h. After the reaction is complete, cool the reaction solution to room temperature, then remove excess SOCl2 by rotary evaporation, and recrystallize with 20 mL of n-hexane to obtain 2-chloroselenobenzoyl chloride, with a yield of 90%.

[0156] (2) Weigh 1.37 g (10 mmol) of p-aminobenzoic acid into a 50 mL round-bottom flask, add 30 mL of anhydrous dichloromethane to dissolve it, then add 2.8 mL of triethylamine, stir and mix well, and protect with N2. Then dissolve 2-chloroselenobenzoyl chloride in 5 mL of dichloromethane, add it dropwise to the above reaction solution at 0 °C, and react at 25 °C for 12 h. Subsequently, remove the solvent by rotary evaporation to obtain the crude product, dissolve it in 10 mL of Na2CO3 (0.5 M) aqueous solution, filter the filtrate, adjust the pH of the filtrate to less than 1 with 1 M HCl, filter the precipitate, wash it with water 3 times, and vacuum dry to obtain a light yellow powder, which is the ebs-COOH derivative containing the carboxylic acid functional group, with a yield of 85%.

[0157] (3) Weigh 319.0 mg (1 mmol) of Ebs-COOH into a 10 mL round-bottom flask and dissolve it in 2 mL of anhydrous DMSO. Weigh 243.2 mg (1.5 mmol) of CDI, dissolve it in 1 mL of anhydrous DMSO, and add it dropwise to the above flask. Under nitrogen protection, activate at room temperature for 3 h. Then, add 149.4 μL (1 mmol) of DBU and 257.2 mg (1 mmol) of GPC to the system sequentially. Under nitrogen protection, stir at room temperature for 4 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v). The corresponding phospholipid compounds GPC-(Ebs)2 and GPC-Ebs are eluted sequentially. The organic phase is removed by rotary evaporation, and the product is obtained by freeze-drying.

[0158] (4) Weigh 319.0 mg (1 mmol) of Ebs-COOH into a 10 mL round-bottom flask and add 5 mL of anhydrous chloroform to disperse it evenly. Weigh 243.2 mg (1.5 mmol) of CDI, dissolve it in 2 mL of anhydrous chloroform, and add it dropwise to the above flask. At this time, the solution becomes clear. Under nitrogen protection, activate at room temperature for 3 h. Then, add 149.4 μL (1 mmol) of DBU and 261.8 mg (0.5 mmol) of 18:0 Lyso PC to the system in sequence. Under nitrogen protection, stir at 30 °C for 24 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v) and freeze-dried to obtain the corresponding phospholipid product 18:0-Ebs PC.

[0159] (5) Weigh 427 mg (1.5 mmol) stearic acid into a 10 mL round-bottom flask and add 2 mL of anhydrous dichloromethane to disperse it evenly. Weigh 325 mg (2.0 mmol) CDI, dissolve it in 2 mL of anhydrous DMSO, and add it dropwise to the above flask. The solution becomes clear at this point. Activate at 35 °C for 3 h, and then add 224 μL (1.5 mmol) DBU and 279 mg (0.5 mmol) GPC-Ebs to the system sequentially. Under nitrogen protection, stir at 35 °C for 24 h. The crude product is purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v), the organic phase is removed by rotary evaporation, and finally, pure Ebs-18:0PC is obtained by lyophilization.

[0160] The NMR and mass spectra of Ebs and their corresponding representative ferroptosis-inhibiting phospholipid compounds GPC-Ebs, GPC-(Ebs)2, 18:0-Ebs PC, and Ebs-18:0PC are shown below. Figure 14 :

[0161] (a) Characterization results of Ebs-COOH are as follows Figure 14 China A: 1 ¹H NMR (DMSO-d⁶, 400MHz) δ 12.93 (s, 1H), 8.13 (d, 1H), 8.00 (d, 2H), 7.94 (d, 1H), 7.86 (d, 2H), 7.70 (t, 1H), 7.49 (t, 1H). HRMS (ESI): m / z calculation results: C 14 H9NO3Se[M+H] + 319.97; Analytical measured result: 319.89.

[0162] (b) Characterization results of GPC-Ebs are as follows Figure 14 B: 1 ¹H NMR (DMSO-d⁶, 400MHz) δ 8.17 (d, J = 8.0Hz, 1H), 8.09–8.00 (m, 2H), 7.91 (dd, J = 16.3, 8.1Hz, 3H), 7.69 (t, J = 7.7Hz, 1H), 7.49 (t, J = 7.6Hz, 1H), 6.26 (d, J = 5.0Hz, 1H), 4.30–4.16 (m, 2H), 4.08 (d, J = 8.4Hz, 2H), 3.98–3.86 (m, 1H), 3.80 (ddd, J = 11.4, 8.7, 4.5Hz, 2H), 3.56–3.51 (m, 2H), 3.13 (s, 9H). HRMS (ESI): m / z calculation results: C 22 H 27 N₂O₈PSe[M+H]+ 559.07; Analytical measurement result: 559.06.

[0163] (c) Characterization results of GPC-(Ebs)2 are as follows Figure 14 C: 1 ¹H NMR (DMSO-d⁶, 400MHz) δ 8.13 (d, J = 8.1Hz, 2H), 8.09–7.97 (m, 4H), 7.96–7.85 (m, 6H), 7.70 (ddd, J = 8.4, 7.2, 1.5Hz, 2H), 7.49 (t, J = 7.3Hz, 2H), 5.51 (dt, J = 8.5, 4.9Hz, 1H), 4.73–4.52 (m, 2H), 4.10–3.97 (m, 4H), 3.49 (t, J = 5.0Hz, 2H), 3.11 (s, 9H). HRMS (ESI): m / z calculation results: C 36 H 34 N3O 10 PSe2[M+H] + 860.03; Analytical measurement result: 859.97.

[0164] (d) The characterization results of 18:0-EbsPC are as follows Figure 14 D: 1 H NMR(DMSO-d6,400MHz)δ8.16(d,J=8.1Hz,1H),8.07–7.96(m,2H),7.96–7.85(m,3H),7.76– 7.62(m,1H),7.48(t,J=7.5Hz,1H),5.39–5.23(m,1H),4.47–4.26(m,2H),4.11–3.98(m,2H ), 3.89 (dt, J = 21.9, 6.0 Hz, 2H), 3.51 (dq, J = 14.3, 4.8 Hz, 2H), 3.20–3.04 (m, 9H), 2.35–2.22 (m, 2H), 1.52–1.40 (m, 2H), 1.29–1.06 (m, 28H), 0.86–0.82 (m, 3H). HRMS(ESI): m / z calculation results: C 40 H 61 N₂O₉PSe[M+H] + 825.33; Analytical measurement result: 825.28.

[0165] (e)Characteristics results of Ebs-18:0PC are as follows Figure 14 E: 1H NMR(400MHz, DMSO-d6)δ8.13(d,J=8.1Hz,1H),8.05–7.97(m,2H),7.95–7.88(m,3H),7.74–7.66(m ,1H),7.49(t,J=7.5Hz,1H),5.35–5.24(m,1H),4.52(dd,J=11.9,3.1Hz,1H),4.45–4.34(m,1H),4 .04(s,2H), 3.87(dt,J=22.4,6.1Hz,2H), 3.56–3.47(m,2H), 3.12(d,J=10.5Hz,9H), 2.28(q,J=6.2,5.4Hz,2H), 1.46(q,J=7.6Hz,2H), 1.28–1.07(m,28H), 0.86–0.81(m,3H). HRMS(ESI): m / z calculation results: C 40 H 61 N₂O₉PS e[M+H] + 825.33; Analytical measurement result: 825.27.

[0166] Example 7

[0167] This embodiment selects the iron chelating agent Dfx as the active group to illustrate how to prepare a biomimetic phospholipid ferroptosis inhibitor. The specific synthetic route is as follows: Figure 15 As shown:

[0168] 373.4 mg (1 mmol) of deferasirox was weighed into a 10 mL round-bottom flask and dissolved in 2 mL of anhydrous DMSO. 243.2 mg (1.5 mmol) of CDI was weighed, dissolved in 1 mL of anhydrous DMSO, and added dropwise to the flask. The mixture was activated at 35 °C for 3 h. Then, 224.1 μL (1.5 mmol) of DBU and 257.2 mg (1 mmol) of GPC were added sequentially to the system under nitrogen protection, and stirring was continued for 24 h. The crude product was purified by silica gel column chromatography (dichloromethane:methanol:water = 65:25:0 → 65:25:2 → 65:25:4, v / v / v), and finally freeze-dried to obtain the phospholipid compound GPC-Dfx corresponding to Dfx.

[0169] The characterization results of GPC-Dfx are as follows Figure 16 : 1¹H NMR (400MHz, DMSO-d⁶) δ 10.79 (s, 1H), 10.18 (s, 1H), 8.12–7.98 (m, 3H), 7.66–7.52 (m, 3H), 7.38 (tdd, J = 6.9, 5.0, 1.7Hz, 2H), 7.07–6.95 (m, 3H), 6.87 (d, J = 8.2Hz, 1H), 4.24 (qd, J = 11.0, 5.5Hz, 2H), 4.06 (s, 2H), 3.89 (p, J = 5.5Hz, 1H), 3.77 (dd, J = 10.1, 4.8Hz, 2H), 3.56–3.46 (m, 2H), 3.11 (s, 9H). HRMS (ESI): m / z calculation results: C 29 H 33 N4O9P[M+H] + 613.20; Analysis of measured results: 613.19.

[0170] Example 8

[0171] This embodiment selects the iron chelating agent Dfo as the active group to illustrate how to prepare a biomimetic phospholipid ferroptosis inhibitor. The specific synthetic route is as follows: Figure 17 As shown:

[0172] Weigh out 28.6 mg (0.01 mmol) of carboxylated distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). 2000 3.9 mg (0.02 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2.8 mg (0.02 mmol) of 1-hydroxybenzotriazole (HOBt) were dissolved in a 25 mL round-bottom flask with 1.0 mL of anhydrous DMF. The mixture was activated at room temperature under argon protection with stirring for 2 h. After activation, 31.3 μL (0.18 mmol) of N,N-diisopropylethylamine (DIPEA) and 26.2 mg (0.04 mmol) of deferoxamine (Dfo) were added to the above reaction solution, and the reaction was carried out at 35 °C for 108 h. After the reaction, the reaction solution was dialyzed against DMF and distilled water (molecular weight cutoff: 2,000 Da) for 24 h. After dialyzing, the Dfo-corresponding phospholipid compound DSPE-PEG was obtained by lyophilization. 2000 -Dfo.

[0173] DSPE-PEG 2000 The characterization results of -Dfo are as follows Figure 18-19 : 1¹H NMR (400 MHz, DMSO-d⁶) δ 4.39–3.81 (m, 10H), 3.43 (s, 191H), 3.00 (s, 4H), 2.25 (d, J = 12.6 Hz, 12H), 1.99 (d, J = 20.2 Hz, 3H), 1.57–1.33 (m, 16H), 1.23 (s, 62H), 0.84 (d, J = 8.3 Hz, 6H). The molecular weight was characterized by MALDI-TOF-MS. DSPE-PEG 2000 After covalently linking -COOH with Dfo, its molecular weight is 3518, consistent with the expected result.

[0174] Example 9

[0175] This embodiment examines the partially radical-scavenging biomimetic ferroptosis inhibitors GPC-Fer, GPC-(Fer)2, 18:0-Fer PC, 18:0-(Fer-C2)PC, 18:0-(Fer-C4)PC, 18:0-(Fer-C6)PC, 18:0-(Fer-C8)PC, and 18:0-(Fer-C4)PC prepared in Examples 1-5. 10 )PC, GPC-AA9, GPC-(AA9)2, 18:0-AA9 PC, GPC-Pnx, GPC-(Pnx)2, 18:0-Pnx PC, 18:0-(Pnx-C2)PC, 18:0-(Pnx-C4)PC, 18:0-(Pnx-C6)PC, 18:0-(Pnx-C8)PC, 18:0-(Pnx-C 10 The biomimetic ferroptosis inhibitors GPC-Ebs, GPC-(Ebs)2, and 18:0-Ebs PC, which mimic the characteristics of glutathione peroxidase 4, prepared in Example 6, and the representative iron-chelating ferroptosis inhibitory phospholipid materials GPC-Dfx and DSPE-PEG-Dfo prepared in Examples 7 and 8, and their effects on the activity of rat cardiomyocyte H9c2 cells.

[0176] Rat H9c2 cardiomyocytes were selected, and the cytotoxicity of biomimetic phospholipid ferroptosis inhibitors was investigated using the MTT assay with cell viability as an indicator. The specific steps were as follows: H9c2 cells in logarithmic growth phase were seeded into 96-well plates at 4500 cells per well, and cultured in 100 μL of medium (89% DMEM + 10% FBS + 1% penicillin and streptomycin) for 24 hours in an adherent culture environment (5% CO2, 37℃). Subsequently, the following representative free radical scavenging ferroptosis inhibitors were added to the 96-well plates: (I) GPC-Fer and GPC-(Fer)2 (Fer concentration: 0-500 μM), 18:0-Fer PC, 18:0-(Fer-C2)PC, 18:0-(Fer-C4)PC, 18:0-(Fer-C6)PC, 18:0-(Fer-C8)PC, and 18:0-(Fer-C8)PC. 10 )PC(0-100μM); GPC-AA9, GPC-(AA9)2 and 18:0-AA9 PC(0-100μM); GPC-Pnx, GPC-(Pnx)2, 18:0-Pnx PC, 18:0-(Pnx-C2)PC, 18:0-(Pnx-C4)PC, 18:0-(Pnx-C6)PC, 18:0-(Pnx-C8)PC, 18:0-(Pnx-C 10 (I) Biomimetic phospholipids mimicking GPX4 characteristics: GPC-Ebs (0-200μM), GPC-(Ebs)2 (0-100μM) and 18:0-Ebs PC (Ebs concentration: 0-200μM); (II) Iron-chelated phospholipids: GPC-Dfx (relative Dfx concentration: 0-1000μM) and DSPE-PEG 2000 -Dfo (0-500μM); after drug addition, continue adherent culture. After 24 hours, wash cells with 100μL PBS, add 100μL of 0.5mg / mL MTT medium solution under light protection, and continue culturing for 4 hours. Then, aspirate the culture medium from the wells, add 100μL DMSO to each well, and incubate in the dark at room temperature with a shaker for 20 minutes until all purple crystals dissolve. Then, measure the absorbance of the sample at 570nm using a microplate reader. Calculate cell viability using the following formula: Viability = (OD value of experimental group / OD value of blank control group) × 100%. Plot cell viability against drug concentration. Results are as follows. Figure 20-21 As shown.

[0177] In vitro cytotoxicity assays showed that after 24 hours of incubation with H9c2 cells, 18:0-Fer PC exhibited greater cytotoxicity than GPC-Fer and GPC-(Fer)2, possibly due to its higher cellular uptake. GPC-Fer and GPC-(Fer)2 had no effect on H9c2 cell viability at Fer concentrations below 500 μM and 200 μM, respectively. 18:0-Fer PC was non-toxic at concentrations below 20 μM. However, extending the carbon chain length of the sn-2 linker arm reduced its cytotoxicity. The following PC types were observed: 18:0-(Fer-C2)PC, 18:0-(Fer-C4)PC, 18:0-(Fer-C6)PC, 18:0-(Fer-C8)PC, and 18:0-(Fer-C... 10 At concentrations below 100 μM, GPC had no effect on cell viability. Furthermore, the free radical scavenging biomimetic ferroptosis inhibitors GPC-AA9 and GPC-Pnx did not affect H9c2 cell activity at concentrations below 100 μM. Compared to free radical scavenging biomimetic ferroptosis inhibitors, GPC-Ebs, GPC-(Ebs)2, and 18:0-Ebs PC exhibited greater cytotoxicity, showing cytotoxicity at concentrations above 50 μM. Finally, regarding the iron-chelating biomimetic ferroptosis inhibitors, GPC-Dfx had no effect on cell viability at concentrations below 50 μM, and DSPE-PEG-Dfo had no effect on cell viability at concentrations below 500 μM. Figure 20-21 ).

[0178] Example 10

[0179] The purpose of this embodiment is to investigate the in vitro efficacy and dose-dependency of biomimetic phospholipid ferroptosis inhibitors containing different active groups. RSL3 is a classic ferroptosis inducer that can induce ferroptosis in cells by inhibiting GPX4. In this embodiment, RSL3 was used as an inducer to construct an H9c2 cardiomyocyte ferroptosis model. The ferroptosis inhibitors included: GPC-Fer, GPC-(Fer)2, 18:0-Fer PC, GPC-AA9, GPC-(AA9)2, 18:0-AA9 PC, GPC-Pnx, GPC-(Pnx)2, 18:0-Pnx PC, GPC-Ebs, GPC-(Ebs)2, 18:0-Ebs PC, GPC-Dfx, and DSPE-PEG-Dfo.

[0180] First, we determined the cytotoxicity of RSL3 on H9c2 cells, selecting a 50% inhibitory concentration (0.5 μM) of RSL3 for subsequent experiments. Dosage regimen: RSL3 (0.5 μM) and various biomimetic ferroptosis inhibitors at different concentrations were co-incubated in a cell culture incubator for 24 h. Subsequent cell culture and activity assays were performed using the same methods as in Example 9. The results are as follows: Figure 22 As shown.

[0181] Experimental results showed that, using cell viability as an indicator, all selected biomimetic phospholipid ferroptosis inhibitors could reverse RSL3-induced ferroptosis in H9c2 cells. Within a certain concentration range, the inhibitory effect on ferroptosis gradually increased with increasing inhibitor concentration. Overall, biomimetic phospholipid ferroptosis inhibitors containing fatty chains were superior to those without, because the presence of fatty chains allowed for a longer membrane retention time, thus exhibiting a better inhibitory effect on ferroptosis. Furthermore, the efficacy of the active group also determined the efficacy of the biomimetic inhibitors. For example, the free radical scavenging active group Pnx had the highest efficacy, and its corresponding biomimetic inhibitor was also more effective than other inhibitors. Overall, free radical scavenging biomimetic ferroptosis inhibitors were more effective than ferroptosis inhibitors mimicking GPX4 function, while iron chelation ferroptosis inhibitors were slightly less effective. Figure 22 ).

[0182] Example 11

[0183] The purpose of this embodiment is to investigate the relationship between the in vitro efficacy of the biomimetic phospholipid ferroptosis inhibitor and the position (sn-1 or sn-2) of its active group on the glycerophospholipid. Phospholipids have glycerol as their structural center, with R1, R2, and a phosphate group attached to the three carbon atoms of glycerol. R1 and R2 can be fatty acids or ferroptosis inhibitory active groups, and their positions correspond to the sn-1 or sn-2 positions of the phospholipid. The group or molecule located at the sn-2 position can be hydrolyzed and released by phospholipase A2 (PLA2). PLA2 is an enzyme that catalyzes the hydrolysis of the ester group at the sn-2 position of the phospholipid glycerol molecule and has important physiological functions; it is overexpressed in many diseases, including inflammation and cancer. Currently, more than 30 subtypes of PLA2 in 6 major classes have been identified, and various PLA2 types are distributed on the cell membrane. When the biomimetic phospholipid ferroptosis inhibitor is retained on the cell membrane, its active group at the sn-2 position can be released through the hydrolysis of PLA2. The latter then diffuses further within the cell membrane to inhibit lipid peroxidation and ferroptosis. The inability to release the active group at the sn-1 position restricts its diffusion across the cell membrane, thus potentially limiting its ferroptosis inhibitory ability. To verify this hypothesis, this embodiment uses two in vitro models: an RSL3-induced cardiomyocyte (H9c2) ferroptosis model and a sodium iodate-induced retinal pigment epithelial cell (ARPE-19) model. Representative free radical-scavenging phospholipid ferroptosis inhibitors 18:0-Fer PC and Fer-18:0PC, and representative GPX4-like phospholipid ferroptosis inhibitors 18:0-Ebs PC and Ebs-18:0PC were selected. Simultaneously, a PLA2 inhibitor was used to reduce the ability of PLA2 to hydrolyze the ester group at the sn-2 position, examining whether the release of the active group affects its ferroptosis inhibitory effect.

[0184] First, we constructed an RSL3 (0.5 μM)-induced ferroptosis model in H9c2 cardiomyocytes, following the same method as in Example 10. Next, we co-incubated the cells for 24 h with the PLA2 inhibitor Darapladib (1 μM) and different concentrations of 18:0-Fer PC or Fer-18:0 PC, and set up a control group without PLA2 inhibitor treatment. Finally, cell viability was detected by MTT assay, and the results are as follows: Figure 23 As shown in the figure. In addition, we constructed an in vitro model of age-related macular degeneration (ARPE-19) cells. First, the modeling concentration was determined by measuring the cytotoxicity of sodium iodate to human retinal pigment epithelial cells (ARPE-19). Second, in this experiment, cells were incubated for 2 hours using medium containing different concentrations of Ebs / 18:0-Ebs PC / Ebs-18:0PC and mixed medium containing darapladib (1 μM) and different concentrations of Ebs / 18:0-Ebs PC / Ebs-18:0PC. Then, sodium iodate was added to bring the final concentration to 20 mM, and the cells were cultured for another 24 hours. Cell viability was then detected using the MTT assay. The results are shown in the figure. Figure 24 As shown.

[0185] Experimental results show that the biomimetic phospholipid ferroptosis inhibitor is more effective than its counterpart small molecule inhibitor. The biomimetic phospholipid ferroptosis inhibitor is more effective when its active group is located at the sn-2 position of the phospholipid than when it is located at the sn-1 position, and this enhanced efficacy can be counteracted by the PLA2 inhibitor darapladib. This confirms that the active group at the sn-2 position, after being hydrolyzed and released by PLA2, may diffuse freely within or near the cell membrane, thus expanding its range for scavenging lipid peroxides and enhancing its ferroptosis inhibitory efficacy. Conversely, the active group at the sn-1 position cannot be released, limiting its diffusion across the cell membrane and resulting in a relatively lower ferroptosis inhibitory capacity. Figure 23-24 ).

[0186] Example 12

[0187] The purpose of this embodiment is to investigate the relationship between the in vitro efficacy of biomimetic phospholipid ferroptosis inhibitors and the carbon chain length of their sn-2 linker arm. In this embodiment, RSL3 (0.5 μM) was used as an inducer to construct an H9c2 cardiomyocyte ferroptosis model. Representative ferroptosis inhibitors included: 18:0-Fer PC, 18:0-(Fer-C2)PC, 18:0-(Fer-C4)PC, 18:0-(Fer-C6)PC, 18:0-(Fer-C8)PC, and 18:0-(Fer-C4)PC. 10)PC; 18:0-Pnx PC, 18:0-(Pnx-C2)PC, 18:0-(Pnx-C4)PC, 18:0-(Pnx-C6)PC, 18:0-(Pnx-C8)PC, 18:0-(Pnx-C 10 PC. The cell culture and drug administration protocol was the same as in Example 9, and the results were as follows. Figure 25-26 As shown.

[0188] Experimental results show that as the carbon chain length of the sn-2 linker increases, the in vitro efficacy of the biomimetic phospholipid ferroptosis inhibitor first increases and then decreases in a concentration-dependent manner. The strongest ferroptosis inhibitory ability is observed when the linker arm is a 6-carbon aliphatic chain, indicating a relatively balanced membrane retention and diffusion capacity. Figure 25-26 ).

[0189] Example 13

[0190] Since doxorubicin-induced cardiomyocyte iron overload and ferroptosis are important mechanisms of doxorubicin cardiotoxicity, inhibiting ferroptosis can significantly reduce doxorubicin-induced cardiac damage. This example demonstrates the efficacy of various ferroptosis-inhibiting phospholipids in reducing doxorubicin cardiotoxicity at the cellular level. An H9c2 cell model co-incubated with doxorubicin and ferric ammonium citrate (FAC) was constructed to simulate and amplify the state of cardiomyocyte ferroptosis after doxorubicin treatment under in vitro conditions. The effects of the three types of phospholipids prepared in Examples 1-8 on inhibiting doxorubicin-induced H9c2 cell ferroptosis, and their dose-dependent relationships, were investigated.

[0191] First, we determined the cytotoxicity of H9c2 cells co-incubated with doxorubicin hydrochloride and FAC, selecting the 50% inhibitory concentration (2 μM doxorubicin hydrochloride and 1 mM FAC) for subsequent experiments. The administration regimen was as follows: 2 μM doxorubicin hydrochloride and 1 mM FAC were co-incubated with different concentrations of various biomimetic ferroptosis inhibitors in a cell culture incubator for 24 h. Subsequent cell culture and activity assays were performed using the same methods as in Example 9 (the cells were protected from light during the experiment). The results are as follows: Figure 27 As shown.

[0192] Experimental results showed that all three types of biomimetic phospholipid ferroptosis inhibitors effectively alleviated cardiomyocyte ferroptosis induced by the combined use of doxorubicin hydrochloride and FAC, and the biomimetic phospholipid ferroptosis inhibitors containing fatty chains were superior to traditional ferroptosis inhibitors without fatty chains. Furthermore, representative free radical scavenging biomimetic inhibitors and GPX4-mimicking biomimetic inhibitors exerted ferroptosis inhibitory effects at lower doses (below 10 μM), while iron chelating biomimetic inhibitors required higher doses to significantly enhance cell viability. Figure 27 ).

[0193] Example 14

[0194] Besides doxorubicin, other anthracyclines also have serious toxic side effects, especially dose-dependent cardiotoxicity. This embodiment illustrates that the synthesized biomimetic ferroptosis inhibitor can broadly alleviate the cardiotoxicity of anthracycline chemotherapy drugs. H9c2 cells were used, and various anthracycline chemotherapy drugs, including doxorubicin, desmethyldaunorubicin, epirubicin, daunorubicin, mitoxantrone, and aclarubicin, were used as inducers to simulate the anthracycline-induced cardiomyocyte damage state under in vitro conditions. The rescue effect of the biomimetic ferroptosis inhibitor prepared in this patent on anthracycline-induced myocardial injury was investigated.

[0195] First, we determined the cytotoxicity of various anthracycline chemotherapeutic drugs on H9c2 cells. The 40% inhibitory concentrations (IICs) of each drug (THP 1 μM, IDA 1 μM, EPI 1 μM, DNR 1 μM, MTX 1 μM, and Aclarubicin ACM 2 μM) were selected for subsequent experiments. The cell culture method was the same as in Example 9. The dosing regimen was as follows: a certain concentration of anthracycline chemotherapeutic drugs and different concentrations of the free radical scavenging biomimetic ferroptosis inhibitor 18:0-Pnx PC were co-incubated in a cell culture incubator for 24 h. Subsequent cell culture and activity detection methods were the same as in Example 9 (the cells were protected from light during the experiment). The results are as follows: Figure 28 As shown.

[0196] Experimental results show that the Pnx-based biomimetic phospholipid ferroptosis inhibitor (18:0-Pnx PC) has broad-spectrum efficacy, not only reducing doxorubicin-induced cardiotoxicity but also effectively reducing H9c2 cardiocytosis induced by other anthracycline chemotherapy drugs, including doxorubicin, desmethyldaunorubicin, epirubicin, daunorubicin, mitoxantrone, and aclarubicin. Furthermore, the Pnx-based biomimetic phospholipid ferroptosis inhibitor (18:0-Pnx PC) exhibits strong efficacy, demonstrating cardioprotective effects at the nanomolar level. Figure 28 ).

[0197] Example 15

[0198] Ferroprelation plays a crucial role in cisplatin-induced acute kidney injury, and inhibiting ferroptosis can alleviate cisplatin-induced kidney damage. This example illustrates the efficacy of various ferroptosis inhibitors on phospholipids in reducing cisplatin nephrotoxicity at the cellular level. Cisplatin was used as an inducer, and HK2 renal tubular cells were selected to simulate the ferroptosis state of renal tubular cells after cisplatin treatment under in vitro conditions. The efficacy and dose-dependent relationships of three types of biomimetic ferroptosis inhibitors prepared in Examples 1-8 in inhibiting cisplatin-induced ferroptosis in HK2 cells were investigated.

[0199] First, we determined the cytotoxicity of cisplatin on HK2 cells. A 50% inhibitory concentration of cisplatin (15 μM) was selected for subsequent experiments, and the cell culture method was the same as in Example 9. The dosing regimen was as follows: 15 μM cisplatin and various biomimetic ferroptosis inhibitors at different concentrations were co-incubated in a cell culture incubator for 24 h. Subsequent cell culture and activity detection methods were the same as in Example 9 (the cells were kept in the dark during the experiment). The results are as follows: Figure 29 As shown.

[0200] Experimental results showed that all three types of biomimetic phospholipid ferroptosis inhibitors effectively inhibited cisplatin-induced cytotoxicity in HK2 cells. Within a certain concentration range, the inhibitory effect of ferroptosis gradually increased with increasing inhibitor concentration, and the biomimetic phospholipid ferroptosis inhibitors containing fatty chains were more effective than the control inhibitors without fatty chains. Furthermore, free radical scavenging biomimetic inhibitors and biomimetic inhibitors mimicking GPX4 function could exert ferroptosis inhibitory effects at low doses (below 10 μM); while iron chelating biomimetic inhibitors required higher concentrations to be effective.

[0201] Example 16

[0202] In this embodiment, an H9c2 cell ferroptosis model was constructed using RSL3 (0.5 μM) as an inducer, and the ability of the biomimetic phospholipid ferroptosis inhibitor to clear lipid peroxides was evaluated by the concentration of intracellular lipid peroxides and their degradation product malondialdehyde (MDA).

[0203] (1) The ability of biomimetic phospholipid iron death inhibitors to scavenge lipid peroxides was evaluated using intracellular lipid peroxides as an indicator. The specific steps were as follows: H9c2 cells were cultured in confocal dishes (80,000 cells / dish). 2 μM of RSL3 and (I) free radical scavenging biomimetic inhibitors GPC-Fer (10 μM), GPC-(Fer)2 (5 μM), 18:0-Fer PC (5 μM), GPC-AA9 (10 μM), GPC-(AA9)2 (5 μM), and 18:0-AA9 PC (5 μM) were added to the culture dishes respectively; (II) three phospholipids mimicking GPX4 characteristics: GPC-Ebs (10 μM), GPC-(Ebs)2 (5 μM), and 18:0-Ebs PC (5 μM); (III) two iron-chelated phospholipids: GPC-Dfx (75 μM) and DSPE-PEG. 2000 -Dfo (300 μM), co-incubated for 6 h, then washed with PBS to remove the drug, then added the fluorescent probe Liperfluo (5 μM), washed three times with PBS after 0.5 h, and observed under a confocal laser scanning microscope (E). x =532nm, E m (535-650 nm), fluorescence intensity was quantified using ImageJ software. Results are as follows: Figure 30 As shown in AB.

[0204] (2) The ability of biomimetic inhibitors to scavenge lipid peroxides was evaluated using the content of MDA, a degradation product of lipid peroxides in cells, as an indicator. The specific steps are as follows: H9c2 cells (2 million cells / plate) were cultured in conventional culture dishes with a diameter of 10 cm. 2 μM RSL3 and various biomimetic ferroptosis inhibitors were added to the culture dishes at the same concentrations as in (1) above. After incubation in a cell culture incubator for 12 h, the MDA concentration was determined using the thiobarbituric acid colorimetric method. The results are as follows: Figure 30 As shown in C.

[0205] Experimental results showed that, using lipid peroxides and their degradation product MDA as indicators, the selected biomimetic ferroptosis inhibitors could significantly reduce intracellular lipid peroxide levels and inhibit cardiomyocyte ferroptosis.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A ferroptosis-inhibiting phospholipid-like material, characterized in that, It is one of the following structural formulas (1)-(3): ； In the structural formulas (1)-(3), R is selected from , phenoxazine or ebuseline ; In the general structural formulas (1)-(3), X is a spacer arm selected from saturated or unsaturated aliphatic chains with 0-23 carbon atoms that do not contain heteroatoms; R is connected to X by an amide bond or a covalent bond. In the general structural formulas (1)-(3), L is selected from the hydrophilic head group of phospholipids, which is phosphatidylcholine PC; In the general structural formulas (2)-(3), Y is a saturated or unsaturated aliphatic chain, and the length of the Y carbon chain is 5-25.

2. The ferroptosis-inhibiting phospholipid material according to claim 1, characterized in that: In the general structural formulas (1)-(3), X is a saturated aliphatic chain and a monounsaturated aliphatic chain, with 0-10 carbon atoms; In the general structural formulas (2)-(3), Y is a saturated aliphatic chain and a monounsaturated aliphatic chain, and the length of the Y carbon chain is 13-21.

3. The ferroptosis-inhibiting phospholipid material according to claim 2, characterized in that: In the general structural formulas (1)-(3), the number of carbon atoms in X is 4-8; In the general structural formulas (2)-(3), the length of the Y carbon chain is 15-17.

4. The ferroptosis-inhibiting phospholipid material according to claim 3, characterized in that: In the general structural formulas (1)-(3), X has 6 carbon atoms; In the general structural formulas (2)-(3), the length of the Y carbon chain is 17.

5. The ferroptosis-inhibiting phospholipid material according to claim 1, characterized in that, R is , phenoxazine The structural formula of the ferroptosis-inhibiting phospholipid material is as follows: ， Among them, 18:0-(Fer-C x )PC and 18:0-(Pnx-C x The carbon chain lengths X of the PC spacer arms are 2, 4, 6, 8, and 10, which correspond to the repeating units n = 1, 2, 3, 4, and 5 in the general formula.

6. The ferroptosis-inhibiting phospholipid material according to claim 1, characterized in that, R represents ebuselenide. At that time, the chemical structural formula of the ferroptosis-inhibiting phospholipid material is one of the following: 。 7. The use of a ferroptosis-inhibiting phospholipid material as described in any one of claims 1-6 in the preparation of a medicament for improving ferroptosis-related diseases, characterized in that: Iron death-related diseases include anthracycline-induced cardiotoxicity, myocardial infarction, platinum-based drug-induced kidney injury, acute kidney injury, iron overload, age-related macular degeneration, or osteoarthritis.

8. The application of a ferroptosis-inhibiting phospholipid material as described in any one of claims 1-6 in constructing a ferroptosis-inhibiting drug delivery system, characterized in that, The ferroptosis inhibitor can be used alone or as an excipient; the drug delivery system is a liposome, micelle, lipid nanoparticle, composite hybrid nanoparticle, scaffold, or implant coating; the encapsulated active drug molecule is one of anthracycline drugs, platinum-based drugs, or active molecules for treating neurodegenerative diseases, ophthalmic diseases, or orthopedic diseases; the route of administration is oral, intravenous, nasal, pulmonary, intra-articular, transdermal, ocular, intraperitoneal, or mucosal administration.

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