Active oxygen activated CO donor compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and particularly relates to an active oxygen activated CO donor compound as well as a preparation method and application thereof. The compound shown in the formula I releases CO under the activation of active oxygen (such as ONOO <-> and H2O2), can effectively relieve ferroptosis in vivo and in vitro, and plays a role through the mechanism of removing ROS and adjusting iron homeostasis. In addition, the compound shown in the formula I also has a protective effect on MPTP-induced PD mouse behavior defects and loss of dopaminergic neurons in a nigra region. The novel ROS-triggered CO donor is a novel method for inhibiting PD-related ferroptosis, and has a treatment potential in PD treatment.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and specifically relates to an active oxygen activated CO donor compound and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD), also known as "tremor paralysis", is a common degenerative disease of the nervous system in the elderly with characteristic motor symptoms and non-motor symptoms. It has become the most common central nervous system disease besides Alzheimer's disease (AD). Its main pathological mechanisms involve the formation and spread of Lewy bodies, mitochondrial dysfunction, oxidative stress, and regulation of neuroinflammation. Currently, the treatment of Parkinson's disease mainly focuses on increasing the activity of dopaminergic neurons. Although it can relieve symptoms, it cannot prevent the progression of the disease. Therefore, intervention strategies with therapeutic capabilities are needed.

[0003] Recent studies have demonstrated that ferroptosis is a promising therapeutic target, and ferroptosis inhibitors have shown the potential to reduce dopaminergic neuron death and slow the progression of PD. Summary of the invention

[0004] The purpose of the present invention is to provide an active oxygen activated CO donor compound and a preparation method and application thereof.

[0005] In a first aspect of the present invention, there is provided a compound of formula I, or a stereoisomer, geometric isomer, conformational isomer, tautomer, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, wherein the compound of formula I has a structure XLR 6 ,

[0006] X has the structure shown below:

[0007]

[0008] In formula X, R 1 , R 2 , R 3 , R 4 Each is independently selected from hydrogen, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxyl and amino; R 5 is selected from C1-C10 alkyl, C2-C10 alkenyl and C2-C10 alkynyl; X is connected to L at "*";

[0009] L is selected from:

[0010]

[0011] In formula L, L is respectively connected to X and R at the "*" position. 6 connected;

[0012] R 6 is selected from C1-C10 alkyl and X.

[0013] The second aspect of the present invention provides a compound of formula I described in the first aspect of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds in the preparation of a medicament for treating and / or preventing Parkinson's disease.

[0014] The third aspect of the present invention provides a use of a compound of formula I described in the first aspect of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds in the preparation of ferroptosis inhibitors.

[0015] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises (i) the compound of formula I described in the first aspect of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds, and (ii) a pharmaceutically acceptable carrier.

[0016] The present invention has the following beneficial effects:

[0017] The present invention has developed a new class of reactive oxygen species (ROS) activated carbon monoxide (CO) donor compounds, which have a simplified structure, enhanced blood-brain barrier penetration, and localized CO delivery to neurons undergoing ferroptosis. The compounds of formula I of the present invention are activated by reactive oxygen species (such as peroxynitrite ONOO- and hydrogen peroxide H 2 O 2 ) releases CO under the activation of ferroptosis, which can effectively reduce ferroptosis in vivo and in vitro, and exerts its effects by scavenging ROS and regulating iron homeostasis. In addition, the compound of formula I of the present invention also shows a protective effect on the behavioral defects and loss of dopaminergic neurons in the substantia nigra of PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This innovative ROS-triggered CO donor is a new method to inhibit PD-related ferroptosis and has therapeutic potential in the treatment of PD. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The mechanism of CO release induced by ROS is studied. (A) CO is detected by gas chromatography. (B) F-CO reacts with ONOO- or H 2 O 2 Fluorescence emission spectra before and after incubation, with an excitation wavelength of 490 nm. (C) CO release curve of F-CO after adding ONOO-, (D) CO release curve of F-CO after adding H2 O 2 CO release curve of F-CO after catalysis, recorded using a CO gas detector. (E) Selectivity of F-CO for various compounds. (F) Confirmation of the oxidation products of F-CO by high performance liquid chromatography (HPLC), (G) 1 H-NMR spectrum.

[0019] Figure 2 F-CO alleviates ferroptosis in SHSY5Y cells by scavenging ROS and releasing CO. (A) Fluorescence images of live SHSY5Y human neuroblastoma cells incubated with HK-Green-4I (ONOO-probe) under different treatments. Scale bar, 50 μm. (B) Fluorescence images of live SHSY5Y human neuroblastoma cells incubated with FerroOrange (Fe 2+ (A) Fluorescence images of live SHSY5Y cells after incubation with CO probe COP490 under different treatments. Scale bar, 50 μm. Western blot analysis of 1-methyl-4-phenylpyridinium (MPP + ) and F-CO after (D) glutathione peroxidase (GPX4), (E) divalent metal transporter 1 (DMT1) and (F) 4-hydroxy-2-nonenal (4-HNE) protein expression (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0020] Figure 3F-CO prevented MPTP-induced behavioral deficits and progressive loss of dopaminergic neurons in the substantia nigra (SN). For mice receiving various treatments on day 10: (A) Schematic diagram of the establishment of the MPTP-induced PD mouse model and F-CO administration. (B) Trajectory diagram of the elevated plus maze test (EPM), as well as (C) distance traveled in the open arms, (D) frequency of entering the open arms, and (E) total number of fecal pellets produced within 5 minutes in the novel environment. In panels A to E, data are presented as mean ± SD, n = 10, *P < 0.05, **P < 0.01, ***P < 0.001. For mice receiving various treatments on day 24: (F) rotarod test, (G) pole test, and (H) motor behavior analysis of hindlimb pinch reflex score, (n = 10, **P < 0.01, ***P < 0.001, ****P < 0.0001). (I) Immunostaining of dopaminergic neurons with tyrosine hydroxylase (TH) antibody in the SNpc of mice that received various treatments on day 26 (scale bar = 200 μm) and (J) quantification of the number of TH-positive neurons (***P < 0.001, ****P < 0.0001). Western blot analysis and quantification of (K) TH, (L) phosphorylated Ser129-α-Syn, and (M) total α-Syn protein levels in the midbrain of mice that received various treatments on day 26 (n = 3, *P < 0.05, **P < 0.01).

[0021] Figure 4 F-CO inhibits ferroptosis in PD mice neurons by regulating lipid peroxidation and iron homeostasis. Western blot analysis of (A) GPX4, (B) solute carrier family member 11 (SLC7A11), (C) long-chain acyl-CoA synthetase 4 (ACSL4), and (D) 4-HNE protein expression levels in mouse midbrain (n=3, *P<0.05, **P<0.01). 2+ Quantitative analysis of (n=5, **P<0.01, ***P<0.001). (G) Immunostaining of GPX4 and TH in substantia nigra sections of mice (scale bar=50μm). (H) Western blot analysis of transferrin receptor (TFRC), (I) DMT1, (J) nuclear receptor coactivator 4 (NCOA4) protein expression levels (n=3, *P<0.05, **P<0.01). All midbrain samples were collected from mice on day 21 after model induction. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0024] Herein, “comprising”, “including”, “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of”. For example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to be disclosed herein.

[0025] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0026] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0027] Herein, when describing embodiments or examples, it should be understood that they are not used to limit the present invention to these embodiments or examples. On the contrary, all substitutes, improvements and equivalents of the methods and materials described in the present invention can be included in the scope limited by the claims.

[0028] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0029] Unless otherwise indicated, the definitions of various groups herein are as follows:

[0030] As used herein, the term "halogen" generally refers to fluorine, chlorine, bromine and iodine; preferably fluorine, chlorine or bromine; more preferably fluorine or chlorine.

[0031] As used herein, "alkyl" refers to a straight or branched saturated hydrocarbon group, such as C1-C10 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10) refers to a straight or branched saturated hydrocarbon group containing 1-10 carbon atoms, preferably C1-C6 alkyl, C1-C4 alkyl, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, isohexyl, 3-methylpentyl or n-hexyl, etc., preferably methyl, ethyl, n-propyl, isopropyl, butyl or isobutyl. Herein, the alkyl group may be optionally substituted by 1, 2, 3 or 4 groups selected from hydroxyl, cycloalkyl, oxo, halogen, deuterium, alkoxy.

[0032] As used herein, "C1-C10 alkoxy" refers to a straight or branched alkoxy group containing 1 to 10 carbon atoms (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), "C1-C4 alkoxy", "C1-C3 alkoxy" and so on, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentoxy, isohexoxy, 3-methylpentoxy or n-hexoxy, etc., preferably methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or isobutoxy. Herein, the alkoxy group may be optionally substituted with 1, 2, 3 or 4 groups selected from alkyl, cycloalkyl, hydroxy, halogen, deuterium, alkoxy.

[0033] As used herein, "hydroxy" refers to -OH.

[0034] As used herein, "amino" refers to -NH 2 .

[0035] As used herein, as a group or part of other groups, the term "alkenyl" refers to a straight or branched hydrocarbon chain group consisting of only carbon atoms and hydrogen atoms, containing at least one double bond, connected to the rest of the molecule by a single bond. In some embodiments, the alkenyl contains 2 to 10 carbon atoms ("C2-C10 alkenyl"), preferably contains 2 to 8 carbon atoms ("C2-C8 alkenyl"), and more preferably contains 2 to 6 carbon atoms ("C2-C6 alkenyl"). Non-limiting examples of alkenyl include, but are not limited to, vinyl, propenyl, allyl, butenyl, but-1-enyl, but-2-enyl, pentenyl, penta-1-enyl, pentadienyl, penta-1,4-dienyl, etc. Unless otherwise specifically provided in this specification, alkenyl may be optionally substituted.

[0036] As used herein, as a group or as part of other groups, the term "alkynyl" refers to a straight or branched hydrocarbon chain group consisting of only carbon atoms and hydrogen atoms with one or more carbon-carbon triple bonds (-C≡C-) connected to the rest of the molecule by a single bond. In some embodiments, the alkynyl contains 2-10 carbon atoms ("C2-C10 alkynyl"), preferably contains 2 to 8 carbon atoms ("C2-C8 alkynyl"), and more preferably contains 2 to 6 carbon atoms ("C2-C6 alkynyl"). Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl and 2-butynyl, etc. Unless otherwise specifically provided in this specification, the alkynyl may be optionally substituted.

[0037] As used herein, "optional", "optional" or "optionally" means that the subsequently described event may or may not occur, and the description includes instances where the event occurs as well as instances where the event does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. "Optionally substituted with halogen" includes instances of "substituted with halogen" and instances of "not substituted with halogen", such as substituted with 0-3 halogens. It should be understood by those skilled in the art that for any group containing one or more substituents, the group does not include any sterically impractical, chemically incorrect, synthetically infeasible and / or inherently unstable substitution patterns.

[0038] As used herein, the term "substituted", whether or not preceded by the term "optionally" (i.e., equivalent to substituted or unsubstituted), refers to the replacement of one or more hydrogens of a specified group or part by a "suitable substituent". Herein, the number of substituents may be one or more, i.e., 1, 2, 3, 4, 5 or 6 or more, depending on the nature of the substituted group and the substituent. For example, when the substituent of an ethyl group is a halogen, the group may be replaced by 1, 2, 3, 4 or 5 substituents, such as trifluoroethyl, pentafluoroethyl, etc., depending on the structure of the substituted group. In some embodiments, the number of the substituents is 1, 2 or 3. In some embodiments, the number of the substituents is 1 or 2. In some embodiments, the number of the substituents is 1. It will be understood that "substituted" or "substituted by..." includes implicit conditions, i.e., such substitutions are carried out according to the allowed valence of the substituted atom, and the substitution produces a stable or chemically feasible compound, such as a compound that will not spontaneously transform, such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from the specified group, the substituent may be the same or different at each position. It will be understood by those skilled in the art that the substituent itself may be substituted if appropriate.

[0039] For the entire application, the above-mentioned "suitable substituents" should be understood to include but are not limited to the alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, cyano, hydroxyl, amino, monoalkylamino, dialkylamino, nitro, aryl, heteroaryl, cycloalkyl (e.g., cycloalkyl, cycloalkenyl, etc.), carboxyl, triphenylphosphine, azido, aldehyde, R a CO-(R a As defined in the specification), heterocyclic group, etc.; these groups as substituents, including alkyl, alkenyl, alkynyl, alkyl in haloalkyl, alkenyl in haloalkenyl, alkynyl in haloalkynyl, alkoxy, alkyl in monoalkylamino, alkyl in dialkylamino, aryl, heteroaryl, cycloalkyl and heterocyclic group themselves are also optionally substituted, for example, they can also be optionally substituted by one or more groups selected from alkyl, halogen, haloalkyl, alkoxy, hydroxyl, amino, monoalkylamino, dialkylamino, nitro, carboxyl, triphenylphosphino, azido, aryl, heteroaryl, cycloalkyl and heterocyclic group.

[0040] The compounds represented by formula I of the present invention also include solvate forms, such as hydrates, alcoholates, etc., and the solvates are also included in the scope of the present invention.

[0041] Herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0042] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts may be prepared by methods known in the art.

[0043] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the profession.

[0044] The term "pharmaceutically acceptable salts" used above also includes their solvates, and the solvates are included in the scope of the present invention. Examples of solvates include, for example, hydrates, alcoholates, and the like.

[0045] Those skilled in the art will recognize that the compounds of the present invention may contain chiral centers and as such may exist in different isomeric forms. "Isomers" as used herein refer to different compounds that have the same molecular formula but differ in arrangement and configuration of the atoms.

[0046] "Stereoisomers" refer to isomers produced by different spatial arrangements of atoms in molecules. They can be divided into cis-trans isomers and enantiomers, or they can be divided into two major categories: enantiomers and diastereomers.

[0047] "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. When appropriate, the term is used to refer to a racemic mixture. When indicating the stereochemistry of the compounds of the present invention, the conventional RS system is used to specify single stereoisomers with known relative and absolute configurations of two chiral centers (e.g., (1S, 2S)); single stereoisomers with known relative configurations but unknown absolute configurations are marked with an asterisk (e.g., (1R*, 2R*)); racemates with two letters (e.g., (1RS, 2RS) is a racemic mixture of (1R, 2R) and (1S, 2S); (1RS, 2SR) is a racemic mixture of (1R, 2S) and (1S, 2R)). "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is indicated according to the Cahn-Ingold-Prelog RS system. When the compound is enantiomerically pure, the stereochemistry at each chiral carbon can be specified by R or S. Resolved compounds whose absolute configuration is unknown can be assigned as (+) or (-) based on the direction (right-handed or left-handed) that they rotate plane polarized light at the wavelength of the sodium D line. Alternatively, the resolved compounds can be defined by the respective retention times of the corresponding enantiomeric diastereomers via chiral HPLC.

[0048] Geometric isomers can occur when a compound contains a double bond or some other feature that imparts a certain amount of structural rigidity to the molecule. If the compound contains a double bond, the substituents can be in the E or Z conformation. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent can have a cis or trans configuration.

[0049] "Conformational isomers" are isomers that differ by rotation about one or more valence bonds.

[0050] "Tautomer" refers to an isomer formed when a proton is shifted from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the present invention are intended to be encompassed within the scope of the present invention.

[0051] "Polymorph" refers to crystalline forms having the same chemical structure / composition but different spatial arrangements of the molecules and / or ions that form the crystals. The compounds of the invention may be provided as amorphous solids or crystalline solids. Freeze drying may be used to provide solid compounds of the invention.

[0052] "Solvate" refers to a physical association of a compound of the invention with one or more organic or inorganic solvent molecules. Such physical associations include hydrogen bonds. In some cases, the solvate will be able to separate, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The solvent molecules in the solvate can exist in a regular arrangement and / or a non-ordered arrangement. The solvate can contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" includes solution phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are well known in the art.

[0053] The present invention also includes all suitable isotopic variants of the compounds of the present invention or their pharmaceutically acceptable salts. Isotopic variants of the compounds of the present invention or their pharmaceutically acceptable salts are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass commonly found in nature. Isotopes that can be incorporated into the compounds of the present invention and their pharmaceutically acceptable salts include, but are not limited to, isotopes of H, C, N and O, such as 2H, 3 H. 12 C. 13 C. 14 C. 15 N. 17 O. 18 O. 35 S. 18 F. 36 C1 and 125 I. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof can be prepared by conventional techniques using appropriate isotopic variations of suitable reagents.

[0054] Herein, "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0055] As used herein, "pharmaceutically acceptable" refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing adverse biological reactions or interacting in an adverse manner with any components contained in the composition.

[0056] As used herein, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

[0057] As used herein, the terms "alleviate," "prevent," and "preventing" include reducing the likelihood that a disease or condition will occur or become worse in a patient.

[0058] As used herein, the term "treatment" and other similar synonyms include the following meanings:

[0059] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0060] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0061] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0062] (iv) alleviating the symptoms caused by the disease or condition.

[0063] As used herein, "inhibit" refers to a reduction or suppression of a particular condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0064] The "therapeutically effective amount" of the compound of formula I or pharmaceutical composition of the present invention refers to the amount of the compound of formula I or pharmaceutical composition of the present invention that can cause a biological or medical response in an individual or improve symptoms, slow down or delay the progression of a disease, or prevent a disease, etc. The "therapeutically effective amount" can be determined by the attending physician or veterinary practitioner and will vary with factors such as the compound, the disease state being treated, the severity of the disease being treated, the age and relevant health conditions of the individual, the route and form of administration, and the judgment of the attending physician or veterinary practitioner.

[0065] As used herein, "subject" refers to an animal. Preferably, the animal is a mammal. Subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0066] As used herein, the terms "administering", "administering", "dosing", etc. refer to methods that can deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used for the compounds and methods described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

[0067] It will also be appreciated by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g. tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable amino, amidino and guanidino protecting groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable sulfhydryl protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, etc. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

[0068] Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, TW and PGM Wuts, Protective Groups in OrganiSynthesis, (1999), 4th Ed., Wiley. The protecting group can also be a polymer resin.

[0069] The compound of formula I of the present invention is a carbon monoxide (CO) donor compound that can be activated by reactive oxygen species (ROS), can penetrate the blood-brain barrier, and deliver CO to neurons undergoing ferroptosis, and can effectively treat Parkinson's disease. Therefore, the present invention provides a compound of formula I, or a stereoisomer, geometric isomer, conformational isomer, tautomer, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, wherein the compound of formula I has a structure XLR 6 ,in:

[0070] X has the structure shown below:

[0071]

[0072] In formula X, R 1 , R 2 , R 3 , R 4 Each is independently selected from hydrogen, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxyl and amino; R 5 is selected from C1-C10 alkyl, C2-C10 alkenyl and C2-C10 alkynyl; X is connected to L at "*";

[0073] L is selected from:

[0074]

[0075] In formula L, L is respectively connected to X and R at the "*" position. 6 connected;

[0076] R 6 is selected from C1-C10 alkyl and X.

[0077] In some embodiments, R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, halogen and C1-C10 alkyl. 1 , R 2 , R 3 , R 4 Each is independently selected from hydrogen, fluorine and C1-C6 alkyl (eg methyl).

[0078] In some embodiments, R 5 Selected from C1-C10 alkyl.

[0079] In some embodiments, the compound of formula I is selected from the group consisting of:

[0080]

[0081] The present invention also provides a pharmaceutical composition, comprising (i) a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, and (ii) a pharmaceutically acceptable carrier.

[0082] Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, corn oil, salad oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween, gum arabic, xanthan gum, tragacanth gum), wetting agents (such as sodium lauryl sulfate), colorants, flavorings (such as vanillin, sweet glucose), stabilizers, antioxidants, preservatives (such as sorbic acid, benzoic acid), pyrogen-free water, etc. In some embodiments, the carrier is selected from one or more of vegetable oil, soybean oil, corn oil, salad oil, gum arabic, xanthan gum, tragacanth gum, sorbic acid, benzoic acid, vanillin, sweet glucose, colloidal silicon dioxide, starch, hydroxypropyl cellulose, lactose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, povidone K-30, polyvinyl pyrrolidone, sodium saccharin, sodium lauryl sulfate, sodium starch glycolate, talc and titanium dioxide.

[0083] In some embodiments, the carrier includes (but is not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvant, and combinations thereof. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0084] Suitable carriers can be selected according to different routes of administration and dosage forms. For example, for solid dosage forms, suitable carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and white clay, etc. Carriers suitable for liquid dosage forms include: sterile water, polyethylene glycol, nonionic surfactants, edible oils (such as corn oil, soybean oil, salad oil, peanut oil and sesame oil), emulsifiers (such as gum arabic, xanthan gum, tragacanth gum). In some embodiments, the liquid dosage form contains a preservative (such as sorbic acid, benzoic acid). Preferably, the liquid dosage form also contains a flavoring agent (such as vanillin, sweet glucose).

[0085] The pharmaceutical composition of the present invention can be prepared into suitable dosage forms, including but not limited to injections, capsules, emulsions, tablets, pills, powders and granules.

[0086] The present invention has no particular limitation on the administration method of the compound or pharmaceutical composition. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

[0087] In some embodiments, the pharmaceutical composition of the present invention also includes other drugs for the treatment of Parkinson's disease. Exemplarily, other drugs can be selected from one or more of compound levodopa (dopasil, carbodopa), dopamine receptor agonists (pramipexole, piribedil, rotigotine), anticholinergics (benzhexol), catechol-o-methyltransferase inhibitors (entacapone) and MAO-B inhibitors (selegiline, rasagiline).

[0088] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds in the preparation of drugs for treating and / or preventing Parkinson's disease.

[0089] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds in the preparation of ferroptosis inhibitors.

[0090] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds, or the pharmaceutical composition of the present invention in inhibiting cell ferroptosis.

[0091] The present invention also provides a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing ONOO in cells undergoing ferroptosis. - and / or Fe 2+ Application of concentration.

[0092] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in improving the level of tyrosine hydroxylase in cells undergoing ferroptosis.

[0093] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or its pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition of the present invention in improving dopaminergic neuron loss. In some embodiments, the dopaminergic neurons are dopaminergic neurons in the substantia nigra compacta in Parkinson's disease.

[0094] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds, or the pharmaceutical composition of the present invention in reducing the level of α-synuclein in the brain of individuals with Parkinson's disease.

[0095] The present invention also provides use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in increasing the expression level of SLC7A11 in individuals with Parkinson's disease.

[0096] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in increasing the expression level of GPX4 in individuals suffering from Parkinson's disease.

[0097] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of ACSL4 in individuals with Parkinson's disease.

[0098] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of 4-HNE in individuals with Parkinson's disease.

[0099] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of MDA in individuals suffering from Parkinson's disease.

[0100] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of TFRC in individuals with Parkinson's disease.

[0101] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of DMT1 in individuals with Parkinson's disease.

[0102] The present invention also provides the use of a compound of formula I of the present invention, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention in reducing the expression level of NCOA4 in individuals with Parkinson's disease.

[0103] The present invention also provides the use of the compound of formula I of the present invention, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds, or the pharmaceutical composition of the present invention in inhibiting oxidative stress and / or lipid peroxidation in brain tissue of individuals with Parkinson's disease.

[0104] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0105] In one or more embodiments, the cell is a neural cell.

[0106] The present invention also provides a method for treating Parkinson's disease, which comprises administering a therapeutically effective amount of the compound of formula I of the present invention or the pharmaceutical composition of the present invention to an individual suffering from Parkinson's disease.

[0107] Preparation Example 1: Synthesis of F-CO, H-CO and Me-CO

[0108] 1. The present invention provides a synthetic route of compound F-CO:

[0109]

[0110] (1) Synthesis of compound 2-1: 4-fluorophenol (3.0 g, 26.76 mmol, 1.0 equivalent) was weighed into a dried two-necked flask, 30 mL of anhydrous dichloroethane was taken to dissolve 4-fluorophenol, methyl pyruvate (3.28 g, 32.11 mmol, 1.2 equivalent) was added, argon was protected, the gas was vented three times, titanium tetrachloride (5.58 g, 29.44 mmol, 1.1 equivalent) was added dropwise under ice bath, the solution was brown viscous liquid, reacted at 0-25°C for 6 h, water was added to quench the reaction, the solution turned yellow. The organic phase was extracted with water and dichloromethane three times, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude product, which was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 20:1, V:V) to obtain 2.02 g of light yellow oily liquid, namely compound 2-1, with a yield of 35%.

[0111] (2) Synthesis of compound 2-2: Compound 2-1 (1.50 g, 7.00 mmol, 1.0 equivalent) was weighed into a 100 ml round-bottom flask with a stirrer, 15 ml of toluene was extracted to fully dissolve the compound, and 1 ml of trifluoroacetic acid was added, and the mixture was reacted at 90°C for 4 h. After cooling to room temperature, most of the trifluoroacetic acid was removed with saturated sodium bicarbonate, and the mixture was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 1.15 g of a white solid, namely compound 2-2, with a yield of 90%.

[0112] (3) Synthesis of compound F-CO: Compound 2-2 (0.50 g, 2.74 mmol, 1.0 equivalent) was weighed into a 100 mL round-bottom flask with a stirrer, 10 mL of anhydrous dichloromethane was drawn to fully dissolve the compound, pyridine (0.22 g, 2.74 mmol, 1.0 equivalent) and DMPA (0.40 g, 3.29 mmol, 1.2 equivalent) were added, and monomethyl oxalyl chloride (0.67 g, 5.49 mmol, 2.0 equivalent) was slowly added dropwise under argon protection and ice bath conditions. The mixture was heated to room temperature for 2 h. After the raw materials were completely reacted, water was added to quench the reaction, and the mixture was extracted with water and dichloromethane three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 0.63 g of pure white solid, i.e., compound F-CO, with a yield of 85%.

[0113] 1 H NMR (400 MHz, CDCl 3 )δ7.15–7.11(m,2H),7.07(dd,J=6.9,2.1Hz,1H),3.91(s,3H),1.87(s,3H). 13C NMR (150 MHz, CDCl 3 )δ151.25,148.32,127.98,110.11,107.25,98.53,44.35,43.85,27.94,12.68. 19 F NMR (377 MHz, CDCl 3 )δ-116.14.ESI-HRMS,m / z,[M+Na] + ,calcd.for C 12 H 9 FO 6 Na + ,291.0275;found291.0282.

[0114] 2. The present invention provides a synthetic route for compound H-CO:

[0115]

[0116] (1) Synthesis of compound 2-3: Phenol (3.0 g, 31.88 mmol, 1.0 equivalent) was weighed into a dried two-necked flask, 30 mL of ultra-dry dichloroethane was extracted to dissolve the phenol, and then methyl pyruvate (3.91 g, 38.25 mmol, 1.2 equivalent) was added. The mixture was ventilated three times under argon protection, and titanium tetrachloride (6.65 g, 35.06 mmol, 1.1 equivalent) was added dropwise under ice bath. The solution was a brown viscous liquid. The reaction was continued for 2 h, and water was added to quench the reaction. The solution turned yellow. The mixture was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 20:1, V:V) to obtain 4.68 g of a light yellow oily liquid, namely compound 2-3, with a yield of 75%.

[0117] (2) Synthesis of compound 2-4: Compound 2-3 (1.50 g, 7.65 mmol, 1.0 equivalent) was weighed into a 100 mL round-bottom flask with a stirrer, 15 mL of toluene was drawn to fully dissolve the compound, and 1 mL of trifluoroacetic acid was added, and the mixture was reacted at 90°C for 4 h. After cooling to room temperature, most of the trifluoroacetic acid was removed with saturated sodium bicarbonate, and the mixture was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 1.16 g of a white solid, namely compound 2-4, with a yield of 92%.

[0118] (3) Synthesis of compound H-CO: Weigh compound 2-4 (0.50 g, 3.05 mmol, 1.0 equivalent) into a 100 mL round-bottom flask with a stirrer, extract 10 mL of anhydrous dichloromethane to fully dissolve the compound, add pyridine (0.24 g, 3.05 mmol, 1.0 equivalent) and DMPA (0.45 g, 3.65 mmol, 1.2 equivalent), and add monomethyl oxalyl chloride (0.75 g, 6.09 mmol, 2.0 equivalent) dropwise under argon protection and ice bath conditions. Warm to room temperature for 2 h. After the raw materials react completely, add water to quench the reaction, extract with water and dichloromethane three times, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate the solvent to obtain a crude product. Separate by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 0.69 g of pure white solid, i.e., compound H-CO, with a yield of 90%.

[0119] 1 H NMR (400 MHz, CDCl 3 )δ7.42(t,J=7.9Hz,1H),7.34(d,J=7.5Hz,1H),7.22–7.14(m,2H),3.89(s,3H),1.87(s,3H). 13 C NMR (100 MHz, CDCl 3 )δ172.44,156.67,155.50,153.56,131.70,125.72,125.11,123.40,111.69,77.37,54.05,23.57.ESI-HRMS,m / z,[M+K] + ,calcd.for C 12 H 10 O 6 K + ,289.0109; found 289.0113.

[0120] 3. The present invention provides a synthetic route for the compound Me-CO:

[0121]

[0122] (1) Synthesis of compound 2-5: Weigh 4-methylphenol (3.0 g, 27.74 mmol, 1.0 equivalent) into a dried two-necked flask, extract 10 mL of ultra-dry dichloroethane to dissolve phenol, then add methyl pyruvate (3.40 g, 33.29 mmol, 1.2 equivalent), purify with argon, ventilate three times, and drop titanium tetrachloride (5.79 g, 30.52 mmol, 1.1 equivalent) in an ice bath. After reacting for 2 hours, the solution becomes a brown viscous liquid. Water is added to quench the reaction, and the solution turns yellow. Extract with water and dichloromethane three times, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate to remove the solvent to obtain a crude product. After separation by column chromatography (solvent system: petroleum ether: ethyl acetate = 20:1, V:V), 4.65 g of light yellow oily liquid, namely compound 2-5, is obtained with a yield of 80%.

[0123] (2) Synthesis of compound 2-6: Compound 2-5 (1.50 g, 7.14 mmol, 1.0 equivalent) was weighed into a 100 mL round-bottom flask with a stirrer, 15 mL of toluene was drawn to fully dissolve the compound, and 1 mL of trifluoroacetic acid was added, and the mixture was reacted at 90°C for 4 h. After cooling to room temperature, most of the trifluoroacetic acid was removed with saturated sodium bicarbonate, and the mixture was extracted three times with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 1.08 g of a white solid, namely compound 2-6, with a yield of 85%.

[0124] (3) Synthesis of compound H-CO: Compound 2-6 (0.50 g, 2.81 mmol, 1.0 equivalent) was weighed into a 100 mL round-bottom flask with a stirrer, 10 mL of anhydrous dichloromethane was drawn to fully dissolve the compound, pyridine (0.22 g, 2.81 mmol, 1.0 equivalent) and DMPA (0.41 g, 3.37 mmol, 1.2 equivalent) were added, and monomethyl oxalyl chloride (0.69 g, 5.61 mmol, 2 equivalent) was added dropwise under argon protection and ice bath conditions. The mixture was heated to room temperature for 2 h. After the raw materials were completely reacted, water was added to quench the reaction, and DCM / H2O was extracted three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude product. The crude product was separated by column chromatography (solvent system: petroleum ether: ethyl acetate = 50:1, V:V) to obtain 0.66 g of pure white solid, i.e., compound Me-CO, with a yield of 89%.

[0125] 1 H NMR (400 MHz, CDCl 3 )δ7.20(dd,J=8.1,1.9Hz,1H),7.13(d,J=1.8Hz,1H),7.04(d,J=8.2Hz,1H),3.89(s,3H),2.34(s,3H),1.85(s,3H).13 C NMR (100 MHz, CDCl 3 )δ172.69,156.65,155.41,151.36,134.88,132.01,125.43,123.69,111.19,77.99,53.94,23.49,21.08.ESI-HRMS,m / z,[M+K] + ,calcd.for C 13 H 12 O 6 K + ,303.0265; found 303.0272.

[0126] Preparation Example 2: Synthesis of F-2CO, H-2CO and Me-2CO

[0127] 1. The present invention provides a synthetic route of compound F-2CO:

[0128]

[0129] Synthesis of compound F-2CO: Weigh compound 2-2 (0.50 g, 2.74 mmol, 1.0 equivalent) in a 100 mL round-bottom flask with a stirrer, extract 10 mL of anhydrous dichloromethane to fully dissolve the compound, add pyridine (0.22 g, 2.74 mmol, 1.0 equivalent) and DMPA (0.40 g, 3.29 mmol, 1.2 equivalent), and slowly drop oxalyl chloride (0.60 g, 1.65 mmol, 0.6 equivalent) under argon protection and ice bath conditions. Warm to room temperature for 2 hours. After the raw material reacts completely, add water to quench the reaction, extract with water and dichloromethane three times, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate the solvent, recrystallize with dichloromethane and petroleum ether to obtain a crude product, separate by column chromatography (pure dichloromethane), and obtain 0.8 g of pure white solid, i.e., compound F-2CO, with a yield of 70%.

[0130] 1 H NMR (400MHz, DMSO) δ7.50 (dd, J=7.7, 2.6Hz, 2H), 7.43–7.30 (m, 4H), 1.84 (s, 6H). 13 C NMR (600MHz, DMSO) δ172.76,160.05,158.50,153.88,149.23,126.64,118.29,112.88,78.04,22.80. 19 F NMR(377MHz,DMSO)δ-117.28.ESI-HRMS,m / z,[M+Na] + ,calcd.forC20 H 12 F 2 O 8 Na + ,441.0392; found 441.0399.

[0131] 2. The present invention provides a synthetic route for compound H-2CO:

[0132]

[0133] Synthesis of compound H-2CO: Weigh compound 2-4 (1.00 g, 6.09 mmol, 1.0 equivalent) in a 100 mL round-bottom flask with a stirrer, extract 15 mL of anhydrous dichloromethane to fully dissolve the compound, add pyridine (0.48 g, 6.10 mmol, 1.0 equivalent) and DMPA (0.90 g, 7.30 mmol, 1.2 equivalent), and slowly drop oxalyl chloride (0.46 g, 3.65 mmol, 0.6 equivalent) under argon protection and ice bath conditions. Warm to room temperature for 2 hours. After the raw material reacts completely, add water to quench the reaction, extract with water and dichloromethane three times, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate the solvent, recrystallize with dichloromethane and petroleum ether to obtain a crude product, separate by column chromatography (pure dichloromethane), and obtain 2.0 g of pure white solid, i.e., compound H-2CO, with a yield of 86%.

[0134] 1 H NMR (400MHz, DMSO) δ7.63–7.43(m,4H),7.35–7.19(m,4H),1.86(s,6H). 13 C NMR(600MHz,DMSO)δ178.45,157.73,155.62,130.18,127.92,125.85,122.06,95.68,21.78.ESI-HRMS,m / z,[M+Na] + ,calcd.for C 20 H 14 O 8 Na + ,405.0581; found 405.0585.

[0135] 3. The present invention provides a synthetic route for the compound Me-2CO:

[0136]

[0137] Synthesis of compound Me-2CO: Weigh compound 2-6 (1.00 g, 5.61 mmol, 1.0 equivalent) in a 100 mL round-bottom flask with a stirrer, extract 15 mL of anhydrous dichloromethane to fully dissolve the compound, add pyridine (0.44 g, 5.62 mmol, 1.0 equivalent) and DMPA (0.82 g, 6.74 mmol, 1.2 equivalent), and slowly drop oxalyl chloride (0.43 g, 3.37 mmol, 0.6 equivalent) under argon protection and ice bath conditions. Warm to room temperature for 2 hours. After the raw materials react completely, add water to quench the reaction, extract with water and dichloromethane three times, dry the organic phase over anhydrous sodium sulfate, filter, and evaporate the solvent, recrystallize with dichloromethane and petroleum ether to obtain a crude product, separate by column chromatography (pure dichloromethane), and obtain 1.8 g of pure white solid, i.e., compound H-2CO, with a yield of 78%.

[0138] 1 H NMR (400 MHz, CDCl 3 )δ7.22(ddd,J=8.3,1.9,0.9Hz,2H),7.14(d,J=1.9Hz,2H),7.04(d,J=8.2Hz,2H),2.37(s,6H),1.79(s,6H). 13 C NMR (100 MHz, CDCl 3 )δ172.56,153.92,151.28,135.06,132.16,124.94,123.97,111.10,111.69,78.39,23.34,21.08.ESI-HRMS,m / z,[M+Na] + ,calcd.for C 22 H 18 O 8 Na + ,433.0899; found 433.0900.

[0139] Peroxynitrite (ONOO -) Solution preparation: prepare acidic hydrogen peroxide solutions, wherein the hydrochloric acid concentration is 0.7M, the hydrogen peroxide concentration is 0.6M (mix 14.5mL concentrated hydrochloric acid with 17.0mL of 30% hydrogen peroxide, and dilute to 250mL with deionized water); 0.6M sodium nitrite solution (10.4g sodium nitrite is made into 250mL aqueous solution) and 3M sodium hydroxide solution (30g sodium hydroxide is made into 250mL aqueous solution). Take 10mL of the above three solutions respectively in a small measuring cylinder, and then add the three solutions to a 100mL beaker while stirring under ice bath conditions to obtain a golden transparent peroxynitrite solution. The solution concentration needs to be diluted with a sodium hydroxide aqueous solution with a pH of 12, and then calibrated by ultraviolet spectrophotometry, according to the molar extinction coefficient of peroxynitrite at 302nm is 1670M -1 cm -1 .

[0140] Herein, the PBS solution is a PBS aqueous solution (pH=7.4, 10 mM).

[0141] In this article, ONOO - PBS solution was prepared by mixing the above-prepared ONOO - The aqueous solution was diluted to the required concentration using PBS buffer.

[0142] In this paper, H 2 O 2 The treatment solution is H 2 O 2 of PBS solution.

[0143] In this paper, CO fluorescent probe COP490 was commercially available.

[0144] Example 1: CO Donor to ONOO - Response to CO release

[0145] Test method: A CO electrochemical detector was used to detect the generation of CO after the reaction between the donor compound and ROS. A 20 mL glass bottle was prepared containing CO donor (40 μM) and ONOO - A 10 mL PBS solution of (40 μM) was prepared, a magnetic particle was added, and the CO detector reading was recorded over time.

[0146] Table 1: Kinetics and total amount evaluation of CO release by donor compounds in response to reactive oxygen species

[0147] CO Donor Name CO release (ppm) CO release time (min) F-CO 202 130 H-CO 57 240 Me-CO 38 200 F-2CO 286 60 H-2CO 88 160 Me-2CO 68 180

[0148] Example 2: Study on the mechanism of CO release activated by reactive oxygen species

[0149] To verify the ROS-induced CO release of F-CO, all measurements were performed at 25 °C (e.g. Figure 1 shown).

[0150] (A) Prepare 10 mL of F-CO (100 μM) and ONOO- (100 μM) or H 2 O 2 A PBS solution (10 mM, pH = 7.4, containing 30% MeCN) (400 μM) was placed in a 15 mL two-way tube connected to a GC2060 gas chromatograph (Shanghai Ruimin Instrument Co., Ltd.) equipped with a 5A molecular sieve column (column length 2 m, column diameter 3 mm). The solution was stirred for 30 minutes and then gas injection was performed. The released carbon monoxide (CO) was recorded and analyzed by gas chromatography (GC). Two peaks were observed with retention times of 1.96 minutes and 3.86 minutes, corresponding to oxygen and carbon monoxide (CO), respectively ( Figure 1 A).

[0151] (B) CO release analysis using CO fluorescent probe COP490. A mixture containing CO fluorescent probe COP490 (5 μM), PdCl2 (10 μM), and F-CO (5 μM) was incubated with ONOO- (10 μM) or H 2 O 2 Fluorescence emission spectra before and after incubation with 50 μM COP490, with an excitation wavelength of 490 nm. In the presence of F-CO, COP490 showed only weak fluorescence. However, in the presence of ONOO- or H 2 O 2 After 30 min of incubation, the fluorescence intensity of COP490 increased significantly, indicating CO release ( Figure 1 B).

[0152] (C) PBS mixed solution (10 mL) of F-CO (40 μM) and ONOO- (40 μM), or (D) F-CO (40 μM) and H 2 O 2 The mixed solution of PBS (200 μM) was placed in a 400 ml glass jar, and the CO concentration in the glass jar was monitored by an electrochemical detector and a release curve was prepared. It was observed that the CO concentration gradually increased until it reached a plateau, indicating that F-CO and ONOO-( Figure 1 C) or H 2 O 2 ( Figure 1 D) The reaction produces CO.

[0153] (E) F-CO (10 μM) and PBS solution (10 mL) of peroxynitrite / hydrogen peroxide / hypochlorous acid / superoxide anion / singlet oxygen / glutathione / cysteine / homocysteine / calcium chloride / zinc chloride / sodium chloride / magnesium chloride / potassium chloride (40 μM) were prepared respectively and placed in a 400 mL glass jar. After reacting at 25°C for 60 min, the CO electrochemical detector reading was read to evaluate the selectivity of F-CO for the above-mentioned various biologically related compounds. The results showed that only ONOO - and H 2 O 2 Effectively trigger F-CO to release CO ( Figure 1 E).

[0154] (F) Dissolve 2 mg of F-CO in 10 ml of MeCN and add 10 ml of 1 mM ONOO - or 10 mL of 1 mM H 2 O 2 , react for 30 minutes. The obtained solution was subjected to HPLC analysis, and it was found that the F-CO peak disappeared and a new peak appeared, corresponding to 5'-fluoro-2'-hydroxyacetophenone ( Figure 1 F).

[0155] (G) Dissolve 2 mg of F-CO in 10 ml of MeCN, add 10 ml of 1 mM concentration of ONOO- and react for 30 minutes. Extract the reaction solution three times with water and dichloromethane, collect the organic phase, dry it, and spin dry it. Take 4 mg and dissolve it in deuterated chloroform, collect it 1 H-NMR spectra, and the commercially available 5'-fluoro-2'-hydroxyacetophenone 1 H-NMR spectrum (deuterated chloroform as solvent). By comparison, it was found that the product of the reaction between F-CO and ROS was indeed 5'-fluoro-2'-hydroxyacetophenone ( Figure 1 G).

[0156] Example 3: F-CO inhibits ferroptosis in neuroblastoma SH-SY5Y cells

[0157] After passing through the blood-brain barrier, the neurotoxin 1-methyl-4-phenylpyridine (MPP + ) specifically damages dopaminergic neurons and induces ferroptosis, leading to pathological changes similar to those of Parkinson's disease (PD). + (3 mM) for 24 h to establish an in vitro PD cell model for studying the potential therapeutic benefits of F-CO as a ferroptosis inhibitor. All drug incubation experiments were carried out in a normoxic incubator at 37°C.

[0158] Intracellular ONOO - and Fe2+ Fluorescence imaging of the blank group: no drug stimulation; MPP+ group: SH-SY5Y cells and MPP + (3 mM) for 24 h; MPP+ and F-CO treatment groups: SH-SY5Y cells were first incubated with F-CO (10 μM) for 2 h, followed by the addition of MPP + (3mM) and continued incubation for 24 hours. Subsequently, the cells were incubated with HK-Green-4I (10μM) or FerroOrange (1μM) probes at 37°C for 30 minutes. Fluorescence images were taken by confocal microscopy (Stellaris, Leica, Wetzlar, Germany). HK-Green-4I (Catalog number: 1448821-82-6, MedChemExpress, New Jersey, USA). FerroOrange (Catalog number: F374, Dojindo, Japan). MPP + Significantly increased intracellular ONOO - and Fe 2+ The concentration of , respectively, was increased by ONOO-probe HK-Green 4i ( Figure 2 A) and Fe 2+ Probe FerroOrange( Figure 2 B) The fluorescence intensity increase was confirmed. F-CO significantly reduced the ONOO - and Fe 2+ The concentration ( Figure 2 A and 2B), indicating its effect on MPP + Induced ferroptosis has a protective effect.

[0159] CO fluorescent probe COP-490 was used to evaluate the concentration of carbon monoxide (CO) released into cells. Cell group settings: blank group: no drug stimulation; MPP+ group: SH-SY5Y cells and MPP + (3 mM) for 24 h; MPP+ and F-CO treatment groups: SH-SY5Y cells were first incubated with F-CO (10 μM) for 2 h, followed by the addition of MPP + (3 mM) and continued incubation for 24 h; MPP+, F-CO and FeTPPs groups: SH-SY5Y cells were first incubated with F-CO (10 μM) for 2 h, and then MPP was added + (3 mM) and FeTTPs (10 μM, an ONOO- scavenger) were incubated for 24 h; in the CORM-3 group, cells were incubated with CORM-3 (10 μM) for 30 min. 2(2 μM) was incubated at 37°C for 30 min. Confocal microscopy (Stellaris, Leica, Wetzlar, Germany) was then used to visualize the intracellular fluorescence in living cells. + The release of CO in SH-SY5Y cells under the action of β-actin was reflected in the significant increase in the fluorescence intensity of COP490, which was comparable to that of CORM-3, a known CO-releasing molecule ( Figure 2 C). However, FeTPPs, a known ONOO-decomposition catalyst, significantly suppressed the increase in CO release, indicating that F-CO promotes CO release in response to cellular oxidative stress.

[0160] Western blot analysis was then performed to confirm several key features of ferroptosis. The experimental method was as follows: MPP+ group: SH-SY5Y cells and MPP + (3 mM) for 24 h; MPP+ and F-CO treatment groups: SH-SY5Y cells were first incubated with F-CO (10 μM) for 2 h, followed by the addition of MPP + (3mM) and continued incubation for 24 hours. Whole cells of different groups were lysed with RIPA buffer (Catalog No.: WB3100, NCM Biotech, China), and 1% protease inhibitor cocktail (Catalog No.: 491248, VicMed, China) and 1% phosphatase inhibitor (Catalog No.: PR20015, Proteintech, USA) were added to the lysate. Protein concentration was determined using a BCA protein quantification kit (Catalog No.: WB6501A, NCM Biotech, China). Equal amounts of total protein were separated by 7.5% or 12.5% ​​sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with primary antibodies against the following target proteins: GPX4 (1:5000, Abcam, USA), DMT1 (1:2000, ABclonal, China), 4-HNE (1:1000, Thermo Fisher Scientific, China). The membrane was then incubated with secondary antibodies for 1-2 hours, including goat anti-rabbit IgG (1:8000, Proteintech, USA) and goat anti-mouse IgG (1:8000, Proteintech, USA) at room temperature. Protein bands were developed using the ChemiDoc touch system (Bio-Rad, USA), and protein signals were normalized to the internal reference protein α-tubulin signal.

[0161] From the experimental results, cells and MPP + (3 mM) for 24 hours, GPX4 was downregulated in cells ( Figure 2 D) DMT1 upregulation ( Figure 2 E) and 4-HNE rise ( Figure 2 F) Prove MPP + Indeed, ferroptosis was induced in SH-SY5Y cells. SH-SY5Y cells were first incubated with F-CO (10 μM) for 2 h, followed by the addition of MPP + (3 mM) for 24 h significantly inhibited ferroptosis, as shown by the + Compared with the control group, the GPX4 level increased, and the DMT1 and 4-HNE levels decreased ( Figure 2 DF). Taken together, these results indicate that F-CO inhibits ferroptosis in SH-SY5Y cells by scavenging ROS and releasing CO.

[0162] Example 4: Evaluation of the activity of F-CO in treating Parkinson's disease in mice

[0163] MPTP easily crosses the blood-brain barrier (BBB) ​​and is converted to MPP+, a potent neurotoxin that disrupts mitochondrial function in dopaminergic neurons. This mechanism makes MPTP a widely used tool for creating Parkinson's disease (PD) mouse models that exhibit significant movement disorders, α-synuclein accumulation, and a significant loss of dopaminergic neurons. In this article, a PD mouse model was established by daily intraperitoneal injection of MPTP (30 mg / kg) on ​​days 1, 2, 3, 4, and 5 for 5 days. Subsequently, F-CO (6 mg / kg) was intravenously injected for treatment on days 1, 2, 3, 4, 5, 7, 9, and 11. For example, a mouse weighing 25 g needs to be injected with 250 uL of MPTP (3 mg / mL) aqueous solution each time for modeling and administration, and injected with F-CO aqueous solution (0.6 mg / mL) for treatment to evaluate its efficacy in preventing early PD in vivo ( Figure 3 A) MPTP-induced PD mice effectively reproduce various movement disorder symptoms of Parkinson's disease. To evaluate whether F-CO can alleviate the behavioral deficits of these MPTP-induced early PD mice, a series of behavioral tests were performed within 3 weeks after model induction.

[0164] The specific method of behavioral testing of Parkinson's disease mouse model is as follows:

[0165] Figure 3B, C, D, elevated plus maze (EPM) test, assessing anxiety-like behavior, which is a standard method for assessing anxiety in rodents. The apparatus consists of a four-arm maze (length 35 cm, width 5 cm, height 15 cm), which is 50 cm above the ground. Experimental procedures: Each mouse was placed individually in the central area of ​​the maze, facing an open arm. Animal behavior was recorded for 5 minutes using the ANY-maze video imaging system as described in the literature. Two main indicators were analyzed: Travel distance: the total distance traveled by the mouse in each arm of the maze. Open arm entry frequency: the number of times the mouse entered the open arm.

[0166] Figure 3 E, fecal pellet excretion in a new environment, is used to assess the amount of fecal pellets excreted by mice in a new environment and is a commonly used indicator for assessing stress and anxiety in rodents. Before the experiment, mice were adapted to a standard home cage for at least two weeks to ensure that they were familiar with the environment. On the day of the experiment, each mouse was transferred individually from its home cage to a clean, empty cage (new environment). The new cage was the same size and shape as the home cage, but without any bedding or familiar cues. The mice were allowed to rest in the new cage for 5 minutes without any restraint. After a 5-minute observation period, the number of all fecal pellets in the new cage was counted. This number reflects the number of fecal pellets produced by mice under the stress of a new environment. The number of fecal pellets produced by each mouse in the new environment was recorded and statistically analyzed.

[0167] Figure 3 F, Rotarod test, the test evaluates the motor coordination of mice. Before MPTP administration, all mice were trained on the rotarod apparatus, and the training conditions were an average speed of 10rpm for 10 minutes. This training regimen was performed twice a day for 3 consecutive days. On the day of the experiment, the rotarod test was performed at a uniformly accelerated speed, ranging from 0 to 45rpm, for 2 minutes, with a total time of 300 seconds, and the latency of the mouse to fall was recorded.

[0168] Figure 3 G, Pole test, assesses the motor coordination of mice. The apparatus consists of a wooden pole with a height of 50-100 cm and a diameter of 0.5 cm. A wooden ball is placed on the top of the pole as a starting platform. The pole is wrapped with gauze to prevent slipping, and the bottom is covered with padding to protect the mice from injury. All mice were pre-trained at least three times before the experiment until they could consistently show the behavior of turning their heads downward and getting down to the bottom of the pole when placed on the wooden ball. In the pole test, the total time required for the mouse to get from the top of the pole to the bottom of the pole was recorded.

[0169] Figure 3H, hind limb clamping test score, reflecting the function of the striatum. The mouse was gently lifted, the middle of the tail was grasped, and observed for 10 seconds. A scoring system of 0, 1, 2 or 3 points was assigned according to the degree of inward clamping of the hind limbs. 0 points means no clamping, given to mice that can move freely and stretch both hind limbs. 1 point means that the mouse clamped one hind limb or partially clamped both hind limbs during the restriction period. 2 points means that the mouse clamped both hind limbs inward during most of the observation period, but still showed some flexibility. 3 points means that the mouse showed complete paralysis of the hind limbs, clamped inward immediately and had no flexibility.

[0170] Compared with the control group, MPTP-induced PD mice showed significantly reduced exploratory behavior in the open arms of the elevated plus maze ( Figure 3 B), which is achieved by the open arm travel distance ( Figure 3 C) and the number of open arm entries ( Figure 3 In addition, these mice produced significantly less total feces within 5 minutes of exposure to the novel environment ( Figure 3 E), reflecting the tendency of constipation and depression. In the F-CO treatment group, the EPM test showed that the distance traveled in the open arm ( Figure 3 C) and the frequency of entering the open arm ( Figure 3 D) was significantly higher than that of the MPTP-treated group. In addition, after F-CO treatment, the total number of feces discharged increased significantly ( Figure 3 E) These results collectively indicate that F-CO alleviates movement disorders and anxiety in PD mice.

[0171] On the 19th day after the model was successfully induced (i.e. Figure 3 On day 24 of A), the motor function of mice was assessed by three tests, namely the rotarod test, the pole test, and the hindlimb grasping reflex. MPTP treatment resulted in impaired behavior, as evidenced by a shortened latency in the rotarod test ( Figure 3 F) The mice's sliding time was prolonged in the pole test ( Figure 3 G), and hindlimb grasp test scores significantly improved ( Figure 3 H), all of which indicate striatal dysfunction. In contrast, F-CO treatment reversed these MPTP intoxication-induced impairments. F-CO-treated mice showed prolonged latency in the rotarod test compared with the PD group ( Figure 3 F), and the time to descend the pole is shortened ( Figure 3 G). In addition, F-CO treated mice showed significant improvement in hind limb grasping reflex ( Figure 3 H) The improvements observed in F-CO treated mice suggest that F-CO has a positive effect on motor function and striatal dysfunction that can offset the adverse effects of MPTP.

[0172] Motor deficits in PD are closely associated with the aggregation of α-synuclein. Dopaminergic neurons in the substantia nigra pars compacta are particularly susceptible to the deleterious effects of αSyn aggregation, which exacerbates motor dysfunction. To investigate whether F-CO restores behavioral deficits by ameliorating MPTP-induced neuronal damage in the early stages of PD, midbrain and cerebral tissue sections at day 21 (i.e., day 26) after model induction were analyzed. Immunofluorescence staining of tyrosine hydroxylase (TH) was performed in the SNpc to identify dopamine neurons. To analyze the expression of TH and GPX4 in the substantia nigra, especially the pars compacta, in Parkinson's mice, 20 μm sagittal brain sections were cut using a freezing microtome and subjected to floating immunofluorescence staining. Brain sections were baked at 60°C for 1 hour and subsequently hydrated in PBS (pH 7.4) overnight. To reduce autofluorescence, sections were treated in a PBS solution containing 0.1 M glycine for 1 hour. After a 1-hour blocking step, PBS solution containing 0.3% Triton X-100 and 5% normal goat serum was used, followed by incubation with primary antibodies (such as anti-TH antibody (1:200, Proteintech, USA) and anti-GPX4 antibody (1:50, SantaCruz, China)) at 4°C overnight. The next day, the sections were washed three times with PBS for 10 minutes each, followed by incubation with secondary antibodies for 1-2 hours, including anti-rabbit IgG (Alexa Fluor 488, 1:400, Abcam, USA) and Alexa Fluor TM 594-labeled goat anti-mouse IgG (1:400, Thermo Fisher Scientific, China) at room temperature. Finally, after a series of PBS washes, the fluorescence intensity was quantified using a confocal microscope (Stellaris5, Leica, Wetzlar, Germany), and the cell nuclei were stained with DAPI. Compared with the control group, the MPTP-induced PD group showed a significant decrease in TH levels, while F-CO treatment significantly improved TH levels ( Figure 3 IJ). Similar findings were further confirmed by Western blot analysis of TH ( Figure 3 K). These results revealed that F-CO significantly alleviated MPTP-induced loss of dopaminergic neurons in the SNpc.

[0173] Lewy bodies, mainly composed of α-synuclein (α-syn), are another key pathological feature of Parkinson's disease. Therefore, the level of α-syn in the midbrain on day 26 was evaluated. Midbrain extracts were lysed using RIPA buffer (Catalog No.: WB3100, NCM Biotech, China), and 1% protease inhibitor cocktail (Catalog No.: 491248, VicMed, China) and 1% phosphatase inhibitor (Catalog No.: PR20015, Proteintech, USA) were added to the lysate. Protein concentration was determined using the BCA protein quantification kit (Catalog No.: WB6501A, NCM Biotech, China). Equal amounts of total protein were separated by 7.5% or 12.5% ​​sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with primary antibodies against the following target proteins: TH (1:3000, Proteintech, USA), T-α-Syn (1:1000, Cell Signaling Technology, USA), P-α-Syn (1:1000, Cell Signaling Technology, USA). α-Tubulin (1:8000, Proteintech, USA). The membrane was then incubated with secondary antibodies for 1-2 hours, including goat anti-rabbit IgG (1:8000, Proteintech, USA) and goat anti-mouse IgG (1:8000, Proteintech, USA) at room temperature. The protein bands were developed using the ChemiDoc touch system (Bio-Rad, USA), and the protein signals were normalized to the internal reference protein α-tubulin signal. Compared with the control group, the levels of phosphorylated (P-α-syn) and total α-syn (T-α-syn) were significantly increased after MPTP induction. However, their levels returned to normal after F-CO treatment ( Figure 3 LM). These results suggest that F-CO alleviates abnormal α-syn accumulation in the midbrain of PD mice. In conclusion, F-CO improves MPTP-induced neurological dysfunction by providing neuroprotection to dopaminergic neurons and reducing α-synuclein levels in the early stages of Parkinson's disease.

[0174] Example 5: Evaluation of the activity of F-CO in inhibiting ferroptosis of neurons in Parkinson's mice

[0175] In this paper, the PD mouse model was established by daily intraperitoneal injection of MPTP (30 mg / kg) on ​​days 1, 2, 3, 4, and 5 for 5 days. Subsequently, F-CO (6 mg / kg) was intravenously injected for treatment on days 1, 2, 3, 4, 5, 7, 9, and 11. The midbrain of mice was harvested on day 26 to test the expression of ferroptosis-related proteins in the midbrain to evaluate its efficacy in preventing ferroptosis in vivo ( Figure 4 ). The specific method is as follows: midbrain extracts were lysed using RIPA buffer (Catalog No.: WB3100, NCM Biotech, China), and 1% protease inhibitor cocktail (Catalog No.: 491248, VicMed, China) and 1% phosphatase inhibitor (Catalog No.: PR20015, Proteintech, USA) were added to the lysate. Protein concentration was determined using a BCA protein quantification kit (Catalog No.: WB6501A, NCM Biotech, China). Equal amounts of total protein were separated by 7.5% or 12.5% ​​sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with primary antibodies against the following target proteins: TH (1:3000, Proteintech, USA), SCL7A11 (1:1000, ABclonal, China), GPX4 (1:5000, Abcam, USA), ACSL4 (1:4000, ABclonal, China), 4-HNE (1:1000, Thermo Fisher Scientific, China), TFR1 (1:1000, ABclonal, China), DMT1 (1:2000, ABclonal, China), NCOA4 (1:1000, ABclonal, China), α-tubulin (1:8000, Proteintech, USA). The membrane was then incubated with secondary antibodies for 1-2 hours, including goat anti-rabbit IgG (1:8000, Proteintech, USA) and goat anti-mouse IgG (1:8000, Proteintech, USA) at room temperature. Protein bands were developed using the ChemiDoc touch system (Bio-Rad, USA), and protein signals were normalized to the internal reference protein α-tubulin signal. Figure 3 I same.

[0176] From the results, GPX4( Figure 4 A) and SLC7A11( Figure 4 B) expression was significantly reduced, while ASCL4 ( Figure 4C) levels were significantly increased. These changes indicate that the cellular glutathione (GSH) production capacity is significantly impaired and the sensitivity to lipid peroxidation is enhanced. At the same time, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), which are metabolites and key indicators of lipid peroxidation, were significantly increased after MPTP treatment ( Figure 4 ED). These findings provide strong evidence that MPTP treatment leads to significant lipid peroxidation in vivo. The increase in lipid peroxidation markers underscores the ability of MPTP to exacerbate oxidative stress and lipid damage, thereby contributing to pathophysiological processes associated with neurodegeneration. In the F-CO treated group, the expression levels of SLC7A11 and GPX4 were significantly increased ( Figure 4 AB). At the same time, the level of ACSL4 was significantly reduced ( Figure 4 C), lipid peroxidation metabolites (4-HNE and MDA, Figure 4 DE) levels were also significantly reduced, approaching the levels observed in the control group. These findings suggest that F-CO effectively alleviates ferroptosis in the Parkinson's disease mouse model by activating the SLC7A11 / GPX4 axis and inhibiting ACSL4, thereby effectively inhibiting lipid peroxidation. In addition to lipid peroxidation, excessive accumulation of intracellular ferrous iron due to dysregulated iron metabolism represents another important molecular mechanism of ferroptosis. Divalent metal transporter 1 (DMT1), transferrin receptor (TRFC), and nuclear receptor coactivator 4 (NCOA4) play a key role in ferroptosis by regulating iron homeostasis. DMT1 promotes the uptake of ferrous iron from the external environment into cells, increasing intracellular iron levels. On the other hand, TRFC mediates the uptake of iron-bound transferrin, further promoting iron accumulation. NCOA4 regulates the degradation of ferritin, a storage protein, to release iron into cells. The increase in iron levels driven by these three factors leads to increased oxidative stress and lipid peroxidation. On day 21 after model induction, the level of free ferrous iron in brain tissue of MTPT-induced PD mice was significantly increased ( Figure 4 F), iron-related transferrin TFRC ( Figure 4 H), DMT1( Figure 4 I) and NCOA4( Figure 4 F-CO reversed these trends in the PD group and significantly reduced the levels of free ferrous iron and iron-related proteins. Specifically, F-CO treatment led to a significant decrease in the expression of TFRC, DMT1, and NCOA4 compared with the PD group ( Figure 4 HJ). Inactivation of proteins involved in iron metabolism leads to reduced accumulation of free ferrous iron in cells ( Figure 4F). As a result, oxidative stress and lipid peroxidation were significantly reduced in brain tissue of PD mice. These findings suggest that F-CO effectively alleviates ferroptosis by restoring iron homeostasis, thereby protecting against PD-associated neuronal ferroptosis.

[0177] In conclusion, the mechanistic studies showed that F-CO effectively inhibited cellular lipid peroxidation and limited intracellular iron metabolism, thereby successfully inhibiting neuronal ferroptosis in PD mice and ultimately exerting a neuroprotective effect. F-CO may serve as a novel ferroptosis inhibitor and a potential ideal drug for PD.

Claims

1. A compound of formula I, or a stereoisomer, geometric isomer, conformational isomer, tautomer, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, wherein the compound of formula I has structure XL-R6, characterized in that: X has the structure shown below: In formula X, R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxyl, and amino; R5 is selected from C1-C10 alkyl, C2-C10 alkenyl, and C2-C10 alkynyl; formula X is connected to L at "*"; L is selected from: In the formula L, L is connected to X and R6 at the "*" position respectively; R6 is selected from C1-C10 alkyl and X.

2. The compound of formula I according to claim 1, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or its pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, characterized in that: R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, and C1-C10 alkyl.

3. The compound of formula I according to claim 1, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or its pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, characterized in that: R1, R2, R3, and R4 are each independently selected from hydrogen, fluorine, and C1-C6 alkyl.

4. The compound of formula I according to claim 1, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or its pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, characterized in that: R5 is selected from C1-C10 alkyl.

5. The compound of formula I according to claim 1, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or its pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, characterized in that: The compound of formula I is selected from the group consisting of:

6. Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof in the preparation of a medicament for treating and / or preventing Parkinson's disease.

7. Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds in the preparation of ferroptosis inhibitors.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (i) a compound of formula I according to any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, and (ii) a pharmaceutically acceptable carrier.

9. An application selected from the following group: (1) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition according to claim 8 in inhibiting cell ferroptosis; (2) Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition as described in claim 8 for reducing the concentration of ONOO- and / or Fe2+ in cells undergoing ferroptosis; (3) Use of a compound of formula I according to any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition according to claim 8 for improving tyrosine hydroxylase levels in cells undergoing ferroptosis; (4) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomers, geometric isomers, conformational isomers, tautomers, or pharmaceutically acceptable salts, polymorphs, solvates, hydrates or isotope-labeled compounds, or the pharmaceutical composition according to claim 8 in improving dopaminergic neuron loss; (5) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition according to claim 8 for reducing the level of α-synuclein in the brain of an individual suffering from Parkinson's disease; (6) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition according to claim 8 for increasing the expression level of SLC7A11 in individuals with Parkinson's disease; (7) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition according to claim 8 for increasing the expression level of GPX4 in individuals with Parkinson's disease; (8) Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition as described in claim 8 for reducing the expression level of ACSL4 in an individual suffering from Parkinson's disease; (9) Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition as described in claim 8 for reducing the expression level of 4-HNE in an individual suffering from Parkinson's disease; (10) Use of the compound of formula I according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, conformational isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound, or the pharmaceutical composition according to claim 8 for reducing the expression level of MDA in individuals with Parkinson's disease; (11) Use of a compound of formula I according to any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition according to claim 8 for reducing the expression level of TFRC in an individual suffering from Parkinson's disease; (12) Use of a compound of formula I according to any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition according to claim 8 for reducing the expression level of DMT1 in an individual suffering from Parkinson's disease; (13) Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition as described in claim 8 for reducing the expression level of NCOA4 in individuals with Parkinson's disease; (14) Use of a compound of formula I as described in any one of claims 1 to 5, or a stereoisomer, geometric isomer, conformational isomer, tautomer thereof, or a pharmaceutically acceptable salt, polymorph, solvate, hydrate or isotope-labeled compound thereof, or a pharmaceutical composition as described in claim 8 for inhibiting oxidative stress and / or lipid peroxidation in brain tissue of an individual suffering from Parkinson's disease.

10. The use according to claim 9, characterized in that The cell is a nerve cell; and / or the dopaminergic neuron is a dopaminergic neuron in the substantia nigra pars compacta in Parkinson's disease.

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