CARDIOVASCULAR AND CEREBROVASCULAR DRUG AND USE THEREOF

Abstract

A cardiovascular and cerebrovascular drug and the use thereof are provided. Specifically, the compound has a structure as represented by formula (I). The compound can be used in treating thrombotic diseases, resisting inflammation and treating nerve injury.

##STR00001##

Claims

1. A method for preventing and/or treating thrombosis-related diseases, the treatment of anti-inflammatory, and/or treating neuronal damage caused by cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumor surgery, wherein the method comprising a step of: administering the compound of formula I or a pharmaceutically acceptable salt thereof to a subject in need thereof: ##STR00033## wherein, R.sub.1 is selected from the group consisting of: hydroxyl, C1-C6 alkoxy, halogen, substituted or unsubstituted OC1-C6 alkyl, and ##STR00034## wherein the substituted refers to substitution with sulfonic acid group or hydroxyl; wherein, m is an integer selected from 1 to 6; the carbon atom of ##STR00035## in ##STR00036## is a chiral carbon atom, and the chirality is selected form the group consisting of: ##STR00037## R.sub.4 is a metal ion selected from the group consisting of: Na.sup.+, K.sup.+, Li.sup.+, and Cs.sup.+; R.sub.5 is C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or heteroaryl; R.sub.2 and R.sub.3 are each independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C2-C6 hydroxyalkyl, or (C1-C3 alkylene)-COOH; and n is an integer selected from 6 to 12.

2. The method according to claim 1, wherein the thrombosis-related disease is selected from the group consisting of: cerebral thrombosis, cerebral infarction (acute ischemic stroke) and its resulting neuronal injury in nervous tissue, cerebral edema, myocardial infarction, pulmonary embolism, or a combination thereof.

3-5. (canceled)

6. A method for treating thrombosis-related diseases, wherein the method comprising a step of: administering a combination of the compound of formula I of claim 1 and urokinase to a subject in need thereof.

7. A method for treating neurological damages caused by cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumor surgery, wherein the method comprising a step of: administering a combination of the compound of formula I of claim 1 and butylphthalide to a subject in need thereof.

8. A pharmaceutical composition or formulation, wherein comprising (a) active ingredient, the active ingredient includes the compound of formula I, or a pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers, the pharmaceutical composition or formulation is used for: (a) prevention and/or treatment of thrombosis-related diseases; (b) anti-inflammatory treatment; and/or (c) treatment of neuronal damages caused by cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumor surgery, ##STR00038## wherein, R.sub.1 is selected from the group consisting of: hydroxyl, C1-C6 alkoxy, halogen, substituted or unsubstituted OC1-C6 alkyl, and ##STR00039## wherein the substituted refers to substitution with sulfonic acid group or hydroxyl; wherein, m is an integer selected from 1 to 6; the carbon atom of ##STR00040## in ##STR00041## is a chiral carbon atom, and the chirality is selected form the group consisting of: ##STR00042## R.sub.4 is a metal ion selected from the group consisting of: Na.sup.+, K.sup.+, Li.sup.+, and Cs.sup.+; R.sub.5 is C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or heteroaryl; R.sub.2 and R.sub.3 are each independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C2-C6 hydroxyalkyl, or (C1-C3 alkylene)-COOH; and n is an integer selected from 6 to 12.

9-10. (canceled)

11. The method according to claim 2, wherein the thrombosis is selected from the group consisting of: vascular endothelial injury induced thrombosis, arteriovenous bypass thrombosis, cerebral ischemia-reperfusion injury, or a combination thereof.

12. The method according to claim 11, wherein the cerebral ischemia-reperfusion injury comprises: cerebral infarction, cerebral edema, and/or neurocyte ferroptosis.

13. The method according to claim 1, wherein the anti-inflammatory is an anti-vascular inflammation.

14. The method according to claim 13, wherein the vascular inflammation includes vascular inflammation caused by high glucose and high fat.

15. The method according to claim 1, wherein the compound of formula I is selected from the group consisting of: ##STR00043## ##STR00044##

Description

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0063] After extensive and in-depth research, the inventor unexpectedly discovered for the first time that a class of compounds with structures as shown in formula I have significant effects on the prevention and/or treatment of thrombosis-related diseases; anti-inflammatory treatment; the treatment of neuronal damages caused by cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumor surgery. The experiment shows that the compound of formula I has good therapeutic effects on brain nerve damage caused by stroke. The compound of formula I of the present invention can be used for the prevention of thrombosis diseases, thrombolysis, anti-inflammatory treatment, etc. The present invention is completed on this basis.

Terms

[0064] The term halogen refers to F, Cl, Br and I.

[0065] The term C1-C6 alkyl refers to a linear or branched chain alkyl having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl and tert-pentyl and the like.

[0066] The term C2-C6 alkenyl refers to a linear or branched alkenyl having 2-6 carbon atoms and containing a double bond, including, without limitation, vinyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl, and the like.

[0067] The term C2-C6 alkynyl refers to a linear or branched alkynyl having 2-6 carbon atoms and containing a triple bond, including, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like.

[0068] The term C1-C6 hydroxyalkyl refers to a linear or branched alkyl group having 1-6 carbon atoms and containing a hydroxyl group, including, without limitation, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl and the like. Preferably, C1-C3 hydroxyalkyl.

[0069] The term C3-C8 cycloalkyl refers to a cyclic alkyl having 3-8 carbon atoms on the ring, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0070] The term C1-C6 alkoxy refers to a linear or branched alkoxy group having 1-6 carbon atoms, including, without limitation, methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like. Preferably C1-C4 alkoxy.

[0071] The terms aromatic ring and aryl have the same meaning, preferably is C6-C10 aryl. The term C6-C10 aryl refers to an aromatic ring group having 6-10 carbon atoms without any heteroatoms on the ring, such as phenyl, naphthyl and the like.

[0072] The term heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms, wherein the heteroatoms include nitrogen, oxygen, and S(O).sub.r (wherein, r is an integer of 0, 1, 2). For example, 4-8-membered heteroaryl refers to a heteroaromatic system containing 4-8 ring atoms, and 4-10 membered heteroaryl refers to a heteroaromatic system containing 4-10 ring atoms, including but not limited to pyrrolyl, furyl, thienyl, pyrazolyl, thiazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridinyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzimidazolyl, triazolyl, etc.

[0073] Unless specifically stated, the groups described in the present invention are substituted or unsubstituted, the groups of the present invention can be substituted by substituents selected from the group consisting of: halogen, acyloxy, cyano, amino, nitro, carboxyl, amide, carboxymethyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, hydroxy C1-C4 alkyl, C5-C7 cycloalkenyl, phenyl, naphthyl, etc.

[0074] represents the binding position of the group.

Active Ingredient

[0075] As used herein, the terms compound of the present invention, and active ingredient of the present invention can be used interchangeably and refer to the compound of formula I.

##STR00012##

[0076] As used herein, the term also comprises pharmaceutically acceptable salt of the compound of formula I. The term pharmaceutically acceptable salt refers to salts that are formed of the compound of the invention and acids or bases, and suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A class of preferred salts are salts formed of the polymer of the present invention and the acids. Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and the like; organic acids such as methanoic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, and the like; and acidic amino acids such as aspartic acid, glutamic acid and the like.

[0077] The compound of formula I of the present invention may be prepared by methods well known to those skilled in the art in the prior art, and there are no particular limitations on the reaction parameters of the individual steps. In addition, typical compounds of the present invention are also commercially available.

[0078] As used herein, in the compound of formula I, the chiral carbon atom, if exist, may be in the R configuration, the S configuration, or a mixture of both.

[0079] In the present invention, the active ingredient is a compound of formula I,

##STR00013## [0080] wherein, [0081] R.sub.1 is selected from the group consisting of: hydroxyl, C1-C6 alkoxy, halogen, substituted or unsubstituted OC1-C6 alkyl, and

##STR00014##

wherein the substituted refers to substitution of sulfonic acid group or hydroxyl; [0082] wherein, m is an integer selected from 1-6; [0083] the carbon atom of

##STR00015##

in

##STR00016##

is chiral carbon atom, and the chirality is selected form the group consisting of:

##STR00017## [0084] R.sub.4 is a metal ion selected from the group consisting of: Na.sup.+, K.sup.+, Li.sup.+, and Cs.sup.+; [0085] R.sub.5 is C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or heteroaryl; [0086] R.sub.2 and R.sub.3 are each independently selected from: H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C2-C6 hydroxyalkyl, and (C1-C3 alkylene)-COOH; [0087] n is an integer selected from 6 to 12.

[0088] In one embodiment, R.sub.1 is selected from the group consisting of: hydroxyl, methoxy,

##STR00018##

[0089] In another embodiment, m=1.

[0090] In another embodiment, R.sub.4 is Na.sup.+.

[0091] In another embodiment, R.sub.5 is CH.sub.3.

[0092] In another embodiment, n is 6, 7, or 8.

[0093] In another embodiment, R.sub.1 is selected from the group consisting of: hydroxyl, methoxy,

##STR00019##

[0094] In another embodiment, R.sub.2 and R.sub.3 are each independently selected from: H, methyl, hydroxypropyl, hydroxyethyl and carboxymethyl.

[0095] In another embodiment, the compound of formula I is selected from the group consisting of: methylcyclodextrin, carboxymethyl cyclodextrin, hydroxyethyl--ethylcyclodextrin, hydroxypropyl--cyclodextrin, and sulfobutylether--cyclodextrin.

[0096] In another example, the parent nucleus of the compound of formula I is cyclodextrin.

[0097] In another embodiment, the compound of formula I is

##STR00020##

[0098] In another embodiment, the compound of formula I is selected from the group consisting of:

##STR00021##

Pharmaceutical Composition and Method of Administration

[0099] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound described in the present invention.

[0100] Due to the excellent anti-thrombotic activity of the compound of the present invention, the compounds of the invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the the compound of the present invention as active ingredients can be used in the treatment, prevention and alleviation of the diseases associated with embolism.

[0101] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients or carriers. Wherein safe and effective amount refers to an amount of compound which is sufficient to significantly improve the condition, and not to generate severe side effects. Generally, the pharmaceutical composition contains 1-2000 mg polymorphs of the invention per dose, preferably, 10-1000 mg polymorphs of the invention per dose. Preferably, the one dose is one capsule or one pill.

[0102] Pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers or gelatinous materials which are suitable for human use and should be of sufficient purity and sufficiently low toxicity. Compatible used herein refers to the ability of each component of a composition can be mixed with the compound of the present invention and can be mixed with each other without appreciably reducing the efficacy of the compound. Examples of pharmaceutically acceptable carrier include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifier (such as Tween), wetting agent (such as lauryl sodium sulfate), colorant, flavoring, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

[0103] The pharmaceutical composition is injections, capsules, tablets, pills, powders or granules.

[0104] There is no special limitation of administration mode for the compound or pharmaceutical compositions of the present invention, and the representative administration mode includes (but is not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0105] The solid dosage forms used for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compounds are mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or CaHPO4, or mixed with any of the following components: (a) fillers or compatibilizer, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and arabic gum; (c) humectant, such as, glycerol; (d) disintegrating agents such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain composite silicates, and sodium carbonate; (e) dissolution-retarding agents, such as paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants such as talc, calciumcetyl alcohol, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or the mixtures thereof. In capsules, tablets, and pills, the dosage form may also include buffers.

[0106] Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coating and shell materials, such as casings and other materials well-known in the art. They can contain opacifiers, and the release of active compounds or compounds in the composition can be delayed in a certain part of the digestive tract. Examples of embedding components that may be employed are polymeric substances and waxes. If necessary, the active compound may also be formed into a microcapsules with one or more of the above excipients.

[0107] Liquid dosage forms for oral administration include pharmaceutically acceptable lotion, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain any conventional inert diluents known in the art such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethyl formamide, as well as oil, in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or the combination thereof.

[0108] In addition to these inert diluents, the composition can also include additives such as wetting agents, emulsifiers and suspensions, sweeteners, correctors, and spices.

[0109] In addition to active compounds, suspensions can include suspending agents such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, methanol aluminum and agar, or mixtures of these substances.

[0110] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersion liquid, suspensions or lotions, and sterile powders for re dissolution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and their suitable mixtures.

[0111] The dosage forms of the compounds of the present invention used for local administration include ointments, powders, patches, sprays, and inhalants. The active ingredients are mixed under sterile conditions with physiologically acceptable carriers and any preservatives, buffers, and propellants if necessary.

[0112] The compound of the present invention may be administered alone or in combination with other pharmaceutically acceptable compound (such as anti-thrombotic drugs).

[0113] The treatment method of the present invention can be administered alone or in combination with other treatment methods or therapeutic drugs.

[0114] When the pharmaceutical compositions are used, a safe and effective amount of compound of the present invention is applied to a mammal (such as human) in need of, wherein the dose of administration is a pharmaceutically effective dose. For a person weighed 60 kg, the daily dose is usually 1-2000 mg, preferably 50-1000 mg. Of course, the particular dose should also depend on various factors, such as the route of administration, patient healthy status, which are within the skills of an experienced physician.

The Main Advantages of the Present Invention Include:

[0115] (a) The compound of formula I of the present invention has excellent therapeutic effects on brain nerve damages caused by stroke.

[0116] (b) The compound of formula I of the present invention can be used for the prevention of thrombosis diseases, and the treatment of thrombolysis, without causing bleeding.

[0117] (c) The compound of formula I of the present invention has anti-inflammatory effect.

[0118] (d) Compound 1 in the compound of formula I of the present invention has excellent safety performance and low toxic side effects.

[0119] The present invention was further described hereafter in combination with specific embodiments. It should be understood that these examples are only used to illustrate the and not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under the conventional conditions, for example, according to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 1989, or according to the manufacture's instructions. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.

Example 1: Evaluation Experiment of the Preventative Effect of Sample on Vascular Endothelial Injury Induced Thrombosis

[0120] Experimental method: 420 tails of 5 dpf wild type AB strain zebrafish were randomly selected and placed in a six-well plate, with 30 fish per well (experimental group). The fish were injected with compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), and compound (11) (structural formula shown in Table 1) at a dose of 4.00 ng/tail, with positive control drug acetylsalicylic acid being administered by dissolving it in water at a concentration of 30.0 g/mL. Normal control group (i.e. zebrafish treated with standard diluted water) and model control group were set up, with a capacity of 3 mL per well (experimental group). Except for normal control group, the remaining experimental groups were all given Punatinib by dissolving it in water to induce vascular endothelial injury induced thrombosis model in zebrafish. The zebrafish were stained with o-dianisidine after co-treatment with ponatinib and samples for a period of time under incubation conditions at 28 C.

[0121] After staining, 10 zebrafish in each group were randomly selected and photographed under the microscope, and the intensity of cardiac erythrocyte staining in zebrafish was analyzed using NIS-Elements D 3.20 advanced image-processing software, and the samples were evaluated for the preventative effect on vascular endothelial injury induced thrombosis by the statistical results of cardiac erythrocyte staining intensity.

[0122] The preventative effect on thrombosis is calculated as follows:

[00001]$\mathrm{Preventative}\mathrm{effect}\mathrm{on}\mathrm{thrombosis}\left(\%\right)=\frac{\left[S\left(\mathrm{tset}\mathrm{product}\mathrm{group}\right)-S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)\right]}{\left[S\left(\mathrm{normal}\mathrm{control}\mathrm{group}\right)-S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)\right]}100\%$

[0123] Statistical analysis was performed using SPSS software, and p<0.05 indicates significant differences

Experimental Results:

TABLE-US-00001 TABLE 1 Preventative effect of samples on vascular endothelial injury induced thrombosis (n = 10) Preventative Cardiac erythrocyte effect on Sample structural Dose staining intensity thrombosis Group formula/name (ng/tail) (pixels, mean SE) (%) Normal 5312 318*** control group Model control 2597 251 group Acetylsalicyclic 30.0 g/mL 5345 228*** 101 acid Compound (1) [00022] 4397 454** 66 CAS: 1309580-41-3 Compound (2) [00023] 3655 318* 39 Compound (3) [00024] 4.00 ng/tail 3045 219 17 Compound (4) [00025] 4300 195*** 63 Compound (5) [00026] 4340 255*** 64 Compound (6) [00027] 4938 436*** 86 Compound (7) [00028] 4584 271*** 73 Compound (8) [00029] 4580 291*** 73 Compound (9) [00030] 4709 296*** 78 Compound (10) [00031] 4326 383** 64 Compound (11) [00032] 4749 12*** 79 compared with model control group, *p < 0.05, **p < 0.01, ***p 0.001.

[0124] Experimental results: the intensity of cardiac erythrocyte staining of zebrafish in the 4.00 ng/tail dose group was 4397, 3655, 3045, 4300, 4340, 4938, 4584, 4580, 4709, 4326 and 4749 pixels respectively for compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), and compound (11).

[0125] Compared to model control group, the preventative effect on thrombosis of compound (3) in 4.00 ng/tail dose group was 17%, with p>0.05. The preventative effect on thrombosis of compound (1), compound (2), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10) and compound (11) in 4.00 ng/tail dose group were 66%, 39%, 63%, 64%, 86%, 73%, 73%, 78%, 64%, and 79%, respectively, with p<0.01, p<0.05, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.01, p<0.001, respectively.

[0126] Conclusion: Compound (1), Compound (2), Compound (4), Compound (5), Compound (6), Compound (7), Compound (8), Compound (9), Compound (10), and Compound (11) showed significant prophylactic effects against endothelial injury induced thrombosis caused by ponatinib in zebrafish under the conditions of this experiment.

[0127] Especially, in 4.00 ng/tail dose group, compounds (6), (11), (9), (7), (8), and (1) had a very significant preventative effect against vascular endothelial injury induced thrombosis caused by ponatinib in zebrafish, with thromboprophylaxis (%) index ranging from 66 to 86%, while the thromboprophylaxis (%) index of 30 ug/ml of acetylsalicylic acid was 101%.

Example 2 Evaluation on Sensitization Risk Induced by the Sample

[0128] Experimental method: 2 dpf wild type AB strain zebrafish were randomly selected and placed in a 24-well plate, with each group 6 parallel wells and 10 zebrafish per well. The zebrafish were injected intravenously with samples at a dose of 500 ng/tail and the positive control C48/80 at a concentration of 1.5 g/mL, while normal control group was set up. Each well has a capacity of 1 mL.

[0129] All experimental groups were given BAPNA by dissolving it in the water. After treatment at 28 C. for 1 day, the liquid was transferred to 96-well plates with 200 L/well, and the relative absorbance values (OD values) of the expression levels of tryptase in each experimental group were detected using an microplate reader, and the results of the statistical analysis of the OD values were used to evaluate the risk of sensitization by sample. Statistical results were expressed as meanSE.

[00002]$\mathrm{sensitization}\mathrm{risk}\left(\%\right)==\frac{\left[\mathrm{OD}\left(\mathrm{sample}\mathrm{group}\right)-\mathrm{OD}\left(\mathrm{normal}\mathrm{control}\mathrm{group}\right)\right]}{\mathrm{OD}\left(\mathrm{normal}\mathrm{control}\mathrm{group}\right)}100\%$

[0130] Statistical analysis was performed using SPSS software, and p<0.05 indicates significant differences.

TABLE-US-00002 TABLE 2 Sensitization risk of each sample Normal Positive Evaluation control control Compound Compound Compound index group group (1) (2) (10) Absorbance 1 0.05085 0.11145 0.06575 0.04785 0.07585 value (OD 2 0.05905 0.09885 0.05845 0.05085 0.06795 value) 3 0.05395 0.09815 0.05605 0.05405 0.06445 4 0.05515 0.09295 0.05725 0.05655 0.07115 5 0.05305 0.09545 0.05895 0.05255 0.06947 6 0.05385 0.09985 0.06195 0.04865 0.06795 mean 0.054 0.099 0.060 0.052 0.069 SE 0.001 0.003 0.001 0.001 0.002 Sensitization 83.3 11.1 3.7 27.8 risk (%) Relative 100% 13% 4% 33% sensitization risk (compared with positive control group) P <0.001 <0.05 >0.05 <0.001

[0131] Experimental results: The sensitization risk of the compounds of the present invention tested was low, even under the condition that the dose was increased by 125 times (i.e. 500 ng/tail) based on the dose of 4 ng/tail in Example 1. Representatively, as shown in Table 2, the sensitization risk of the tested compound (1), compound (2), and compound (10) of the present invention were all significantly lower than the that of the positive control.

[0132] Among them, Compound (2) and Compound (1) have no or substantially no sensitization risk, and Compound (10) has a low sensitization risk.

[0133] Experimental conclusion: Combining the experimental results of Examples 1 and 2, i.e., considering the safety and efficacy in a comprehensive manner, compound (1) was selected as the preferred compound for subsequent experiments.

Example 3 Evaluation of the Preventative Effect of Compound 1 on Vascular Endothelial Injury Induced Thrombosis

[0134] Experimental method: Wild-type AB strain zebrafish were treated with 4 g/mL punatinib for 18 h to establish vascular endothelial-injury thrombosis model in zebrafish. 150 of 5 dpf wild-type AB strain zebrafish were randomly selected and placed in six-well plates with 30 fish per well (experimental group). The zebrafish were injected with compound (1) at a dose of 2.06 ng/tail, and 6.18 ng/tail, and the positive control drug acetylsalicylic acid was given by dissolving it in water with a concentration of 30 g/mL, while the normal control group (i.e., zebrafish treated with standard dilution water) and the model control group were set up, and the volume of each well (experimental group) was 3 mL. Except for normal control group, the remaining experimental groups were all given Punatinib by dissolving it in water to induce vascular endothelial injury thrombosis model in zebrafish. The zebrafish were co-treated with compound (1) and punatinib for a period of time under incubation condition at 28 C., and then stained with o-dianisidine. After staining, 10 zebrafish were randomly selected from each group and photographed under the microscope. The intensity of cardiac erythrocyte staining in zebrafish was analyzed using NIS-Elements D 3.10 advanced image-processing software, and the preventative effect of compound (I) on vascular endothelial injury thrombosis was evaluated based on the statistical results of cardiac erythrocyte staining intensity. The preventative effect on thrombosis is calculated as follows:

[00003]$\mathrm{Thromboprophylactic}\mathrm{effect}\left(\%\right)=\frac{\left[S\left(\mathrm{tset}\mathrm{product}\mathrm{group}\right)-S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)\right]}{\left[S\left(\mathrm{normal}\mathrm{control}\mathrm{group}\right)-S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)\right]}100\%$

[0135] Statistical analysis were performed using Analysis of Variance and Dunnett's t-test, and p<0.05 indicates a significant difference, providing a representative experimental plot.

TABLE-US-00003 TABLE 3 Preventative effect of compound (I) on vascular endothelial injury induced thrombosis (n = 10) Cardiac erythrocyte Thrombopro- Dose staining intensity phylactic Group (ng/tail) (pixels, mean SE) effect (%) Normal control group 5080 155*** 100 Model control group 2300 237 0 Acetylsalicylic acid 30 g/mL 4655 136*** 85 Compound (1) 2.06 4601 265*** 83 6.18 5044 345*** 99 Compared with model control group, ***p < 0.001.

[0136] Experimental results: The intensity of cardiac erythrocyte staining in zebrafish in compound (1) 2.06 ng/tail and 6.18 ng/tail dose groups were 4601 and 5044 pixels, respectively, with p<0.001 compared with the model control group, and the thromboprophylactic effect thereof were 83% and 99%, respectively.

[0137] Experimental Conclusion: Compound (1) had a significant and dose-dependent preventative effect on punatinib-induced vascular endothelial injury thrombosis in zebrafish, and the preventative effect of compound (1) on vascular endothelial injury thrombosis was superior to that of the positive control, acetylsalicylic acid.

Example 4 Effect of Compound (1) on Arteriovenous Bypass Thrombosis in Rats

Experimental Method:

[0138] Grouping: Divided into 5 groups, with 10 animals in each group. The model control group was given with solvent, and the test drug compound (1) and the positive control were given intravenously once a day for consecutive 3 d. The modeling started 30 min after the last dose.

[0139] Model preparation: 50 SD male rats were anesthetized with sodium pentobarbital at a dose of 60 mg/kg via ip. The rat was fixed in the supine position, and the skin was incised at the midline of the neck; the fascia and muscle tissues were separated, the left common carotid artery and the right external jugular vein were dissociated, and the arterio-venous (A-V) bypass surgery was performed using the polyethylene tubing with a built-in thread. The arterial clip was used to clamp the proximal end of the carotid artery, and one end of the polyethylene tubing was inserted into the artery, and another end was inserted into the vein, thereby completing the bypass surgery. 30 minutes after the last dose, the arterial clip was opened, and blood flowed from the left common carotid artery to the right external jugular vein through the bypass tubing, and the blood flow formed a bypass circulation, and the thrombus was taken out 15 min later.

[0140] Detection index: the blood flow was open after 15 min, the thread was taken out, the residual liquid was sucked dry, the thread attached with thrombus was weighed and the wet weight of thrombus was calculated; the dry weight of thrombus was weighed and calculated after bake-drying in the oven at 50 C. Thrombosis inhibition rate was calculated. Tails were clipped at the end of the experiment to detect bleeding time.

TABLE-US-00004 TABLE 4 Effect of compound (1) on arteriovenous bypass thrombosis in rats (x s, n = 10) Wet weight Dry weight Wet weight Dry weight inhibition inhibition Group Dose mg/kg of thrombus of thrombus rate (%) rate (%) Model control 0.0319 0.0033 0.0082 0.0017 Compound (1) 10 0.0254 0.0042** 0.0069 0.0014** 19.4 15.9 Compound (1) 20 0.0246 0.0045** 0.006 0.0014** 21.9 26.8 Compound (1) 40 0.0207 0.0041*** 0.0051 0.0011*** 34.3 37.8 Ozagre 16 0.0208 0.0041*** 0.0054 0.0018** 34 34.1 Note: Compared with model group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0141] Results: Compared with the model control group, intravenous injection of compound (1) at a dose of 10 mg/kg, 20 mg/kg, 40 mg/kg inhibit rat arteriovenous bypass thrombosis in different degrees, and the thrombus wet weight and dry weight inhibition rate (%) were respectively 19.4% (P>0.005) and 15.9% (P>0.005); 21.9% (P<0.001) and 26.8% (P<0.001); 34.3% (P<0.001) and 37.8% (P<0.001). The thrombus wet weight and dry weight inhibition rate (%) of Ozagre administered intravenously at a dose of 16 mg/kg were 34% (P<0.001) and 34.1% (P<0.01).

[0142] Conclusion: The anti-thrombotic effect of Compound (1) is dose-dependent, and is comparable or superior to that of ozagrel.

TABLE-US-00005 TABLE 5 Effect of compound (1) on bleeding time in rats (x s, n = 10) Group Dose (mg/kg) Bleeding time (s) Model control 396.1 143.9 Compound (1) 10 403.4 185.1 Compound (1) 20 422.6 205.7 Compound (1) 40 428.4 185.6 Ozagre 16 740.8 224*** Note: Compared with model group, ***P < 0.001.

[0143] Experimental results: Compared with the model control group, intravenous administration of different doses of compound (1) have no significant effect on the bleeding time in rats; positive control, intravenous administration of Ozagre, significantly prolonged the bleeding time in rats (P<0.01).

[0144] Experimental conclusion: The bleeding experiment of compound (1) shows non dose dependence, which proves that compound (1) has no significant bleeding risk.

Example 5 Effect of Preventative Administration of Compound (1) on Cerebral Ischemia-Reperfusion Injury in Rats

[0145] Experimental method: SD rats were randomly divided into six groups, i.e., pseudosurgery group, model control group, positive control drug ozagre (6 mg/kg) group, and administration drug compound (1) high-dose (67.5 mg/kg) group, medium-dose (22.5 mg/kg) group, and low-dose (7.5 mg/kg) group. The administration group was pre-administered via tail vein injection, while the pseudosurgery group and model control group were given equal volumes of physiological saline once a day for 3 consecutive days. 10 minutes after the last administration, a rat MCAO/R model was established and performed reperfusion 2 hours post ischemia. 24 hours after MCAO reperfusion, the neurobehavioral scores of rats were performed to determine their neurological function; the cerebral infarction rate and cerebral water content in rats were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining method.

TABLE-US-00006 TABLE 6 Effect of preventative administration of compound (1) on cerebral infarction rate in rats with focal cerebral ischemia (MCAO)/reperfusion (The data were expressed in mean SD, n = 8) Cerebral Dose infarction Group (mg/kg) rate (%) Pseudosurgery group 0 Model control group 27.59 6.52 Ozagre group 6 mg/kg 14.39 8.14** Compound (1) high-dose group 67.5 mg/kg 12.28 10.29** Compound (1) medium-dose group 22.5 mg/kg 13.93 6.99** Compound (1) low-dose group 7.5 mg/kg 15.18 4.74* **P < 0.01, compared with model control group; *P < 0.05, compared with model control group.

TABLE-US-00007 TABLE 7 Effect of preventative administration of compound (1) on cerebral water content in rats with focal cerebral ischemia (MCAO)/reperfusion (The data were expressed in mean SD, n = 8) Cerebral water Group Dose (mg/kg) content (%) Pseudosurgery group 77.18 0.51 Model control group 82.12 1.28## Ozagre group 6 mg/kg 78.42 3.73** Compound (1) high-dose group 67.5 mg/kg 78.23 1.84** Compound (1) medium-dose group 22.5 mg/kg 79.01 1.27* Compound (1) low-dose group 7.5 mg/kg 80.16 1.03 ##P < 0.01, compared with pseudosurgery group; **P < 0.01, compared with model control group; *P < 0.05, compared with model control group.

TABLE-US-00008 TABLE 8 Effect of preventative administration of compound (1) on neuroethology of rats with focal cerebral ischemia (MCAO)/reperfusion (The data are expressed in mean SD, n = 8) Group Dose (mg/kg) Behavioral score Pseudosurgery group 0 Model control group 3.00 0.76 Ozagre group 6 mg/kg 1.50 0.56** Compound (1) 67.5 mg/kg 1.50 0.53** high-dose group Compound (1) 22.5 mg/kg 1.87 0.64* medium-dose group Compound (1) 7.5 mg/kg 2.00 1.07 low-dose group **P < 0.01, compared with model control group; *P < 0.05, compared with model control group.

[0146] Results: Compound (1) in all dose groups and the positive drug ozagre group reduced neurological function scores, cerebral infarction rate and cerebral water content of MCAO/R rats.

[0147] Conclusion: preventative administration of compound (1) reduces cerebral infarction rate and cerebral water content of cerebral ischemia-reperfusion injury in rats, and improves neuromotor function in rat.

Example 6 Effect of Combination of Compound (1) and Urokinase (i.v) on Ischemic Brain Injury in Rats Caused by Autologous Thrombus and Thrombin

[0148] Experimental method: A model of cerebral ischemic injury in SD rats was prepared by injecting autologous thrombus and thrombin into the internal carotid via the external carotid artery to embolize the cerebral artery of rats, and the rats were randomly divided into five groups according to behavioral scores: the sham operated group (isolating the external carotid artery only); the model control group (the two groups were administered with equal volume of physiological saline); the urokinase (5,000 U/kg) group, and the compound (1) (22.5 mg/kg) group, with 20 rats for each group (10 for the detection of cerebral infarction rate and 10 for pathological test). The drug was administered once by slow tail vein injection (1 ml per minute) 2 h after modeling, and the decrease in blood flow was monitored after the injection of thrombin, and 120 min after the administration of the drug. Neurobehavioral scoring was performed to determine the neurological function of rats after 24 h. The cerebral infarction rate and cerebral water content of rats were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining method, and the pathological damage of the brain of rats administered with drug 2 h after cerebral ischemia was observed by hematoxylin-eosin (HE) staining.

Experimental Results:

TABLE-US-00009 TABLE 9 Effect of compound (1) (i.v) on cerebral blood flow after cerebral ischemic injury in rats caused by autologous thrombus and thrombin (The data were expressed in mean SD, n = 10) Percentage Percentage decrease after decrease two injection of hours after Group Dosage thrombin (%) administration (%) Blank group 0.04 4.01 0.33 0.33 Model control 44.40 5.22 41.18 5.67 group Compound (1) 22.5 mg/kg 43.67 7.17 27.14 6.38**.sup.## group Urokinase group 5000 U/kg 50.10 7.94 37.12 4.49.sup.## Compound (1) + 22.5 mg/kg + 33.79 3.95 20.14 3.77**.sup.## Urokinase group 5000 U/kg **P < 0.01, compared with model control group; .sup.##P < 0.01, compared with self group after thrombin injection.

TABLE-US-00010 TABLE 10 Effect of compound (1) (i.v) on neurobehavior of rats (The data are expressed in mean SD, n = 10) Group Dosage Behavioral score Model control group 3.00 0.82 Compound (1) group 22.5 mg/kg 1.60 0.70* Urokinase group 5000 U/kg 1.60 0.70* Compound (1) + 22.5 mg/kg+ 1.60 0.52* Urokinase group 5000U/kg *P < 0.05, compared with model control group.

TABLE-US-00011 TABLE 11 Effect of compound (1) (i.v) on cerebral infarction rate and cerebral water content of rate with cerebral ischemic injury caused by autologous thrombus and thrombin Cerebral Cerebral infarction water Group Dosage rate (%) content (%) Blank group 79.32 0.63 Model control 29.94 6.18 82.20 1.33.sup.## group Compound (1) 22.5 mg/kg 13.91 10.78* 79.93 1.98** group Urokinase group 5000 U/kg 9.72 9.89** 79.78 0.72** Compound (1) + 22.5 mg/kg + 6.61 6.14** 79.51 0.81** Urokinase group 5000 U/kg .sup.##P < 0.01, compared with blank group; *P < 0.05, **P < 0.01, compared with model control group.

TABLE-US-00012 TABLE 12 Effect of compound (1) (i.v) on the pathological damage score of cerebral ischemic area in rats with cerebral ischemic injury induced by autologous thrombus and thrombin (mean SD, n = 10) Pathological Group Dosage damage score Model control group 3.70 0.48 Compound (1) group 22.5 mg/kg 1.90 1.10* Urokinase group 5000 U/kg 1.75 0.86* Compound (1) + 22.5 mg/kg + 1.55 0.60* Urokinase group 5000 U/kg *P < 0.05, **P < 0.01, compared with model control group.

[0149] Intravenous administration of compound (1), urokinase and compound (1)+urokinase significantly reduced the percentage decrease in cerebral blood flow in the model rats, the area of cerebral infarction and cerebral water content of the rats, and improved the pathological injury and behavioral changes after the model of cerebral ischemic injury caused by autologous thrombus and thrombin, with the compound (1)+urokinase group showing the most significant level of improvement in cerebral ischemic injury caused by autologous thrombus and thrombin.

[0150] Conclusion: compound (1) (i.v) and its co-administration with urokinase reduce the cerebral infarction rate, cerebral water content and pathologic damage in rats with ischemic brain injury induced by autologous thrombus and thrombin, and improved the neuromotor function of rats, which shows good anti-ischemic brain injury effect.

Example 7 Effect of Therapeutic Administration of Compound (1) on Cerebral Ischemia-Reperfusion Injury in Rats

[0151] Experimental method: SD rats were randomly divided into 11 groups, including pseudosurgery group, a model control group, positive control drug edaravone via intravenous injection (6 mg/kg, i.v.) group, positive control drug butylphthalide injection via intravenous injection (5 mg/kg, i.v.) group, compound (1) high-dose (24 mg/kg, i.v.), medium-dose (12 mg/kg, i.v.), low-dose (6 mg/kg, i.v.) via intravenous injection group, positive drug butylphthalide soft capsule via intragastric administration (60 mg/kg, i.g.) group, and compound via intragastric administration at (1) high-dose (60 mg/kg, i.g.), medium-dose (30 mg/kg, i.g.), low-dose (15 mg/kg, i.g.) group. A middle cerebral artery occlusion (MCAO) model for cerebral ischemia-reperfusion of male rats was prepared using the internal carotid suture-occluded method with an ischemic time of 90 min followed by reperfusion. Injection and oral administration for once were performed 1 h after reperfusion, and neurobehavioral scoring was performed on rats after 24 h to determine neurological function in rats; the cerebral infarction rate and cerebral water content of rats were determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

TABLE-US-00013 EXAMPLE 13 Effect of therapeutic administration of compound (1) on cerebral infarction rate in rats with focal cerebral ischemia (MCAO)/reperfusion (The data are expressed in mean SD, n = 8) Dose Cerebral Group (mg/kg) infarction rate (%) Pseudosurgery group Model control group 41.33 2.67 Edaravone group 6 mg/kg 10.23 7.87** Butylphthalide 5 mg/kg 19.18 12.94* injection group Compound (1) i.v. 24 mg/kg 11.64 9.38** high-dose group Compound (1) i.v. 12 mg/kg 16.94 9.52** medium-dose group Compound (1) i.v. 6 mg/kg 30.03 7.79 low-dose group Butylphthalide soft 60 mg/kg 15.13 11.43** capsule group i.g Compound (1) i.g. 60 mg/kg 13.61 10.24** high-dose group Compound (1) i.g. 30 mg/kg 21.52 11.04* medium-dose group Compound (1) i.g. 15 mg/kg 29.49 5.93 low-dose group **P < 0.01, compared with model control group; *P < 0.05, compared with model control group.

TABLE-US-00014 TABLE 14 Effect of therapeutic administration of compound (1) on cerebral water content in rats with focal cerebral ischemia (MCAO)/reperfusion (The data are expressed in mean SD, n = 8) Dose Cerebral water Group (mg/kg) content (%) Pseudosurgery 78.55 0.40 group Model control 84.45 0.54## group Edaravone 6 mg/kg 79.81 1.03** group Butylphthalide 5 mg/kg 80.56 2.07* injection group Compound (1) i.v. 24 mg/kg 79.95 1.16** high-dose group Compound (1) i.v. 12 mg/kg 80.74 0.89** medium-dose group Compound (1) i.v. 6 mg/kg 82.30 1.04* low-dose group Butylphthalide soft 60 mg/kg 80.10 1.49** capsule group i.g Compound (1) i.g. 60 mg/kg 79.87 1.71** high-dose group Compound (1) i.g. 30 mg/kg 80.61 1.21* medium-dose group Compound (1) i.g. 15 mg/kg 82.24 1.49 low-dose group ##P < 0.01, compared with pseudosurgery group; **P < 0.01, compared with model control group; *P < 0.05. compared with model control group.

TABLE-US-00015 TABLE 15 Effect of preventative administration of compound (1) on neuroethology in rats with focal cerebral ischemia (MCAO)/reperfusion (The data were expressed in mean SD, n = 8) Dose Behavioral Group (mg/kg) score Pseudosurgery group Model control group 3.00 0.76 Edaravone group 6 mg/kg 1.62 0.51** Butylphthalide 5 mg/kg 1.62 0.52* injection group Compound (1) i.v. 24 mg/kg 1.62 0.52** high-dose group Compound (1) i.v. 12 mg/kg 1.88 0.35** medium-dose group Compound (1) i.v. 6 mg/kg 2.13 0.35 low-dose group Butylphthalide soft 60 mg/kg 1.50 0.53** capsule group i.g Compound (1) i.g. 60 mg/kg 1.62 0.52** high-dose group Compound (1) i.g. 30 mg/kg 2.00 0.53* medium-dose group Compound (1) i.g. 15 mg/kg 2.25 0.71 low-dose group **P < 0.01, compared with model control group; *P < 0.05, compared with model control group.

[0152] Results: All dose groups of compound (1) and all positive drug groups reduced the neurological function scores, cerebral infarction rate and cerebral water content of MCAO/R rats to different degrees, with compound (1) (24 mg/kg, i.v.) and compound (1) (60 mg/kg, i.g.) groups showing the most significant effect.

[0153] Conclusion: therapeutic administration of compound (1) reduces the cerebral infarction rate, cerebral water content in rats with cerebral ischemia/reperfusion injury, and improves neuromotor function in rats.

Example 8 Study on Inhibition of Compound 1, Compound 3 and Compound 5 Against Neurocyte Ferroptosis During Cerebral Ischemia/Reperfusion

[0154] Experimental method: Primary neuron cells were incubated in a medium containing Erastin (final concentration of 50 M) for 24 hours, and then incubated in a medium containing different concentrations of the test drug and Erastin (final concentration of 50 M) for another 24 hours. After incubation, Real time PCR was used to detect mRNA expression of GPX4 in each group of cells, CCK8 assay was used to detect cell viability in each group of cells, C11-BODIPY probe method was used to detect intracellular lipid ROS levels in each group of cells, and PGSK probe method was used to detect iron content in cells.

[0155] The experimental data were expressed in meansSD, and statistical differences between groups were determined using one-way ANOVA and Tukey's test, with p-values less than 0.05 considered significant.

Experimental Results:

TABLE-US-00016 TABLE 16 Effect of compound 1, compound 3 and compound 5 with different concentrations on cell viability of Erastin-induced neuronal cells Compound 3 (%) Compound 5 (%) Compound 1 (%) Control 100 8.4 100 9.66 100 6.18 group Model control 48.24 5.73*** 50.37 4.9*** 49.85 4.14*** group 1 M 52.87 2.55 58.95 7.21 57.01 2.82.sup. 2.5 M 56.6 4.76 62.63 7.82 61.77 6.56.sup. 5 M 62.52 8.51 67.38 6.73 65.4 5.81 10 M 66.77 6.27 .sup.73.74 7.33.sup.# 76.62 5.99.sup.## 20 M 73.21 8.1.sup.# 79.85 6.34.sup.## 80.62 5.39.sup.## 40 M 77.95 6.34.sup.## 84.2 4.71.sup.## 83.43 6.21.sup.##

[0156] After incubation, the CCK8 experiment was used to detect the cell viability of each group of cells. The results were presented as MeanSD. Compared with control group, ***p<0.0001. Compared with model control group, .sup.#P<0.05, .sup.##P<0.01.

[0157] As can be seen from Table 16, the cell viability of the model group was significantly reduced compared with the control group (p<0.0001); when the concentration of compound 3 is greater than 10 M, the cell viability was significantly increased compared with the model group (p<0.05); and when the concentrations of compound 5 and compound 1 are greater than 5 M, the cell viability was significantly increased compared with the model group (p<0.05).

TABLE-US-00017 TABLE 17 Effect of compound 1, compound 3 and compound 5 with different concentrations on lipid ROS levels in Erastin-induced nerve cells Compound 3(%) Compound 5 (%) Compound 1 (%) Control group 100 10.41 100 8.24 100 10.71 Model control 151.96 5.14*** 152.74 3.88*** 157.42 5.12*** group 1 M 146.56 7.67 145.89 5.33 150.18 6.05.sup. 2.5 M 141.34 4.22 141.47 6.53 142.58 3.01.sup. 5 M 136.81 7.9 133.09 9.69 137.9 6.72 10 M 132.38 2.23 .sup.128.99 9.84.sup.# 132.33 6.57.sup.## 20 M .sup.125.33 9.79.sup.# 122.71 8.5.sup.## 121.82 5.9.sup.## 40 M 120.37 10.16.sup.## 114.82 7.13.sup.## 114.13 8.82.sup.##

[0158] After incubation, the C11-BODIPY probe method was used to detect the lipid ROS levels in each group of cells. The results were presented as MeanSD. Compared with the control group, ***p<0.0001; compared with the model group, #p<0.05, ##p<0.01.

[0159] As can be seen from Table 17, the intracellular lipid ROS levels in the model group increased significantly compared with the control group (p<0.0001); when the concentration of compound 3 is greater than 10 M, the intracellular lipid ROS levels decreased significantly compared with the model group (p<0.05); and when the concentrations of compound 5 and compound 3 are greater than 5 M, the intracellular lipid ROS levels decreased significantly compared with the model control group (p<0.01).

TABLE-US-00018 TABLE 18 Effect of compound 1, compound 3 and compound 5 with different concentrations on iron content in Erastin-induced neuronal cells Compound 3 Compound 5 Compound 1 Control group 100 9.28 100 9.79 100 10.88 Model control 49 5.12*** 50.83 7.37*** 51.49 4.73*** group 1 M 56.48 3.32.sup. 56.75 9.31.sup. 61.06 7.12 2.5 M 62.3 7 68.32 6.01.sup. 66.81 7.21 5 M 67.06 11.62 72.65 7 71.85 4.47 10 M 71.61 9.86.sup. 76.52 7.86.sup.# .sup.77.52 12.88.sup.# 20 M 76.67 7.25.sup.# 80.28 6.96.sup.## 82.16 6.86.sup.## 40 M 83.81 9.91.sup.## 87.37 9.57.sup.## 86.69 8.01.sup.##

[0160] After incubation, the PGSK probe method was used to detect the intracellular iron content. The results were presented as MeanSD. Compared with the control group, ***p<0.0001; Compared with the model group, #p<0.05, ##p<0.01.

[0161] As can be seen from Table 18, compared with the control group (p<0.0001), the fluorescence intensity of intracellular PGSK probe in the model group was significantly weakened and the iron content was significantly elevated (p<0.0001); when the concentration of compound 3 is greater than 10 M, the intracellular iron content was significantly decreased compared with the model group (p<0.05); when the concentrations of compound 5 and compound 3 are greater than 5 M, the intracellular iron content was significantly decreased compared with the model group (p<0.05).

TABLE-US-00019 TABLE 19 Effect of Compound 1, Compound 3 and Compound 5 with different concentrations on the expression of GPX4 mRNA in Erastin-induced neuronal cells Compound 3 Compound 5 Compound 1 Control group 1.0016 0.07 1.0031 0.1 1.0029 0.09.sup. Model control 0.4008 0.04*** 0.3796 0.03*** 0.3804 0.04*** group 1 M 0.4359 0.05 0.4777 0.07.sup. 0.4845 0.06.sup. 2.5 M 0.4875 0.01 0.5373 0.08.sup. 0.5759 0.07.sup. 5 M 0.5407 0.06 0.6033 0.06.sup.# 0.6469 0.08.sup.# 10 M 0.6152 0.07.sup.## 0.6942 0.01.sup.## 0.7093 0.15.sup.## 20 M 0.6765 0.07.sup.## 0.7693 0.1.sup.## 0.7664 0.08.sup.## 40 M 0.7421 0.08.sup.## 0.8368 0.07.sup.## 0.8503 0.06.sup.##

[0162] After incubation, real time PCR was used to detect the mRNA expression of GPX4 in each group of cells. The results were presented as MeanSD. Compared with the control group, ***p<0.0001; Compared with the model group, #p<0.05, #p<0.01.

[0163] As can be seen from Table 19, GPX4 mRNA expression in cells of the model group was significantly decreased compared with the control group (p<0.0001); when the concentration of compound 3 is greater than 5 M, the cellular GPX4 mRNA expression level was significantly elevated compared with the model group (p<0.05); and when the concentrations of compound 5 and compound 3 are greater than 2.5 M, the cellular GPX4 mRNA expression level was significantly increased compared with the model group (p<0.01).

[0164] Conclusion: Compound 1, Compound 3 and Compound 5 all significantly increased Erastin-induced rat neuronal cell viability and GPX4 mRNA expression, and significantly decreased intracellular lipid ROS levels and iron content. The above findings indicated that Compound 1, Compound 3 and Compound 5 all inhibited Erastin-induced ferroptosis in rat neuronal cells.

Example 9 Evaluation of Regressive Effect of Compound 1, Compound 3, and Compound 5 on Vascular Inflammation

[0165] Experimental method: 360 tails of 5 dpf macrophage fluorescent zebrafish were randomly selected and placed in beakers with 30 tails in each beaker, compound (1), compound (3), and compound (5) were given intravenously, and the positive control group was given atorvastatin calcium by dissolving it in water at a concentration of 30 g/mL, while the normal control group (fish farming water-treated zebrafish) and the model control group were set up, and the volume of each beaker (experimental group) was 25 mL. Except for the normal control group, all other groups were given high-lipids and high-sucrose feed by dissolving it in water. Compound (1), compound (3) and compound (5) group were co-treated with high-lipids and high-sucrose feed for 30 h. The treatment was carried out up to 9 dpf, and 10 zebrafish were randomly selected from each experimental group to collect the intensity of the macrophage fluorescence within the tail vein and around the tail vein of the zebrafish under fluorescence microscope, and the results of the statistical analysis were used to evaluate the regressive effect of compound (1), compound (3), compound (5) on zebrafish vascular inflammation in the high sugar and high fat model.

[00004]$\mathrm{Anti}-\mathrm{inflammatory}\mathrm{effect}\left(\%\right)=\frac{\left[S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)-S\left(\mathrm{test}\mathrm{product}\mathrm{group}\right)\right]}{S\left(\mathrm{model}\mathrm{control}\mathrm{group}\right)}100\%$

Experimental Results:

TABLE-US-00020 TABLE 20 Regressive effect of the test product on vascular inflammation in high-glucose and high-fat zebrafish (n = 10) Fluorescence intensity of perivascular Anti- Dosage macrophage (pixels, inflammatory Group (ng/tail) Mean SE) effect (%) Normal 116966 7407 control group Model 184631 3800 control group Atorvastatin 30 g/mL 106352 6663 42*** calcium 11.1 113809 7995*** 38*** Compound (3) 33.3 118747 6434*** 36*** 100 118504 10539*** 36*** 27.8 113678 4811*** 38*** Compound (5) 83.3 118039 9198*** 36*** 250 115627 5934*** 37*** 222 134308 6117*** 27*** Compound (1) 667 116848 5036*** 37*** 2000 108154 4723*** 41*** Compared with model control group, ***p < 0.001.

[0166] Conclusion: Compound (1), compound (3) and compound (5) showed significant elimination effect on vascular inflammation in high-glucose and high-fat zebrafish, under the dosage conditions of this experiment.

Example 10 Preparation of Injection

TABLE-US-00021 TABLE 21 Formula table for preparing injection of compound (1) Prescription Prescription dosage Compound (1) 1000 mg Glycerol 10 g Sodium chloride 3.5 g Sodium citrate dihydrate 150 mg 0.1M hydrochloric acid Appropriate amount or sodium hydroxide Injection water 500 ml was added to pH range of solution 5.0-6.5

[0167] 80% of the total amount of injection water was taken and added with the prescribed amount of sodium chloride, sodium citrate dihydrate, glycerol, and compound (1) in sequence. The mixture was stirred to dissolve, and adjusted to pH 5.0-6.5 with 0.1M hydrochloric acid or sodium hydroxide. The product was obtained by adding injection water to the volume. The resulting solution was subpackaged into 10 ml glass vials and sterilized under heat and pressure to obtain the compound injection. Each vial contains 20 mg of compound (1).

Example 11 Preparation of Lyophilized Powder for Injection

TABLE-US-00022 TABLE 22 Formula table for preparing lyophilized powder for injection of compound (1) Prescription Prescription dosage Function Compound (1) 0.1~0.5g Active Ingredients Sodium hydroxide Appropriate PH or hydrochloric acid amount regulator Water for injection Add to 2 mL Solvent

[0168] A prescribed amount of compound (1) was added into 80% of the prescribed amount of water for injection, and stirred to dissolve; the mixture was added with water for injection to the prescribed amount. The above solution was added with activated carbon (0.1-0.5%) and stirred for dozens of minutes. After decolorization by activated carbon and filtration to remove the activated carbon, the resulting solution was added with sodium hydroxide solution (or hydrochloric acid solution) to adjust pH value. Samples were taken for semi-finished product testing. After secondary filtration through a 0.22 m filter, bottle-filling was performed. Performing lyophilization according to the set lyophilization parameters, plugging, capping, and packaging.

[0169] All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that, after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.