Edaravone homologues and methods and uses

Abstract

The invention discloses an edaravone homologue as well as a method and application thereof. The edaravone homologue has a structure shown in a formula (I), in the formula (I), R1 and R2 are substituent groups at any position on a benzene ring, and R1-R2 are the same or different and are independently selected from hydrogen or C1-C3 alkyl. Compared with edaravone, the edaravone homologue disclosed by the invention has the advantages that the oxidative stress level can be more effectively reduced, neuronal apoptosis is inhibited, and cerebral ischemia reperfusion injury is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an edaravone homologue and a method and use thereof. Background Art

[0002] According to the different pathogenesis, stroke can be divided into hemorrhagic stroke and ischemic stroke. Ischemic stroke accounts for about 85% of all stroke cases and is the most common type. The treatment methods for ischemic stroke mainly include surgical treatment and antithrombotic drugs. Antithrombotic drugs mainly include thrombolytic drugs, antiplatelet aggregation drugs and anticoagulants. Restoring blood supply to the ischemic area of ​​the brain through mechanical thrombectomy and drug thrombolysis is a key link in the survival of brain tissue after ischemia. In most cases, the restoration of blood supply to the ischemic area of ​​the brain will lead to cerebral ischemia-reperfusion injury (CIRI). The pathological process of cerebral ischemia-reperfusion injury is a complex cascade reaction, mainly including oxidative stress, inflammatory response, neuronal apoptosis, blood-brain barrier destruction, etc., which will cause more serious brain damage and even lead to the death of the patient.

[0003] CN117919254A discloses the use of a triazole compound in the preparation of a drug for preventing and treating ischemic stroke. CN110204477A discloses a diterpene alkaloid with antioxidant effect and its use in the preparation of a drug. CN111793032A discloses a pyrazolone compound and its preparation method and use.

[0004] The prior art also reports the effective effect of edaravone (EDA, chemical name 3-methyl-1-phenyl-2-pyrazol-5-one) in combating cerebral ischemia-reperfusion injury. CN117224551A discloses a compound solution composition formed by edaravone, sodium aescinate, cosolvent, latent solvent and water.

[0005] Current studies have found that the main mechanism of action of edaravone in treating ischemic stroke is to inhibit neuronal apoptosis, inhibit the generation of reactive oxygen species and enhance the body's ability to scavenge free radicals. In the case of treating severe cerebral ischemia-reperfusion injury, edaravone's ability to inhibit neuronal apoptosis and its antioxidant effect appear to be relatively weak, thus limiting its widespread clinical application. Summary of the invention

[0006] One object of the present invention is to provide an edaravone homologue or a pharmaceutically acceptable salt thereof, which can be used to treat cerebral ischemia-reperfusion injury. Compared with edaravone (EDA), it can more effectively reduce cerebral infarction volume, improve neurological function scores, reduce the level of oxidative stress markers and inhibit neuronal apoptosis, and improve cerebral ischemia-reperfusion injury. Another object of the present invention is to provide a method for preparing the above-mentioned edaravone homologue or a pharmaceutically acceptable salt thereof, and its process is stable. Another object of the present invention is to provide the use of the above-mentioned edaravone homologue or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned purpose.

[0008] In one aspect, the present invention provides an edaravone homologue or a pharmaceutically acceptable salt thereof, wherein the edaravone homologue has a structure shown in formula (I):

[0009]

[0010] In formula (I), R1 and R2 are substituents at any position on the benzene ring, and R1 to R2 are the same or different and are independently selected from hydrogen or C1-C3 alkyl.

[0011] According to the edaravone homologue or a pharmaceutically acceptable salt thereof of the present invention, preferably, R1 to R2 are the same or different and are independently selected from hydrogen or methyl.

[0012] According to the edaravone homologue or a pharmaceutically acceptable salt thereof of the present invention, preferably, the edaravone homologue represented by formula (I) is:

[0013]

[0014] On the other hand, the present invention also provides a method for preparing the above-mentioned edaravone homologue or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0015] (1) reacting the compound represented by formula (A) with dimethyl carbonate in the presence of sodium hydride to obtain a compound represented by formula (B);

[0016] (2) reacting the compound represented by formula (B) with 3,5-dimethoxybenzyl halide in the presence of sodium hydride to obtain a compound represented by formula (C);

[0017] (3) reacting the compound represented by formula (C) with phenylhydrazine substituted with R2 to obtain the compound represented by formula (D); wherein R2 is selected from hydrogen or C1-C3 alkyl;

[0018] (4) reacting the compound represented by formula (D) with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain an edaravone homologue represented by formula (I);

[0019]

[0020] In formula (A), formula (B), formula (C) and formula (D), R1 is a substituent at any position on the benzene ring, and R1 is selected from hydrogen or C1-C3 alkyl; in formula (D), R2 is a substituent at any position on the benzene ring, and R2 is selected from hydrogen or C1-C3 alkyl.

[0021] According to the method of the present invention, preferably:

[0022] In step (1), the compound represented by formula (A) is added dropwise to a mixture of sodium hydride, dimethyl carbonate and a first solvent, and reacted under reflux conditions;

[0023] In step (2), a solution containing the compound represented by formula (B) is added dropwise to a mixture of sodium hydride and a second solvent, and then 3,5-dimethoxybenzyl halide is added to react at 20 to 40°C.

[0024] According to the method of the present invention, preferably, the first solvent is tetrahydrofuran or 2-methyltetrahydrofuran; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran; and the 3,5-dimethoxybenzyl halide is 3,5-dimethoxybenzyl bromide.

[0025] According to the method of the present invention, preferably, in step (3), the reaction temperature is 110-125°C.

[0026] According to the method of the present invention, preferably, in step (4), the phase transfer catalyst is tetrabutylammonium bromide.

[0027] In another aspect, the present invention also provides a use of the edaravone homologue or a pharmaceutically acceptable salt thereof as described above in the preparation of a medicament for treating cerebral ischemia-reperfusion injury.

[0028] According to the use of the present invention, preferably, in an in vivo experiment on mice, the dosage of the edaravone homologue is 5-20 mg / kg.

[0029] The present invention found that the edaravone homologue of the present invention can significantly reduce the volume of cerebral infarction, improve neurological function scores, reduce reactive oxygen levels and inhibit neuronal apoptosis. Compared with edaravone, the edaravone homologue of the present invention showed more excellent effects on the above indicators. In cell experiments, the edaravone homologue of the present invention can protect neurons from oxidative stress damage and improve neuronal survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It indicates the protective effect of homologue E31 on cerebral infarction and behavioral disorders in mice after MCAO / R injury. Figure 1 A is the result that E31 and EDA can significantly reduce cerebral infarction caused by MCAO / R. Figure 1 B represents Zea-Longa behavioral score, Figure 1 C represents G&B behavioral score. *** p<0.001vs Sham group; # p<0.05, ## p<0.01, ### p<0.001vs MCAO / R group mice; @ p<0.05, @@ p<0.01, @@@ p<0.001vs MCAO / R+EDA 10mg group mice.

[0031] Figure 2 This is the result of the protective effect of homologue E31 on neuronal morphological damage caused by MCAO / R.

[0032] Figure 3 This is a display of Tunel coloring.

[0033] Figure 4 is the proportion of Tunel-positive cells in each group. *** p<0.001vs Sham group; # p<0.05, ### p<0.001vsMCAO / R group; @@@ p<0.001vs MCAO / R+EDA 10mg group.

[0034] Figure 5 The results show that the homologue E31 has a protective effect on the ultrastructural damage of neuronal cells induced by MCAO / R.

[0035] Figure 6 The results show that the homologue E31 has a protective effect on the morphological damage of HT22 cells induced by OGD / R.

[0036] Figure 7 The protective effect of homologue E31 on the survival rate of HT22 cells induced by OGD / R is shown. *** p<0.001vs Control group; ## p<0.01, ### p<0.001vs MCAO / R group; @ p<0.05,@@ p<0.01, @@@ p<0.001 vs EDA.

[0037] Figure 8 The homologue E31 was shown to protect HT22 cells from OGD / R-induced impairment of cell viability.

[0038] Fig. 9 The homologue E31 was shown to protect HT22 cells from apoptosis induced by OGD / R.

[0039] Fig.10 The inhibitory effect of homologue E31 on ROS levels in HT22 cells is shown.

[0040] Fig.11 The protective effect of homologue E31 on ultrastructural damage of HT22 cells after OGD / R injury is shown. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0042] <Term Explanation>

[0043] In the present invention, Cm-Cn represents a group having m to n carbon atoms; for example, C1-C3 alkyl represents an alkyl group having 1 to 3 carbon atoms.

[0044] <Edaravone homologue or pharmaceutically acceptable salt thereof>

[0045] Based on edaravone (EDA), the inventors of the present application carefully designed and optimized the synthesis of the edaravone homologues of the present invention, which can be used to prepare drugs for treating cerebral ischemia-reperfusion injury. The edaravone homologues of the present invention have a structure shown in formula (I):

[0046]

[0047] Such a structure can more effectively reduce the volume of cerebral infarction, improve neurological function scores, reduce the level of oxidative stress markers and inhibit neuronal apoptosis. The present invention surprisingly found that compared with edaravone (EDA), the edaravone homologue of the present invention has a better effect of improving cerebral ischemia-reperfusion injury.

[0048] In the present invention, R1 is a substituent at any position on the benzene ring, and R1 is independently selected from hydrogen or C1-C3 alkyl. Preferably, R1 is selected from hydrogen or methyl. More preferably, R1 is hydrogen.

[0049] In the present invention, R2 is a substituent at any position on the benzene ring, and R2 is independently selected from hydrogen or C1-C3 alkyl. Preferably, R2 is selected from hydrogen or methyl. More preferably, R2 is hydrogen.

[0050] In the present invention, examples of the C1-C3 alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl.

[0051] According to a specific embodiment of the present invention, the edaravone homologue of the present invention has the structure shown below:

[0052]

[0053] The edaravone homologues of the present invention can form pharmaceutically acceptable salts, which refer to salts formed by the edaravone homologues shown in formula (I) and inorganic acids such as phosphoric acid, sulfuric acid, and hydrochloric acid, or organic acids such as acetic acid, tartaric acid, citric acid, and malic acid, or acidic amino acids such as aspartic acid and glutamic acid, or salts formed by forming esters or amides with the above acids and then with inorganic bases, such as sodium, potassium, calcium, aluminum or ammonium salts.

[0054] <Preparation method>

[0055] The method for preparing the edaravone homologue or a pharmaceutically acceptable salt thereof of the present invention comprises: (1) a synthesis step of the compound represented by formula (B); (2) a synthesis step of the compound represented by formula (C); (3) a synthesis step of the compound represented by formula (D); and (4) a synthesis step of the final product. The following is a detailed description.

[0056] Synthesis steps of the compound represented by formula (B)

[0057] In step (1) of the present invention, the compound represented by formula (A) is reacted with dimethyl carbonate in the presence of sodium hydride to obtain a compound represented by formula (B).

[0058]

[0059] In formula (A) and (B), the definition of R1 is as mentioned above and will not be repeated here.

[0060] According to one embodiment of the present invention, the compound represented by formula (A) is added dropwise to a mixture of sodium hydride, dimethyl carbonate and a first solvent, and reacted under reflux conditions; the first solvent is tetrahydrofuran or 2-methyltetrahydrofuran, preferably tetrahydrofuran.

[0061] The molar ratio of the compound represented by formula (A), dimethyl carbonate and sodium hydride (NaH) is 1:2-2.05:2-2.05, preferably 1:2-2.03:2-2.03, for example 1:2:2.

[0062] The reaction is carried out under reflux conditions, and the reaction time may be 1.5 to 3.5 hours, preferably 2 to 3.5 hours, and more preferably 2 to 3 hours.

[0063] In the present invention, after the reaction is completed, the temperature is lowered, a saturated ammonium chloride solution is added at 0 to 10°C to quench the reaction, and the pH value is adjusted to 6 to 7 with hydrochloric acid. Ethyl acetate is added for liquid separation, the aqueous layer is extracted once or twice with ethyl acetate, the organic phases are combined, the combined organic phases are washed with water once or twice, washed with saturated brine once or twice, then dried with anhydrous sodium sulfate, and concentrated to remove the solvent. The obtained concentrate is separated with a silica gel column, and the eluent is a mixed solvent of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 19 to 21:1, for example, it can be 20:1.

[0064] Synthesis steps of the compound represented by formula (C)

[0065] In step (2) of the present invention, the compound represented by formula (B) is reacted with 3,5-dimethoxybenzyl halide in the presence of sodium hydride to obtain a compound represented by formula (C).

[0066]

[0067] In formula (C), the definition of R1 is as mentioned above and will not be repeated here.

[0068] A solution containing the compound represented by formula (B) is added dropwise to a mixture formed by sodium hydride and a second solvent, and then 3,5-dimethoxybenzyl halide is added to react at 20-40°C; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran, preferably tetrahydrofuran. The 3,5-dimethoxybenzyl halide can be 3,5-dimethoxybenzyl chloride or 3,5-dimethoxybenzyl bromide, preferably 3,5-dimethoxybenzyl bromide (also known as 3,5-dimethoxybenzyl bromide). The solvent used in the solution containing the compound represented by formula (B) is the second solvent.

[0069] In the present invention, the molar ratio of the compound represented by formula (B), sodium hydride and benzyl halide is 1:2-2.05:1.5-1.6, preferably 1:2-2.02:1.5-1.55, for example 1:2:1.5.

[0070] The reaction temperature may be 20 to 40° C. The reaction time may be 15 to 30 h, preferably 18 to 25 h, and more preferably 20 to 25 h.

[0071] In the present invention, after the reaction is completed, a saturated ammonium chloride solution is added to the reactant to quench the reaction, the liquid is separated, the aqueous phase is extracted twice or three times with ethyl acetate, and the organic phase is combined. The organic phase is washed once with water, once with saturated brine, dried with anhydrous sodium sulfate, and concentrated to remove the solvent. The concentrate is separated with a silica gel column, and the eluent used is a mixed solvent of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 24 to 26:1, for example, it can be 25:1.

[0072] Synthesis steps of the compound represented by formula (D) (i.e., cyclization reaction)

[0073] In step (3), the compound represented by formula (C) is reacted with phenylhydrazine substituted with R2 to obtain the compound represented by formula (D); wherein R2 is selected from hydrogen or C1-C3 alkyl.

[0074]

[0075] The molar ratio of the compound represented by formula (C) to the phenylhydrazine substituted by R2 is 1:1.5 to 2, preferably 1:1.5 to 1.9, and more preferably 1:1.6 to 1.8.

[0076] In this step, no solvent may be added. The reaction temperature may be 110 to 120° C., preferably 115 to 120° C., more preferably 115 to 118° C. The reaction time may be 2 to 6 hours, preferably 3 to 5 hours, more preferably 4 to 4.5 hours.

[0077] In the present invention, after the reaction is completed, the mixture is cooled to room temperature, ethyl acetate and water are added to separate the liquids, the aqueous phase is extracted with ethyl acetate after separation, and the organic phases are combined. The combined organic phases are washed once or twice with water, washed once or twice with saturated brine, and then dried with anhydrous sodium sulfate and concentrated to remove the solvent. The concentrate is recrystallized with ethanol / petroleum ether, and the volume ratio of ethanol to petroleum ether is 1:2 to 3, for example, 1:2.

[0078] Synthesis steps of the final product

[0079] In step (4) of the present invention, the compound represented by formula (D) is reacted with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain the compound represented by formula (I).

[0080] The molar ratio of the compound represented by formula (D), N-fluorobisbenzenesulfonamide and phase transfer catalyst is 1:1-1.5:0.05-0.06, preferably 1:1-1.3:0.05-0.055. The phase transfer catalyst is preferably tetrabutylammonium bromide.

[0081] In this step, an alkali metal hydroxide, such as sodium hydroxide or potassium hydroxide, preferably potassium hydroxide, is also added. The molar ratio of the compound represented by formula (D) to the alkali metal hydroxide can be 1:2.9 to 3.1, preferably 1:3.0 to 3.1, and more preferably 1:3.0 to 3.05. The solvent used can be diethyl ether.

[0082] The reaction temperature may be 20 to 40° C., and the reaction time may be 2 to 5 h, preferably 2.5 to 4 h, and more preferably 3 to 3.5 h.

[0083] According to a specific embodiment of the present invention, the compound represented by formula (D), N-fluorobisbenzenesulfonamide, tetrabutylammonium bromide, potassium hydroxide and diethyl ether are added to a reaction bottle and stirred for reaction for 2 to 5 hours.

[0084] In the present invention, ethyl acetate and water are added to separate after the reaction is completed, and the aqueous phase after separation is extracted with ethyl acetate, and the organic phases are combined. The combined organic phase is washed with water once or twice, washed with saturated brine once or twice, dried with anhydrous sodium sulfate, and concentrated to remove the solvent. The concentrate is separated with a silica gel column, and the eluent used is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 1:2 to 5, preferably 1:2 to 4, for example, it can be 1:2.

[0085] <Application>

[0086] The present invention also provides a use of the above-mentioned edaravone homologue or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cerebral ischemia-reperfusion injury. The edaravone homologue of the present invention exhibits a more significant effect than edaravone (EDA) in inhibiting oxidative stress, inhibiting neuronal apoptosis and improving cerebral ischemia-reperfusion injury.

[0087] The present invention selects a suitable animal model (such as a mouse cerebral ischemia-reperfusion model) for in vivo experiments. During the experiment, the present invention sets a plurality of dosage groups and a control group to ensure the accuracy and reliability of the results. The present invention also uses a variety of detection means to conduct in-depth research on the effects of the drug. The present invention does not make any particular limitation on the detection means.

[0088] In the in vivo experiment of mice, the dosage of the edaravone homologue of the present invention is 5 to 20 mg / kg, preferably 7 to 15 mg / kg, and more preferably 10 to 15 mg / kg. The dosage of 5 to 20 mg / kg specifically means that the dosage of the edaravone homologue is 5 to 20 mg per kilogram of body weight of the mouse. Such a dosage has a better effect on improving cerebral ischemia-reperfusion injury.

[0089] <Test Method>

[0090] 1 H NMR, 13 C NMR, 19 F NMR was measured using a Bruker DPX400 UltraShield nuclear magnetic resonance spectrometer, using deuterated chloroform as solvent.

[0091] Example 1

[0092] Step 1: Preparation of ethyl benzoylacetate (i.e., preparation of the compound represented by formula (B))

[0093]

[0094] Under argon protection, sodium hydride (NaH, 60% mass fraction, 8.4g) was added to the reaction bottle, followed by adding 17.6mL of dimethyl carbonate and 30mL of dried tetrahydrofuran to the reaction bottle, and then heated to reflux. Then 10mL of acetophenone (i.e., the compound shown in formula (A)) was added dropwise to the system, and bubbles were observed to be released. The addition was completed in half an hour, and the stirring reaction was continued for 2h. The reaction was determined to be complete by TLC detection of acetophenone reaction, and the reaction was judged to be over. After the reaction was completed, the temperature was lowered, and 20mL of saturated ammonium chloride was added to the reactant under an ice bath, and the pH was adjusted to 6-7 with 3mol / L hydrochloric acid. Then ethyl acetate was added for liquid separation, and the water layer was extracted once with ethyl acetate, and the organic phase was combined, and the combined organic phase was washed once with water and once with saturated brine, and then dried over anhydrous sodium sulfate and concentrated to remove the solvent. The obtained concentrate was separated by a silica gel column (the eluent was a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 20:1) to obtain 10.7 g of a colorless oil, which was ethyl benzoylacetate product, with a yield of 77%.

[0095] Step 2: Preparation of benzyl-substituted ethyl benzoyl acetate (i.e., the compound represented by formula (C))

[0096] Among them, Me is methyl.

[0097] Under argon protection, NaH (60% mass fraction, 1g) and 10mL of dried tetrahydrofuran were added to the reaction bottle and mixed, and then the 10mL tetrahydrofuran solution of ethyl benzoyl acetate (2.6g, 15mmol) prepared above was added dropwise, and stirred at room temperature (rt) until no bubbles were released. Then, 3.3g of 3,5-dimethoxybenzyl bromide was added to the reaction system at one time, and the reaction was stirred at room temperature for 20h. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to the reactant, the liquid was separated, the aqueous phase was extracted with ethyl acetate twice, and the organic phase was combined. The organic phase was washed once with water, once with saturated brine, dried with anhydrous sodium sulfate, and concentrated to remove the solvent. The concentrate was separated with a silica gel column (the eluent used was a mixed solvent formed by a volume ratio of petroleum ether and ethyl acetate of 25:1), and 4.2g of white solid was obtained, which was the benzyl-substituted ethyl benzoyl acetate product with a yield of 88%.

[0098] Step 3: Ring-forming reaction (i.e., preparation of the compound represented by formula (D))

[0099]

[0100] Here, Me represents a methyl group.

[0101] Under argon protection, 1.55g of phenylhydrazine and 3.28g of the benzyl-substituted ethyl benzoyl acetate prepared above were added to the reaction bottle in sequence, and the temperature was raised to 115°C for 4h under stirring to complete the reaction of the benzyl-substituted ethyl benzoyl acetate (TLC detection). After the reaction was completed, it was cooled to room temperature, ethyl acetate and water were added to separate the liquids, and the aqueous phase was extracted with ethyl acetate after separation, and the organic phases were combined. The combined organic phase was washed once with water, washed once with saturated brine, and then dried over anhydrous sodium sulfate and concentrated to remove the solvent. The concentrate was recrystallized from ethanol / petroleum ether to obtain 2.3g of a white solid, recorded as P2, i.e., the compound shown in formula (D), with a yield of 63%.

[0102] Step 4: Preparation of the final product (i.e., preparation of the compound represented by formula (I))

[0103]

[0104] Wherein, Me is a methyl group;

[0105] P2 (0.38 g) prepared above, 0.35 g N-fluorobisbenzenesulfonamide, 0.03 g tetrabutylammonium bromide, 0.12 g solid potassium hydroxide, 10 mL ether were added to the reaction flask in sequence, and the reaction was vigorously stirred for 3 h at room temperature. After the reaction was completed, ethyl acetate and water were added to separate the liquids, and the aqueous phase after separation was extracted with ethyl acetate, and the organic phases were combined. The combined organic phase was washed once with water, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. The concentrate was separated by silica gel column (the eluent used was petroleum ether / ethyl acetate, and the volume ratio of the two was 5:1) to obtain the final product, which was recorded as E31. The yield was 87%. The final product was structurally characterized.

[0106] The structure of the final product is as follows:

[0107]

[0108] Wherein, Me represents a methyl group and is denoted as E31.

[0109] The structural identification data of the final product E31 (i.e., the compound represented by formula (I)) are as follows:

[0110] 1 H NMR (400 MHz, chloroform-d) δ 7.94-7.85 (m, 2H), 7.77-7.65 (m, 2H), 7.56-7.45 (m, 3H), 7.42-7.33 (m, 2H), 7.24-7.13 (m, 1H), 6.22 (t, J = 2.3 Hz, 1H), 6.00 (d, J = 2.3 Hz, 2H), 3.68-3.37 (m, 8H). 13 C NMR (101 MHz, chloroform-d) δ 168.07 (d, J = 21.3 Hz), 160.50, 154.21 (d, J = 13.7 Hz), 136.96, 131.87 (d, J = 12.1 Hz), 131.11, 129.58 (d, J = 1.7 Hz), 129.05, 128.87, 126.67, 126.66, 125.88, 119.17, 107.40, 100.87, 96.15, 94.17, 55.03, 41.36 (d, J = 26.3 Hz). 19 F NMR (376 MHz, chloroform-d) δ-161.82 (s, 1F).

[0111] Experimental Example 1-Animal Experiment

[0112] 1.1 Experimental drugs

[0113] The experiment used SPF-grade KM mice weighing about 25 g, purchased from Sikebes Co., Ltd.

[0114] 1.2 Protective effects of different doses of E31 on brain damage induced by MCAO / R in mice

[0115] In order to verify the protective effect of homologue E31 on MCAO / R injury and its administration concentration in vivo, the cerebral infarction volume of each group of mice was evaluated by TTC staining. In addition, the Zea Longa scoring method and G&B scoring method were used to evaluate the behavioral dysfunction of each group of mice.

[0116] The establishment of the mouse MCAO / R model includes the following steps: (1) preparation of materials and equipment: KM mice, electrocoagulation pen, stereo microscope, sutures (25-35g mice), surgical tools (scalpels, tweezers, scissors, sutures, etc.), surgical gloves, white coats and masks, etc.; (2) preparation process of the mouse MCAO / R injury model: mice were fasted for 12 hours before surgery, anesthetized and fixed on the operating table, a midline incision was made on the neck, the right common carotid artery, external carotid artery and internal carotid artery were exposed, the external carotid artery was cauterized, and a knot was tied at the proximal end of the common carotid artery with sutures. A slipknot was tied at the distal end of the common carotid artery with suture thread, and the distal end of the internal carotid artery was clamped with a hemostatic clamp. A small incision was made between the two knots of the common carotid artery, and the mouse embolism line was inserted from the incision to the hemostatic clamp. The slipknot was tightened, and the hemostatic clamp was released. The insertion was continued. If a slight resistance could be felt, it indicated that the embolism line had penetrated deep into the middle cerebral artery and blocked the blood flow. At this time, the slipknot at the distal end of the common carotid artery was tied into a dead knot and the surgical wound was sutured. After 1 hour, the embolism line was pulled out to achieve reperfusion, and the common carotid artery was cauterized from the incision of the common carotid artery to avoid bleeding, and then the surgical wound was sutured. Relevant indicators were tested after 24 hours.

[0117] 1.2.1 Experimental groups: Sham group mice, MCAO / R group mice, MCAO / R+E315mg group mice (MCAO / R injured mice were administered E31 at a dose of 5 mg / kg), MCAO / R+E3110mg group mice (MCAO / R injured mice were administered E31 at a dose of 10 mg / kg), MCAO / R+E3120mg group mice (MCAO / R injured mice were administered E31 at a dose of 20 mg / kg) and MCAO / R+EDA10mg group mice (MCAO / R injured mice were administered EDA at a dose of 10 mg / kg).

[0118] Among them, EDA refers to edaravone (3-methyl-1-phenyl-2-pyrazol-5-one), and its structural formula is as follows:

[0119]

[0120] 1.2.2TTC staining method: conventional method was used.

[0121] 1.2.3 Experimental Results

[0122] The brain tissue sections of mice in the sham group were red. The mice in the MCAO / R group had obvious white cerebral infarction areas. Figure 1 As shown in A, compared with the MCAO / R group, the cerebral infarction volume of the MCAO / R+E31 5 mg group, MCAO / R+E31 10 mg group, MCAO / R+E31 20 mg group and MCAO / R+EDA10 mg group was significantly reduced. Among them, the cerebral infarction volume of the MCAO / R+E31 10 mg group and the MCAO / R+E3120 mg group was significantly smaller than that of the MCAO / R+EDA 10 mg group, and the cerebral infarction volume of the MCAO / R+E31 10 mg group was the smallest.

[0123] Reference Figure 1 B and Figure 1 As shown in C, the mice in the Sham group did not show obvious behavioral disorders. The mice in the MCAO / R group showed obvious behavioral disorders, which were manifested by a significant decrease in the comprehensive evaluation of limbs, limb motor ability, and coordination ability of the mice (a significant increase in the Zea Longa score and a significant decrease in the G&B score). Compared with the mice in the MCAO / R group, the behavioral disorders of the mice in the MCAO / R+E31 5mg group, the MCAO / R+E31 10mg group, the MCAO / R+E31 20mg group, and the MCAO / R+EDA10mg group were significantly improved, which were manifested by the enhancement of the comprehensive evaluation of limbs, limb motor ability, and coordination ability (a significant decrease in the Zea Longa score and a significant increase in the G&B score).

[0124] Among them, the behavioral performance of mice in the MCAO / R+E31 10mg group and the MCAO / R+E31 20mg group was better than that of mice in the MCAO / R+EDA 10mg group. This is also consistent with the conclusion of the above cerebral infarction evaluation. As for the fact that the efficacy of 20mg / kg E31 is not as good as 10mg / kg E31, we speculate that the reason may be that the maximum effective dose of E31 is exceeded, and the effect no longer increases with the increase of dose, and even the efficacy may be weakened or toxic reactions may occur. Therefore, we used 10mg / kg as the dosage of E31 in subsequent in vivo experiments on mice.

[0125] 1.3 Protective effect of homologue E31 on pathological damage of brain tissue in mice induced by MCAO / R

[0126] In order to visually present the effect of E31 administration on pathological damage of brain tissue in MCAO / R mice, we used Nissl staining to observe the changes in neuronal cell morphology and Nissl bodies in the penumbra region of the brain tissue of each group of mice.

[0127] 1.3.1 Nissl staining method: conventional method was used.

[0128] 1.3.2 Experimental Results

[0129] like Figure 2 As shown in the figure, the neurons of the mice in the Sham group were clearly identifiable, with round or oval cell bodies, neatly arranged, uniform in size, and clear boundaries. Dark blue Nissl bodies were observed in the cell bodies, and significantly longer dendrites or axons were observed around them. In contrast, the neurons of the mice in the MCAO / R group were arranged in a disordered and loose manner, with wrinkled and irregular cell bodies, weakened Nissl staining, and broken, deformed, or disappeared nerve fibers. The morphological damage of neuronal cells in the brain tissue of the mice in the MCAO / R+E31 10mg group was significantly improved, with basically normal morphology of neuronal cell bodies, clear boundaries, more obvious Nissl body staining, and nerve fibers that were damaged but still visible. Although the morphology of neurons in the brain tissue of the mice in the MCAO / R+EDA 10mg group was also improved, the improvement effect was not as good as that of the mice in the MCAO / R+E31 10mg group.

[0130] 1.4 Protective effect of homologue E31 on neuronal apoptosis in mice after MCAO / R injury

[0131] To further verify whether the homologue E31 can alleviate MCAO / R injury by alleviating cell apoptosis response, we used Tunel fluorescence staining to observe the changes in the proportion of apoptotic cells in the penumbra area of ​​the brain tissue of each group of mice.

[0132] 1.4.1 Tunel fluorescent staining method: conventional method was used.

[0133] 1.4.2 Experimental Results

[0134] like Figure 3 and Figure 4 As shown, it was found that there was no apoptosis in the brain tissue of the Sham group mice. However, obvious apoptosis occurred in the penumbra area of ​​the brain tissue of the MCAO / R group mice. The apoptosis of neurons in the penumbra area of ​​the brain tissue of the MCAO / R+E31 10mg group mice and the MCAO / R+EDA 10mg group mice was significantly reduced, among which, the apoptosis of neurons in the penumbra area of ​​the brain tissue of the MCAO / R+E31 10mg group mice was significantly lighter than that of the MCAO / R+EDA 10mg group mice.

[0135] Figure 3In the figure, DAPI is 4',6-diamidino-2-phenylindole, a common fluorescent dye that stains cell nuclei blue. MERGE means to merge DAPI and TUNEL staining into one picture.

[0136] 1.5 Effects of homologue E31 on ultrastructure of neurons after MCAO / R injury in mice

[0137] In order to further observe the effect of administration of homologue E31 on the ultrastructure of cells in the penumbra region of brain tissue of MCAO / R injured mice, we performed ultrathin sectioning on the penumbra region of brain tissue of each group of mice (the sham group mice had no penumbra, and the position was close to that of other groups) and displayed the results by transmission electron microscopy.

[0138] Results Figure 5 .like Figure 5 As shown, the neurons of mice in the Sham group were full and had intact membranes, and the nuclei were normal in morphology ( Figure 5 The red arrow of the Sham group) shows that the organelles are arranged in an orderly manner, and the mitochondria are oval and complete in shape ( Figure 5 The blue arrows in the Sham group). The cell membranes of the neurons in the MCAO / R group were damaged and the cell bodies were shrunken ( Figure 5 MCAO / R group (green arrow), the nucleus is shrunken and the chromatin in the nucleus is condensed and deposited on the inner side of the nuclear membrane ( Figure 5 In the MCAO / R group (red arrow), the mitochondria in the cells were shortened, the volume was reduced, and the mitochondrial cristae were blurred ( Figure 5 MCAO / R group (blue arrow), while the nerve cells of MCAO / R+EDA 10mg group and MCAO / R+E31 10mg group recovered to a relatively full and intact state ( Figure 5 Green arrows in the MCAO / R+EDA 10mg group and the MCAO / R+E31 10mg group), and the shrinkage of the cell nucleus was also alleviated ( Figure 5 The red arrows in the MCAO / R+EDA 10mg group and the MCAO / R+E31 10mg group) show that the cell organelles are arranged in an orderly manner, and a small amount of chromatin is deposited in the nucleus, but it is not concentrated on the inner side of the nuclear membrane ( Figure 5 The mitochondrial morphology was improved in the MCAO / R+EDA 10mg group and the MCAO / R+E31 10mg group (blue arrows), with larger mitochondrial volumes and clearer internal cristae ( Figure 5 The above results confirmed that E31 can effectively reduce the volume of cerebral infarction, relieve the neurobehavioral disorders of animals, improve neuronal morphology, reduce the occurrence of apoptosis, and improve the damage induced by MCAO / R.

[0139] Experimental Example 2-Cell Experiment

[0140] 2.1 Experimental drugs

[0141] HT22 cells were used and purchased from Wuhan Pronocell Life Science Co., Ltd.

[0142] 2.2 Protective effect of different concentrations of homologue E31 on HT22 cell morphology after OGD / R injury

[0143] In order to screen the concentration of homologue E31 that alleviates OGD / R injury (oxygen glucose deprivation / reoxygenation injury) of HT22 cells in vitro, we first observed the concentrations of different concentrations (10 -5 ~10 -8 mol / L, mol / L can be abbreviated as M) homologue E31 on the protective effect of HT22 cell morphology after OGD / R treatment.

[0144] Among them, the preparation of HT22 cell OGD / R model includes: culturing HT22 in DMEM high-glucose medium containing 10% special grade fetal bovine serum and 1% double antibody. Before formal modeling, discard the normal DMEM high-glucose complete medium, wash the cells twice with sugar-free Earle's, add Earle's solution containing sodium dithionite, and place them in a hypoxic chamber to achieve oxygen-glucose deprivation. After 1.5 hours, replace it with normal DMEM high-glucose complete medium to achieve the reperfusion process, and perform relevant tests after 24 hours.

[0145] 2.2.1 Experimental groups: Control group (blank group), OGD / R group, OGD / R+10 -5 MEDA group (OGD / R damaged cells were given EDA at a concentration of 10 -5 M), OGD / R+10 -5 ME31 group (OGD / R damaged cells were treated with E31 at a concentration of 10 -5 M), OGD / R+10 -6 ME31 group (OGD / R damaged cells were treated with E31 at a concentration of 10 -6 M), OGD / R+10 -7 ME31 group (OGD / R damaged cells were treated with E31 at a concentration of 10 -7 M), OGD / R+10 -8 ME31 group (OGD / R damaged cells were treated with E31 at a concentration of 10 -8 M).

[0146] 2.2.2 Experimental results

[0147] See Figure 6 .like Figure 6 As shown in the figure, the cells in the control group (i.e., blank group) were plump, tightly arranged, and had obvious cell processes. In the OGD / R group, the number of adherent cells decreased, the cell synapses shortened or disappeared, the shrinkage was obvious, and obvious apoptosis occurred. -8 ME31, 10 -7 ME31,10 -6 ME31, 10 -5 ME31 and 10 -5 M EDA can improve the morphological damage of HT22 cells caused by OGD / R to a certain extent, increase the number of adherent cells, and reduce the number of apoptotic cells, but the differences among the groups cannot be observed with the naked eye.

[0148] 2.3 Effects of different concentrations of homologue E31 on the survival rate of HT22 cells after OGD / R injury

[0149] In order to determine the effective concentration of homologue E31 on OGD / R injury of HT22 cells, we also used CCK-8 assay to observe the effect of different concentrations (10 -5 ~10 -8 M) Effect of the homologue E31 on the survival rate of HT22 cells after OGD / R treatment.

[0150] 2.3.1 Experimental grouping is the same as 2.2.1 above.

[0151] 2.3.2CCK-8 detection method: conventional method was used.

[0152] 2.3.3 Experimental results

[0153] See Figure 7 .Depend on Figure 7 As shown in the figure, the cell survival rate of the Control group (i.e., blank group) was 100%, while the cell survival rate of the OGD / R group was significantly reduced. -8 ME31 group and OGD / R+10 -5 The cell survival rate in the ME31 group increased slightly, while that in the OGD / R+10 -6 ME31, OGD / R+10 -7 ME31 group and OGD / R+10 -5 The cell survival rate in the M EDA group increased significantly. -6 ME31 and 10 -7 The protection effect of ME31 is better than 10 -5 M EDA, where 10 -6 ME31 has the best protection effect. -5 The efficacy of ME31 is not as good as 10 -6We speculate that the reason may be that the maximum effective dose of E31 has been exceeded, and the effect no longer increases with the increase of dose, and even the drug efficacy may be weakened or toxic reactions may occur. Therefore, in subsequent cell experiments, we used 10 -6 M was used as the administration concentration of E31.

[0154] 2.4 Protective effect of homologue E31 on HT22 cell viability after OGD / R injury

[0155] To determine the effect of homologue E31 on cell viability after OGD / R-induced injury in HT22 cells, we performed Calcein-AM and PI co-staining.

[0156] 2.4.1 Calcein-AM and PI co-staining method: conventional method was used.

[0157] 2.4.2 Experimental Results

[0158] See Figure 8 . Figure 8 In the , Calcein AM is based on Calcein (calcein), with an acetylmethoxymethyl ester (AM) group introduced. PI is propidium iodide. Calcein-AM and PI are used together to stain and mark live cells and dead cells. MERGE means to merge Calcein-AM and PI staining into one picture.

[0159] like Figure 8 As shown in the figure, the HT22 cells in the Control group showed bright green fluorescence and no obvious red fluorescence, indicating that the cell viability was high and there was no cell damage. The green fluorescence of the HT22 cells in the OGD / R group was significantly weakened and the red fluorescence intensity was significantly increased, indicating that the cell viability was significantly weakened and the cells were significantly damaged. -6 HT22 cells in ME31 group and OGD / R+10 -5 The green fluorescence intensity of HT22 cells in the M EDA group increased, and the red fluorescence intensity decreased significantly, indicating that cell viability was significantly restored and cell damage was significantly reduced. -6 The protection effect of ME31 is better than 10 -5 MED A.

[0160] 2.5 Protective effect of homologue E31 on apoptosis of HT22 cells after OGD / R injury

[0161] To further determine whether homologue E31 can alleviate OGD / R injury by alleviating excessive cell apoptosis, we detected the Annexin V-PI co-staining of cells in each group by flow cytometry.

[0162] 2.5.1 Annexin V-PI co-staining: conventional method was used.

[0163] 2.5.2 Experimental Results

[0164] See Fig. 9 .like Fig. 9 As shown in the figure, the proportion of normal cells in the Control group was 78.16%, while the proportion of normal cells in the OGD / R group HT22 cells was only 57.67%, with a large number of early apoptotic cells (6.56%) and late apoptotic or necrotic cells (33.67%). -5 MEDA group cells and OGD / R+10 -6 The proportion of surviving cells in the ME31 group increased significantly (66.69%, 72.75%), the proportion of late apoptotic or necrotic cells decreased significantly (26.28%, 19.90%), and the early apoptosis rate did not change significantly. This result shows that E31 can significantly alleviate cell death caused by OGD / R, and 10 -6 The protection effect of ME31 is better than 10 -5 MED A.

[0165] 2.6. Inhibitory effect of homologue E31 on ROS in HT22 cells after OGD / R injury

[0166] To further determine whether homologue E31 could alleviate OGD / R injury by inhibiting ROS levels, we performed DCFH-DA staining.

[0167] 2.6.1 DCFH-DA staining: conventional method was used.

[0168] 2.6.2 Experimental Results

[0169] See Fig.10 .like Fig.10 As shown in the figure, the cells in the Control group showed extremely weak green fluorescence, indicating that the ROS level was low. The green fluorescence of the cells in the OGD / R group was significantly enhanced, proving that OGD / R caused a significant increase in the ROS level in the cells. - 5 MEDA group and OGD / R+10 -6 The ROS level in the ME31 group was significantly reduced, and the OGD / R+10 -6 The ROS level in ME31 group was higher than that in OGD / R+10 -5 The ROS level in the M EDA group was even lower.

[0170] 2.7 Effects of homologue E31 on the ultrastructure of HT22 cells after OGD / R injury

[0171] In order to further observe the effect of compound E31 administration on the ultrastructure of HT22 cells damaged by OGD / R, we performed ultrathin sectioning on HT22 cells in each group and observed them under transmission electron microscopy.

[0172] Results Fig.11 .like Fig.11 As shown, the cells in the Control group were plump and had intact membranes, and the nuclei were normal in morphology ( Fig.11 The light red arrow of the Control group shows that the organelles are arranged in an orderly manner, and the mitochondria are oval and complete in shape ( Fig.11 The cell membrane of cells in the OGD / R group was damaged and the cell body was shrunken ( Fig.11 OGD / R group (green arrow), the nucleus is shrunken and the chromatin in the nucleus is condensed and deposited on the inner side of the nuclear membrane ( Fig.11 In the OGD / R group (red arrow), the mitochondria in the cells were shortened, the volume was reduced, and the mitochondrial cristae were blurred ( Fig.11 OGD / R group (blue arrow), while OGD / R+10 -5 HT22 cells in M ​​EDA group and OGD / R+10 -6 The HT22 cells in the ME31 group were relatively plump and had intact membranes ( Fig.11 Medium OGD / R+10 -5 MEDA group and OGD / R+10 -6 Green arrows in the ME31 group), and the shrinkage of the cell nucleus was also alleviated ( Fig.11 Medium OGD / R+10 -5 MEDA group and OGD / R+10 -6 The red arrows in the ME31 group show that the cell organelles are arranged in an orderly manner, and a small amount of chromatin is deposited in the nucleus, but it is not concentrated on the inner side of the nuclear membrane ( Fig.11 Medium OGD / R+10 -5 MEDA group and OGD / R+10 -6 The blue arrow in the ME31 group shows that the mitochondrial morphology has improved, the volume is larger, and the internal cristae are clearer ( Fig.11 Medium OGD / R+10 -5 MEDA group and OGD / R+10 -6 The above results confirmed that E31 can effectively improve neuronal morphology, reduce the occurrence of apoptosis, and improve OGD / R-induced damage.

[0173] In summary, in animal experiments, we found that the homologue E31 of the present invention can significantly reduce cerebral infarction volume, improve neurological function scores, reduce the level of oxidative stress markers and inhibit neuronal apoptosis. Compared with Edaravone EDA, the Edaravone homologue of the present invention showed more excellent effects on these indicators. In cell experiments, we observed that the homologue E31 of the present invention can protect neurons from oxidative stress damage and reduce cell apoptosis rate.

[0174] These experimental data not only confirm the effectiveness of the present invention, but also provide clues for further research on its mechanism of action, and provide important references and lessons for the development and optimization of related drugs in the future. The inventors believe that the drugs prepared from the edaravone homologues of the present invention are expected to play a more important role in clinical practice and bring good news to the majority of patients.

[0175] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. An edaravone homologue or a pharmaceutically acceptable salt thereof, characterized in that: The edaravone homologue has a structure shown in formula (I): In formula (I), R1 and R2 are substituents at any position on the benzene ring, and R1 to R2 are the same or different and are independently selected from hydrogen or C1-C3 alkyl.

2. The edaravone homologue or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R1 to R2 are the same or different and are independently selected from hydrogen or methyl.

3. The edaravone homologue or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The edaravone homologues shown in formula (I) are:

4. A method for preparing the edaravone homologue or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The steps include: (1) reacting the compound represented by formula (A) with dimethyl carbonate in the presence of sodium hydride to obtain the compound represented by formula (B); (2) reacting the compound represented by formula (B) with 3,5-dimethoxybenzyl halide in the presence of sodium hydride to obtain the compound represented by formula (C); (3) reacting the compound represented by formula (C) with phenylhydrazine substituted with R2 to obtain the compound represented by formula (D); wherein R2 is selected from hydrogen or C1-C3 alkyl; (4) reacting the compound represented by formula (D) with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain an edaravone homologue represented by formula (I); In formula (A), formula (B), formula (C) and formula (D), R1 is a substituent at any position on the benzene ring, and R1 is selected from hydrogen or C1-C3 alkyl; in formula (D), R2 is a substituent at any position on the benzene ring, and R2 is selected from hydrogen or C1-C3 alkyl.

5. The method according to claim 4, characterized in that: In step (1), the compound represented by formula (A) is added dropwise to a mixture of sodium hydride, dimethyl carbonate and a first solvent, and reacted under reflux conditions; In step (2), a solution containing the compound represented by formula (B) is added dropwise to a mixture of sodium hydride and a second solvent, and then 3,5-dimethoxybenzyl halide is added to react at 20 to 40°C.

6. The method according to claim 5, characterized in that The first solvent is tetrahydrofuran or 2-methyltetrahydrofuran; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran; and the 3,5-dimethoxybenzyl halide is 3,5-dimethoxybenzyl bromide.

7. The method according to claim 4, characterized in that In step (3), the reaction temperature is 110-125°C.

8. The method according to claim 4, characterized in that In step (4), the phase transfer catalyst is tetrabutylammonium bromide.

9. Use of the edaravone homologue or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating cerebral ischemia-reperfusion injury.

10. The use according to claim 9, characterized in that In the in vivo experiment on mice, the dosage of the edaravone homologue was 5 to 20 mg / kg.

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