Pyrazolone compound as well as preparation method and application thereof

Abstract

The invention discloses a pyrazolone compound as well as a preparation method and application thereof. The pyrazolone compound has a structure as 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-C6 alkyl. Compared with 3-methyl-1-phenyl-2-pyrazoline-5-ketone (EDA), the pyrazolone compound disclosed by the invention can be used for more effectively inhibiting oxidative stress, inhibiting neuronal apoptosis and improving cerebral ischemia reperfusion injury. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a pyrazolone compound and a preparation method and application thereof. Background Art

[0002] Stroke is a sudden acute cerebrovascular disease characterized by cerebral ischemia and hypoxia caused by cerebral blood supply disorder. It has the characteristics of high morbidity, high disability rate and high mortality rate, which seriously threatens human life and health. According to 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 of ischemic stroke mainly include surgical treatment and antithrombotic drugs. Antithrombotic drugs mainly include thrombolytic drugs, antiplatelet aggregation drugs and anticoagulant drugs. 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 patients.

[0003] CN111329852A discloses the use of a 4-phenylbutyric acid derivative in the preparation of a drug for treating cerebral ischemia-reperfusion injury. 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. Although edaravone has been widely used in the treatment of diseases such as ischemic stroke, the limitations of its mechanism of action in clinical applications still limit its widespread clinical application. Current studies have found that the main mechanism of action of edaravone in the treatment of 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 more severe cerebral ischemia-reperfusion injury, the ability of edaravone to inhibit neuronal apoptosis and its antioxidant effect appear to be relatively weak, thereby limiting its widespread clinical application. Summary of the invention

[0005] An object of the present invention is to provide a pyrazolone compound or a pharmaceutically acceptable salt thereof, which can be used to treat cerebral ischemia-reperfusion injury. Compared with edaravone (chemical name 3-methyl-1-phenyl-2-pyrazol-5-one, denoted as EDA), it can more effectively reduce cerebral infarction volume, improve neurological function scores, reduce oxidative stress marker levels and inhibit neuronal apoptosis.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned pyrazolone compound or a pharmaceutically acceptable salt thereof, which has a stable process.

[0007] Another object of the present invention is to provide the use of the above-mentioned pyrazolone compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cerebral ischemia-reperfusion injury.

[0008] In one aspect, the present invention provides a pyrazolone compound or a pharmaceutically acceptable salt thereof, wherein the pyrazolone compound 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-C6 alkyl.

[0011] According to the pyrazolone compound 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 C1-C3 alkyl.

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

[0013]

[0014] On the other hand, the present invention also provides a method for preparing the pyrazolone compound or a pharmaceutically acceptable salt thereof as described above, comprising the following steps:

[0015] The compound represented by formula (A) is reacted with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain the compound represented by formula (I);

[0016]

[0017] In formula (A), 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-C6 alkyl.

[0018] According to the preparation method of the present invention, preferably, the phase transfer catalyst is tetrabutylammonium bromide.

[0019] According to the preparation method of the present invention, preferably, the compound represented by formula (A) is prepared by the following steps:

[0020] (1) reacting the compound represented by formula (B) with dimethyl carbonate in the presence of sodium hydride to obtain the compound represented by formula (C);

[0021] (2) reacting the compound represented by formula (C) with a benzyl halide in the presence of sodium hydride to obtain a compound represented by formula (D);

[0022] (3) reacting the compound represented by formula (D) with phenylhydrazine substituted with R2 to obtain the compound represented by formula (A); wherein R2 is selected from hydrogen or C1-C6 alkyl;

[0023]

[0024] In 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-C6 alkyl.

[0025] According to the preparation method of the present invention, preferably:

[0026] In step (1), the compound represented by formula (B) 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;

[0027] In step (2), a solution containing the compound represented by formula (C) is added dropwise to a mixture formed by sodium hydride and a second solvent, and then a benzyl halide is added to react at 20 to 40° C.; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran; the benzyl halide is benzyl bromide;

[0028] In step (3), the reaction temperature is 110-120°C.

[0029] In another aspect, the present invention also provides a use of the pyrazolone compound or a pharmaceutically acceptable salt thereof as described above in the preparation of a medicament for treating cerebral ischemia-reperfusion injury.

[0030] According to the use of the present invention, preferably, in an in vivo experiment on mice, the dosage of the pyrazolone compound is 5 to 20 mg / kg.

[0031] According to the use of the present invention, preferably, in an in vivo experiment on mice, the dosage of the pyrazolone compound is 7-15 mg / kg.

[0032] The present invention found that the pyrazolone compound of the present invention can significantly reduce the volume of cerebral infarction, improve neurological function scores, reduce the level of reactive oxygen and inhibit neuronal apoptosis. Compared with edaravone, the pyrazolone compound of the present invention showed a more excellent effect on the above indicators. In cell experiments, the pyrazolone compound of the present invention can protect neurons from oxidative stress damage and improve neuronal survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results show that the pyrazolone compound E28 has a protective effect on cerebral infarction and behavioral disorders in mice after MCAO / R injury. Figure 1 A is the display of TTC staining of each group. Figure 1 B shows that E28 and EDA can significantly reduce the cerebral infarction caused by MCAO / R. Figure 1 C and Figure 1 D indicates Zea-Longa behavioral score and DG&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.

[0034] Figure 2 This is the protective effect of pyrazolone compound E28 on neuronal morphological damage caused by MCAO / R.

[0035] Figure 3 This is a display of Tunel coloring.

[0036] Figure 4 is the proportion of Tunel-positive cells in each group. *** p<0.001vs Sham group; ## p<0.01vs MCAO / R group; @@ p<0.01vs MCAO / R+EDA 10mg group.

[0037] Figure 5 The results show that the pyrazolone compound E28 has a protective effect on the ultrastructural damage of neuronal cells induced by MCAO / R.

[0038] Figure 6 The results show that the pyrazolone compound E28 has a protective effect on the morphological damage of HT22 cells induced by OGD / R.

[0039] Figure 7 The protective effect of pyrazolone compound E28 on the survival rate of HT22 cells induced by OGD / R is shown. *** p<0.001vs Control group; # p<0.05, ### p<0.001vs MCAO / R group; @ p<0.05, @@@ p<0.001vs EDA.

[0040] Figure 8 The results show that the pyrazolone compound E28 has a protective effect on the damage of HT22 cell viability caused by OGD / R.

[0041] Fig. 9 The results show that the pyrazolone compound E28 has a protective effect on the apoptosis of HT22 cells induced by OGD / R.

[0042] Fig.10 The inhibitory effect of pyrazolone compound E28 on ROS levels in HT22 cells is shown.

[0043] Fig.11 The protective effect of pyrazolone compound E28 on ultrastructural damage of HT22 cells after OGD / R injury is shown. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0045] <Term Explanation>

[0046] In the present invention, Cm-Cn represents an alkyl group having m to n carbon atoms; for example, C1-C6 alkyl represents an alkyl group having 1 to 6 carbon atoms.

[0047] <Pyrazolone compound or pharmaceutically acceptable salt thereof>

[0048] Based on 3-methyl-1-phenyl-2-pyrazol-5-one, the inventors of the present application carefully designed and optimized the synthesis of the pyrazolone compound of the present invention, which can be used to prepare a drug for treating cerebral ischemia-reperfusion injury. The pyrazolone compound of the present invention has a structure shown in formula (I):

[0049]

[0050] 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 3-methyl-1-phenyl-2-pyrazolidine-5-one, the pyrazolone compound of the present invention has a better effect of improving cerebral ischemia-reperfusion injury.

[0051] In the present invention, 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-C6 alkyl.

[0052] Preferably, R1 is selected from hydrogen or C1-C3 alkyl, more preferably, R1 is hydrogen.

[0053] Preferably, R2 is selected from hydrogen or C1-C3 alkyl, more preferably, R2 is hydrogen.

[0054] In the present invention, C1-C6 alkyl may include but is not limited to straight chain alkyl or branched chain alkyl; preferably C1-C3 alkyl, more preferably C1-C3 straight chain alkyl. Examples of C1-C6 alkyl include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, etc.

[0055] According to a specific embodiment of the present invention, the pyrazolone compound of the present invention has the structure shown below:

[0056]

[0057] The pyrazolone compound of the present invention can form a pharmaceutically acceptable salt, which refers to a salt formed by the pyrazolone compound represented by formula (I) and an inorganic acid such as phosphoric acid, sulfuric acid, and hydrochloric acid, or an organic acid such as acetic acid, tartaric acid, citric acid, and malic acid, or an acidic amino acid such as aspartic acid and glutamic acid, or a salt formed by forming an ester or amide with the above-mentioned acid and then with an inorganic base, such as a sodium, potassium, calcium, aluminum or ammonium salt.

[0058] <Preparation method>

[0059] The method for preparing the pyrazolone compound or a pharmaceutically acceptable salt thereof of the present invention comprises: (1) a step of synthesizing the compound represented by formula (C); (2) a step of synthesizing the compound represented by formula (D); (3) a step of synthesizing the compound represented by formula (A); and (4) a step of synthesizing the final product. The following is a detailed description.

[0060] Synthesis steps of the compound represented by formula (C)

[0061] In step (1) of the present invention, the compound represented by formula (B) is reacted with dimethyl carbonate in the presence of sodium hydride to obtain a compound represented by formula (C).

[0062]

[0063] In formula (B) and (C), the definition of R1 is as described above and will not be repeated here.

[0064] According to one embodiment of the present invention, the compound represented by formula (B) 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.

[0065] The molar ratio of the compound represented by formula (B), dimethyl carbonate and sodium hydride (NaH) is 1:2 to 2.05:2 to 2.05, preferably 1:2 to 2.02:2 to 2.03, for example, 1:2:2.

[0066] 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.

[0067] After the reaction is completed, the temperature is lowered, saturated ammonium chloride is added in an ice bath to quench the reaction, and the pH value is adjusted to 6-7 with hydrochloric acid. Ethyl acetate is added for separation, and the aqueous layer is extracted once or twice with ethyl acetate, and the organic phases are combined, and the combined organic phases are washed with water once or twice, washed with saturated brine once or twice, and 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 petroleum ether and ethyl acetate in a volume ratio of 19-21:1, for example, 20:1.

[0068] Synthesis steps of the compound represented by formula (D)

[0069] In step (2) of the present invention, the compound represented by formula (C) is reacted with a benzyl halide in the presence of sodium hydride to obtain a compound represented by formula (D).

[0070]

[0071] In formula (D), the definition of R1 is as mentioned above and will not be repeated here.

[0072] A solution containing the compound represented by formula (C) is added dropwise to a mixture of sodium hydride and a second solvent, and then a benzyl halide is added to react at 20-40°C; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran, preferably tetrahydrofuran. The benzyl halide can be benzyl chloride or benzyl bromide, preferably benzyl bromide (also known as benzyl bromide).

[0073] In the present invention, the solution containing the compound represented by formula (C) is a solution formed by dissolving the compound represented by formula (C) in a second solvent.

[0074] The molar ratio of the compound represented by formula (C), sodium hydride and benzyl halide is 1:2 to 2.05:1 to 1.5, preferably 1:2 to 2.03:1 to 1.3, and more preferably 1:2 to 2.02:1 to 1.2.

[0075] 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.

[0076] 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 with ethyl acetate 2 or 3 times, and the organic phases are combined. The organic phase is washed once with water and 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 petroleum ether and ethyl acetate in a volume ratio of 24 to 26:1, for example, 25:1.

[0077] Synthesis steps of the compound represented by formula (A) (i.e., cyclization reaction)

[0078] In step (3), the compound represented by formula (D) is reacted with phenylhydrazine substituted with R2 to obtain the compound represented by formula (A); wherein R2 is selected from hydrogen or C1-C6 alkyl.

[0079]

[0080] The molar ratio of the compound represented by formula (D) to the phenylhydrazine substituted with R2 is 1:1.5 to 2, preferably 1:1.5 to 1.9, and more preferably 1:1.6 to 1.8.

[0081] 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.

[0082] 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 1:1 to 3, for example, 1:3.

[0083] Synthesis steps of the final product

[0084] In step (4) of the present invention, the compound represented by formula (A) is reacted with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain the compound represented by formula (I).

[0085] The molar ratio of the compound represented by formula (A), N-fluorobisbenzenesulfonamide and the phase transfer catalyst is 1:1-1.5:0.05-0.06, preferably 1:1-1.3:0.05-0.06. The phase transfer catalyst is preferably tetrabutylammonium bromide.

[0086] 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 (A) to potassium hydroxide is 1:2.9-3.1, preferably 1:3.0-3.1, and more preferably 1:3.0-3.05.

[0087] 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.

[0088] After the reaction is completed, ethyl acetate and water are added to separate the liquids, 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 petroleum ether / ethyl acetate, and the volume ratio of the two is 4 to 5:1, for example, it can be 5:1.

[0089] <Application>

[0090] The present invention also provides a use of the pyrazolone compound or a pharmaceutically acceptable salt thereof as described above in the preparation of a drug for treating cerebral ischemia-reperfusion injury. The pyrazolone compound of the present invention exhibits more significant effects than 3-methyl-1-phenyl-2-pyrazolin-5-one in inhibiting oxidative stress, inhibiting neuronal apoptosis, and improving cerebral ischemia-reperfusion injury.

[0091] 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.

[0092] In the in vivo experiment of mice, the dosage of the pyrazolone compound 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 pyrazolone compound is 5 to 20 mg per kilogram of the body weight of the mouse. Such a dosage has a better effect on improving cerebral ischemia-reperfusion injury.

[0093] <Test Method>

[0094] 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.

[0095] Example 1

[0096] Step 1: Preparation of ethyl benzoylacetate (i.e., preparation of the compound represented by formula (C))

[0097]

[0098] Under argon protection, sodium hydride (NaH, 60% mass fraction, 8.4g) was added to the reaction flask, followed by adding 17.6mL of dimethyl carbonate and 30mL of dried tetrahydrofuran to the reaction flask and mixing, and then heated to reflux. Subsequently, 10mL of acetophenone (i.e., the compound shown in formula (B)) 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 completely detected by TLC, 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 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%.

[0099] Step 2: Preparation of benzyl-substituted ethyl benzoyl acetate (i.e., the compound represented by formula (D))

[0100]

[0101] Under argon protection, NaH (60% mass fraction, 1g) and 10mL 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, benzyl bromide (2.57g, 1.8mL) 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 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, washed 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 petroleum ether and ethyl acetate in a volume ratio of 25:1), and 3.4g of colorless oil was obtained, which was the benzyl-substituted ethyl benzoyl acetate product, with a yield of 85%.

[0102] Step 3: cyclization reaction

[0103]

[0104] Among them, Ph represents a phenyl group; Bn represents a benzyl group.

[0105] Under argon protection, 1.55g of phenylhydrazine and 1.93g 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 until the benzyl-substituted ethyl benzoyl acetate was basically reacted (TLC detection). After the reaction was completed, it was cooled to room temperature, ethyl acetate and water were added to separate, and the aqueous phase was extracted with ethyl acetate after separation, and the organic phase was 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 1.35g of light yellow solid, recorded as P1, i.e., the compound shown in formula (A), with a yield of 52%.

[0106] Step 4: Preparation of the final product (i.e., preparation of the compound represented by formula (I))

[0107]

[0108] Wherein, Ph represents a phenyl group; Bn represents a benzyl group;

[0109] The above prepared P1 (0.32 g), 0.35 g N-fluorobisbenzenesulfonamide, 0.03 g tetrabutylammonium bromide, 0.12 g solid potassium hydroxide, and 10 mL ether were added to the reaction bottle in sequence, and the reaction was vigorously stirred at room temperature for 3 h. After the reaction was completed, ethyl acetate and water were added to separate the liquids. 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 and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. The concentrate was separated with a 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 E28. The final product was structurally characterized.

[0110] The structure of the final product is as follows:

[0111]

[0112] Wherein, Ph represents phenyl group and Bn represents benzyl group. It is denoted as E28.

[0113] The structural identification data of the final product E28 (i.e., the compound represented by formula (I)) are as follows:

[0114] 1 H NMR (400 MHz, chloroform-d) δ 7.85-7.66 (m, 2H), 7.61-7.46 (m, 2H), 7.35 (dq, J = 9.3, 2.6, 1.8 Hz, 3H), 7.26-7.12 (m, 2H), 7.08-6.87 (m, 4H), 6.81-6.65 (m, 2H), 3.61-3.21 (m, 2H). 13 C NMR (101 MHz, chloroform-d) δ 166.88 (d, J = 21.5 Hz), 152.99 (d, J = 13.9 Hz), 135.78, 130.05, 128.82, 128.72, 128.29, 128.27, 128.01, 127.74, 127.29, 126.84, 125.54, 125.53, 124.80, 118.19, 95.27, 93.28, 40.05 (d, J = 26.0 Hz). 19 F NMR (376 MHz, chloroform-d) δ-162.21 (s, 1F).

[0115] Experimental Example 1-Animal Experiment

[0116] 1.1 Experimental drugs

[0117] The mice used in the experiment were SPF-grade KM mice weighing about 25 g, purchased from Sikebes Co., Ltd.

[0118] 1.2 Protective effects of different doses of E28 on brain damage induced by MCAO / R in mice

[0119] In order to verify the protective effect of pyrazolone compound E28 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.

[0120] 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 (25g-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. A slipknot was tied at the distal end of the common carotid artery, 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 suture was inserted from the incision to the hemostatic clamp. The slipknot was tightened, and the hemostatic clamp was released. The insertion continued. If a slight resistance could be felt, it indicated that the suture 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 suture 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.

[0121] 1.2.1 Experimental groups: Sham group mice (sham operation group), MCAO / R group mice, MCAO / R+E28 5 mg group mice (MCAO / R injured mice were administered E28 at a dose of 5 mg / kg), MCAO / R+E28 10 mg group mice (MCAO / R injured mice were administered E28 at a dose of 10 mg / kg), MCAO / R+E28 20 mg group mice (MCAO / R injured mice were administered E28 at a dose of 20 mg / kg) and MCAO / R+EDA 10 mg group mice (MCAO / R injured mice were administered EDA at a dose of 10 mg / kg).

[0122] Among them, EDA refers to edaravone (3-methyl-1-phenyl-2-pyrazol-5-one), and its structural formula is shown below:

[0123]

[0124] 1.2.2TTC staining method: conventional method was used.

[0125] 1.2.3 Experimental Results

[0126] Reference Figure 1 A and Figure 1 As shown in B, the brain tissue sections of mice in the sham group were red. Obvious white cerebral infarction areas appeared in mice in the MCAO / R group. Compared with mice in the MCAO / R group, the cerebral infarction volumes of mice in the MCAO / R+E28 5mg group, MCAO / R+E28 10mg group, MCAO / R+E28 20mg group, and MCAO / R+EDA10mg group were significantly reduced. Among them, the cerebral infarction volumes of mice in the MCAO / R+E28 10mg group and MCAO / R+E2820mg group were significantly smaller than those of mice in the MCAO / R+EDA10mg group, and the cerebral infarction volume of mice in the MCAO / R+E28 10mg group was the smallest.

[0127] Reference Figure 1 C and Figure 1 As shown in D, 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 movement 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+E28 5mg group, the MCAO / R+E28 10mg group, the MCAO / R+E28 20mg group, and the MCAO / R+EDA10mg group were significantly improved, which were manifested by the enhancement of the comprehensive evaluation of limbs, limb movement ability, and coordination ability (a significant decrease in the Zea Longa score and a significant increase in the G&B score).

[0128] Among them, the behavioral performance of mice in the MCAO / R+E28 10mg group and the MCAO / R+E28 20mg group was better than that of mice in the MCAO / R+EDA10mg group. This is also consistent with the conclusion of the above cerebral infarction evaluation. As for the fact that the efficacy of 20mg / kg E28 is not as good as 10mg / kg E28, we speculate that the reason may be that the maximum effective dose of E28 is exceeded, and the effect is no longer enhanced 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 E28 in subsequent in vivo experiments on mice.

[0129] 1.3. Protective effect of compound E28 on pathological damage of brain tissue in mice induced by MCAO / R

[0130] In order to visually present the effect of E28 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.

[0131] 1.3.1 Nissl staining method: conventional method was used.

[0132] 1.3.2 Experimental Results

[0133] 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+E28 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+E28 10mg group.

[0134] 1.4. Protective effect of compound E28 on neuronal apoptosis in mice after MCAO / R injury

[0135] In order to further verify whether compound E28 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.

[0136] 1.4.1 Tunel fluorescent staining method: conventional method was used.

[0137] 1.4.2 Experimental Results

[0138] 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+E28 10mg group mice and the MCAO / R+EDA10mg group mice was significantly alleviated, among which the apoptosis of neurons in the penumbra area of ​​the brain tissue of the MCAO / R+E28 10mg group mice was significantly lighter than that of the MCAO / R+EDA 10mg group mice.

[0139] 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.

[0140] 1.5 Effects of compound E28 on the ultrastructure of neurons after MCAO / R injury in mice

[0141] In order to further observe the effect of compound E28 administration 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.

[0142] Results Figure 5 .like Figure 5 As shown, the neurons of the Sham group mice were full and had intact membranes, and the nuclei were normal in morphology ( Figure 5 The red arrows of the Sham group show 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).

[0143] The nerve cells of the mice in the MCAO / R+EDA10mg group and the MCAO / R+E2810mg group recovered to a relatively full and intact state ( Figure 5 Green arrows in the MCAO / R+EDA 10mg group and the MCAO / R+E28 10mg group), and the shrinkage of the cell nucleus was also alleviated ( Figure 5 The red arrows in the MCAO / R+EDA10mg group and the MCAO / R+E2810mg 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 blue arrows in the MCAO / R+EDA10mg group and the MCAO / R+E2810mg group showed that the mitochondrial morphology was improved, the volume was larger, and the internal cristae were clearer ( Figure 5The yellow arrows in the MCAO / R+EDA 10 mg group and the MCAO / R+E28 10 mg group). The above results confirmed that compound E28 can effectively reduce cerebral infarction volume, alleviate animal neurobehavioral disorders, improve neuronal morphology, reduce the occurrence of cell apoptosis, and improve MCAO / R-induced damage.

[0144] Experimental Example 2-Cell Experiment

[0145] 2.1 Experimental drugs

[0146] HT22 cells were purchased from Wuhan Pronocell Life Science Co., Ltd.

[0147] 2.2 Protective effect of compound E28 at different concentrations on HT22 cell morphology after OGD / R injury

[0148] In order to screen the concentration of compound E28 that alleviates OGD / R injury (oxygen glucose deprivation / reoxygenation injury) in HT22 cells in vitro, we first observed the concentrations of E28 at different concentrations (10 -5 ~10 -8 mol / L, mol / L can be abbreviated as M) of compound E28 on the morphology of HT22 cells after OGD / R treatment.

[0149] Among them, the method for making the in vitro OGD / R injury model of HT22 cells is as follows: HT22 cells are cultured in DMEM high-glucose medium containing 10% special grade fetal bovine serum and 1% double antibody. Before formal modeling, the normal DMEM high-glucose complete medium is discarded, the cells are washed twice with sugar-free Earle's, Earle's solution containing sodium dithionite is added, and placed in a hypoxic chamber to achieve oxygen-glucose deprivation. After 1.5 hours, it is replaced with normal DMEM high-glucose complete medium to achieve the reperfusion process, and relevant tests are performed after 24 hours.

[0150] 2.2.1 Experimental groups: Control group (blank group), OGD / R group, OGD / R+10 -5 MEDA group (HT22 cells injured by OGD / R were given EDA at a concentration of 10 -5 M), OGD / R+10 -5 ME28 group (HT22 cells injured by OGD / R were given E28 at a concentration of 10 -5 M), OGD / R+10 -6 ME28 group (HT22 cells injured by OGD / R were given E28 at a concentration of 10 -6 M), OGD / R+10 -7 ME28 group (HT22 cells injured by OGD / R were given E28 at a concentration of 10-7 M), OGD / R+10 -8 ME28 group (HT22 cells injured by OGD / R were given E28 at a concentration of 10 -8 M).

[0151] 2.2.2 Experimental results

[0152] 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 ME28, 10 -7 ME28,10 -6 ME28, 10 -5 ME28 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.

[0153] 2.3 Effects of different concentrations of compound E28 on the survival rate of HT22 cells after OGD / R injury

[0154] In order to determine the effective concentration of compound E28 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 compound E28 on the survival rate of HT22 cells after OGD / R treatment.

[0155] 2.3.1 Experimental groups are the same as above.

[0156] 2.3.2CCK-8 detection method: conventional method was used.

[0157] 2.3.3 Experimental results

[0158] 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%, and the cell survival rate of the OGD / R group was significantly reduced. -8 ME28 group and OGD / R+10 -5 The cell survival rate of ME28 group was slightly increased, while that of OGD / R+10 -6 ME28 group, OGD / R+10 -7 ME28 group and OGD / R+10 -5The cell survival rate in the M EDA group increased significantly. -6 ME28 and 10 -7 The protection effect of ME28 is better than 10 -5 MEDA, of which 10 -6 ME28 has the best protection effect. -5 ME28 is not as effective as 10 -6 We speculate that the reason may be that the maximum effective dose of compound E28 is exceeded, and the effect no longer increases with the increase of dose, and even the drug effect may be weakened or toxic. Therefore, in subsequent cell experiments, we used 10 -6 M was used as the administration concentration of compound E28.

[0159] 2.4 Protective effect of compound E28 on HT22 cell viability after OGD / R injury

[0160] To determine the effect of compound E28 on the cell viability of HT22 cells after OGD / R-induced injury, we performed Calcein-AM and PI co-staining.

[0161] 2.4.1 Calcein-AM and PI co-staining method: conventional method was used.

[0162] 2.4.2 Experimental Results

[0163] See Figure 8 In the figure, Calcein AM is based on Calcein (calcein), with an acetyl methoxymethyl ester (AM) group introduced. PI is propidium iodide. Calcein-AM and PI are used together to stain and mark living cells and dead cells. MERGE means to merge Calcein-AM and PI into one picture. 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 ME28 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 ME28 is better than 10 -5 MED A.

[0164] 2.5 Protective effect of compound E28 on apoptosis of HT22 cells after OGD / R injury

[0165] In order to further determine whether compound E28 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.

[0166] 2.5.1 Annexin V-PI co-staining: conventional method was used.

[0167] 2.5.2 Experimental Results

[0168] See Fig. 9 .like Fig. 9 As shown in the figure, the proportion of normal cells in the control group (i.e., blank group) was 78.86%, while the proportion of normal cells in the HT22 cells in the OGD / R group was only 58.16%, with a large number of early apoptotic cells (37.08%) and late apoptotic or necrotic cells (26.03%). -6 Surviving cells in ME28 group and OGD / R+10 -5 The proportion of surviving cells in the M EDA group increased significantly, to 71.05% and 65.46%, respectively, and the proportion of late apoptotic or necrotic cells decreased significantly, to 21.61% and 27.01%, respectively, while the proportion of early apoptotic cells did not change significantly. This result shows that compound E28 can significantly alleviate cell death caused by OGD / R, and 10 -6 The protection effect of ME28 is better than 10 -5 MED A.

[0169] 2.6 Inhibitory effect of compound E28 on ROS in HT22 cells after OGD / R injury

[0170] To further determine whether compound E28 could alleviate OGD / R injury by inhibiting ROS levels, we performed DCFH-DA staining.

[0171] 2.6.1 DCFH-DA staining: conventional method was used.

[0172] 2.6.2 Experimental Results

[0173] See Fig.10 .like Fig.10 As shown in the figure, the cells in the control group (i.e., blank 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 intracellular ROS level, while OGD / R+10 -5 MEDA group and OGD / R+10 -6The level of ROS in the cells of ME28 group was significantly reduced, and OGD / R+10 -6 The level of ROS in ME28 group was higher than that in OGD / R+10 -5 The cellular ROS level in the M EDA group was even lower.

[0174] 2.7 Effects of compound E28 on the ultrastructure of HT22 cells after OGD / R injury

[0175] In order to further observe the effect of compound E28 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.

[0176] 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 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 ME28 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 ME28 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 of the ME28 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 ME28 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 -6The above results confirmed that E28 can effectively improve neuronal morphology, reduce the occurrence of apoptosis, and improve OGD / R-induced damage.

[0177] In summary, in animal experiments, we found that the pyrazolone compound E28 of the present invention can significantly reduce the volume of cerebral infarction, improve neurological function scores, reduce the level of oxidative stress markers and inhibit neuronal apoptosis. Compared with compound EDA, the pyrazolone compound of the present invention showed more excellent effects on these indicators. In cell experiments, we observed that the pyrazolone compound E28 of the present invention can protect neurons from oxidative stress damage and reduce cell apoptosis rate. These experimental data not only confirm the effectiveness of the pyrazolone compound of the present invention, but also provide clues for further research on its mechanism of action, and provide important references and references for the development and optimization of related drugs in the future. The inventor believes that the medicine prepared by the pyrazolone compound of the present invention is expected to play a more important role in clinical practice and bring good news to the majority of patients.

[0178] 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. A pyrazolone compound or a pharmaceutically acceptable salt thereof, characterized in that: The pyrazolone compound 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-C6 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R1 to R2 are the same or different and are independently selected from hydrogen or C1-C3 alkyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound represented by formula (I) is:

4. A method for preparing the pyrazolone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The steps include: The compound represented by formula (A) is reacted with N-fluorobisbenzenesulfonamide in the presence of a phase transfer catalyst to obtain the compound represented by formula (I); In formula (A), 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-C6 alkyl.

5. The preparation method according to claim 4, characterized in that: The phase transfer catalyst is tetrabutylammonium bromide.

6. The preparation method according to claim 4, characterized in that: The compound represented by formula (A) is prepared by the following steps: (1) reacting the compound represented by formula (B) with dimethyl carbonate in the presence of sodium hydride to obtain the compound represented by formula (C); (2) reacting the compound represented by formula (C) with a benzyl halide in the presence of sodium hydride to obtain a compound represented by formula (D); (3) reacting the compound represented by formula (D) with phenylhydrazine substituted with R2 to obtain the compound represented by formula (A); wherein R2 is selected from hydrogen or C1-C6 alkyl; In 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-C6 alkyl.

7. The preparation method according to claim 6, characterized in that: In step (1), the compound represented by formula (B) 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; In step (2), a solution containing the compound represented by formula (C) is added dropwise to a mixture formed by sodium hydride and a second solvent, and then a benzyl halide is added to react at 20 to 40° C.; the second solvent is tetrahydrofuran or 2-methyltetrahydrofuran; the benzyl halide is benzyl bromide; In step (3), the reaction temperature is 110-120°C.

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

9. The use according to claim 8, characterized in that In an in vivo experiment on mice, the dosage of the pyrazolone compound is 5 to 20 mg / kg.

10. The use according to claim 9, characterized in that In an in vivo experiment on mice, the dosage of the pyrazolone compound is 7 to 15 mg / kg.

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