WO2020169054A1

WO2020169054A1 - CRYSTALLINE FORM OF (E)-α,β-UNSATURED AMIDE COMPOUND, PREPARATION METHOD THEREFOR, AND USE THEREOF

Info

Publication number: WO2020169054A1
Application number: PCT/CN2020/075918
Authority: WO
Inventors: 李娜; 陈连蔚; 吴忠伟; 陈磊; 杨志清; 张鑫龙; 龚永祥
Original assignee: 浙江海正药业股份有限公司
Priority date: 2019-02-22
Filing date: 2020-02-20
Publication date: 2020-08-27
Also published as: CN111606828B; CN111606828A

Abstract

The present invention relates to crystalline forms I and II of a (E)-α,β-unsatured amide compound, preparation methods therefor, and uses thereof. The crystalline forms all have excellent properties in physical and chemical stability and processing adaptability.

Description

(E)-α,β-Unsaturated amide compound crystal form and its preparation method and application

Technical field

The invention relates to the field of chemical pharmacy. More specifically, the present invention relates to polymorphs of (E)-α,β-unsaturated amide compounds and methods for preparing the crystal forms, pharmaceutical compositions containing them, and their pharmaceutical uses.

technical background

The applicant’s previous patent application (PCT/CN2018/102824, the entire application is cited as a reference in this application) reports a new class of (E)-α,β-unsaturated amide compounds, which are Nrf2 pathways The activator can effectively protect nerve cells from oxidative damage. Such compounds also have certain immunomodulatory activity.

The compound of formula I is a representative of this class of new (E)-α,β-unsaturated amide compounds, which have been demonstrated in animal models of stroke, Alzheimer’s disease, Parkinson’s disease and multiple sclerosis It has very good efficacy and has a bright future for treating multiple diseases with one medicine.

The compound of formula I is a polymorphic compound. For a polymorphic compound, different crystal forms have different physical and chemical properties, including melting point, chemical stability, apparent solubility, dissolution rate, optical and mechanical properties, etc. These physical and chemical properties directly determine the efficacy of a particular crystal form, and directly affect the quality of raw materials and preparations.

Therefore, it is necessary to study the properties of each crystal form of the drug compound to meet the needs of drug preparation.

Summary of the invention

The chemical name of the compound of formula I is N-[(E)-4-[methoxy(methyl)amino]-4-oxo-but-2-enoyl]carbamate methyl ester, and the code is MS-77.

One of the objectives of the present invention is to provide the crystal form I (hereinafter referred to as "crystal form I") of the compound of formula I with good chemical and physical stability. The crystal form I has excellent properties in terms of chemical stability and processing (filtration, drying) adaptability.

The X-ray powder diffraction pattern of the crystal form I of the present invention has characteristic peaks at the following diffraction angles 2θ: 8.82±0.2°, 14.56±0.2°, 16.82±0.2°, 17.72±0.2°, 20.22±0.2°, 22.50± 0.2°, 26.24±0.2°, 29.40±0.2°.

Further, the X-ray powder diffraction pattern of the crystal form I has characteristic peaks at the following diffraction angles 2θ: 10.06±0.2°, 24.22±0.2°, 25.00±0.2°, 26.76±0.2°, 28.16±0.2°, 30.56±0.2°, 31.14±0.2°, 33.74±0.2°, 38.66±0.2°, 40.20±0.2°, 44.84±0.2°, 46.04±0.2°.

Furthermore, the X-ray powder diffraction spectrum of crystal form I of the present invention has 2θ, d and relative intensity data as shown in Table 1 below:

Table 1

Without limitation, the X-ray powder diffraction spectrum of the crystal form I of the present invention is basically as shown in FIG. 1.

The peak value of the differential scanning calorimetry (DSC) spectrum of the crystalline form I of the present invention is 192.7°C.

Without limitation, the crystal form I of the present invention has a DSC spectrum as shown in FIG. 2.

Without limitation, the crystal form I of the present invention has a TGA pattern as shown in FIG. 3.

Another object of the present invention is to provide a method for preparing the crystal form I, and the method is selected from any one of the following methods:

Method (1), which includes the following steps:

1) Dissolve the compound of formula I in any solvent of alcohol, ketone, ester, acetonitrile and dioxane, dissolve it, and evaporate and crystallize; the alcohol is a C2-C4 alcohol; the ketone is a C3-C6 Ketone; the ester is a C3-C6 ester; the dissolution temperature is 25-45°C; the mass-volume ratio of the compound of formula I to the solvent is (mg/ml) 10:1.5-30;

2) Filter to obtain crystal form I.

Method (2), which includes the following steps:

1) The compound of formula I is dissolved in any solvent of alcohol, ketone, ester, acetonitrile, dioxane, dimethylacetamide, dimethylformamide, and dimethylsulfoxide, and the temperature is lowered to crystallize; The alcohol is a C2-C4 alcohol; the ketone is a C3-C6 ketone; the ester is a C3-C6 ester; the dissolution temperature is 40-70°C; the mass-volume ratio of the compound of formula I to the solvent is (mg /ml) 10: 0.4～3; the temperature of said crystallization is 0～20℃;

Or the compound of formula I is dissolved in a mixed solvent of any organic solvent among ethanol, isopropanol, dioxane, acetone, and acetonitrile and water, and the temperature is lowered to crystallize; the dissolution temperature is 40-70°C; The volume ratio of the organic solvent to water is 1:0.1-9; the mass-volume ratio of the compound of formula I and the mixed solvent is (mg/ml) 10:1-1.5; the crystallization temperature is 0-5°C ；

2) Filter to obtain crystal form I.

Another object of the present invention is to provide crystal form II of the compound of formula I (hereinafter referred to as "crystal form II"), the X-ray powder diffraction (XRD) pattern of the crystal form II has characteristic peaks at the following diffraction angle 2θ : 7.34±0.2°, 14.72±0.2°, 16.98±0.2°, 20.30±0.2°, 22.16±0.2°, 25.84±0.2°, 38.44±0.2°.

Further, the X-ray powder diffraction pattern of the crystal form II has characteristic peaks at the following diffraction angles 2θ: 9.20±0.2°, 13.90±0.2°, 18.46±0.2°, 23.74±0.2°, 29.72±0.2°, 34.42±0.2°.

Furthermore, the X-ray powder diffraction spectrum of the crystal form II of the present invention has the 2θ data shown in Table 2 below:

Table 2

Without limitation, the X-ray powder diffraction spectrum of the crystal form II of the present invention is basically shown in FIG. 4.

Without limitation, the peak value of the differential scanning calorimetry (DSC) spectrum of the crystalline form II of the present invention is 188.3°C.

Without limitation, the crystal form II of the present invention has a DSC spectrum as shown in FIG. 5.

Without limitation, the crystal form II of the present invention has a TGA pattern as shown in FIG. 6.

Another object of the present invention is to provide a method for preparing the crystal form II, the method is selected from any one of the following methods:

Method (1), which includes the following steps:

1) Add the compound of formula I to CH2Cl2 and dissolve under reflux; the mass-volume ratio (mg/ml) of the compound of formula I to CH2Cl2 is 10:1.4-1.6;

2) Add the above solution to ethyl acetate (EA) at -15～-10℃; the volume ratio of the EA and CH2Cl2 is 0.9～1.1:1;

3) Stir to crystallize, filter to obtain crystal form II.

Method (2), which includes the following steps:

1) The compound of formula I is dissolved in dioxane; the temperature of the dissolution is 45-60°C; the mass-volume ratio (mg/ml) of the compound of formula I to dioxane is 10:1.4-1.6 ；

2) Add the above solution to n-heptane at 4-6°C; the volume ratio of n-heptane to dioxane is 3 to 3.5:1;

3) Stir to crystallize, filter to obtain crystal form II.

In the above method, the unit of the mass-volume ratio of the compound of formula I to the solvent may be mg/ml, g/L, etc., depending on the specific scale of operation.

Another object of the present invention is to provide a pharmaceutical composition, which comprises an effective dose of crystal form I or crystal form II; the pharmaceutical composition also comprises one or more pharmaceutically acceptable carriers.

The present invention also relates to the application of crystal form I or crystal form II, or their pharmaceutical composition in the preparation of a medicine for treating diseases related to Nrf2 activation, wherein the diseases related to Nrf2 activation are stroke and neurodegeneration Sexual diseases, diabetes, diabetic nephropathy, coronary heart disease, atherosclerosis or non-alcoholic fatty liver.

In another aspect, the present invention also relates to the use of crystal form I or crystal form II, or their pharmaceutical composition in the preparation of a medicament for the treatment of stroke.

In another aspect, the present invention also relates to the application of the crystal form I or the crystal form II, or their pharmaceutical composition in the preparation of a medicament for the treatment of neurodegenerative diseases.

In a preferred embodiment, the neurodegenerative disease is selected from multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), muscle atrophy Lateral sclerosis (ALS), Friedreich’s ataxia (FRDA), spinal muscular atrophy (SMA), optic neuromyelitis (NMO) and spinocerebellar ataxia (SCA).

In another aspect, the present invention also relates to the use of crystal form I or crystal form II, or their pharmaceutical composition in the preparation of a medicament for the treatment of diseases related to immune regulation, wherein the diseases related to immune regulation are preferably From psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, transplant rejection and inflammatory diseases.

In another aspect of the present invention, there is provided a method for treating diseases associated with Nrf2 activation, the method comprising administering an effective dose of crystal form I or crystal form II, or a pharmaceutical composition thereof, to a patient in need.

In another aspect of the present invention, there is provided a method for treating stroke, the method comprising administering an effective dose of crystal form I or crystal form II, or a pharmaceutical composition thereof to a patient in need.

In another aspect of the present invention, there is provided a method of treating neurodegenerative diseases, the method comprising administering an effective dose of crystal form I or crystal form II, or a pharmaceutical composition thereof to a patient in need.

In another aspect of the present invention, there is provided a method for treating diseases related to immune regulation, the method comprising administering an effective dose of crystal form I or crystal form II, or a pharmaceutical composition thereof, to a patient in need.

After a lot of research, the inventors have discovered the crystal form I and crystal form II of the compound of formula I, which have good solubility, simple crystallization process, easy operation, low pollution, can realize industrial production, and have crystal form I or crystal form II. The compound of formula I has the advantages of high product purity, excellent physical and chemical properties, good chemical stability, and reproducible processing (filtering, drying,).

Description of the drawings:

Fig. 1 is an X-ray powder diffraction pattern of the crystal form I of the compound of formula I obtained in Example 1.

2 is a DSC chart of the crystal form I of the compound of formula I obtained in Example 1.

3 is a TGA spectrum of the crystal form I of the compound of formula I obtained in Example 1.

4 is an X-ray powder diffraction pattern of the crystal form II of the compound of formula I obtained in Example 5.

5 is a DSC chart of the crystal form II of the compound of formula I obtained in Example 5.

6 is the TGA spectrum of the crystal form II of the compound of formula I obtained in Example 5.

Fig. 7 shows that the compound of formula I induces Nrf2 to enter the nucleus in HT22 cells (4h).

Figure 8 shows that the compound of formula I up-regulates the expression of HO-1 protein in HT22 cells (4h).

Figure 9 shows the inhibitory effect of the compound of formula I on mouse EAE (***p<0.001, **p<0.01, *p<0.05, compared with the solvent group (one-way ANOVA/Dunnett)).

Figure 10 is the effect of Morris water maze experiment formula I compound on the escape latency of AD rat model (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one-way ANOVA/Dunnett) )).

Figure 11 is the effect of Morris water maze experiment formula I compound on the number of times the AD rat model crosses the target platform (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one-way ANOVA) /Dunnett)).

Figure 12 shows the effect of the compound of formula I on the latency in the 6-OHDA-induced PD rat rotarod experiment (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one-way) ANOVA/Dunnett)).

Figure 13 is the effect of the compound of formula I on the climbing time in the 6-OHDA-induced PD rat climbing experiment (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one -way ANOVA/Dunnett)).

Figure 14 is the effect of formula I compound on the rotation speed of PD rats induced by apomorphine (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one-way ANOVA/Dunnett) )).

Figure 15 is the effect of the compound of formula I on the neurological score of acute cerebral artery ischemia-reperfusion injury in rats (***p<0.001, **p<0.01, *p<0.05, compared with the model group (one-way) ANOVA/Dunnett)).

Figure 16 shows the effect of compound of formula I on the weight ratio of cerebral infarction area/whole brain in rats with acute cerebral artery ischemia reperfusion injury (***p<0.001, **p<0.01, *p<0.05, similar to the model group Than (one-way ANOVA/Dunnett)).

Figure 17 shows the sensitization rate of the compound of formula I to the skin of guinea pigs.

Specific embodiment

The following examples are intended to further explain the present invention, but not to limit or limit the scope of the present invention.

The raw materials of the compound of formula I used in the method of the present invention are derived from the following synthetic methods:

Preparation example: Synthesis method of the compound of formula I

(E) Preparation of-N'-methoxy-N'-methyl-but-2-ene diamide (compound Ia)

Step 1: Add 250g (1.92mol) (E)-4-methoxy-4-oxo-but-2-enoic acid, 225g (2.31mol) N,O-dimethylhydroxylamine into the reaction flask Hydrochloride and 2500 ml of dichloromethane, reduce the temperature to 0～10℃, add 550 g (2.88 mol) of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) , Warm up to room temperature, react for 1 hour, the reaction is complete. Wash with 1500 ml of water, collect the organic phase, decolorize with activated carbon, filter, and concentrate the filtrate to dryness.

Step 2: Add 1250 ml of ammonia to the concentrate obtained above, cool to 0-10°C, react for 20 minutes, after the reaction is complete, filter, and recrystallize the solid with ethanol and ethyl acetate to obtain 142.3 g of compound Ia.

Preparation of methyl N-[(E)-4-[methoxy(methyl)amino]-4-oxo-but-2-enoyl]carbamate (compound of formula I)

Step 1: Put 5.0 g (31.6 mmol) (E)-N'-methoxy-N'-methyl-but-2-ene diamide (compound Ia) and 50 ml 1,2-into the reaction flask Dichloroethane, cooled to 0-10°C, added 6 ml (70.9 mmol) of oxalyl chloride, reacted at room temperature for 6 hours, heated to 65°C, reacted for 15 minutes, concentrated to dryness, and set aside.

Step 2: Add 30 milliliters of anhydrous methanol to another reaction flask, lower the temperature to 0-10°C, add the concentrated solution obtained in Step 1, and react for 10 minutes to precipitate a solid to obtain 3.56 g of formula I compound.

The solvent used in the present invention is not particularly limited, and commercially available conventional solvents can be used.

Unless otherwise specified, the "stirring" described in the method of the present invention can adopt conventional methods in the art. For example, the stirring method includes magnetic stirring and mechanical stirring, and the stirring speed is 50-300 rpm/min, preferably 100-200 rpm/min.

The X-ray powder diffraction instrument and test conditions involved in the present invention are: X-ray diffraction instrument model MiniFlex600Cu target; operation method: scanning speed 20°/min, scanning step width 0.02°.

The DSC test conditions involved in the present invention are: DSC detector model: NETZSCH DSC 214 Polyma; operation method: heating rate 10°C/min, temperature range: 25-250°C.

The TGA test conditions involved in the present invention are: the model of the TGA detector is: METTLER TOLEDO TGA2; the operation method: the heating rate is 10°C/min, and the temperature range: 25-250°C.

The HPLC purity test conditions of the compound of formula I involved in the present invention are: chromatographic column: Welch Ultimate XB-C18 250*4.6mm 5μm; mobile phase A: 0.01% trifluoroacetic acid-water; mobile phase B: acetonitrile; filtration and degassing ; Detection wavelength: 226nm; Flow rate: 1.0ml/min; Injection volume: 10μl; Column temperature: 25°C; Mobile phase conditions are shown in Table 3:

table 3

t(min)t(min)	流动相A(％)Mobile phase A (%)	流动相B(％)Mobile phase B (%)
00	8383	1717
3030	8383	1717

It should be emphasized that the meaning or intended protection scope of the numerical value or numerical endpoint involved in the technical solution of the present invention is not limited to the number itself. Those skilled in the art can understand that they include those that have been widely used in the art. Acceptable allowable error ranges, such as experimental errors, measurement errors, statistical errors and random errors, etc., and these error ranges are all included in the scope of the present invention.

Example 1

Add 1 g of the raw material obtained in the preparation example to 300 ml of ethanol, dissolve at 45°C, evaporate and crystallize at 45°C, and filter to obtain 0.52g crystals, HPLC=99.89%, by measuring X-ray powder diffraction pattern (XRD), it is confirmed that the crystal form I.

The X-ray powder diffraction, DSC and TGA spectra of this crystal form are shown in Figures 1-3, respectively, and are named crystal form I in the present invention.

Example 2

Add 10 mg of the raw material obtained in the preparation example to 1.5 ml of dioxane, dissolve at 40°C, evaporate and crystallize at 40°C, and filter to obtain 5.3 mg of crystals, HPLC=99.84%, measured X-ray powder diffraction pattern (XRD) , Confirmed as crystal form I.

Example 3

Add 10 mg of the raw materials obtained in the preparation example to 30 ml of isopropyl acetate, dissolve at 25°C, evaporate and crystallize at 25°C, and filter to obtain 6.4 mg of crystals, HPLC=99.76%, measured X-ray powder diffraction pattern (XRD), Confirmed as crystal form I.

Example 4

Add 10 mg of the raw material obtained in the preparation example to 3 ml of ethanol, dissolve at 70°C, cool to 20°C to crystallize, and filter to obtain 4.5 mg of crystals. HPLC=99.86%. The X-ray powder diffraction pattern (XRD) was measured and confirmed to be crystals. Type I.

Example 5

Add 50mg of the raw material obtained in the preparation example to 2ml of dimethylacetamide, dissolve at 40°C, cool to 0°C to crystallize, and filter to obtain 3.8mg crystals, HPLC=99.86%, measured X-ray powder diffraction pattern (XRD) , Confirmed as crystal form I.

Example 6

Add 10 mg of the raw material obtained in the preparation example to 1.5 ml of methanol-water mixture, dissolve at 40°C, the volume ratio of methanol-water is 10:1, cool to 0°C to crystallize, filter to obtain 4.9mg crystals, HPLC=99.81%, The X-ray powder diffraction pattern (XRD) was measured, and it was confirmed to be crystal form I.

Example 7

Add 10 mg of the raw material obtained in the preparation example to 1 ml of acetonitrile water mixture, dissolve at 70°C, the volume ratio of acetonitrile water is 1:9, cool to 5°C to crystallize, filter to obtain 6.3mg crystals, HPLC=99.76%, measured X-ray powder diffraction pattern (XRD), confirmed to be crystal form I.

Example 8

Add 1g of the raw material obtained in the preparation example to 150ml CH2Cl2, dissolve under reflux, add the solution to 150ml EA at -10°C, stir and crystallize, filter to obtain 0.21g crystals, HPLC=99.62%, measured by X-ray powder diffraction The spectrum (XRD) confirmed that it was form II.

The X-ray powder diffraction, DSC and TGA spectra of this crystal form are shown in Figures 4-6, respectively, and are named crystal form II in the present invention.

Example 9

Add 10mg of the raw material obtained in the preparation example to 1.4ml CH2Cl2, dissolve under reflux, add the solution to 1.26ml EA at -10°C, stir and crystallize, filter to obtain 2.9mg crystals, HPLC=99.61%, measured by X-ray Powder diffraction pattern (XRD), confirmed to be crystal form II.

Example 10

Add 10mg of the raw material obtained in the preparation example to 1.6ml CH2Cl2, dissolve under reflux, add the solution to 1.76ml EA at -15°C, stir and crystallize, filter to obtain 3.5mg crystals, HPLC=99.62%, measured by X-ray Powder diffraction pattern (XRD), confirmed to be crystal form II.

Example 11

Add 10mg of the raw material obtained in the preparation example to 1.4ml dioxane, dissolve at 45°C, add the solution to 4.2ml n-heptane at 5°C, stir and crystallize and filter to obtain 3.6mg crystals, HPLC=99.61%, After measuring the X-ray powder diffraction pattern (XRD), it was confirmed to be crystal form II.

Example 12

Add 10 mg of the raw material obtained in the preparation example to 1.5 ml of dioxane, dissolve at 50°C, add the solution to 5 ml of n-heptane at 6°C, stir and crystallize, and filter to obtain 3.4 mg of crystals. HPLC=99.62%. The X-ray powder diffraction pattern (XRD) was measured, and it was confirmed to be crystal form II.

Example 13

Add 10 mg of the raw material obtained in the preparation example to 1.6 ml of dioxane, dissolve at 60°C, add the solution to 5.6 ml of n-heptane at 4°C, stir and crystallize, filter to obtain 3.9 mg of crystals, HPLC=99.61%, After measuring the X-ray powder diffraction pattern (XRD), it was confirmed to be crystal form II.

Stability experiment

Take the crystalline form I prepared in Example 1 and the crystalline form II prepared in Example 8, and conduct a stability test at 60° C. for 10 days. The HPLC purity and maximum single impurity content of the compound before and after storage were tested, and the results are shown in Table 4; the crystal form changes of the samples after 10 days of storage were analyzed by X-ray powder diffraction detection, and the results are shown in Table 5.

Table 4

From the stability data of 10 days at 60°C in Table 4, it can be seen that after 10 days, the HPLC purity and maximum single impurity content of crystal form I and crystal form II are both small, and the chemical stability of the two crystal forms Better; and the HPLC purity and maximum single impurity content changes of crystal form I are smaller than the HPLC purity and maximum single impurity content changes of crystal form II. This result shows that the crystal form I obtained by the present invention has excellent stability at 60°C于晶型II.

table 5

To	10天10 days
晶型IForm I	XRD图谱无变化No change in XRD pattern

Crystal Form II

XRD shows a mixed crystal of crystalline form I and II

From the XRD analysis results of 10 days at 60℃ in Table 5, it can be seen that the crystal form I has not changed after 10 days; while the crystal form II has been transformed, and some of the crystals are transformed into the crystal form I. This result shows that The physical stability of the invented crystalline form I at 60°C is better than that of the crystalline form II.

Hygroscopicity test

Take a dry stoppered glass weighing bottle (outer diameter of 50mm, height of 15mm), place it in a suitable 25°C±1°C constant temperature desiccator (place a saturated sodium chloride solution at the bottom) the day before the test, and take the same weight The crystal form I prepared in Example 1 and the crystal form II prepared in Example 8 were laid flat in the above weighing bottle at a temperature of 25℃±1℃ and a relative humidity of 75%±2%. Place it for 24h and conduct a moisture absorption test. The results are shown in Table 6.

Table 6

To	晶型IForm I	晶型IICrystal Form II
吸湿性(％)Hygroscopicity (%)	00	00

It can be seen from the results of the hygroscopicity experiment in Table 6 that the crystal form I and crystal form II obtained in the present invention have no hygroscopicity.

Homogenization experiment

Take 30 mg of the crystal form II prepared in Example 8, add 4 ml of ethanol, stir at 20° C. for 1 hour, and filter. The X-ray powder diffraction pattern (XRD) is measured, and the crystal form I is confirmed.

From the homogenization experiment results, it is known that the crystal form II obtained in the present invention is easily transformed into crystal form I in an ethanol solution, indicating that the stability of the crystal form I is better than that of the crystal form II.

Solubility test

Sample solution: Take an appropriate amount of the crystal form I obtained in Example 1 and the crystal form II obtained in Example 8 to a 20ml measuring flask, and add 10ml of solvent (aqueous hydrochloric acid, pH=1.0, pH=2.0, pH=4.5, pH=6.8) Mix well, shake for 10 min at 37°C, filter immediately, take the filtrate and dry it to constant weight at 60°C, and obtain the solubility of the corresponding crystal form by gravimetric method. The results are shown in Table 7.

Table 7

It can be seen from the results in Table 7 that the solubility of crystal form I and crystal form II in the above-mentioned solvent both increase with the increase of pH value, but the solubility of crystal form II is better than that of crystal form I.

Active example

The activity experiment of the compound of formula I is carried out in the following way

1. The compound induces Nrf2 to enter the nucleus and HO-1 expression in the cytoplasm of HT22 cells

Take the mouse hippocampal neuron cells HT22 cells that proliferate well and are in the logarithmic growth phase, and count them after trypsinization and dispersion. The cell density is adjusted with RPMI 1640 medium containing 5% fetal bovine serum, and 1×10 ⁶ cells are inoculated Into the T25 culture flask, place it in a 37°C, 5% CO ₂ , 100% relative humidity incubator for 24 hours, and then add a certain concentration of compound. Continue to culture, and harvest the cells after 4 hours, extract nuclear protein and total cell protein, Western Blot to detect the content of Nrf2 in the nucleus and the expression of HO-1 in the cytoplasm. The experimental results show that the compound of formula I can significantly increase the content of Nrf2 protein in the nucleus of HT22 cells, suggesting that the compound of formula I has the effect of inducing Nrf2 protein in the cytoplasm to enter the nucleus (Figure 7). At the same concentration, the compound of formula I is stronger than fumar Dimethyl Acid (DMF); and the compound of formula I can induce the expression of HO-1 in the cytoplasm, thereby significantly increasing the expression of HO-1 in the cytoplasm (Figure 8).

2. The protective effect of the compound on HT22 cells injured by sodium L-glutamate

Take HT22 cells that proliferate well and are in the logarithmic growth phase, and count after trypsin digestion and dispersion. The cell density is adjusted with RPMI 1640 medium containing 5% fetal bovine serum, and 1000 cells/well are inoculated into a 96-well plate. Incubate in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity for 24 hours. Compounds of different concentrations were added to each well, after 24 hours of continuous cultivation, a certain concentration of L-glutamate monosodium salt was added, and after another 24 hours of cultivation, the CellTiter-Glo kit was used to detect cell viability. The experimental results show that in the HT22 cell injury model induced by sodium glutamate, the compound of the present invention has a protective effect on HT22 cells injured by sodium L-glutamate (Table 8).

Table 8 The protective effects of compounds on the HT22 cell injury model induced by sodium glutamate

CmpsCmps	EC ₅₀(μM) EC ₅₀ (μM)
DMFDMF	0.330.33
式I化合物Formula I compound	0.180.18

3. The compound's inhibitory effect on IFN-γ-induced Hacat cells to secrete CXCL9

Take Hacat cells that proliferate well and are in the logarithmic growth phase. After trypsin digestion and dispersion, they are counted to prepare a cell suspension. The cell density is adjusted with MEM medium containing 10% fetal bovine serum to 1.2×10 ⁵ cells/well Inoculate into a 24-well plate, incubate in a 37°C, 5% CO ₂ , 100% relative humidity incubator for 16 hours, then add IFN-γ and different concentrations of compounds at the same time, continue to incubate for 24 hours, collect the cell supernatant, and use Human CXCL9/MIG Elisa kit detects the secretion of CXCL9. The experimental results showed that in the cell model where IFN-γ induced Hacat cells to secrete CXCL9, the compound of formula I had a certain inhibitory effect on the secretion of CXCL9 (Table 9).

Table 9 Inhibitory effects of compounds on IFN-γ-induced Hacat cells to secrete CXCL9

CmpsCmps	IC ₅₀(μM) IC ₅₀ (μM)
DMFDMF	30.0430.04
式I化合物Formula I compound	15.9715.97

4. The inhibitory effect of the compound on LPS-induced TNF-α secretion in Ana-1 cells

Take Ana-1 cells that proliferate well and are in the logarithmic growth phase, and count them after trypsin digestion and dispersion to prepare a cell suspension. Use RPMI1640 culture medium containing 10% fetal bovine serum to adjust the cell density to 0.8×10 ⁵ cells Inoculate each well into a 24-well plate, incubate in a 37°C, 5% CO ₂ , 100% relative humidity incubator for 24 hours, add LPS and compound solutions of different concentrations, continue to incubate for 3 hours, collect the cell supernatant, and use Mouse TNF -αElisa kit detects the secretion of TNF-α. The experimental results show that in the cell model of Ana-1 cells secreting TNF-α induced by LPS, the compound of formula I has a certain inhibitory effect on the secretion of TNF-α (Table 10).

Table 10 Inhibitory effect of compounds on LPS-induced TNF-α secretion in Ana-1 cells

CmpsCmps	IC ₅₀(μM) IC ₅₀ (μM)
DMFDMF	52.6252.62
式I化合物Formula I compound	3.183.18

5. The inhibitory effect of the compound on MOG-induced experimental allergic encephalomyelitis (EAE) in C57BL/6 mice

Female C57BL/6 mice aged 6-8 weeks were randomly grouped. On day 0, 100μL of MOG35-55 (1mg/mL) and Freund's complete adjuvant (2mg/mL) were injected into the hind limbs and back muscles of the mice on day 0. The immune emulsion was injected with pertussis toxin (200ng) into the abdominal cavity 48h later to induce the development of EAE model. From the 3rd day to the 30th day, different doses of compounds were given by gavage, and the EAE progress of the mice was scored according to the clinical symptoms, the inhibitory effect of the drugs on the EAE progress of the mice was investigated and the inhibition rate was calculated (inhibition rate = (1- (Mean AUC of clinical score(Test/Vehicle)))*100). The experimental results show that in the mouse EAE model, compared with the model group, the compound of formula I (10mg/kg, qd) can significantly inhibit the development and progress of mouse EAE, the inhibition rate is 61.37%, while DMF (15mg/kg , bid) The inhibition rate was 41.39%. Obviously, the inhibitory effect of the compound of formula I (10 mg/kg, qd) is stronger than that of DMF (15 mg/kg, bid) (Figure 9).

6. The compound improves the learning and memory of Alzheimer's disease (AD) rats injected with Aβ into the lateral ventricle

Male wistar rats, 12 weeks old, were surgically injected with condensed oligomer Aβ25-35 (10 nM) into the lateral ventricle to prepare AD rat models. From the second day onwards, they were given different doses of compounds by gavage. Donepezil was used as the control drug. Ten days later, a behavioral experiment (Morris water maze) was started to evaluate the improvement effect of the drugs on the learning and memory ability of rats. The Morris water maze experiment is divided into two parts: positioning navigation and space exploration. Positioning and sailing training began on the first day, training for a total of 3 days, repeated training twice a day. On the 4th day, the test escaped the incubation period for the last time, and then the underwater platform was removed for space exploration experiments.

In the Morris water maze experiment, on the 4th day, animals in the sham operation group had no significant changes in academic performance compared with the animals in the blank group, suggesting that the operation did not affect the learning and memory abilities of rats (p>0.05). Compared with the sham operation group, the latency of model rats to reach the platform was significantly longer (p<0.001), indicating that the AD animal model was successfully prepared. Compared with the model group, the compound of formula I (15mg/kg, qd) and donepezil (3mg/kg, qd) can significantly shorten the latency of rats to reach the platform (Figure 10); in the space exploration experiment on day 4, the drug The treatment had a significant effect on the number of times the animals crossed the target platform. Compared with the model group, the number of crossings of the rats in the compound of formula I and donepezil group was significantly increased (Figure 11).

Morris water maze experiment showed that the compound of formula I (15mg/kg, qd) can significantly improve the learning and memory of AD model rats.

Seven. Compounds improve the behavior of rats with Parkinson's disease (PD) induced by 6-OHDA

Male wistar rats, 12 weeks old, were injected with 6-OHDA into the medial forebrain tract to prepare a PD rat model. 21 days after 6-OHDA injection, apomorphine was used to induce rotation to verify the success of the model. Mice were randomly divided into groups and given different doses of compound by gavage. The sham operation group and model group were given double distilled water, L-dopa was used as the control drug, and behavioral testing (including the rotating rod test) was performed after 10 days. , Pole climbing experiment and apomorphine-induced rotation experiment) to evaluate the efficacy of the test compound.

In the rotating rod experiment, compared with the sham operation group, the rats in the model group had a significantly shorter fall latency (p<0.001), indicating that their motor function was impaired and the PD model was successfully prepared. Compared with the model group, the control drugs levodopa (10mg/kg, qd) and the compound of formula I (10mg/kg, qd) can significantly prolong the incubation period of rats' fall (Figure 12);

In the rod climbing experiment, compared with the sham operation group, the rod climbing time of rats in the model group was significantly longer (p<0.01), indicating that their motor function was impaired and the PD model was successfully prepared. Compared with the model group, the control drug levodopa (10mg/kg, qd) and the compound of formula I (10mg/kg, qd) can significantly shorten the climbing time of rats (Figure 13).

In the rotation experiment of PD rats induced by apomorphine, compared with the sham operation group, the rotation speed of rats in the model group was significantly increased (p<0.01), suggesting that the DA system function was impaired and the PD model was successfully prepared. Compared with the model group, the control drug levodopa (10mg/kg, qd) and the compound of formula I (10mg/kg, qd) can significantly reduce the rat's rotation speed (Figure 14).

Based on the above results, the compound of formula I can significantly alleviate the PD-like behavioral disorder in rats induced by 6-OHDA.

8. The neuroprotective effect of the compound on acute cerebral artery ischemia-reperfusion injury in rats

After SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital, a suture method was used to prepare an acute cerebral artery ischemia-reperfusion injury model (MCAO). After 2 hours of ischemia, the model thread plug was pulled out to cause reperfusion injury, and intravenous drug intervention was given within 5 minutes, with edaravone as the positive control drug. The administration was continued on the second day. On the third day, Zea-Longa 5-level standard scoring was used to score the postoperative rats' behavior (0 points: normal, no neurological deficit; 1 point: left front paw incomplete extension, mild neurological deficit; 2 points : When walking, the rat turns to the left side (paralyzed side), with moderate neurological deficit; 3 points: When walking, the rat's body falls to the left side (paralyzed side), with severe neurological deficit; 4 points: unable to walk spontaneously , Loss of consciousness). After the test, the rats were anesthetized, and then the brain was decapitated, and the brain tissue was fixed with paraformaldehyde. After washing the whole brain with normal saline, place it in a clean petri dish and quickly freeze it in a refrigerator at -20°C for 30 min. Starting from the midpoint of the line between the anterior pole of the brain and the optic chiasm, slice the brain every 2 mm, and cut the brain into 5-6 slices, then put the brain slices in 1% TTC solution, incubate at 37°C for 10-15 minutes . The non-stained infarct area tissue was cut with a scalpel, and the weight ratio of the infarct area to the whole brain was calculated to evaluate the neuroprotective effect of the compound on acute cerebral artery ischemia-reperfusion injury in rats. The behavioral score results showed that: after the intervention of the compound of formula I (10mg/kg), the behavior score of SD rats was significantly lower than that of the model group, and lower than the control drug edaravone (6mg/kg) group (Figure 15). In terms of the infarct area/whole brain weight ratio, both the edaravone group and the formula I compound group were significantly smaller than the model group. The above results indicate that both edaravone and the compound of formula I can effectively protect the nerves of rats with cerebral artery ischemia-reperfusion injury, and the effect of the compound of formula I is better than that of edaravone (Figure 16).

9. Compound sensitization test

On the day before the experiment, the male guinea pigs were shaved off the areas where they were in contact with the drug. Stir 0.1 g of the test drug (inducing dose) and appropriate amount of petroleum jelly, and smear the skin on the 0th, 7th and 14th day, each induction time is 6h, while the guinea pigs in the control group are smeared with petroleum jelly. 14 days after the last induction of smearing, 0.08g of the test drug (provocation dose) was applied to the contralateral skin of the guinea pig induction site, while the contralateral skin of the corresponding site of the guinea pig in the control group was still smeared with petroleum jelly, which was removed after 6 hours. The test drug in the administration group and the petroleum jelly in the control group were washed and washed to observe whether there was skin reaction, and the degree of reaction was scored and photographed and recorded, and the sensitization rate of each group was counted. There was no significant reaction in the Control group. DMF caused 90% of the guinea pigs to have obvious skin erythema and edema. The formula I compound group had no obvious skin reaction, only slight edema (Figure 17). The results show that DMF has extremely strong allergenicity, while the compound of formula I only has extremely weak allergenicity.

Claims

A crystal form I of a compound of formula I,

It is characterized in that its X-ray powder diffraction pattern has characteristic peaks at the following diffraction angles 2θ: 8.82±0.2°, 14.56±0.2°, 16.82±0.2°, 17.72±0.2°, 20.22±0.2°, 22.50±0.2° , 26.24±0.2°, 29.40±0.2°.
The crystalline form I of claim 1, wherein the X-ray powder diffraction pattern further has characteristic peaks at the following diffraction angles 2θ: 10.06±0.2°, 24.22±0.2°, 25.00±0.2°, 26.76± 0.2°, 28.16±0.2°, 30.56±0.2°, 31.14±0.2°, 33.74±0.2°, 38.66±0.2°, 40.20±0.2°, 44.84±0.2°, 46.04±0.2°.
The crystalline form I according to claim 1 or 2, characterized in that its X-ray powder diffraction spectrum is basically as shown in Fig. 1.
A method for preparing the crystal form I according to any one of claims 1 to 3, the method being selected from any of the following methods:

Method (1), which includes the following steps:

1) Dissolve the compound of formula I in any solvent of alcohol, ketone, ester, acetonitrile and dioxane, dissolve it, and evaporate and crystallize; the alcohol is a C2-C4 alcohol; the ketone is a C3-C6 Ketone; the ester is a C3-C6 ester; the dissolution temperature is 25-45°C; the mass-volume ratio of the compound of formula I to the solvent is (mg/ml) 10:1.5-30;

2) Filter to obtain crystal form I;

Method (2), which includes the following steps:

1) The compound of formula I is dissolved in any solvent of alcohol, ketone, ester, acetonitrile, dioxane, dimethylacetamide, dimethylformamide, and dimethylsulfoxide, and the temperature is lowered to crystallize; The alcohol is a C2-C4 alcohol; the ketone is a C3-C6 ketone; the ester is a C3-C6 ester; the dissolution temperature is 40-70°C; the mass-volume ratio of the compound of formula I to the solvent is (mg /ml) 10: 0.4～3; the temperature of said crystallization is 0～20℃;

Or the compound of formula I is dissolved in a mixed solvent of any organic solvent among ethanol, isopropanol, dioxane, acetone, and acetonitrile and water, and the temperature is lowered to crystallize; the dissolution temperature is 40-70°C; The volume ratio of the organic solvent to water is 1:0.1-9; the mass-volume ratio of the compound of formula I and the mixed solvent is (mg/ml) 10:1-1.5; the crystallization temperature is 0-5°C ；

2) Filter to obtain crystal form I.
A crystal form II of the compound of formula I,

It is characterized in that its X-ray powder diffraction pattern has characteristic peaks at the following diffraction angles 2θ: 7.34±0.2°, 14.72±0.2°, 16.98±0.2°, 20.30±0.2°, 22.16±0.2°, 25.84±0.2° , 38.44±0.2°.
The crystal form II according to claim 5, wherein the X-ray powder diffraction pattern further has characteristic peaks at the following diffraction angles 2θ: 9.20±0.2°, 13.90±0.2°, 18.46±0.2°, 23.74± 0.2°, 29.72±0.2°, 34.42±0.2°.
The crystal form II according to claim 5 or 6, characterized in that its X-ray powder diffraction spectrum is basically as shown in Fig. 4.
A method for preparing the crystal form II according to any one of claims 5 to 7, the method is selected from any one of the following methods:

Method (1), which includes the following steps:

1) Add the compound of formula I to CH 2 Cl 2 and dissolve under reflux; the mass-volume ratio (mg/ml) of the compound of formula I to CH 2 Cl 2 is 10:1.4-1.6;

2) Add the above solution to ethyl acetate (EA) at -15~-10°C; the volume ratio of the EA to CH 2 Cl 2 is 0.9-1.1:1;

3) Stir and crystallize, filter to obtain crystal form II;

Method (2), which includes the following steps:

1) The compound of formula I is dissolved in dioxane; the temperature of the dissolution is 45-60°C; the mass-volume ratio (mg/ml) of the compound of formula I to dioxane is 10:1.4-1.6 ；

2) Add the above solution to n-heptane at 4-6°C; the volume ratio of n-heptane to dioxane is 3 to 3.5:1;

3) Stir to crystallize, filter to obtain crystal form II.
A pharmaceutical composition comprising an effective dose of the crystal form I according to any one of claims 1 to 3 or the crystal form II according to any one of claims 5 to 7.
The crystal form I according to any one of claims 1 to 3 or the crystal form II according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 9 is prepared for treatment and activation of Nrf2 Application in medicine for related diseases, wherein the diseases related to Nrf2 activation are stroke, neurodegenerative diseases, diabetes, diabetic nephropathy, coronary heart disease, atherosclerosis or non-alcoholic fatty liver.
The crystal form I according to any one of claims 1 to 3 or the crystal form II according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 9 is prepared for the treatment of stroke Application in medicine.
The crystal form I according to any one of claims 1 to 3 or the crystal form II according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 9 is prepared for the treatment of neurodegeneration Application of medicines for diseases.
The use according to claim 10 or 12, wherein the neurodegenerative disease is selected from multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease ( HD), Amyotrophic Lateral Sclerosis (ALS), Friedreich’s Ataxia (FRDA), Spinal Muscular Atrophy (SMA), Optic Neuromyelitis (NMO) and Spinocerebellar Ataxia (SCA).
The crystal form I according to any one of claims 1 to 3 or the crystal form II according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 9 is prepared for treatment and immunomodulation Application in medicines for related diseases, wherein the diseases related to immune regulation are preferably selected from psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, transplant rejection and inflammatory diseases.