RU2784538C2 - CRYSTAL FORM OF COMPOUND OF 1H-IMIDAZO[4,5-b]PYRIDINE-2(3H)-ONE AND ITS PRODUCTION METHOD - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a crystal Form A of a compound 1, where the crystal Form A is characterized by X-ray powder diffractogram having characteristic diffraction peaks at following angles 2θ: 10.69±0.2°, 12.31±0.2°, 13.45±0.2°, 14.10±0.2°, 14.62±0.2°, 19.07±0.2°, 20.33±0.2°, 21.79±0.2°. The crystal Form A of the compound 1 is obtained by addition of the compound 1 to an alcohol solvent, a ketone solvent, a mixed solvent consisting of the alcohol solvent and water, a mixed solvent consisting of the ketone solvent and water; the alcohol solvent is selected from a group consisting of methanol, ethanol; the ketone solvent is selected from a group consisting of acetone, and heating for dissolution, and then cooling for crystallization to obtain the crystal Form A. At the same time, the mixed solvent consisting of the alcohol solvent and water consists of a mixed solvent consisting of ethanol and water.

EFFECT: crystal Form A of a compound of 1H-imidazo[4,5-b]pyridine-2(3H)-one (1), having good stability, intended for the use in the production of a drug for the treatment of a disease related to PDE4 receptor.

10 cl, 4 dwg, 10 tbl, 9 ex

Description

Cross-reference to related application

The present application claims priority for the following application:

Chinese Application No. 201810085704.1 filed on January 29, 2018.

The field of technology to which the invention belongs

A crystalline form of the compound 1 H -imidazo[4,5- b ]pyridin-2(3H)-one and a method for its preparation, as well as the use of a crystalline form for the preparation of a drug for the treatment of a disease associated with PDE4, are presented.

Prerequisites for the creation of the invention

Tumor necrosis factor (TNFα) is a cytokine released predominantly by monocytes and macrophages in response to immune stimulation. TNFα can promote most of the processes of cell differentiation, recruitment, proliferation, and protein degradation. TNFα has a protective effect against infectious agents, tumors and tissue damage at low levels. However, excessive release of TNFα can also cause disease. For example, when administered to mammals or humans, TNFα can cause or exacerbate inflammation, fever, cardiovascular problems, bleeding, blood clotting, and acute reactions like acute infection and shock. Excessive or uncontrolled TNFα production in animals or humans is often indicative of the following diseases: endotoxemia and/or toxic shock syndrome, cachexia, adult respiratory stress syndrome, cancer (such as solid tumors and hematologic tumors), heart disease (such as congestive heart deficiency), viral infection, genetic disease, inflammatory disease, allergic disease, or autoimmune disease.

Cancer is a disease of particular destructiveness, and an increase in the level of TNFα in the blood indicates a risk of cancer or metastasis. As a rule, cancer cells cannot survive in the circulatory system of a healthy subject, and one reason is that the inner wall of blood vessels serves as a barrier to extravasation of cancer cells. Studies have shown that ELAM-1 on endothelial cells can mediate adhesion of colon cancer cells to cytokine-exposed endothelium.

Cyclic adenosine monophosphate (cAMP) plays a role in many diseases and disorders. Increasing the concentration of cAMP in leukocytes during inflammation suppresses leukocyte activation and subsequently releases inflammatory regulatory factors, including TNFα and NF-κB. Elevated cAMP also leads to relaxation of airway smooth muscle.

The main cellular mechanism of cAMP inactivation is due to the destruction of cAMP by a family of isozymes called cyclic nucleotide phosphodiesterases (PDEs). The PDE family is known to include 11 members. To date, inhibition of the PDE4 enzyme has been shown to be particularly effective in inhibiting the release of inflammatory mediators and relaxing airway smooth muscle, and therefore the PDE4 enzyme has become one of the popular drug targets. According to different genetic coding, the PDE-4 family can be divided into 4 subtypes (PDE-4A, B, C, D). Among them, the expression of PDE-4A, PDE-4B and PDE-4D in inflammatory cells (such as B cells, T cells and neutrophils) is stronger than in PDE-4C. Inhibition of the PDE4 enzyme results in an increase in cAMP levels, thereby regulating TNFα levels so as to treat diseases.

The essence of the invention

In one aspect, crystalline Form A of Compound 1 is provided, wherein crystalline Form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 14.10±0.2°, 19.07±0.2°, 21.79± 0.2°.

Compound 1

In some embodiments, in accordance with the present disclosure, the crystalline Form A of compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 10.69±0.2°, 12.31±0.2°, 13.45± 0.2°, 14.10±0.2°, 14.62±0.2°, 19.07±0.2°, 20.33±0.2°, 21.79±0.2°.

In some embodiments, in accordance with the present disclosure, the crystalline Form A of compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.25±0.2°, 8.93±0.2°, 10.69± 0.2°, 12.31±0.2°, 13.45±0.2°, 14.10±0.2°, 14.62±0.2°, 18.16±0.2°, 19.07±0.2°, 20.33±0.2°, 21.79±0.2°.

In some embodiments, in accordance with the present disclosure, the crystalline Form A of Compound 1 has the X-ray powder diffraction pattern shown in FIG. one.

In some embodiments, in accordance with the present disclosure, the crystalline Form A of compound 1 has an X-ray powder diffraction pattern with the analysis data shown in Table 1

Table 1
X-ray powder diffraction analysis data of the crystalline Form A of compound 1 No. Angle 2θ (°) interplanar
distance (Å) Relative intensity (%) No. Angle 2θ (°) interplanar
distance (Å) Relative intensity (%) one 6.250 14.1293 21.2 21 24.713 3.5995 17.3 2 8.932 9.8927 19.2 22 24,930 3.5686 14.4 3 9.425 9.3754 9.8 23 25.622 3.4739 14.8 four 10,690 8.2687 45.3 24 26.922 3.3090 14.6 5 12.306 7.1868 45.9 25 27.220 3.2734 10.6 6 12.660 6.9865 29.3 26 28.010 3.1829 22.1 7 13.449 6.5783 38.0 27 28.324 3.1483 26.0 eight 14.098 6.2769 100.0 28 29.410 3.0344 7.5 9 14.615 6.0558 28.8 29 29.942 2.9817 11.6 ten 15.162 5.8386 11.5 thirty 30.854 2.8957 5.3 eleven 17.417 5.0874 5.7 31 31.459 2.8414 2.1 12 18.162 4.8805 24.3 32 32.250 2.7735 3.0 13 18.796 4.7173 28.0 33 32.725 2.7342 2.5 fourteen 19.072 4.6496 72.4 34 33.082 2.7056 2.2 fifteen 20.333 4.3639 32.1 35 33.379 2.6822 3.6 16 20.728 4.2817 27.7 36 34.167 2.6221 3.6 17 21.794 4.0746 68.2 37 35.525 2.5249 2.7 eighteen 22.739 3.9074 9.9 38 35.902 2.4993 3.0 19 22.998 3.8639 13.1 39 36.988 2.4283 2.6 twenty 24.261 3.6656 20.0 40 37.462 2.3987 3.3

In some embodiments according to the present disclosure, the crystalline Form A of compound 1 has a differential scanning calorimetry curve having an endothermic peak start point at 201.70°C±2°C.

In some embodiments according to the present disclosure, the crystalline Form A of Compound 1 has the DSC curve shown in FIG. 2.

In some embodiments, in accordance with the present disclosure, the crystalline Form A of compound 1 has a thermogravimetric analysis curve, where the weight loss at 100.00±2°C is 0.02039%.

In some embodiments according to the present disclosure, the crystalline Form A of Compound 1 has the TGA curve shown in FIG. 3.

In another aspect, a process is provided for preparing crystalline Form A, comprising adding Compound 1 to an alcohol solvent, a ketone solvent, an ether solvent, a mixed solvent of an alcohol solvent and water, a mixed solvent of a ketone solvent and water, or a mixed solvent of ethereal solvent and water; heating to dissolve and then cooling to crystallize to obtain crystalline Form A.

In some embodiments, in accordance with the present disclosure, the alcoholic solvent is selected from the group consisting of methanol, ethanol, and isopropanol.

In some embodiments according to the present disclosure, the ketone solvent is selected from the group consisting of acetone and butanone.

In some embodiments according to the present disclosure, the ether solvent is selected from the group consisting of glycol dimethyl ether.

In some embodiments, in accordance with the present disclosure, the mixed solvent consisting of an alcoholic solvent and water is selected from the group consisting of a mixed solvent consisting of ethanol and water.

In some embodiments according to the present disclosure, in a mixed solvent consisting of an alcoholic solvent and water, the volume ratio of the alcoholic solvent to water is selected from the group consisting of 1:0.2-1.5.

In yet another aspect, the use of crystalline form A of Compound 1 is provided for the preparation of a medicament for the treatment of a disease associated with the PDE4 receptor.

In some embodiments according to the present disclosure, a PDE4 related disease includes psoriasis, psoriatic arthritis, chronic obstructive pneumonia, ankylosing spondylitis, inflammatory bowel disease.

technical effect

The crystalline Form A of Compound 1 has good stability, low hygroscopicity, and drug potential. The crystalline Form A of Compound 1 shows good stability in an alcohol solvent, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, a mixed solvent of an alcohol solvent and water, a mixed solvent of acetonitrile and water, or a mixed solvent of acetone and water. Crystal Form A of Compound 1 exhibits good stability under accelerated test conditions of 40° C./75% RH. crystalline Form A of compound 1 shows good stability under long-term storage conditions at 25ºC/60% relative humidity.

Compound 1 shows excellent in vitro activity to inhibit phosphodiesterase subtype 4B (PDE4B). In addition, Compound 1 shows excellent in vitro activity to inhibit TNFα production in hPBMC, which is higher than that of Apremilast. Compound 1 in three dose groups of 0.3, 1 and 3 mg/kg significantly improves the symptoms of collagen-induced arthritis. In addition, Compound 1 in the 1 mg/kg and 3 mg/kg dose groups showed a significant improvement in the pathology of arthritis. These three dose groups show a clear dose-response relationship in assessing the pathology of arthritis. The therapeutic effect of compound 1 at 3 mg/kg (clinical evaluation and evaluation of the pathology of arthritis) is better than that of Apremilast at 5 mg/kg.

General definitions

Unless otherwise noted, the following terms and phrases have the following definitions. A particular term or phrase should not be considered vague or obscure without a specific definition and should be understood according to its usual meanings. The trade name used must refer to the respective product or active ingredient.

Intermediates can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthesis methods, and equivalent alternatives well known to those skilled in the art. Preferred embodiments include, but are not limited to, the Examples below.

In specific embodiments, the chemical reaction is carried out in a suitable solvent, and the solvent must be suitable for the chemical changes of the present disclosure and the required reagents and materials. To obtain the compound of the present invention, a person skilled in the art may modify or select a synthetic step or reaction scheme based on the presented embodiments.

The present disclosure will be described in detail and the Examples should not be construed as limiting it.

The solvents used in the present invention are commercially available and can be used without further purification.

The following abbreviations are used: DMF: dimethylformamide; MsOH: methanesulfonic acid; EtOH: ethanol; NaOH: sodium hydroxide.

Compounds are named manually or using ChemDraw® software. Providers use directory connection names.

X-ray powder diffractometer, XRPD

Device: X-ray diffractometer BRUKER D8 advance

Test method: Approximately 10-20 mg of sample is used for XRPD detection.

The specific XRPD parameters are as follows:

Radiating tube: Cu, kα, (λ=1.54056Ǻ).

Radiating tube voltage: 40KV, Radiating tube current: 40mA

Slit divergence: 0.60mm

Detector slit: 10.50mm

Anti-scatter gap: 7.10 mm

Scan range: 4-40 deg.

Step size: 0.02 deg.

Time/step: 0.12 sec

Stage rotation speed: 15 rpm

Differential Scanning Calorimeter, DSC

Device: TA Q2000 Differential Scanning Calorimeter

Test method: A sample (about 1 mg) is placed in a DSC aluminum test dish, under 50 ml/min N ₂ flow, heated from 25°C to 350°C at a heating rate of 10°C/min.

Thermogravimetric analyzer, TGA

Device: Thermogravimetric analyzer TA Q5000IR

Test method: Sample (2-5 mg) is placed in a TGA platinum test dish, under 25 ml/min N ₂ flow, heated from room temperature to 350°C at a heating rate of 10°C/min.

Dynamic vapor sorption, DVS

Device: SEM Advantage-1 Dynamic Vapor Sorption Analyzer

Test Conditions: A sample (10-20 mg) is placed in a DVS sample disc for testing.

The specific DVS parameters are as follows:

Temperature: 25°C

Scale: dm/dt=0.01%/min (min: 10 min, max: 180 min)

Drying: drying at 0% RH for 120 min.

RH (%) tested gradient: 10%

RH (%) gradient test range: 0% - 90% - 0%

Hygroscopicity is divided into the following categories:

Hygroscopic category ΔW% Transition to liquid state Absorbing enough water to form liquid Very hygroscopic ΔW% ≥ 15% hygroscopic 15% > ∆W% ≥ 2% slightly hygroscopic 2% > ∆W% ≥ 0.2% Non-hygroscopic or slightly hygroscopic ∆W% < 0.2% Note: ΔW% refers to the hygroscopic weight gain of the test sample at 25±1°C and 80±2% RH

Content determination method

Device: Agilent 1260 HPLC with DAD detector or Shimadzu LC-20A HPLC with PDA detector

Specific chromatographic parameters are as follows:

Chromatography column: Agilent Eclipse plus C18 (4.6mm × 150mm, 3.5µm)

Column temperature: 40°C

Flow rate: 1.0 ml/min

Detector wavelength: 230nm

Injection volume: 10 µl

Chromatogram registration time: 60 min

Mobile phase A: 0.04% aqueous trifluoroacetic acid (v/v)

Mobile phase B: acetonitrile

Thinner: acetonitrile:purified water=3:1(v/v)

Washing solution: acetonitrile: purified water = 3:1 (v/v)

Gradient elution procedure:

Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 85 fifteen 30.00 70 thirty 50.00 thirty 70 55.00 fifteen 85 55.01 85 fifteen 60.00 85 fifteen

Brief description of the drawings

Fig. 1 shows an X-ray powder diffraction pattern obtained using Cu-Kα radiation, crystalline form A of compound 1.

Fig. 2 shows the DSC curve of the crystalline form A of compound 1.

Fig. 3 shows the TGA curve of crystalline form A of compound 1.

Fig. 4 shows the DVS curve of crystalline form A of compound 1.

Examples

The following Examples are presented to better illustrate for a better understanding of the present invention. Specific embodiments should not be construed as limiting the present invention.

Example 1 Preparation of Crystal Form A of Compound 1

Step 1 : Synthesis of Compound 3

At room temperature, compound b (10.00 g, 39.77 mmol), compound 2 (9.78 g, 35.79 mmol) and diisopropylamine (10.28 g, 79.53 mmol, 13.89 ml) were dissolved in N,N-dimethylformamide (200.00 ml), the mixture was purged with nitrogen three times and the reaction mixture was heated to 120° C. under nitrogen protection with stirring for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, water (400 ml) and extracted with ethyl acetate (200 ml ×3). The organic phases were combined and washed with brine (100 ml×3), dried over anhydrous sodium sulfate and filtered to remove the drying agent. The filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (eluent: ethyl acetate/petroleum ether=1/4-1/2, v/v) to obtain the target compound 3 .

MS-ESI m/z : 509.8 [M+Na] ⁺ , 511.8 [M+Na+2] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.27 (s, 1H), 7.27 (d, J =6.8 Hz, 1H), 6.90-6.86 (m, 2H), 6 .81 (d, J = 8.0 Hz, 1H), 5.72 (sq., J = 6.4 Hz, 1H), 4.04 (sq., J = 6.8 Hz, 2H), 3 .80 (s, 3H), 3.68 (dd, J =6.6, 14.6 Hz, 1H), 3.40 (dd, J =6.4, 14.8 Hz, 1H), 2, 52 (s, 3H), 2.45 (s, 3H), 1.40 (t, J =7.0 Hz, 3H).

Step 2 : Synthesis of Compound 4

At room temperature, compound 3 (12.10 g, 24.78 mmol) and o-fluorophenylboronic acid (5.20 g, 37.17 mmol) were dissolved in dioxane (150.00 ml) and water (50.00 ml), then potassium carbonate (10.27 g, 74.34 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride-dichloromethane complex (2.02 g, 2.48 mmol) were added under nitrogen protection. The reaction mixture was heated under nitrogen protection to 80° C. with stirring for 14 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, water (500 ml) was added and extracted with ethyl acetate (300 ml×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered to remove the drying agent. The filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (eluent: ethyl acetate/petroleum ether=1/10-1/4, v/v) to obtain the target compound 4 .

MS-ESI m/z : 504.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.07 (s, 1H), 7.42 (d, J =6.8 Hz, 1H), 7.38-7.30 (m, 1H), 7 .18-7.06 (m, 3H), 6.98-6.89 (m, 2H), 6.83 (d, J = 8.4 Hz, 1H), 5.82 (sq., J = 6.6 Hz, 1H), 4.13-4.00 (m, 3H), 3.81 (s, 3H), 3.43 (dd, J =6.4, 14.7 Hz, 1H), 2.55 (s, 3H), 2.23 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H).

Step 3 : Synthesis of Compound 5

At room temperature, methanol (200.00 ml) was added to compound 4 (9.20 g, 18.27 mmol) and ammonium chloride (9.77 g, 182.70 mmol) and zinc powder (11.95 g, 182 .70 mmol) in 20 batches at 0°C. The reaction mixture was stirred at 0° C. for 16 hours. After completion of the reaction, the reaction mixture was filtered to remove zinc powder, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was dissolved in dichloromethane (200 ml). The suspension was filtered to remove insolubles, and the filtrate was concentrated under reduced pressure to give compound 5 .

MS-ESI m/z : 474.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.55 (s, 1H), 7.42-7.32 (m, 1H), 7.20 (d, J =4.8 Hz, 2H), 7 .13 (t, J = 9.0 Hz, 1H), 7.08-7.03 (m, 2H), 6.88 (d, J = 8.8 Hz, 1H), 5.70 (s, 1H), 4.19-4.07 (m, 2H), 4.00-3.90 (m, 1H), 3.87 (s, 3H), 3.58 (dd, J =6.0, 14.4 Hz, 1H), 2.78 (s, 3H), 2.05 (s, 3H), 1.47 (t, J =7.2 Hz, 3H).

Step 4 : Synthesis of Compound 1

At room temperature, compound 5 (9.30 g, 19.64 mmol) and triethylamine (19.87 g, 196.40 mmol) were dissolved in tetrahydrofuran (200.00 ml). The reaction mixture was cooled to 0° C. and a solution of triphosgene (2.33 g, 7.86 mmol) in tetrahydrofuran (50.00 ml) was added dropwise. A tetrahydrofuran (50.00 ml) solution was added dropwise to the above reaction solution. After the addition, the reaction mixture was stirred at 0°C for 3 h under a protective nitrogen atmosphere. After completion of the reaction, water (200 ml) was added to the reaction mixture and extracted with ethyl acetate (100 ml×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered to remove the drying agent. The filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (eluent: ethyl acetate/petroleum ether=1/3-2/1, v/v) to obtain compound 1 .

¹ H NMR (400 MHz, CDCl ₃ ) δ: 10.13-10.01 (m, 1H), 7.87 (s, 1H), 7.39-7.32 (m, 1H), 7.31 ( d, J = 1.6 Hz, 1H), 7.18-7.15 (m, 3H), 7.11 (t, J= 9.2 Hz, 1H), 6.74 (d, J= 8 .4 Hz, 1H), 6.14 (dd, J =4.8, 9.6 Hz, 1H), 4.86 (dd, J =9.4, 14.6 Hz, 1H), 4.09 -3.97 (m, 2H), 3.88 (dd, J =4.4, 14.8 Hz, 1H), 3.76 (s, 3H), 2.70 (s, 3H), 2, 16 (s, 3H), 1.35 (t, J =7.0 Hz, 3H).

Step 5 : Obtaining crystalline Form A of Compound 1

At room temperature, water (160.00 ml) and ethanol (170.00 ml) were added to compound 1 (6.45 g, 12.91 mmol), and the reaction mixture was stirred at 90° C. for 0.5 h. The reaction solution gradually became transparent during mixing. The reaction mixture was slowly cooled to 20° C. with stirring and stirring was continued at 20° C. for 16 h, during which time many white solids precipitated. White solids were collected by filtration and dried in a vacuum oven at 45° C. for 18 hours to give crystalline Form A of compound 1 .

MS-ESI m/z : 500.2 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.97 (s, 1H), 7.94 (s, 1H), 7.46-7.39 (m, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.26-7.22 (m, 3H), 7.17 (t, J= 9.0 Hz, 1H), 6.81 (d, J= 8.0 Hz, 1H), 6.22 (dd, J =4.8, 9.6 Hz, 1H), 4.93 (dd, J =9.6, 14.8 Hz, 1H), 4.13-4.03 (m, 2H), 3.95 (dd, J =4.8, 14.8 Hz, 1H), 3.83 (s, 3H), 2.77 (s, 3H), 2.23 (s, 3H), 1.42 (t, J =7.0 Hz, 3H).

Experimental Example 1: Stability Testing of Crystalline Form A in Various Solvents

50 mg of crystalline Form A was added in several portions to one solvent or mixed solvents indicated in the following table, stirred at 40° C. for 2 days, and centrifuged. The solids in all samples were collected and dried in a vacuum oven (40° C.) overnight. Crystal forms were tested for XRPD. The results are shown in Table 2.

table 2
Stability tests of crystalline Form A in various solvents No. Solvent Added solvent (ml) Condition (after 2 days) crystalline form one methanol 0.4 Suspension Crystal Form A 2 ethanol 0.4 Suspension Crystal Form A 3 Acetonitrile 0.4 Suspension Crystal Form A four Acetone 0.4 Suspension Crystal Form A 5 ethyl acetate 0.4 Suspension Crystal Form A 6 Tetrahydrofuran 0.3 Suspension Crystal Form A 7 Methanol:Water (3:1) 0.4 Suspension Crystal Form A eight Ethanol:Water (3:1) 0.4 Suspension Crystal Form A 9 Acetonitrile:Water (1:1) 0.4 Suspension Crystal Form A ten Acetone:Water (1:2) 0.4 Suspension Crystal Form A

Conclusion: The crystalline Form A of Compound 1 shows good stability in alcohol solvent, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, mixed solvent of alcohol solvent and water, mixed solvent of ketone solvent and water, mixed solvent of acetonitrile and water , or a mixed solvent consisting of acetone and water.

Experimental Example 2: Hygroscopicity test of the crystalline Form A of Compound 1

Materials:

Dynamic vapor sorption analyzer SEM DVS Advantage-1

Procedures:

10-20 mg of Compound 1 crystalline Form A was placed in a DVS sample disc for testing.

Results:

The DVS curve of the crystalline Form A of Compound 1 is shown in FIG. 4, △W= 0.08%.

Conclusion:

The crystalline Form A of Compound 1 had a hygroscopic weight gain of 0.08% at 25° C. and 80% RH, which was less than 0.2%, indicating no or little hygroscopicity.

Experimental Example 3: Solid State Stability Test of Compound 1 Crystal Form A at High Temperature, High Humidity, and Strong Light Conditions.

According to the "Guidelines for Stability Test of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition Four General Principles 9001), compound 1 crystalline Form A was tested for stability at high temperature (60°C, open sample), high humidity (room temperature/ relative humidity 92.5%, open sample) and strong illumination (4500 ± 500 lux, 90 μW/cm ² , tightly closed).

Weighed 1.5 g of crystalline Form A of compound 1 and placed on an open glass slide and distributed in a thin layer. Samples were placed under high temperature and high humidity conditions in a control desiccator and samples were taken on days 5, 10 and 30 for testing and the test results were compared with the original day 0 test results. film and samples were taken on days 5 and 10 for testing and the test results were compared with the original test results on day 0. The test results are shown in the following Table 3.

Table 3
Solid State Stability Test Results of Compound 1 Crystalline Form A Under High Temperature, High Humidity, High Light Conditions Test conditions Sample time Appearance Content total impurities - Day 0 White powder 99.5% 0.11% High temperature (60°C, open sample) Day 5 White powder 99.2% 0.16% Day 10 White powder 98.8% 0.16% Day 30 White powder 99.0% 0.16% High humidity (room temperature/92.5% RH, open sample) Day 5 White powder 99.0% 0.16% Day 10 White powder 98.9% 0.16% Day 30 White powder 99.0% 0.16% Strong illumination (4500±500lux, 90µw/ ^cm2 , tightly closed) Day 5 White powder 98.8% 0.11% Day 10 White powder 98.6% 0.11%

Conclusion: Crystal Form A of Compound 1 showed good stability under conditions of high temperature, high humidity, or strong light.

Experimental Example 4 Solid State Stability Test of Compound 1 Crystalline Form A Under Accelerated Test Conditions

According to "Guidelines for Stability Test of APIs and Preparations" (Chinese Pharmacopoeia 2015, Volume IV, General Principles 9001), Compound 1 crystalline Form A was tested for stability under accelerated test conditions at high temperature and high humidity (40°C/RH humidity 75%, sample tightly closed).

Weighed to 1.4 g of the crystalline Form A of compound 1 and placed in a two-layer low density polyethylene bag. Each layer of the low density polyethylene bag was folded and sealed respectively, and then the bag was placed in an aluminum foil bag and sealed by heat sealing. Samples were taken at 1, 2, 3 and 6 months for testing and the test results were compared with the original day 0 test results. The test was repeated three times, each time with a different batch of Compound 1 crystalline Form A. The test results are shown in the following Table 4.

Table 4
Solid State Stability Test Results of Compound 1 Crystalline Form A Under Accelerated Test Conditions (40°C/75% RH, Closed Sample) Test conditions The consignment Sample time Appearance Content total impurities Crystal form (XRPD) - one Day 0 White powder 99.4% 0.09% Crystal Form A 40°C/75% RH, sealed sample ^1st month White powder 99.3% 0.09% Not determined ^2nd month White powder 99.1% 0.09% Not defined ^3rd month White powder 99.5% 0.09% Not determined ^6th month White powder 100.1% 0.10% Crystal Form A - 2 Day 0 White powder 99.4% 0.10% Crystal Form A 40°C / 75% RH, sealed sample ^1st month White powder 99.1% 0.11% Not determined ^2nd month White powder 99.0% 0.11% Not defined ^3rd month White powder 99.2% 0.10% Not determined ^6th month White powder 100.5% 0.12% Crystal Form A - 3 Day 0 White powder 99.5% 0.11% Crystal Form A 40°C / 75% RH, sealed sample ^1st month White powder 98.8% 0.16% Not determined ^2nd month White powder 98.9% 0.16% Not defined ^3rd month White powder 99.2% 0.16% Not determined ^6th month White powder 100.0% 0.11% Crystal Form A

Conclusion: Crystal Form A of Compound 1 showed good stability under accelerated test conditions of 40° C./75% relative humidity.

Experimental Example 5 Solid State Stability Test of Compound 1 Crystalline Form A Under Long-Term Storage Conditions

According to the "Guidelines for Stability Test of APIs and Preparations" (Chinese Pharmacopoeia 2015, Volume IV, General Principles 9001), Compound 1 crystalline Form A was tested for stability under long-term storage conditions (25°C/60% relative humidity, tightly closed sample).

Weighed to 1.4 g of the crystalline Form A of compound 1 and placed in a two-layer low density polyethylene bag. Each layer of the low density polyethylene bag was folded and sealed respectively, and then the bag was placed in an aluminum foil bag and sealed by heat sealing. Samples were taken at 3, 6, 9, 12 and 18 months for testing and the test results were compared with the original day 0 test results. The test was repeated three times, each time with a different batch of Compound 1 crystalline Form A. The test results are shown in the following Table 5.

Table 5
Solid State Stability Test Results of Compound 1 Crystalline Form A Under Long Term Storage Conditions (25°C/60% RH, Tightly Sealed Sample) Test conditions The consignment Sample time Appearance Content total impurities Crystal form (XRPD) - one Day 0 White powder 99.4% 0.09% Crystal Form A 25°C/60% RH, sealed sample ^3rd month White powder 99.0% 0.09% Not defined ^6th month White powder 101.4% 0.09% Crystal Form A ^9th month White powder 99.6% 0.09% Not determined ^12th month White powder 98.8% 0.08% Crystal Form A ^18th month White powder 97.3% 0.10% Not determined - 2 Day 0 White powder 99.4% 0.10% Crystal Form A 25°C/60% RH, sealed sample ^3rd month White powder 100.5% 0.11% Not determined ^6th month White powder 100.1% 0.12% Crystal Form A ^9th month White powder 99.6% 0.11% Not defined ^12th month White powder 98.2% 0.09% Crystal Form A ^18th month White powder 99.1% 0.11% Not determined - 3 Day 0 White powder 99.5% 0.11% Crystal Form A 25°C/60% RH, sealed sample ^3rd month White powder 98.9% 0.16% Not determined ^6th month White powder 98.5% 0.12% Crystal Form A ^9th month White powder 99.2% 0.11% Not determined ^12th month White powder 98.4% 0.10% Crystal Form A ^18th month White powder 99.7% 0.17% Not determined

Conclusion: Crystal Form A of compound 1 showed good stability under long-term storage conditions of 25°C/60% relative humidity.

Test Example 1: Phosphodiesterase Subtype 4B Inhibitory Activity of Compound 1 (PDE4B Enzyme)

This biological experiment based on fluorescence polarization was used to determine the expression of AMP/GMP, i.e. to demonstrate enzyme activity by monitoring AMP/GMP antibody binding.

Agents:

Experimental buffer solution: 10 mM trihydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.5), 5 mM MgCl ₂ , 0.01% polyoxyethylene lauryl ether (Brij 35), 1 mM dithiothreitol (DTT) and 1% DMSO .

Enzymes: Recombinant humanized PDE4B (Genebank Accession Number NM_002600; amino acids 305 terminal) was expressed using baculovirus in Sf9 insect cells using an N-terminal GST tag. Mol.mass=78 kDa.

Enzyme substrate: 1 µM cAMP

Detection: Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 label.

Procedures:

1. Dissolution of recombinant human PDE4B enzyme and enzyme substrate (1 μM cAMP) in freshly prepared experimental buffer;

2. Transfer the resulting PDE4B enzyme buffer solution to the reaction wells;

3. Adding compound 1, dissolved with 100% DMSO, to the reaction well with PDE4B enzyme buffer solution by acoustic method (550 nanoliter echo) and incubating for 10 minutes at room temperature;

4. Then adding the enzyme substrate buffer solution to the indicated reaction wells to initiate the reaction;

5. Implementation of incubation for 1 hour at room temperature;

6. Add detection mix (Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 label) to stop the reaction and incubate for 90 minutes with slow stirring. Fluorescence polarization determination range excitation/emission=620/688.

Data Analysis: The fluorescence polarization signal was converted to nM according to the AMP/GMP standard curve and the reference % enzyme activity relative to DMSO calculated using Excel. Curve fitting was performed using the GraphPad Prism program (graphically representing medical data).

Table 6
Results of an in vitro screening test for a compound of the present disclosure Compound IC50 (nM) Compound 1 0.685

Conclusion: Compound 1 showed excellent in vitro phosphodiesterase subtype 4B (PDE4B) inhibitory activity.

Test Example 2: In Vitro Evaluation of TNFα Production Inhibition in Human Peripheral Blood Mononuclear Cells (hPBMC)

Compound 1 activity to inhibit lipopolysaccharide (LPS)-induced TNFα production in human peripheral blood mononuclear cells.

Procedures:

1. Collection of normal human whole blood that has been anticoagulated using an EDTA-K2 anticoagulant tube;

2. PBMC separation by ficoll density gradient centrifugation, counting and bringing the cell concentration to 2×10 ⁶ /ml;

3. U-bottom 96-well plate was added 2×10 ⁵ cells/well, LPS 1 ng/ml was added, various compound concentrations at 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM , 10 pM, 200 µl system/well, in duplicate wells;

4. Implementation of incubation for 24 h, collection of supernatant;

5. Detection of the TNFα level in the supernatant by ELISA, fitting of the inhibition curve and calculation of IC50 using the Graphpad Prism program.

Table 7
Inhibitory activity of a compound of the present disclosure on TNFα production in hPBMC Compound IC50(nM) Compound IC50(nM) Apremilast 16.5 (n=3) Compound 1 0.56 (n=2) n represents the number of trials

Conclusion: Compound 1 showed excellent in vitro activity of inhibiting TNFα production in hPBMC, which was higher than that of Apremilast.

Test Example 3: CIA in vivo model

Target:

The collagen-induced arthritis mouse model is an animal model used to evaluate the effectiveness of drug treatment of psoriatic arthritis, and the pathogenesis and symptoms are highly correlated with psoriatic arthritis. A number of symptoms similar to human psoriatic arthritis, such as joint redness and swelling, articular cartilage damage, and damage to the joint capsule, are induced in the model by injection of type II collagen to activate the reactivity of B cells, T cells to bone collagen, and activated B cells and T cells invade the joints, causing joint inflammation. Preclinical evaluation of candidate compounds for the treatment of psoriatic arthritis often uses collagen-induced arthritis in mice to evaluate efficacy.

The purpose of this experiment was to investigate the therapeutic effect of compound 1 on collagen-induced arthritis in mice in order to provide relevant preclinical pharmacodynamic information for subsequent clinical studies.

Procedures:

1. Immunization with type II collagen/Freund's complete adjuvant

Preparation of acetic acid: Dilution of acetic acid to 100 mM, filtration through a 0.22 μm membrane filter and storage at 4°C.

Bovine Type II Collagen Solution: Dissolving bovine type II collagen (CII) in acetic acid solution that has been stored at 4° C. overnight.

Preparation of the emulsion: Mixing the CII solution, which has been stored overnight, with an equal volume of complete Freund's adjuvant and homogenizing the mixture using a high speed homogenizer until a stable emulsion is formed from the solution.

Preparation of Lipopolysaccharide (LPS): Weighing LPS, adding normal saline and mixing until a stable solution is obtained at a concentration of 0.3 mg/kg.

2. Arthritis induction:

Mice were randomly divided into different treatment groups. The day of the first immunization was defined as day 0, and subsequent days were designated consecutively.

After DBA/1 mice were anesthetized with isoflurane, the resulting collagen emulsion was injected subcutaneously into the tail.

On day 23, 100 μl of LPS solution was administered intraperitoneally.

Mice in the normal group were not immunized.

3. Administration and Dosing Schedule

On day 27, when the mean clinical score was about 1, 60 mild-onset mice were selected and again randomized according to body weight and score, 10 mice per group.

The CIA model typically uses dexamethasone as a positive control drug in the 0.3 mg/kg dose group. In addition, the appropriate dosing regimen for compound 1 and the control compound Apremilast was determined according to the results of preliminary experiments. Group 1 included normal mice without any treatment; Group 2 was the vehicle control group; Group 3 was the 0.3 mg/kg dexamethasone administration group; Group 4 was the 5 mg/kg Apremilast administration group; Group 5, Group 6 and Group 7 were Compound 1 administration groups at doses of 0.3, 1 and 3 mg/kg, respectively. The administration was carried out once a day for a total of 11 days. The volume of intragastric administration was 10 ml/kg.

Table 8
Groups and dosing regimen Group Tested medicinal product Quantity Route of administration Concentration Dose Administration frequency mg/ml mg/kg one normal group 5 N/A. N/A N/A N/A 2 Carrier ten Through a stomach tube N/A N/A 1/day, 11 days 3 Dexamethasone ten Through a stomach tube 0.03 0.3 1/day, 11 days four Apremilast ten Through a stomach tube 0.5 5 1/day, 11 days 5 Compound 1 ten Through a stomach tube 0.03 0.3 1/day, 11 days 6 Compound 1 ten Through a stomach tube 0.1 one 1/day, 11 days 7 Compound 1 ten Through a stomach tube 0.3 3 1/day, 11 days

4. Determination of the incidence rate of arthritis

Clinical observation: From 7 days before immunization to 23 days after immunization, the main health status and changes in body weight of DBA/1 mice were observed daily (recorded once a week). After the 23rd day, the health status, morbidity and change in body weight of mice were observed daily (recorded at least three times a week) until the end of the experiment.

Clinical assessment: after LPS injection, the incidence of mice was observed every day. After the onset of disease in mice (clinical symptoms of arthritis), they were scored according to the severity of the condition (redness and swelling, joint deformity) according to a 0-4 point standard, with a maximum score of 4 for each limb and a maximum score of 16 for each animal. The evaluation standard is shown in Table 9. The evaluation was carried out at least three times a week.

Table 9
Arthritis Clinical Assessment Standard Grade Clinical symptoms 0 No erythema, redness and swelling one Erythema or slight redness and swelling near the tarsal or ankle or metatarsal, 1 finger with redness and swelling 2 Slight erythema and swelling of the ankle and metatarsal, or more than two fingers with redness and swelling 3 Moderate erythema and swelling of the ankle, carpal joint, and metatarsal four Severe redness and swelling of all ankle joints, carpal joints, metatarsus and fingers

Pathology: On day 38, the mouse was sacrificed. Two mouse hind limbs were taken, soaked in 10% formalin solution, decalcified with formic acid solution, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE) and observed by microphotographic method. The degree of joint damage was assessed by four parameters: inflammatory cell infiltration, pannus formation, cartilage damage, and bone resorption, and was assessed by a 0-4 point standard. The assessment standard is presented in table 10:

Table 10
Arthritis Pathology Assessment Standard Defeat Characteristics of the lesion Grade Infiltration by inflammatory cells No visible inflammatory cells 0 Fibrosis of cells under the synovium, with minimal cellular infiltration one Synovial cell hyperplasia, with little monocyte infiltration 2 Hyperplasia of synovial cells, with massive infiltration of monocytes, plasma cells, lymphocytes 3 Infiltration of large numbers of inflammatory cells around the joint, tissue fibrosis, synovial thickening four Pannus formation No visible pannus formation 0 Minimal pannus formation at the cartilage margin one Hyperplasia of fibrous tissue between cartilages, with slight pannus formation at the joint margin 2 Pannus formation on 50% of the articular cartilage surface 3 Pannus formation over the entire surface of the articular cartilage four cartilage damage No visible cartilage damage 0 Hyperplasia of articular chondrocytes one Loss of chondrocyte matrix, with few destroyed chondrocytes 2 Hyperplasia of fibrous tissue around the joints, with a large number of destroyed chondrocytes 3 Large hyperplasia of fibrous tissue between articular cartilage, with cartilage erosion four Bone resorption No visible bone resorption 0 Minimal bone resorption at the edge of the synovial membrane one Formation of a small number of osteoclasts for a small amount of bone tissue 2 Bone tissue under focal articular cartilage, with bone resorption 3 Bone resorption for a wide range of bone tissues, with cartilage erosion four

5. Statistical processing

Experimental data were expressed as mean ± standard error (Mean ± SEM), body weight and clinical score were analyzed using two-way analysis of variance, pathological score and AUC were analyzed using t-test, and a p value <0.05 was considered significant.

Experimental results:

1. Clinical assessment:

On day 25 after the first immunization (Day 2 after the second immunization), the mice began to develop clinical symptoms of arthritis. Administration started on day 27. The median clinical score of the vehicle control group gradually increased and reached 8.3 points on day 36, indicating a successful establishment of the collagen-induced arthritis model.

Compared to the vehicle control group, Compound 1 at 0.3, 1, and 3 mg/kg could significantly reduce the clinical score of arthritis in mice at the end point of the experiment (Day 37), and the clinical mean scores at three doses dropped to 3.6 ( p <0.0001), 4.3 ( p <0.001) and 3.5 ( p <0.0001). Therefore, Compound 1 can effectively reduce collagen-induced arthritis at a dose as low as 0.3 mg/kg. In the 0.3 mg/kg dexamethasone administration group, the clinical assessment of collagen-induced arthritis could be significantly suppressed. From day 30, the clinical score was maintained at 0, which was significantly different from the vehicle control group ( p < 0.0001), and remained at this level until the end of the experiment. In the Apremilast 5 mg/kg administration group, the increase in clinical score was also suppressed and showed a significant difference from the vehicle control group from day 33 and continued until the end of the experiment. Until day 37, the mean clinical symptom score was 4.2, which was 3.7 ( p < 0.001) lower than the vehicle control group.

By analyzing the clinical score curve of each animal in each group, the area under the curve (AUC) was calculated. Using the mean area under the curve between groups, the percent inhibition in each dose group relative to the vehicle control group was calculated. Compared with the vehicle control group, the dexamethasone group and the Apremilast group significantly reduced the clinical scores of arthritis in animals, and the inhibition percentages were 96.4% ( p <0.0001) and 41.3% ( p <0.05 ), respectively. Compound 1 at three doses of 0.3, 1 and 3 mg/kg can significantly reduce the area under the curve for the clinical assessment of arthritis in animals, and the inhibition percentages were 43.9% ( p <0.05), 39.4% ( p < 0.05) and 51.7% ( p <0.01), respectively. The 1 mg/kg Compound 1 group had a percentage of inhibition comparable to the 5 mg/kg Apremilast group ( p <0.05 for both groups), while the 3 mg/kg Compound 1 group had better inhibition percentages, than the Apremilast 5 mg/kg group ( p values were <0.01 and <0.05, respectively)

2. Histopathological evaluation

Two hind limbs from each group of mice were sectioned for HE staining and an overall score for both hind limbs was obtained. Arthritis in mice in the vehicle control group had an overall pathological score of 20.20±1.15. Compared to the vehicle control group, the control compound Apremilast at 5 mg/kg also significantly reduced the arthritis pathological score in mice, which could be reduced to 13.90±1.89 ( p <0.05). Compound 1 at 1 and 3 mg/kg could significantly reduce the pathological scores of arthritis in mice, which could decrease to 14.00±2.43 ( p <0.05) and 9.20±1.83 ( p <0. 0001), respectively. The Compound 1 at 1 mg/kg group had a pathological arthritis score comparable to the Apremilast 5 mg/kg group (p<0.05 for both groups), while the Compound 1 at 3 mg/kg group had a better pathological score arthritis than in the Apremilast 5 mg/kg group ( p values <0.0001 and <0.05, respectively).

3. Conclusion

Compound 1 in three dose groups of 0.3, 1 and 3 mg/kg significantly improved the symptoms of collagen-induced arthritis.

There was a significant improvement in the pathology of arthritis in the 1 mg/kg and 3 mg/kg dose groups, and these three dose groups showed a clear dose-response relationship in the pathological assessment of arthritis. The therapeutic effect of compound 1 at 3 mg/kg (clinical assessment and pathological assessment of arthritis) was better than that of Apremilast at 5 mg/kg.

Claims (20)

1. Crystal Form A of compound 1, where crystalline Form A is characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 10.69±0.2°, 12.31±0.2°, 13.45±0.2°, 14.10±0, 2°, 14.62±0.2°, 19.07±0.2°, 20.33±0.2° 21.79±0.2°

Connection 1. 2. Crystalline Form A of compound 1 according to claim 1, where crystalline Form A is characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.25±0.2°, 8.93±0.2°, 10.69±0.2°, 12.31±0, 2°, 13.45±0.2°, 14.10±0.2°, 14.62±0.2°, 18.16±0.2°, 19.07±0.2°, 20, 33±0.2°, 21.79±0.2°. 3. Crystal Form A of compound 1 according to claim 2, where crystalline Form A is characterized by the X-ray powder diffraction pattern shown in FIG. one. 4. Crystalline Form A of compound 1 according to any one of paragraphs. 1-3, where crystalline Form A is characterized by a differential scanning calorimetry curve having an endothermic peak start point at 201.70°C±2°C. 5. Crystalline Form A of compound 1 according to claim 4, wherein the crystalline Form A is characterized by the DSC curve shown in FIG. 2. 6. Crystal Form A of compound 1 according to any one of paragraphs. 1-3, where the crystalline Form A is characterized by a thermogravimetric analysis curve, where the weight loss at 100.00±2°C is 0.02039%. 7. Crystalline Form A of compound 1 according to claim 6, wherein the crystalline Form A is characterized by the TGA curve shown in FIG. 3. 8. A method for obtaining crystalline Form A of compound 1 according to claim 1, including adding compound 1 to an alcohol solvent, a ketone solvent, a mixed solvent consisting of an alcohol solvent and water, a mixed solvent consisting of a ketone solvent and water; the alcohol solvent is selected from the group consisting of methanol, ethanol; the ketone solvent is selected from the group consisting of acetone and heating to dissolve and then cooling to crystallize to obtain crystalline Form A. 9. A method for obtaining crystalline Form A of compound 1 according to claim 8, where the mixed solvent consisting of an alcoholic solvent and water is selected from the group consisting of a mixed solvent consisting of ethanol and water. 10. A method for obtaining crystalline Form A of compound 1 according to claim 9, where in a mixed solvent consisting of an alcoholic solvent and water, the volume ratio of the alcoholic solvent to water is selected from the group consisting of 1:0.2-1.5.

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