Crystal of boric acid ester derivative, and preparation method and application thereof

Abstract

The invention relates to a crystal of a borate derivative, and a preparation method and application thereof. Specifically, the invention provides a crystal of a compound as shown in a formula I, and a preparation method and application thereof. # imgabs0 #

Description

A crystal of borate derivative, preparation method and use thereof Technical Field

[0001] The present disclosure belongs to the field of pharmacy and relates to a crystal of a borate derivative, a preparation method and use thereof. Background Art

[0002] Phosphodiesterases (PDEs) are a class of intracellular enzymes that cleave the phosphodiester bonds of the second messenger molecules 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). The cyclic nucleotides cAMP and cGMP act as second messengers in various cellular pathways. Among them, PDE4 is highly specific for cAMP and has four isoforms: PDE4A, PDE4B, PDE4C, and PDE4D. PDE4 is involved in physiological and pathological processes such as monocyte and macrophage activation, neutrophil infiltration, vascular smooth muscle proliferation, vasodilation, and myocardial contraction, affecting central nervous system function, cardiovascular function, inflammation / immune system, and cell adhesion. PDE4 plays a major role in regulating the expression of pro- and anti-inflammatory mediators. PDE4 inhibitors can inhibit the release of harmful mediators from inflammatory cells.

[0003] Many PDE4 inhibitors have been discovered in recent years. For example, roflumilast is approved for the treatment of severe chronic obstructive pulmonary disease (COPD) to reduce the number of flare-ups or prevent worsening of COPD symptoms; apremilast is approved for the treatment of adults with active psoriatic arthritis. While PDE4 inhibitors exhibit promising pharmacological activity, they can also cause side effects, such as induced gastrointestinal symptoms like vomiting and diarrhea. There remains a need for the development of selective PDE4 inhibitors, particularly those with affinity for both PDE4B and PDE4D.

[0004] The boron (B)-containing drug crisaborole was approved by the FDA on December 14, 2016, as a topical treatment for mild to moderate atopic dermatitis. The boron atom facilitates skin penetration and binds to the bimetallic center of phosphodiesterase 4 (PDE4). Other boron-containing small molecules that inhibit PDEs have been reported, such as CN102014927A and WO2020070651. However, the compounds disclosed herein have not been previously reported in any literature, and these compounds exhibit specific PDE4 inhibition.

[0005] PCT / CN2021 / 141010 provides a new type of PDE4 inhibitor, whose chemical name is (R)-4-(6-(8-methoxy-2,2-dimethylchroman-5-yl)pyrazin-2-yl)-1,2-oxaborolan-2-ol, and has the structure shown in Formula I.

[0006] The crystal form of a pharmaceutically active ingredient often affects the chemical stability of the drug. Differences in crystallization and storage conditions can lead to variations in the compound's crystal structure, sometimes resulting in the formation of alternative morphologies. Generally speaking, amorphous drug products lack a regular crystal structure and often exhibit other drawbacks, such as poor product stability, fine crystallization, difficulty in filtration, susceptibility to agglomeration, and poor flowability. Drug polymorphs impose varying requirements on product storage, production, and scale-up. Therefore, in-depth research into the crystal forms of these compounds is essential to improve their various properties.

[0007] Summary of the Invention

[0008] The present disclosure provides a crystalline form A of a compound represented by formula I, which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 7.537°, 15.219°, 21.798°, and 22.911°.

[0009] In some embodiments, the crystalline form A of the compound represented by formula I has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and has characteristic peaks at 7.537°, 11.366°, 14.297°, 15.219°, 16.190°, 17.623°, 19.134°, 21.798°, 22.911°, and 24.735°.

[0010] In some embodiments, the crystalline form A of the compound represented by the formula I has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and has characteristic peaks at 7.537°, 11.366°, 14.018°, 14.297°, 15.219°, 16.190°, 17.623°, 19.134°, 21.062°, 21.798°, 22.911°, 24.735°, 25.123°, and 26.884°.

[0011] In some embodiments, the crystalline form A of the compound represented by the formula I has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and has characteristic peaks at 7.537°, 11.366°, 14.018°, 14.297°, 15.219°, 16.190°, 17.623°, 18.412°, 19.134°, 20.010°, 21.062°, 21.798°, 22.911°, 23.889°, 24.735°, 25.123°, 26.884°, 28.343°, 29.645°, 33.499°, and 36.362°.

[0012] In some embodiments, the X-ray powder diffraction pattern of Form A of the compound represented by Formula I, expressed in terms of a diffraction angle 2θ, is shown in FIG7 .

[0013] In some embodiments, the crystalline form A of the compound represented by Formula I described in the present disclosure, wherein the error range of the 2θ angle value is ±0.2°.

[0014] The present disclosure also provides a method for preparing the crystalline form A of the compound represented by formula I as described above, which is method 1 or method 2:

[0015] Method 1 includes the following steps:

[0016] (a1) mixing the compound represented by formula I with a solvent (1);

[0017] (b1) stirring crystallization;

[0018] wherein the solvent (1) is selected from one or more of water, nitromethane, N,N-dimethylformamide (DMF), acetonitrile, methanol, ethanol, acetone, isopropyl alcohol (IPA), methyl ethyl ketone (MEK), tetrahydrofuran (THF), 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether (MTBE), 1,2-xylene, toluene, dioxane and hexane;

[0019] Method 2 includes the following steps:

[0020] (a2) mixing the compound represented by formula I with a solvent (2);

[0021] (b2) Volatilization and crystallization;

[0022] Wherein, the solvent (2) is selected from one or more of acetone, acetonitrile, dichloromethane (DCM), dioxane, ethanol, ethyl acetate, heptane, isopropanol, methanol, methyl ethyl ketone, methyl tert-butyl ether, toluene and water.

[0023] In some embodiments, the solvent (1) is water, nitromethane, N,N-dimethylformamide, acetonitrile, ethanol, acetone, isopropanol, methyl ethyl ketone, tetrahydrofuran, 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether, 1,2-xylene, toluene, dioxane, hexane, or a mixed solvent of water and methanol.

[0024] In some embodiments, when the solvent (1) is a mixed solvent of water and methanol, the molar ratio of the water to the methanol is 0.37-0.90, and in non-limiting embodiments can be 0.37, 0.40, 0.47, 0.50, 0.57, 0.60, 0.66, 0.70, 0.74, 0.80, 0.82, or 0.90.

[0025] In some embodiments, the temperature during the stirring crystallization is 10-60°C, and in non-limiting embodiments can be 10°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0026] In some embodiments, the temperature during the stirring crystallization is room temperature.

[0027] In some embodiments, when the temperature during the stirring crystallization is 10-40°C (e.g., room temperature), the solvent (1) is water, nitromethane, acetonitrile, ethanol, acetone, isopropanol, methyl ethyl ketone, tetrahydrofuran, 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether, 1,2-xylene, toluene, dioxane, hexane, or a mixed solvent of water and methanol.

[0028] In some embodiments, when the temperature during the stirring crystallization is 41-60°C (e.g., 50°C), the solvent (1) is water, N,N-dimethylformamide, acetonitrile, acetone, methyl ethyl ketone, 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether, 1,2-xylene, toluene, hexane, or a mixed solvent of water and methanol.

[0029] In some embodiments, the stirred crystallization is slurry crystallization.

[0030] In some embodiments, the solvent (2) is acetone, acetonitrile, dichloromethane, dioxane, ethyl acetate, heptane, isopropanol, methyl ethyl ketone, methyl tert-butyl ether, toluene, water, or any of the following mixed solvents: acetone and acetonitrile, acetone and dichloromethane, acetone and dioxane, acetone and ethanol, acetone and ethyl acetate, acetone and heptane, acetone and isopropanol, acetone and methanol, acetone and methyl ethyl ketone, acetone and methyl tert-butyl ether Butyl ether, acetone and tetrahydrofuran, acetone and toluene, acetone and water, acetonitrile and dichloromethane, acetonitrile and dioxane, acetonitrile and ethanol, acetonitrile and ethyl acetate, acetonitrile and isopropanol, acetonitrile and methyl ethyl ketone, acetonitrile and methyl tert-butyl ether, acetonitrile and tetrahydrofuran, acetone and toluene, dichloromethane and dioxane, dichloromethane and ethanol, dichloromethane and ethyl acetate, dichloromethane and heptane, dichloromethane and isopropanol, dichloromethane and Methyl ethyl ketone, dichloromethane and methyl tert-butyl ether, dichloromethane and tetrahydrofuran, dichloromethane and toluene, dioxane and methyl ethyl ketone, dioxane and tetrahydrofuran, dioxane and toluene, ethanol and ethyl acetate, ethanol and methanol, ethanol and tetrahydrofuran, ethanol and toluene, ethanol and water, ethyl acetate and heptane, ethyl acetate and isopropanol, ethyl acetate and methanol, ethyl acetate and methyl ethyl ketone, ethyl acetate and methyl tert-butyl ether, ethyl acetate and toluene, heptane and methyl ethyl ketone, heptane and methyl tert-butyl ether, heptane and tetrahydrofuran, heptane and toluene, methanol and methyl ethyl ketone, methanol and methyl tert-butyl ether, methanol and toluene, methanol and water, methyl ethyl ketone and methyl tert-butyl ether, methyl ethyl ketone and tetrahydrofuran, methyl ethyl ketone and toluene, methyl tert-butyl ether and tetrahydrofuran, methyl tert-butyl ether and toluene, tetrahydrofuran and toluene, tetrahydrofuran and water.

[0031] In some embodiments, when the solvent (2) is a mixed solvent, the volume ratio of the two is 1:3-3:1, preferably 1:1.

[0032] In some embodiments, the temperature during the volatilization and crystallization is 10-40°C, and in non-limiting embodiments can be 10°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0033] In some embodiments, the temperature during the volatilization and crystallization is room temperature.

[0034] In some embodiments, the volume (μl) of the solvents (1) and (2) can be 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 or any value between two numbers.

[0035] In certain embodiments, the stirring crystallization or volatilization crystallization further comprises a filtration, washing or drying step.

[0036] The present disclosure also provides a pharmaceutical composition, which comprises the above-mentioned crystal form A or the crystal form A prepared by the above-mentioned preparation method, and optionally a pharmaceutically acceptable excipient.

[0037] The present disclosure also provides a method for preparing the above-mentioned pharmaceutical composition, which comprises the step of mixing the above-mentioned crystalline form A or the crystalline form A prepared by the above-mentioned method with a pharmaceutically acceptable excipient.

[0038] The present disclosure also provides a use of the above-mentioned Form A, the Form A prepared by the above-mentioned preparation method, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with PDE. In some embodiments, the disease associated with PDE is asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

[0039] The present disclosure also provides a use of the above-mentioned crystalline form A or the crystalline form A prepared by the above-mentioned preparation method or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative enteritis or rheumatism.

[0040] The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.2 (including the case where the numbers exceeding 1 decimal place are rounded off), which can be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0041] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "9103 Guiding Principles for Hygroscopicity of Drugs" in Part IV of the 2015 edition of the Chinese Pharmacopoeia,

[0042] Deliquescent: Absorbs sufficient water to form a liquid;

[0043] Highly hygroscopic: weight gain due to moisture absorption is not less than 15%;

[0044] Hygroscopic: weight gain due to moisture absorption is less than 15% but not less than 2%;

[0045] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0046] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0047] The "differential scanning calorimetry or DSC" described in this disclosure refers to measuring the temperature difference and heat flow difference between a sample and a reference object during the process of heating or maintaining the sample at a constant temperature to characterize all physical and chemical changes related to thermal effects and obtain phase change information of the sample.

[0048] The drying temperature in the present disclosure is generally 25°C to 100°C, preferably 40°C to 70°C. Drying can be performed at normal pressure or reduced pressure, with a pressure of less than -0.08 MPa.

[0049] The "room temperature" described in this disclosure is 25°C±5°C.

[0050] The "excipients" described in this disclosure include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent or emulsifier that has been approved by the U.S. Food and Drug Administration for use by humans or livestock animals.

[0051] The "beating" mentioned in the present disclosure refers to a purification method that utilizes the property that a substance has poor solubility in a solvent but impurities have good solubility in a solvent. Beating purification can remove color, change the crystal form or remove a small amount of impurities.

[0052] The starting material used in the crystal form preparation method of the present invention can be any form of compound, including but not limited to: amorphous, any crystal form, hydrate, solvate, etc.

[0053] Numerical values ​​in this disclosure, such as data related to the determination and calculation of substance content, inevitably have a certain degree of error. Generally speaking, ±10% is within a reasonable error range. While there may be some degree of error depending on the context in which it is used, this error range does not exceed ±10% and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0054] The compound of formula I disclosed herein, (R)-4-(6-(8-methoxy-2,2-dimethylchroman-5-yl)pyrazin-2-yl)-1,2-oxaborolan-2-ol (amorphous form, hereinafter referred to as compound I), was prepared by referring to the method in PCT / CN2021 / 141010, and this text quotes the relevant content for illustration. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1: XRPD spectrum of amorphous form of Compound 1.

[0056] Figure 2: Comparison of clinical scores of each group of compounds in the disease model in Test Example 4.

[0057] Figure 3: Comparison of clinical scores of each group of compounds in Test Example 4 in the erythema disease model.

[0058] Figure 4: Comparison of clinical scores of each group of compounds in Test Example 4 in the psoriasis model.

[0059] Figure 5: Comparison of the inhibitory effects of various groups of compounds in Test Example 4 on skin thickness increase.

[0060] Figure 6: Comparison of the effects of each group of compounds on the proportion of spleen body weight in Test Example 4.

[0061] Figure 7: XRPD spectrum of Form A.

[0062] Figure 8: DSC spectrum of Form A.

[0063] Figure 9: TGA spectrum of Form A.

[0064] Figure 10: DVS spectrum of Form A.

[0065] In Figures 1-6, * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, *** indicates p < 0.001 compared with the model group, and the symbols ▼, ●, ◆, and # are only used to distinguish groups and have no practical meaning. DETAILED DESCRIPTION

[0066] The present disclosure will be described in more detail below with reference to examples. The examples or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure, and are not intended to limit the essence and scope of the present disclosure.

[0067] The abbreviations used in this disclosure are explained as follows:

[0068] XRPD X-ray powder diffraction

[0069] DSC Differential Scanning Calorimetry

[0070] TGA Thermogravimetric Analysis

[0071] DVS Dynamic Water Sorption

[0072] 1 H-NMR liquid state nuclear magnetic hydrogen spectrum

[0073] DMF N,N-dimethylformamide

[0074] MEK Methyl Ethyl Ketone

[0075] MTBE methyl tert-butyl ether

[0076] THF Tetrahydrofuran

[0077] IPA Isopropyl alcohol

[0078] DCM dichloromethane

[0079] ACN acetonitrile

[0080] Test conditions of the instruments used in the experiments in this disclosure:

[0081] 1. X-ray Powder Diffraction (XRPD)

[0082] Instrument model: Malver Panalytical Aeris X-ray powder diffractometer

[0083] Radiation: Monochromatic Cu-Kα radiation (λ=1.54188)

[0084] Scanning mode: θ / 2θ, scanning range (2θ range): 3.5~50°

[0085] Voltage: 40kV, Current: 15mA.

[0086] 2. Differential Scanning Calorimeter (DSC)

[0087] Instrument model: TA DSC250

[0088] Purge gas: nitrogen; nitrogen purge rate: 50mL / min

[0089] Heating rate: 10℃ / min

[0090] Temperature range: 25℃-300℃.

[0091] 3. Thermogravimetric Analysis (TGA)

[0092] Instrument model: TA TGA550

[0093] Purge gas: nitrogen; nitrogen purge speed: 20ml / min

[0094] Heating rate: 10℃ / min

[0095] Temperature range: 30℃-350℃.

[0096] 4. Dynamic moisture sorption (DVS)

[0097] The test was performed using SMS Intrinsic PLUS at 25°C, with humidity ranging from 50% to 0% to 90% in 10% increments. The judgment standard was that the mass change dM / dT for each gradient was less than 0.0002%, TMAX was 360 min, and the cycle was repeated twice.

[0098] 5. The high performance liquid chromatography (HPLC) described in the stability test of Form A of the present invention was performed on an Agilent 1260 Infinity II. HPLC conditions were as follows: column: Agilen XDB C18 5 μm 4.6×250 mm; mobile phase: A-0.05% TFA (in water), B-ACN; flow rate: 1.0 ml / min; wavelength: 220 nm.

[0099] 6. The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shift (δ) was measured in 10-6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (Methanol-d4). The internal standard was tetramethylsilane (TMS).

[0100] 7. HPLC determination in the preparation examples of Compound I was performed using an Agilent 1100 high pressure liquid chromatograph, a GAS15B DAD UV detector, and a Water Vbridge C18 150×4.6 mm 5 μM column.

[0101] 8. MS determination was performed using an Agilent 6120 triple quadrupole mass spectrometer, a G1315D DAD detector, and a Waters Xbridge C18 4.6×50 mm, 5 μM column. The mass spectra were scanned in positive / negative ion mode with a mass scan range of 80–1200.

[0102] 9. Preparative HPLC conditions: water; column: Sunfire (Prep C18 OBD 19×250 mm 10 μm).

[0103] 10. Chiral column separation conditions: Column: Chiralpak IG 5 μm 30×250 mm; Mobile phase: Hex:EtOH=35:65, 15 mL / min; Temperature: 30°C; Wavelength: 254 nm.

[0104] 11. The thin layer chromatography silica gel plate used is Yantai Huanghai HSGF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.2mm±0.03mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm.

[0105] 12. The flash column purification system used was Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

[0106] 13. Forward column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh or 300-400 mesh silica gel as the carrier, or uses Changzhou Santai pre-filled ultra-pure normal phase silica gel column (40-63μm, 60g, 24g, 40g, 120g or other specifications).

[0107] The known starting materials in the present disclosure can be synthesized by methods known in the art, or can be purchased from companies such as Shanghai Titan Technology, ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Bid Pharmaceuticals.

[0108] Unless otherwise specified in the examples, all reactions were carried out under a nitrogen atmosphere.

[0109] Nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a capacity of about 1L.

[0110] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0111] Hydrogen was produced by a QPH-1L hydrogen generator from Shanghai Quanpu Scientific Instrument Co., Ltd.

[0112] The nitrogen atmosphere or hydrogen atmosphere is usually evacuated and filled with nitrogen or hydrogen, and the operation is repeated three times.

[0113] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0114] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0115] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the column chromatography eluent system used to purify the compound, and the developing solvent system for thin layer chromatography, the volume ratio of the solvent were adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could be added for adjustment.

[0116] Example 1: Preparation of Compound I

[0117] Step 1): Compound 1a (2.0 g, 14.6 mmol) and triethylamine (1.8 g, 17.8 mmol) were dissolved in N,N-dimethylformamide (30 mL), and the solution was cooled to 0°C. Under a nitrogen atmosphere, tert-butyldiphenylsilyl chloride (4.0 g, 14.6 mmol) was added dropwise to the reaction system. The mixture was warmed to room temperature and stirred until the reaction was complete as detected by TLC. The reaction solution was poured into water and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with water (50 mL × 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain compound 1b (5.1 g), which was used directly in the next reaction.

[0118] Step 2): Under a nitrogen atmosphere, a mixture of compound 1b (5.1 g, 13.6 mmol), bipyralidoborane (4.2 g, 16.3 mmol), [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (512 mg, 0.7 mmol), potassium acetate (2.0 g, 20.4 mmol) and dioxane (100 mL) was heated to 80 ° C and stirred overnight. The reaction solution was poured into water and extracted with ethyl acetate (100 mL × 2). Washed with water (30 mL × 2), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain compound 1c (2.0 g), LCMS: m / z 423.2 (M+H) + .

[0119] Step 3): At room temperature, compound a (100 g, 492 mmol), DMF (1000 mL), potassium iodide (92.6 g, 837 mmol), cuprous iodide (3.13 g, 9.85 mmol), and potassium carbonate (136 g, 985 mmol) were added sequentially to a 2000 mL single-necked flask. Under a nitrogen atmosphere, 3-chloro-3-methyl-1-butyne (100 mL, 886 mmol) was added dropwise. The mixture was stirred at 70°C for 16 hours. After cooling to room temperature, water (1000 mL) was added and the mixture was extracted with petroleum ether (1000 mL x 3). The mixture was dried over anhydrous sodium sulfate and filtered. The residue was concentrated under reduced pressure and purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound b (50 g).

[0120] 1 H NMR (400MHz, CDCl3) δ7.57 (d, J = 2.4Hz, 1H), 7.15 (dd, J = 8.8, 2.4Hz, 1H), 6.76 (d, J = 8.8Hz, 1H), 3.79 (s, 3H), 2.58 (s, 1H), 1.65 (s, 6H).

[0121] Step 4): To a 500 mL single-necked flask at room temperature, compound b (20.0 g, 74.4 mmol), n-hexane (200 mL), and palladium calcium carbonate (1.95 g, 18.8 mmol) were added sequentially. Stir at room temperature under a hydrogen atmosphere for 16 hours. Filter and concentrate under reduced pressure to obtain compound c (19 g).

[0122] 1H NMR (400MHz, CDCl3) δ7.15 (d, J=2.4Hz, 1H), 7.09 (dd, J=8.8, 2.4Hz, 1H), 6.73 (d, J=8.8Hz, 1H), 6.12 (dd, J=17.6, 10.8Hz, 1H), 5.14 (dd, J=20.0, 9.2Hz, 2H), 3.79 (s, 3H), 1.46 (s, 6H).

[0123] Step 5): At room temperature, compound c (10.0 g, 36.9 mmol) and diethylaniline (10 mL, 62.5 mmol) were added sequentially to a 50 mL single-necked flask. Stir at 210°C for 1 hour. Cool to room temperature, adjust the pH to neutral with 1 M HCl solution, extract with ethyl acetate (200 mL x 3), and wash with water (200 mL x 3). Concentrate under reduced pressure to obtain compound d (9.1 g).

[0124] 1 H NMR (400MHz, DMSO-d6) δ8.97(s,1H),6.97(d,J=8.8Hz,1H),6.77(d,J=8.8Hz,1H) ,5.14-5.01(m,1H),3.78(s,3H),3.40(d,J=6.8Hz,2H),1.74(s,3H),1.63(s,3H).

[0125] Step 6): At room temperature, compound d (5.00 g, 18.5 mmol), toluene (25 mL), 15 (5.00 g, 15.9 mmol). Stir at 100°C for 2 hours under nitrogen atmosphere, cool to room temperature, filter, and concentrate under reduced pressure to obtain compound e (3.89 g).

[0126] 1 H NMR (400MHz, DMSO-d6) δ7.04 (d, J = 8.8 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 3.71 (s, 3H), 2.63 (t, J = 6.8 Hz, 2H), 1.78 (t, J = 6.8 Hz, 2H), 1.26 (s, 6H).

[0127] Step 7): To a 500 mL single-necked flask at room temperature, compound e (28.4 g, 105 mmol), dioxane (300 mL), pinacol diboron (31.9 g, 126 mmol), potassium acetate (20.6 g, 209 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (7.66 g, 10.5 mmol) were added sequentially. Under a nitrogen atmosphere, the mixture was stirred at 105°C for 16 hours. The mixture was cooled to room temperature and filtered. The residue was concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate) to afford compound f (28.4 g).

[0128] LCMS: m / z 319 (M+H) + .

[0129] Step 8): To a 250 mL single-necked flask at room temperature, compound f (6.20 g, 19.5 mmol), 1,4-dioxane (80 mL), water (16 mL), 2,6-dichloropyrazine (2.90 g, 39.0 mmol), potassium carbonate (5.39 g, 39.0 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (1.43 g, 1.95 mmol) were added sequentially. Under a nitrogen atmosphere, the mixture was stirred at 110°C for 16 hours. The mixture was cooled to room temperature and filtered. The residue was concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate) to afford compound g (5.7 g).

[0130] LCMS: m / z 305 (M+H) + .

[0131] Step 9): To a 100 mL single-necked flask at room temperature, compound g (35.0 g, 115 mmol), compound 1c (44.5 g, 149 mmol), potassium carbonate (23.8 g, 172 mmol), potassium acetate (16.9 g, 172 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (8.41 g, 11.5 mmol) were added in a mixture of 1,4-dioxane (250 mL) and water (5 mL). Stir until dissolved, replace the atmosphere with nitrogen three times, and stir at 110°C for 16 hours. Cool to room temperature, add water (500 mL), extract with ethyl acetate (300 mL x 3), and dry over anhydrous sodium sulfate. Filter and concentrate under reduced pressure. The residue is purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound h (37 g).

[0132] LCMS: m / z 441.1 (M+H) + .

[0133] Step 10): At room temperature, add 1,2-bis(diphenylphosphino)ethane (1.81 g, 4.54 mmol), (1,5-cyclooctadiene)methoxyiridium(I) dimer (1.50 g, 2.27 mmol), and anhydrous 1,2-dichloroethane (100 mL) to a 250 mL three-necked flask. Stir at room temperature for 10 minutes, then add compound h (10.0 g, 22.7 mmol). Heat to 70°C, and add pinacol borane (20.3 g, 159 mmol) dropwise. React at 95°C for 4 hours. Cool to 0°C, and quench with methanol (50 mL). Concentrate, and purify the residue by column chromatography (petroleum ether / ethyl acetate) to obtain compound i (1.4 g).

[0134] LCMS: m / z 569.3 (M+H) + .

[0135] Step 11): In a 50 ml single-necked flask, add compound i (9.00 g, 15.8 mmol) and tetrahydrofuran (30 mL) at room temperature. Cool to 0°C, and add 2 M hydrochloric acid (50 mL). Stir at 50°C for 16 hours. Concentrate under reduced pressure, extract with ethyl acetate (300 mL x 3), dry over anhydrous sodium sulfate, and filter. Concentrate under reduced pressure, and purify the residue by reverse-phase column chromatography (acetonitrile / water / trifluoroacetic acid) to obtain compound IA (3.82 g).

[0136] Chiral separation (column: Chiralpak IG 5 μm 30*250 mm; mobile phase: Hex:EtOH=35:65; flow rate: 15 mL / min; temperature: 30° C.; wavelength: 254 nm) was used to obtain compound I (with a shorter retention time).

[0137] Compound I

[0138] Compound I was analyzed by XRPD, and the results showed that the product was amorphous, and its XRPD spectrum is shown in Figure 1.

[0139] LCMS: m / z 355.0 (M+H) + .

[0140] 1H NMR (400MHz, DMSO-d6) δ8.62(s,1H),8.48(s,1H),6.98(d,J=8.4Hz,1H),6.90(d,J=8.4Hz,1H),4.29(dd,J=8.8,7.6Hz,1H),3.96(dd ,J=8.8,6.8Hz,1H),3.77(s,3H),3.74-3.62(m,1H),2.93-2.67(m,2H),1.68(t,J=6.8Hz,2H),1.38-1.24(m,7H),1.21-1.08(m,1H).

[0141] Example 2: Preparation of Form A of Compound I

[0142] 10 mg of Compound I was added to 0.2 ml of solvent, as shown in Table 1 below. The compound was kept slurried in the solvent and stirred at room temperature (25°C) or 50°C for 7 days. The solvent was quickly removed using filter paper. The resulting solid was detected as Form A by XRPD.

[0143] Table 1. Solvents selected for the preparation of Form A by stirring crystallization and XRPD test results

[0144] Note: N / A means not tested

[0145] Example 3: Preparation of Form A of Compound I

[0146] 5 mg of Compound I was added to a solvent as shown in Table 2. The mixture was evaporated at room temperature and placed in a vacuum drying oven at 50°C for 7 days to remove the solvent. The resulting solid was identified as Form A by XRPD.

[0147] Table 2. Solvents selected for preparing Form A by volatile crystallization and XRPD test results

[0148] The solids obtained in Examples 3 and 4 were analyzed by X-ray powder diffraction, and the product was defined as Form A. The XRPD spectrum is shown in Figure 7 , and the positions of its characteristic peaks are shown in Table 3. The DSC spectrum is shown in Figure 8 , showing an endothermic peak at 140.43°C. The TGA spectrum is shown in Figure 9 , showing a weight loss of 1.53% from 25°C to 100°C.

[0149] DVS analysis, as shown in Figure 10, shows that under normal storage conditions (i.e., 25°C, 60% RH), the sample experienced a moisture gain of approximately 0.08%; under accelerated storage conditions (i.e., 70% RH), the moisture gain was approximately 0.15%; and under extreme conditions (i.e., 90% RH), the moisture gain was approximately 0.70%. During the humidity fluctuations from 0% to 90% RH, the desorption and adsorption processes of the sample overlapped. Furthermore, XRPD analysis of the crystal form after DVS analysis revealed no change in crystal form before and after DVS analysis.

[0150] Table 3. XRPD characteristic diffraction peak data of Form A

[0151] Example 4: Stability Test of Form A 1

[0152] Determine whether temperature and humidity have an effect on the stability of Form A of the compound represented by Formula I.

[0153] Form A was placed in a stabilization chamber and subjected to accelerated stability testing using a range of temperature and humidity conditions. Samples were removed after stabilization for varying days under each temperature and humidity condition, and the resulting solids were compared with those obtained from the initial Form A. The content of the compound represented by Formula I was determined by HPLC and analyzed by XRPD. The results are shown in Table 4 below.

[0154] The results showed that Form A had no change and had good stability.

[0155] Table 4. Temperature / humidity conditions, content and related substance determination results of Form A stability experiment

[0156] Example 5: Stability Experiment 2 of Form A

[0157] Determine whether light has an effect on the stability of Form A of the compound represented by Formula I.

[0158] Form A was placed in a light-stabilizing chamber and removed after reaching ICH intensity. The resulting solid was compared with the initial Form A. The content of the compound represented by Formula I was determined by HPLC and XRPD was performed. The experimental results are shown in Table 5 below.

[0159] Table 5. Light stability of crystal form A

[0160] Biological evaluation

[0161] The present disclosure is further described below in conjunction with test examples, but these test examples are not intended to limit the scope of the present disclosure.

[0162] The structure of compound A is:

[0163] Compound A was prepared using the method disclosed in "Example 4 on page 181 of the specification of patent application WO2020070651A".

[0164] Test Example 1: In vitro PDE4B enzyme activity detection experiment

[0165] 1. Experimental Materials

[0166] 2. Experimental steps

[0167] Compound stock solutions were prepared in test tubes at a concentration of 10 mM in 90% DMSO (10% water) and used to prepare serial dilutions with a 1:5 gradient, starting from a final concentration of 100 μM and going down to 0.05 nM.

[0168] Transfer 0.2 μL of compound solution to a 384-well reaction plate. Transfer 0.2 μL of 100% DMSO to each negative and positive control. Then, add 10 μL of a 2x concentration PDE4B1 enzyme solution (final concentration 0.04 nM) to each well. For the no-enzyme control wells, replace the enzyme solution with 10 μL of 1x reaction buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35). Centrifuge at 1000 rpm for 1 minute and incubate at room temperature for 15 minutes. Next, add 10 μL of a 2x FAM-cAMP substrate solution (final concentration 0.1 μM) to each well of the 384-well reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 30 minutes. After completion of the reaction, add 60 μL of stop solution to each well of the 384-well reaction plate and incubate at room temperature for 60 minutes on a shaker at 600 rpm in the dark.

[0169] After the incubation, read the RLU data and calculate the inhibition rate. The IC is calculated based on the concentration and inhibition rate fitting curve. 50 The maximum value refers to the reading of the DMSO control, and the minimum value refers to the reading of the no enzyme activity control.

[0170] The inhibitory effect of compound I on PDE4B1 enzyme activity in vitro was determined by the above test. The IC 50 See Table 6 for values.

[0171] Table 6

[0172] Test Example 2: Inhibitory effect of compounds on the release of proinflammatory cytokines from peripheral blood mononuclear cells (PBMCs)

[0173] Thaw frozen PBMCs and detect cell viability and number using trypan blue staining. Wash the thawed PBMCs with RPMI1640 complete medium (RPMI1640 + 10% FBS + 1% PS), centrifuge and discard the supernatant. Resuspend the PBMCs in RPMI1640 complete medium and adjust the cell density to 2×10 6 cells / mL. 2×10 5 PBMC cells were cultured in 96-well cell culture plates, and different concentrations of the test compound were added. Starting from the maximum concentration of 100 μM of the compound, 9 concentration gradient dilutions were made at a ratio of 1:5, and double-well detection was performed. LPS was added at a final concentration of 0.1 ng / mL, and the total volume was 200 μL. Negative and positive controls were set up. Only LPS and a final concentration of DMSO were added to the negative control wells. In addition to cells and LPS, 1 μg / mL of dexamethasone was added to the positive control wells as a positive control. The cells were incubated in a 37°C incubator for 24 hours. After incubation, 100 μL of cell culture supernatant was collected and the level of TNF-α was detected by ELISA. 100 μL of CellTiter-Glo was added to the remaining cells in each well to detect the cell viability level. Calculate the IC value of the compound for inhibiting TNF-α release 50 value.

[0174] The inhibition of the release of proinflammatory cytokines from PBMC by compound I of the present disclosure in vitro was determined by the above test. The measured IC 50 See Table 7 for values.

[0175] Table 7

[0176] Test Example 3: In vitro inhibition of IL-23 secretion by DC cells differentiated from human monocytes by compounds

[0177] Day 0: Mononuclear cells were isolated and purified from fresh human peripheral blood and resuspended in complete RPMI-1640 culture medium. When differentiating DC cells, 50 ng / ml IL-4 and 100 ng / ml GM-CSF were added to the culture medium at a concentration of 1×10 6 Differentiation culture was performed in a 100 mm diameter dish at a density of 10 cells / mL in an incubator at 37°C and a carbon dioxide concentration of 5%. Day 3: Replace half the volume of complete RPMI culture medium with fresh complete RPMI culture medium, maintaining the IL-4 concentration at 50 ng / ml and the GM-CSF concentration at 100 ng / ml. Day 6: Collect the non-adherent cells (DCs) in the dish and wash them with PBS. Resuspend the washed DCs in complete RPMI culture medium at a cell density of 1×10 6 Then add 1×10 cells / mL to each well of a 96-well cell culture plate.5 DCs were pretreated with varying concentrations of the test compound (or a DMSO blank negative control at the same concentration) for 1 hour. DCs were then stimulated with 200 μg / ml of the TLR2 agonist zymosan for 24 hours. Day 7: After 24 hours of zymosan stimulation of DCs, supernatants were collected from each well of the 96-well plate and IL-23 concentrations were measured by ELISA.

[0178] The inhibition of the compounds disclosed herein on the secretion of IL-23 by DC cells differentiated from human monocytes in vitro was determined by the above test, and the measured IC 50 See Table 8 for values.

[0179] Table 8

[0180] Test Example 4: Imiquimod-induced psoriasis inhibition experiment

[0181] An appropriate amount of Compound I was prepared into an ointment with the following ingredients: 0.1% Compound I, 9% hexylene glycol, 78.8% white petrolatum, 5% paraffin, 7% glyceryl mono- and distearate, and 0.1% dihydroxybutyltoluene, and mechanically stirred to form an ointment.

[0182] 1) Modeling and drug administration

[0183] Seven-week-old Balb / c female mice were selected and their backs were shaved the day before the experiment, with a shaved area of ​​2cm×3cm. Six hours after the test substance was applied to the skin on days 1-7 of the experiment, imiquimod (IMQ) ointment (Aldara (5%)) was applied to the back skin of the mice for 7 consecutive days to construct a psoriasis mouse model. The control group was given the same dose of vaseline ointment. The severity of skin inflammation was evaluated on days 3, 5, and 7, including measuring skin thickness, scabs, and erythema, and scoring was performed using a 5-point system (0-5). The total score was used to assess the severity of skin inflammation. On day 7 of the experiment, the spleen was weighed and the percentage of spleen to body weight was calculated to assess the degree of immunosuppressive effect of the drug.

[0184] This experiment set up a normal control group, a model control group, low-, medium- and high-dose groups (0.01%, 0.03% and 0.1%) of Compound I, and a 0.03% reference Compound A group.

[0185] 2) Evaluation indicators

[0186] The total score for skin inflammation is a clinical score, which is relatively subjective; higher scores indicate more severe disease. The degree of skin thickness is an objective evaluation indicator; greater thickness indicates more severe disease. The spleen's proportion to body weight is also an objective evaluation indicator; a smaller spleen-to-body weight ratio indicates a stronger immunosuppressive effect of the drug.

[0187] 3) Experimental results

[0188] 3.1) Clinical scoring

[0189] Each dose of Compound A and Compound I significantly reduced the clinical scores at the end of the experiment. (Figure 2-Figure 5). From the perspective of individual scores, each dose group of Compound I mainly improved the psoriasis crusting of the model and reduced skin thickness. In the skin thickness score, both the low and high dose groups of Compound I significantly inhibited the increase in skin thickness, while Compound A had no significant effect. The inhibitory effect of 0.03% Compound I on the increase in skin thickness was significantly higher than that of the same dose of Compound A (Figure 5). The results show that Compound I is significantly better than the reference Compound A in improving the manifestations of psoriasis. The specific scoring data are shown in Table 9 below.

[0190] Table 9

[0191] 3.2) Spleen body weight ratio

[0192] Under the action of IMQ, there was no significant difference in the body weight of animals in the model group and each drug-treated group, while the spleen in the model group increased significantly (Figure 6), indicating splenomegaly. Compound A had no significant effect on the spleen body weight ratio, while the three dose groups of Compound I significantly reduced the spleen body weight ratio. This suggests that the compound has an immunosuppressive effect, and the effect of Compound I is dose-dependent. At the same dose (0.03%), the effect of Compound I on the spleen body weight ratio was significantly different from that of Compound A, indicating that Compound I has a stronger immune effect than Compound A. Specific data are shown in Table 10 below.

[0193] Table 10

Claims

1. A crystalline form A of a compound of formula I, having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, having characteristic peaks at 7.537°, 15.219°, 21.798°, and 22.911°; preferably having characteristic peaks at 7.537°, 11.366°, 14.297°, 15.219°, 16.190°, 17.623°, 19.134°, 21.798°, 22.911°, and 24.735°; more preferably having characteristic peaks at 7.537°, 11.366°, 14.018°, 14.297°, 15.219°, 16.190°, 17.623°, 19.134°, 21.798°, 22.911°, and 24.735°. 7.537°, 11.366°, 14.018°, 14.297°, 15.219°, 16.190°, 17.623°, 18.412°, 19.134°, 20.010°, 21.062°, 21.798°, 22.911°, 23.889°, 24.735°, 25.123°, 26.884°, 28.343°, 29.645°, 33.499°, and 36.362°; further preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in Figure 7, 2. The crystalline form A according to claim 1, wherein the error range of the 2θ angle value is ±0.2°.

3. A method for preparing Form A according to claim 1 or 2, which is method 1 or method 2: Method 1 includes the following steps: (a1) mixing the compound represented by formula I with a solvent (1); (b1) stirring crystallization; in, The solvent (1) is selected from one or more of water, nitromethane, N,N-dimethylformamide, acetonitrile, methanol, ethanol, acetone, isopropanol, methyl ethyl ketone, tetrahydrofuran, 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether, 1,2-xylene, toluene, dioxane and hexane; Method 2 includes the following steps: (a2) mixing the compound represented by formula I with a solvent (2); (b2) Volatilization and crystallization; Wherein, the solvent (2) is selected from one or more of acetone, acetonitrile, dichloromethane, dioxane, ethanol, ethyl acetate, heptane, isopropanol, methanol, methyl ethyl ketone, methyl tert-butyl ether, tetrahydrofuran, toluene and water.

4. The preparation method according to claim 3, wherein the solvent (1) is water, nitromethane, N,N-dimethylformamide, acetonitrile, ethanol, acetone, isopropanol, methyl ethyl ketone, tetrahydrofuran, 1,2-dimethoxyethane, chloroform, methyl tert-butyl ether, 1,2-xylene, toluene, dioxane, hexane, or a mixed solvent of water and methanol.

5. The preparation method according to claim 4, wherein when the solvent (1) is a mixed solvent of water and methanol, the molar ratio of the water to the methanol is 0.37-0.90, preferably 0.37, 0.40, 0.47, 0.50, 0.57, 0.60, 0.66, 0.70, 0.74, 0.80, 0.82, or 0.

90.

6. The preparation method according to claim 3, wherein the solvent (2) is acetone, acetonitrile, dichloromethane, dioxane, ethyl acetate, heptane, isopropyl alcohol, methyl ethyl ketone, methyl tert-butyl ether, toluene, water or any mixed solvent of the following groups: acetone and acetonitrile, acetone and dichloromethane, acetone and dioxane, acetone and ethanol, acetone and ethyl acetate, acetone and heptane, acetone and isopropyl alcohol, acetone and methanol, acetone and methyl ethyl ketone, acetone and methyl tert-butyl ether Ether, acetone and tetrahydrofuran, acetone and toluene, acetone and water, acetonitrile and dichloromethane, acetonitrile and dioxane, acetonitrile and ethanol, acetonitrile and ethyl acetate, acetonitrile and isopropanol, acetonitrile and methyl ethyl ketone, acetonitrile and methyl tert-butyl ether, acetonitrile and tetrahydrofuran, acetone and toluene, dichloromethane and dioxane, dichloromethane and ethanol, dichloromethane and ethyl acetate, dichloromethane and heptane, dichloromethane and isopropanol, dichloromethane and methyl ethyl ketone, dichloromethane and methyl tert-butyl methyl ether, dichloromethane and tetrahydrofuran, dichloromethane and toluene, dioxane and methyl ethyl ketone, dioxane and tetrahydrofuran, dioxane and toluene, ethanol and ethyl acetate, ethanol and methanol, ethanol and tetrahydrofuran, ethanol and toluene, ethanol and water, ethyl acetate and heptane, ethyl acetate and isopropanol, ethyl acetate and methanol, ethyl acetate and methyl ethyl ketone, ethyl acetate and methyl tert-butyl ether, ethyl acetate and toluene, heptane and methyl ethyl ketone, heptane and methyl tert-butyl ether, heptane and tetrahydrofuran, heptane and toluene, methanol and methyl ethyl ketone, methanol and methyl tert-butyl ether, methanol and toluene, methanol and water, methyl ethyl ketone and methyl tert-butyl ether, methyl ethyl ketone and tetrahydrofuran, methyl ethyl ketone and toluene, methyl tert-butyl ether and tetrahydrofuran, methyl tert-butyl ether and toluene, tetrahydrofuran and toluene, tetrahydrofuran and water; preferably, when the solvent (2) is a mixed solvent, the volume ratio of the two is 1:3-3:1, preferably 1:

1.

7. A pharmaceutical composition comprising the crystalline form A according to claim 1 or 2 or the crystalline form A prepared by the preparation method according to any one of claims 3 to 6, and optionally a pharmaceutically acceptable excipient.

8. A method for preparing the pharmaceutical composition according to claim 7, comprising the step of mixing the crystalline form A according to claim 1 or 2 or the crystalline form A obtained by the preparation method according to any one of claims 3 to 6 with a pharmaceutically acceptable excipient.

9. Use of the crystalline form A according to claim 1 or 2, or the crystalline form A obtained by the preparation method according to any one of claims 3 to 6, or the pharmaceutical composition according to claim 7, in the preparation of a medicament for preventing and / or treating a disease associated with PDE; preferably, the disease associated with PDE is asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

10. Use of the crystalline form A according to claim 1 or 2, or the crystalline form A prepared by the preparation method according to any one of claims 3 to 6, or the pharmaceutical composition according to claim 7, in the preparation of a medicament for preventing and / or treating asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative enteritis, or rheumatism.

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