Medicinal salt of boric acid ester derivative, crystal form and application thereof

Abstract

The present disclosure relates to a pharmaceutically acceptable salt of a borate derivative, a crystalline form and use thereof. Specifically, the present disclosure provides pharmaceutically acceptable salts of a compound as shown in formula (I), crystalline forms and uses thereof. # imgabs0 #

Description

A pharmaceutically acceptable salt of a borate ester derivative, its crystalline form and use Technical Field

[0001] The present disclosure belongs to the field of pharmacy and relates to a pharmaceutically acceptable salt of a borate ester derivative, a crystalline form thereof and uses 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 pharmaceutical products lack a regular crystal structure and often exhibit other drawbacks, such as poor product stability, fine crystallization, difficulty filtering, susceptibility to agglomeration, and poor flowability. Polymorphic drug forms 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 pharmaceutically acceptable salt of a compound represented by formula I,

[0009] In some embodiments, the pharmaceutically acceptable salt is selected from lithium salts.

[0010] In some embodiments, the molar ratio of the compound represented by Formula I to the base molecule or cation is 1:0.5 to 1:3, for example, 1:0.5, 1:1, 1:2 or 1:3.

[0011] In some embodiments, the molar ratio of the compound represented by Formula I to the base molecule or cation is 1:1.

[0012] In some embodiments, the chemical ratio of the compound of Formula I to the acid molecule or acid radical is 1:0.5 to 1:3, for example, 1:0.5, 1:1, 1:2 or 1:3. The present disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound of Formula I, comprising the steps of forming a salt of the compound of Formula I with a base, or forming a salt of the compound of Formula I with an acid.

[0013] In some embodiments, the solvent used in the salt-forming reaction is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone and dioxane.

[0014] In some embodiments, the volume (μl) of the solvent used in the salt-forming reaction is 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments, is 1, 5, 10, 13, 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.

[0015] In some embodiments, the preparation method further comprises centrifugation, washing or drying steps.

[0016] The present disclosure also provides a crystalline form α of the lithium salt of the compound represented by Formula I, which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 13.913°, 17.884°, 19.894°, 21.182°, and 33.454°.

[0017] In some embodiments, the crystalline form α has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 11.855°, 13.913°, 14.383°, 17.884°, 19.894°, 21.182°, 22.148°, 29.948°, 30.310°, and 33.454°.

[0018] In some embodiments, the crystalline form α has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, with characteristic peaks at 11.855°, 13.913°, 14.383°, 17.884°, 19.894°, 21.182°, 22.148°, 23.910°, 29.948°, 30.310°, 33.454°, and 36.812°.

[0019] In some embodiments, the crystalline form α has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and has characteristic peaks at 8.859°, 11.855°, 13.913°, 14.383°, 14.863°, 17.884°, 19.894°, 21.182°, 22.148°, 23.910°, 29.948°, 30.310°, 31.609°, 33.454°, 34.721°, and 36.812°.

[0020] In some embodiments, the X-ray powder diffraction spectrum of the crystalline form α expressed in terms of a diffraction angle 2θ is shown in FIG6 .

[0021] The present disclosure also provides a method for preparing the above-mentioned crystal form α, which comprises the following steps: (a) mixing the compound represented by formula I, lithium hydroxide and a solvent, and stirring; (b) crystallizing;

[0022] In some embodiments, the solvent in step (a) is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone, and dioxane.

[0023] In some embodiments, the volume (μl) of the solvent used in step (a) is 1-200 times the mass (mg) of the compound of Formula I, and in non-limiting embodiments is 1, 5, 10, 13, 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 a value between any two numbers.

[0024] In some embodiments, in step (a), the mass of the lithium hydroxide is 0.5-3 times the mass of the compound represented by Formula I, and in non-limiting embodiments is 0.5, 0.7, 1, 1.5, 2, 2.5, 3 or any value between two numbers.

[0025] In some embodiments, in step (a), the stirring temperature is 10-70°C, and in non-limiting embodiments is 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C or any value between two numbers.

[0026] In some embodiments, in step (a), the stirring temperature is 50°C.

[0027] In some embodiments, in step (b), the crystallization is cooling crystallization, and the temperature of the cooling crystallization is preferably 0°C.

[0028] In some embodiments, the crystallization further includes filtering, washing or drying steps.

[0029] The present disclosure also provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of the compound represented by the above formula I and optionally a pharmaceutically acceptable excipient.

[0030] The present disclosure also provides a pharmaceutical composition, which comprises the above-mentioned crystal form α or the crystal form α prepared by the above-mentioned preparation method, and optionally a pharmaceutically acceptable excipient.

[0031] The present disclosure also provides a method for preparing the above-mentioned pharmaceutical composition, which comprises the step of mixing a pharmaceutically acceptable salt of the compound represented by the above-mentioned formula I or the above-mentioned crystal form α or the crystal form α prepared by the above-mentioned preparation method with a pharmaceutically acceptable excipient.

[0032] The present disclosure also provides use of a pharmaceutically acceptable salt of the compound represented by the above formula I, or the above crystal form α, or the crystal form α prepared by the above preparation method, or the above pharmaceutical composition in preparing a drug for preventing and / or treating PDE-related diseases.

[0033] In some embodiments, the PDE-associated disease is asthma, obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

[0034] The present disclosure also provides a use of a pharmaceutically acceptable salt or crystal form α of the compound represented by the above-mentioned formula I, or crystal form α 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, septicemia, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative enteritis or rheumatism.

[0035] 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 number exceeding 1 decimal place is 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.

[0036] 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,

[0037] Deliquescent: Absorbs sufficient water to form a liquid;

[0038] Highly hygroscopic: weight gain due to moisture absorption is not less than 15%;

[0039] Hygroscopic: weight gain due to moisture absorption is less than 15% but not less than 2%;

[0040] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0041] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0042] 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.

[0043] 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.

[0044] The "room temperature" described in this disclosure is generally 25°C ± 5°C.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Numerical values ​​in this disclosure, such as those regarding the content of a substance, are measured and calculated, and therefore inevitably contain a certain degree of error. Generally speaking, a reasonable error range of ±10% is acceptable. While some degree of error may vary depending on the context in which the value is used, this error range may not exceed ±10% and may include ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0049] 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 (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

[0050] Figure 1: Comparison of clinical scores of each group of compounds in the disease model in Test Example 4.

[0051] Figure 2: Comparison of clinical scores of each group of compounds in Test Example 4 in the erythema disease model.

[0052] Figure 3: Comparison of clinical scores of each group of compounds in Test Example 4 in the psoriasis model.

[0053] Figure 4: Comparison of the inhibitory effects of various groups of compounds in Test Example 4 on skin thickness increase.

[0054] Figure 5: Comparison of the effects of each group of compounds on the proportion of spleen body weight in Test Example 4.

[0055] Figure 6: XRPD spectrum of Form α.

[0056] Figure 7: DSC spectrum of Form α.

[0057] Figure 8: TGA spectrum of Form α.

[0058] In Figures 1 to 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 used only to distinguish between groups. DETAILED DESCRIPTION

[0059] The present disclosure will be described in more detail below with reference to examples. The 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.

[0060] The abbreviations used in this disclosure are explained as follows: XRPD X-ray powder diffraction DSC Differential Scanning Calorimetry TGA Thermogravimetric Analysis DVS Dynamic Water Sorption 1 H-NMR, liquid nuclear magnetic hydrogen spectrum, DMF, N,N-dimethylformamide, MEK, methyl ethyl ketone, MTBE, methyl tert-butyl ether, THF, tetrahydrofuran, IPA, isopropanol, DCM, dichloromethane, ACN, acetonitrile

[0061] Test conditions of the instruments used in the experiments in this disclosure:

[0062] 1. X-ray Powder Diffraction (XRPD)

[0063] Instrument model: Malver Panalytical Aeris X-ray powder diffractometer

[0064] Radiation: Monochromatic Cu-Kα radiation (λ=1.54188)

[0065] Scanning mode: θ / 2θ, scanning range (2θ range): 3.5~50°

[0066] Voltage: 40kV, Current: 15mA.

[0067] 2. Differential Scanning Calorimeter (DSC)

[0068] Instrument model: TA DSC250

[0069] Purge gas: nitrogen; nitrogen purge rate: 50mL / min

[0070] Heating rate: 10℃ / min

[0071] Temperature range: 25℃-300℃.

[0072] 3. Thermogravimetric Analysis (TGA)

[0073] Instrument model: TA TGA550

[0074] Purge gas: nitrogen; nitrogen purge speed: 20ml / min

[0075] Heating rate: 10℃ / min

[0076] Temperature range: 30℃-350℃.

[0077] 4. Dynamic moisture sorption (DVS)

[0078] 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.

[0079] 5. The high performance liquid chromatography (HPLC) described in the stability test 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.

[0080] 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).

[0081] 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.

[0082] 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 samples were scanned in positive / negative ion mode with a mass scan range of 80-1200.

[0083] 9. Preparative HPLC conditions: water; column: Sunfire (Prep C18 OBD 19*250mm 10μm).

[0084] 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.

[0085] 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.

[0086] 12. The flash column purification system used was Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

[0087] 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).

[0088] 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.

[0089] Unless otherwise specified in the examples, all reactions were carried out under a nitrogen atmosphere.

[0090] Nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a capacity of about 1L.

[0091] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0092] Hydrogen was produced by a QPH-1L hydrogen generator from Shanghai Quanpu Scientific Instrument Co., Ltd.

[0093] The nitrogen atmosphere or hydrogen atmosphere is usually evacuated and filled with nitrogen or hydrogen, and the operation is repeated three times.

[0094] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0095] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0096] 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.

[0097] Example 1: Preparation of Compound I

[0098] 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.

[0099] 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) + .

[0100] 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).

[0101] 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).

[0102] Step 4): To a 500 mL single-necked flask at room temperature, add compound b (20.0 g, 74.4 mmol), n-hexane (200 mL), and palladium calcium carbonate (1.95 g, 18.8 mmol). Stir at room temperature under a hydrogen atmosphere for 16 hours. Filter and concentrate under reduced pressure to obtain compound c (19 g).

[0103] 1 H 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).

[0104] 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).

[0105] 1H 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).

[0106] 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).

[0107] 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).

[0108] 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).

[0109] LCMS: m / z 319 (M+H) + .

[0110] 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).

[0111] LCMS: m / z 305 (M+H) + .

[0112] 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).

[0113] LCMS: m / z 441.1 (M+H) + .

[0114] 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).

[0115] LCMS: m / z 569.3 (M+H) + .

[0116] 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, add ethyl acetate (300 mL x 3), extract, 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).

[0117] Compound I was obtained by 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).

[0118] LCMS: m / z 355.0 (M+H) + .

[0119] 1 H 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).

[0120] Example 2: Preparation of lithium salt of compound I

[0121] Compound I (15 mg, 42.3490 μmol) was dissolved in 0.2 ml of methanol, and lithium hydroxide (10.7 mg) was added. The solution was heated to 50°C for 1 hour, stirred at 50°C for 4 hours, then cooled to 0°C within 6 hours, stirred at 0°C for 48 hours, and dried in vacuo to obtain the lithium salt of compound I.

[0122] 1 H NMR (400MHz, DMSO-d6) δ8.75–8.12(m,1H),7.13–6.45(m,1H),3.76(d,J=2.9Hz,3H),3.35( s,2H),2.97–2.63(m,1H),2.10(d,J=13.2Hz,0H),1.66(t,J=7.2Hz,1H),1.41–1.02(m,3H).

[0123] Example 3: Preparation of Form α of the Lithium Salt of Compound I

[0124] Compound I (15 mg, 42.3490 μmol) was dissolved in 0.2 ml of a good solvent (Table 1). Lithium hydroxide (10.7 mg) was added. The solution was heated to 50°C over 1 hour and stirred at 50°C for 4 hours. The temperature was then programmed to 0°C over 6 hours and stirred at 0°C for 48 hours. The solution was filtered and dried under vacuum. The resulting solid was identified as Form α by XRPD analysis. The XRPD spectrum is shown in Figure 6 , the characteristic peak positions are shown in Table 2 , the DSC spectrum is shown in Figure 7 , and the TGA spectrum is shown in Figure 8 .

[0125] Table 1. Solvents used in the preparation of Form α of the lithium salt of Compound I and XRPD test results

[0126] Table 2. XRPD characteristic diffraction peak data of Form α

[0127] Biological evaluation

[0128] 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.

[0129] The structure of compound A is:

[0130] Compound A was prepared using the method disclosed in "Example 4 on page 181 of the specification of patent application WO2020070651A".

[0131] Test Example 1: In vitro PDE4B enzyme activity detection experiment

[0132] 1. Experimental Materials

[0133] 2. Experimental steps

[0134] A 10 mM stock solution of the compound was prepared in a test tube in 90% DMSO (10% water). Serial dilutions were prepared with a 1:5 gradient, starting from 100 uM and ending at 0.05 nM.

[0135] 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 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.

[0136] 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.

[0137] The inhibitory effect of compound I on PDE4B1 enzyme activity in vitro was determined by the above test. The IC 50 See Table 3 for values.

[0138] Table 3

[0139] Test Example 2: Inhibitory effect of compounds on the release of proinflammatory cytokines from peripheral blood mononuclear cells (PBMCs)

[0140] 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 5PBMC 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.

[0141] 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 4 for values.

[0142] Table 4

[0143] Test Example 3: In vitro inhibition of IL-23 secretion by DC cells differentiated from human monocytes by compounds

[0144] 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.

[0145] 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 5 for values.

[0146] Table 5

[0147] Test Example 4: Imiquimod-induced psoriasis inhibition experiment

[0148] 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.

[0149] 1) Modeling and drug administration

[0150] Seven-week-old female Balb / c mice were selected. The day before the experiment, the back of the mice was shaved (2 cm x 3 cm). Six hours after the test substance was applied to the skin, imiquimod (IMQ) ointment (Aldara (5%)) was applied to the back of the mice for 7 consecutive days to establish a psoriasis mouse model. A control group received the same dose of petrolatum ointment. The severity of skin inflammation was assessed on days 3, 5, and 7, including measurement of skin thickness, scab formation, and erythema, and scored on a 5-point scale (0-5). The total score was used to assess the severity of skin inflammation. On day 7, the spleen was weighed, and the percentage of spleen to body weight was calculated to assess the degree of immunosuppressive effect of the drug.

[0151] 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.

[0152] 2) Evaluation indicators

[0153] 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.

[0154] 3) Experimental results

[0155] 3.1) Clinical scoring

[0156] Each dose of Compound A and Compound I significantly reduced the clinical scores at the end of the experiment. (Figure 1-Figure 4). From the perspective of single-item 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, and 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 4). The results showed that Compound I was significantly better than the reference Compound A in improving the manifestations of psoriasis. The specific scoring data are shown in Table 6.

[0157] Table 6

[0158] 3.2) Spleen body weight ratio

[0159] 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 5), 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 each 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 7.

[0160] Table 7

Claims

1. A pharmaceutically acceptable salt of a compound of formula I, wherein the pharmaceutically acceptable salt is selected from lithium salts, 2. The pharmaceutically acceptable salt of the compound of formula I according to claim 1, wherein the molar ratio of the compound of formula I to the base molecule or cation is 1:0.5 to 1:3, preferably 1:0.5, 1:1, 1:2 or 1:

3.

3. A method for preparing a pharmaceutically acceptable salt of the compound of formula I according to claim 1 or 2, comprising the steps of forming a salt of the compound of formula I with a base, 4. The preparation method according to claim 3, wherein the solvent used in the salt-forming reaction is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone and dioxane.

5. A crystalline form α of a lithium salt of a compound represented by formula I, having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, with characteristic peaks at 13.913°, 17.884°, 19.894°, 21.182°, and 33.454°; preferably, characteristic peaks at 11.855°, 13.913°, 14.383°, 17.884°, 19.894°, 21.182°, 22.148°, 29.948°, 30.310°, and 33.454°; more preferably, characteristic peaks at 11.855°, 13.913°, 14.383°, 17.884°, 19.894°, 21.182°, 22.148°, 29.948°, 30.310°, and 33.454°. 6°, 22.148°, 23.910°, 29.948°, 30.310°, 33.454°, and 36.812°; further preferably, there are characteristic peaks at 8.859°, 11.855°, 13.913°, 14.383°, 14.863°, 17.884°, 19.894°, 21.182°, 22.148°, 23.910°, 29.948°, 30.310°, 31.609°, 33.454°, 34.721°, and 36.812°; further preferably, an X-ray powder diffraction pattern represented by a diffraction angle 2θ is shown in FIG6 , 6. The crystalline form α of the lithium salt of the compound represented by formula I according to claim 5, wherein the error range of the 2θ value is ±0.2°.

7. A method for preparing the crystalline form α of the lithium salt of the compound represented by formula I according to claim 5 or 6, comprising the following steps: (a) mixing a compound represented by formula I, lithium hydroxide and a solvent, and stirring; (b) crystallization, 8. The preparation method according to claim 7, wherein the solvent is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone and dioxane.

9. The preparation method according to claim 7, wherein In step (a), the stirring temperature is 10-70°C, such as 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, preferably 50°C.

10. The preparation method according to claim 7, wherein In step (b), the crystallization is cooling crystallization, and the temperature of the cooling crystallization is preferably 0°C.

11. A pharmaceutical composition comprising a pharmaceutically acceptable salt of the compound of Formula I according to claim 1 or 2, or the crystalline form α of the lithium salt of the compound of Formula I according to claim 5 or 6, or the crystalline form α of the lithium salt of the compound of Formula I prepared by the preparation method according to any one of claims 7 to 10, and optionally a pharmaceutically acceptable excipient.

12. A method for preparing the pharmaceutical composition according to claim 11, comprising mixing a pharmaceutically acceptable salt of the compound of Formula I according to claim 1 or 2, or the crystalline form α of the lithium salt of the compound of Formula I according to claim 5 or 6, or the crystalline form α of the lithium salt of the compound of Formula I prepared by the preparation method according to any one of claims 7 to 10, with a pharmaceutically acceptable excipient.

13. Use of a pharmaceutically acceptable salt of the compound of formula I according to claim 1 or 2, or the crystalline form α of the lithium salt of the compound of formula I according to claim 5 or 6, or the crystalline form α of the lithium salt of the compound of formula I prepared by the preparation method according to any one of claims 7 to 10, or the pharmaceutical composition according to claim 11 in the preparation of a medicament for preventing and / or treating a PDE-related disease; preferably, the PDE-related disease is asthma, obstructive pulmonary disease, septicemia, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatism.

14. Use of a pharmaceutically acceptable salt of the compound of formula I according to claim 1 or 2, or the crystalline form α of the lithium salt of the compound of formula I according to claim 5 or 6, or the crystalline form α of the lithium salt of the compound of formula I prepared by the preparation method according to any one of claims 7 to 10, or the pharmaceutical composition according to claim 11 in the preparation of a medicament for preventing and / or treating asthma, obstructive pulmonary disease, septicemia, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative enteritis, or rheumatism.

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