WO2023246858A1 - 一种硼酸酯衍生物的可药用盐、其结晶形式及用途 - Google Patents

一种硼酸酯衍生物的可药用盐、其结晶形式及用途 Download PDF

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WO2023246858A1
WO2023246858A1 PCT/CN2023/101654 CN2023101654W WO2023246858A1 WO 2023246858 A1 WO2023246858 A1 WO 2023246858A1 CN 2023101654 W CN2023101654 W CN 2023101654W WO 2023246858 A1 WO2023246858 A1 WO 2023246858A1
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formula
compound
compound represented
pharmaceutically acceptable
preparation
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French (fr)
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孙龙伟
侯延婷
朱嘉俊
王中利
谢蓝
罗志阳
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瑞石生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds

Definitions

  • the present disclosure belongs to the field of medicine and relates to a pharmaceutically acceptable salt of a borate ester derivative, its crystalline form and uses.
  • Phosphodiesterases are a class of phosphodiesters that cleave the second messenger molecules 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) key intracellular enzymes.
  • the cyclic nucleotides cAMP and cGMP act as second messengers in various cellular pathways.
  • PDE4 is highly specific for cAMP and has 4 subtypes: PDE4A, PDE4B, PDE4C and PDE4D.
  • PDE4 is involved in promoting monocyte and macrophage activation, neutrophil infiltration, vascular smooth muscle proliferation, vasodilation, myocardial contraction and other related physiological and pathological processes. It has important effects on central nervous system function, cardiovascular function, inflammation/immune system, Cell adhesion, etc. are all affected. PDE4 plays a major regulatory role in the expression of pro-inflammatory and anti-inflammatory mediators, and PDE4 inhibitors can inhibit the release of harmful mediators from inflammatory cells.
  • PDE4 inhibitors have been discovered in recent years. For example, roflumilast is approved for use in 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.
  • COPD severe chronic obstructive pulmonary disease
  • apremilast is approved for the treatment of adults with active psoriatic arthritis.
  • PDE4 inhibitors show good pharmacological activity, these PDE inhibitors can cause side effects, such as induced gastrointestinal symptoms such as vomiting and diarrhea.
  • the boron (B)-containing drug criborole was approved by the FDA on December 14, 2016 as a topical treatment for mild to moderate atopic dermatitis. This boron atom aids skin penetration and binds to the bimetallic center of phosphodiesterase 4 (PDE4).
  • PDE4 phosphodiesterase 4
  • other boron-containing PDEs inhibitor small molecules have been reported, such as CN102014927A and WO2020070651.
  • the compounds of the present disclosure have not been disclosed in any literature, and such compounds exhibit specific PDE4 inhibitory effects.
  • PCT/CN2021/141010 provides a new class of PDE4 inhibitors, whose chemical name is (R)-4-(6-(8-methoxy-2,2-dimethylchroman-5) -(yl)pyrazin-2-yl)-1,2-oxaborolan-2-ol, having the structure shown in formula I,
  • crystal form of pharmaceutical active ingredients often affects the chemical stability of the drug. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other forms. Crystal form. Generally speaking, amorphous pharmaceutical products do not have regular crystal structures and often have other defects, such as poor product stability, fine crystallization, difficulty in filtration, easy agglomeration, and poor fluidity. Polymorphic forms of drugs have different requirements for product storage, production and scale-up. Therefore, it is necessary to conduct in-depth research on the crystal forms of the aforementioned compounds and improve various properties of the aforementioned compounds.
  • the pharmaceutically acceptable salt is selected from lithium salts.
  • the molar ratio of the compound represented by Formula I to alkali molecules or cations is 1:0.5 to 1:3, such as 1:0.5, 1:1, 1:2 or 1:3.
  • the molar ratio of the compound represented by Formula I to alkali molecules or cations is 1:1.
  • the chemical ratio of the compound represented by Formula I to acid molecules or acid radicals is 1:0.5 to 1:3, such as 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 represented by Formula I, which includes the step of forming a salt of the compound represented by Formula I with a base, or the step of forming a salt of the compound represented by Formula I with an acid.
  • the solvent used in the salt-forming reaction is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone and dioxane.
  • the volume ( ⁇ l) 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.
  • the preparation method further includes centrifugation, washing or drying steps.
  • the present disclosure also provides the crystal form ⁇ of the lithium salt of the compound represented by Formula I, which has an X-ray powder diffraction pattern expressed as a diffraction angle 2 ⁇ angle at 13.913°, 17.884°, 19.894°, 21.182°, and 33.454°. Characteristic peaks,
  • the crystalline form ⁇ has an There are characteristic peaks at , 30.310° and 33.454°.
  • the crystalline form ⁇ has an There are characteristic peaks at , 29.948°, 30.310°, 33.454°, and 36.812°.
  • the crystalline form ⁇ has an There are characteristic peaks at , 22.148°, 23.910°, 29.948°, 30.310°, 31.609°, 33.454°, 34.721°, and 36.812°.
  • the X-ray powder diffraction pattern expressed as a diffraction angle 2 ⁇ of the crystalline form ⁇ is as shown in Figure 6 .
  • the present disclosure also provides a method for preparing the above crystal form ⁇ , which includes the following steps: (a) mixing the compound of formula I, lithium hydroxide and a solvent and stirring; (b) crystallizing;
  • the solvent in step (a) is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, acetone, and dioxane.
  • the volume ( ⁇ l) of the solvent used in step (a) is 1-200 times the mass (mg) of the compound represented by 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 in between.
  • the mass of the lithium hydroxide is 0.5-3 times the mass of the compound shown in Formula I, and in non-limiting embodiments is 0.5, 0.7, 1, 1.5, 2, 2.5, 3, or any value in between.
  • 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 °C or any value between two numbers.
  • step (a) the stirring temperature is 50°C.
  • the crystallization is cooling crystallization, and the temperature of the cooling crystallization is The temperature is preferably 0°C.
  • the present disclosure also provides a pharmaceutical composition, which includes a pharmaceutically acceptable salt of the compound represented by Formula I above and optionally a pharmaceutically acceptable excipient.
  • the present disclosure also provides a pharmaceutical composition, which includes the above-mentioned crystal form ⁇ or the crystal form ⁇ prepared by the above-mentioned preparation method, and optionally pharmaceutically acceptable excipients.
  • the present disclosure also provides a method for preparing the above-mentioned pharmaceutical composition, which includes combining a pharmaceutically acceptable salt of the compound represented by the above-mentioned formula I or the above-mentioned crystal form ⁇ or the crystal form ⁇ prepared by the above-mentioned preparation method, and a pharmaceutically acceptable salt. Acceptable excipient mixing step.
  • the present disclosure also provides a pharmaceutically acceptable salt of the compound represented by the above formula I or the above crystal form ⁇ or the crystal form ⁇ prepared by the above preparation method or the above pharmaceutical composition for use in the prevention and/or treatment of PDE. Use in medicines for related diseases.
  • the PDE-related disease is asthma, obstructive pulmonary disease, septicemia, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis, or rheumatic disease.
  • the present disclosure also provides a pharmaceutically acceptable salt or crystal form ⁇ of the compound represented by the above-mentioned formula I or a crystal form ⁇ prepared by the above-mentioned preparation method or the above-mentioned pharmaceutical composition for use in the prevention and/or treatment of asthma, Use in medicines for obstructive pulmonary disease, sepsis, nephritis, diabetes, allergic rhinitis, allergic conjunctivitis, ulcerative colitis or rheumatism.
  • the "2 ⁇ or 2 ⁇ angle" mentioned in this disclosure refers to the diffraction angle, ⁇ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2 ⁇ is ⁇ 0.2 (including numbers exceeding 1 decimal place after rounding case), 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.
  • Deliquescence Absorbing enough water to form a liquid
  • the weight gain by attracting moisture is not less than 15%;
  • the weight gain by attracting moisture is less than 15% but not less than 2%;
  • weight gain due to moisture attraction is less than 2% but not less than 0.2%;
  • weight gain due to moisture absorption is less than 0.2%.
  • Differential scanning calorimetry or DSC refers to measuring the temperature difference and heat flow difference between the sample and the reference during the process of heating or constant temperature of the sample to characterize all physical changes related to thermal effects and Chemical changes to obtain phase change information of the sample.
  • the drying temperature mentioned in this disclosure is generally 25°C to 100°C, preferably 40°C to 70°C. Drying can be done under normal pressure or under reduced pressure, and the pressure is ⁇ -0.08MPa.
  • Root temperature as used in this disclosure is generally 25°C ⁇ 5°C.
  • Excipients include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock animals. , preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents or emulsifiers.
  • the "beating” mentioned in this disclosure refers to a method of purification that utilizes the characteristics of a substance that has poor solubility in a solvent but good impurity solubility in the solvent. Beating and purification can remove color, change the crystal form, or remove a small amount of impurities.
  • the starting materials used in the crystal form preparation method of the present disclosure can be compounds in any form. Specific forms include but are not limited to: amorphous, any crystal form, hydrate, solvate, etc.
  • the numerical values in this disclosure for example, the content of relevant substances are measured and calculated, and there is inevitably a certain degree of error. Generally speaking, ⁇ 10% is within the reasonable error range. There is a certain degree of error variation depending on the context where it is used. The error variation does not exceed ⁇ 10% and can be ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2% or ⁇ 1%, preferably ⁇ 5%.
  • Figure 1 Comparison of clinical scores of each group of compounds in disease models in Test Example 4.
  • Figure 2 Comparison of clinical scores of each group of compounds in the erythema disease model in Test Example 4.
  • Figure 3 Comparison of clinical scores of each group of compounds in the psoriasis disease model in Test Example 4.
  • Figure 4 Comparison of the inhibitory effects of each group of compounds on the increase in skin thickness in Test Example 4.
  • Figure 5 Comparison of the effects of each group of compounds on the proportion of spleen body weight in Test Example 4.
  • the detection uses SMS Intrinsic PLUS, at 25°C, the humidity is from 50%-0%-90%, the step is 10%, the judgment standard is that the change in dM/dT of each gradient is less than 0.0002%, TMAX 360min, two cycles.
  • HPLC high-performance liquid chromatography
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). NMR shifts ( ⁇ ) are given in units of 10 -6 (ppm). NMR was measured using a Bruker AVANCE-400 nuclear magnetic instrument. The measurement solvents were deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), and deuterated methanol (Methanol-d 4 ). The internal standard was Tetramethylsilane (TMS).
  • HPLC measurement of Compound I in the preparation examples uses Agilent1100 high-pressure liquid chromatograph, GAS15B DAD UV detector, and Water Vbridge C18 150*4.6mm 5 ⁇ M chromatographic column.
  • MS was measured using Agilent6120 triple quadrupole mass spectrometer, G1315D DAD detector, Waters Xbridge C18 4.6*50mm, 5 ⁇ M chromatographic column, scanning in positive/negative ion mode, and the mass scanning range was 80 ⁇ 1200.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 silica gel plate.
  • the thin layer chromatography (TLC) uses silica gel plate with a specification of 0.2mm ⁇ 0.03mm.
  • the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm. .
  • the rapid column purification system uses Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).
  • 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 prefilled ultra-pure normal phase silica gel column (40-63 ⁇ m, 60g, 24g, 40g, 120g or other specifications).
  • the known starting materials in the present disclosure can be synthesized by methods known in the art, or can be purchased from Shanghai Titan Technology, ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Bide Pharmaceutical and other companies.
  • Nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere refers to the reaction bottle connected to a hydrogen balloon with a volume of about 1L.
  • Hydrogen was produced by a QPH-1L hydrogen generator from Shanghai Quanpu Scientific Instrument Company.
  • the nitrogen atmosphere or hydrogen atmosphere is usually evacuated, filled with nitrogen or hydrogen, and repeated three times.
  • the solution refers to an aqueous solution.
  • the reaction temperature is room temperature, which is 20°C to 30°C.
  • the reaction progress in the examples is monitored by thin layer chromatography (TLC), the developing agent used in the reaction, the eluent system of column chromatography and the developing agent system of thin layer chromatography used to purify the compound, and the volume of the solvent.
  • TLC thin layer chromatography
  • the ratio is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.
  • Step 1) Compound 1a (2.0g, 14.6mmol) and triethylamine (1.8g, 17.8mmol) were dissolved in N,N-dimethylformamide (30mL), and the solution was cooled to 0°C. Under nitrogen atmosphere, tert-butyldiphenylsilyl chloride (4.0g, 14.6mmol) was added dropwise to the reaction system. Raise to room temperature and continue stirring until TLC detects the completion of the reaction. Pour the reaction solution into water and extract with ethyl acetate (100 mL ⁇ 3). The organic phase was washed with water (50mL ⁇ 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 (5.1g), which was directly used in the next step. reaction.
  • Step 2) Under nitrogen atmosphere, combine compound 1b (5.1g, 13.6mmol), pinacol diborate (4.2g, 16.3mmol), [1,1'-bis(diphenylphosphino)ferrocene ]
  • a mixture of palladium dichloride (512 mg, 0.7 mmol), potassium acetate (2.0 g, 20.4 mmol) and dioxane (100 mL) was warmed to 80°C and stirred overnight.
  • the reaction solution was poured into water, and extracted with ethyl acetate (100 mL ⁇ 2). Wash with water (30mL ⁇ 2), dry over anhydrous sodium sulfate, and concentrate.
  • the residue is purified by silica gel column chromatography (petroleum ether/ethyl acetate) to obtain Compound 1c (2.0g), LCMS: m/z 423.2 (M+H) + .
  • Step 3) At room temperature, add compound a (100g, 492mmol), DMF (1000mL), potassium iodide (92.6g, 837mmol), copper iodide (3.13g, 9.85mmol), potassium carbonate ( 136g, 985mmol). Under nitrogen atmosphere, 3-chloro-3-methyl-1-butyne (100 mL, 886 mmol) was added dropwise. Stir at 70°C for 16 hours. Cool to room temperature, add water (1000mL), and extract with petroleum ether (1000mL ⁇ 3). Dry over anhydrous sodium sulfate and filter. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound b (50 g).
  • Step 4) At room temperature, add compound b (20.0g, 74.4mmol), n-hexane (200mL), and palladium calcium carbonate (1.95g 18.8mmol) in sequence to a 500mL single-mouth bottle. Stir at room temperature for 16 hours under a hydrogen atmosphere. Filter and concentrate under reduced pressure to obtain compound c (19g).
  • Step 5) At room temperature, add compound c (10.0g, 36.9mmol) and diethylaniline (10mL, 62.5mmol) in sequence to a 50mL single-neck bottle. Stir at 210°C for 1 hour. Cool to room temperature, adjust the pH to neutral with 1M HCl solution, extract with ethyl acetate (200mL ⁇ 3), and wash with water (200mL ⁇ 3). Concentrate under reduced pressure to obtain compound d (9.1g).
  • Step 6) At room temperature, add compound d (5.00g, 18.5mmol), toluene (25mL), and 15 (5.00g, 15.9mmol). Under nitrogen atmosphere, stir at 100°C for 2 hours. Cool to room temperature and filter. Concentrate under reduced pressure to obtain compound e (3.89g).
  • Step 7) At room temperature, add compound e (28.4g, 105mmol), dioxane (300mL), pinacol diborate (31.9g, 126mmol), and potassium acetate (20.6g) into a 500mL single-mouth bottle in sequence. 209mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (7.66g, 10.5mmol). Under nitrogen atmosphere, stir at 105°C for 16 hours. Cool to room temperature and filter. It was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound f (28.4g).
  • Step 8) At room temperature, add compound f (6.20g, 19.5mmol), 1,4-dioxane (80mL), water (16mL), and 2,6-dichloropyrazine ( 2.90g, 39.0mmol), potassium carbonate (5.39g, 39.0mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (1.43g, 1.95mmol). Under nitrogen atmosphere, stir at 110°C for 16 hours. Cool to room temperature and filter. Concentrated under reduced pressure, the residue was subjected to column chromatography. Purification method (petroleum ether/ethyl acetate) gave compound g (5.7g).
  • Step 9) At room temperature, add compound g (35.0g, 115mmol), compound 1c (44.5g, 149mmol), potassium carbonate (23.8g, 172mmol), potassium acetate (16.9g, 172mmol) and [ 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (8.41g, 11.5mmol) in a mixed solvent of 1,4-dioxane (250mL) and water (5mL). Stir until dissolved, replace 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 ⁇ 3), and dry over anhydrous sodium sulfate. It was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound h (37g).
  • Step 10) At room temperature, add 1,2-bis(diphenylphosphine)ethane (1.81g, 4.54mmol) and (1,5-cyclooctadiene)methoxyiridium (I) into a 250mL three-necked flask. ) dimer (1.50 g, 2.27 mmol) and anhydrous 1,2-dichloroethane (100 mL). Stir at room temperature for 10 minutes, and add compound h (10.0g, 22.7mmol). The temperature was raised to 70°C, and pinacolborane (20.3g, 159mmol) was added dropwise. React at 95°C for 4 hours.
  • Step 11 Add compound i (9.00g, 15.8mmol) and tetrahydrofuran (30mL) into a 50mL single-neck bottle at room temperature. Cool to 0°C and add 2M hydrochloric acid (50 mL). Stir at 50°C for 16 hours. Concentrate under reduced pressure, add ethyl acetate (300 mL ⁇ 3) for extraction, dry over anhydrous sodium sulfate, and filter. Concentrate under reduced pressure, and the residue was purified by reverse-phase column chromatography (acetonitrile/water/trifluoroacetic acid) to obtain compound I-A (3.82g).
  • Dissolve compound I (15 mg, 42.3490 ⁇ mol) in 0.2 ml methanol, add lithium hydroxide (10.7 mg), heat the solution to 50°C for 1 hour, stir at 50°C for 4 hours, and then program the temperature to 0°C within 6 hours. , stirred at 0°C for 48 hours, continued the reaction, and dried under vacuum to obtain the lithium salt of compound I.
  • Dissolve compound I (15 mg, 42.3490 ⁇ mol) in 0.2 ml good solvent (Table 1), add hydrogen Lithium oxide (10.7 mg), heat the solution to 50°C for 1 hour, stir at 50°C for 4 hours, program to cool down to 0°C within 6 hours, stir at 0°C for 48 hours, filter, and vacuum dry, the solid obtained is defined by XRPD detection For crystal form ⁇ . Its XRPD spectrum is shown in Figure 6, its characteristic peak positions are shown in Table 2, its DSC spectrum is shown in Figure 7, and its TGA spectrum is shown in Figure 8.
  • Compound A was prepared using the method disclosed in the patent application "Example 4 on page 181 of the specification in WO2020070651A”.
  • IC 50 value where the maximum value refers to the reading value of DMSO control, and the minimum value refers to the reading value of no enzyme activity control.
  • Test Example 2 Inhibitory effect of compounds on the release of pro-inflammatory cytokines from peripheral blood mononuclear cells (PBMC)
  • PBMC frozen PBMC and detect cell viability and number by trypan blue staining. Wash the thawed PBMC with RPMI1640 complete medium (RPMI1640+10% FBS+1% PS), centrifuge and discard the supernatant. Resuspend PBMC in RPMI1640 complete medium and adjust the cell density to 2 ⁇ 10 6 cells/mL. Spread 2 ⁇ 10 5 PBMC cells in a 96-well cell culture plate, add different concentrations of the compound to be tested, starting from the maximum compound concentration of 100 ⁇ M, make 9 concentration gradient dilutions at a ratio of 1:5, and detect in duplicate wells. Add LPS at a final concentration of 0.1ng/mL, with a total volume of 200 ⁇ L.
  • Test Example 3 In vitro experiment of compounds inhibiting the secretion of IL-23 by DC cells differentiated from human monocytes
  • Day 0 Isolate and purify monocytes from fresh human peripheral blood and resuspend them in complete RPMI-1640 culture medium.
  • IL-4 at a concentration of 50ng/ml and 100ng/ml to the culture medium.
  • GM-CSF was cultured in a culture dish with a diameter of 100 mm at a density of 1 ⁇ 10 6 cells/mL.
  • the incubator temperature was 37°C and the carbon dioxide concentration was 5%.
  • Day 3 Replace half the volume of complete RPMI culture medium with fresh complete RPMI culture medium, keeping the concentration of IL-4 at 50ng/ml and the concentration of GM-CSF at 100ng/ml.
  • DC cells non-adherent cells
  • the washed DC cells are resuspended in complete RPMI culture medium.
  • the resuspended cell density is 1 ⁇ 10 6 cells/mL.
  • 1 ⁇ 10 5 DC cells were added to each well of the 96-well cell culture plate, and different concentration gradients were added.
  • the test compound (or equivalent concentration of DMSO blank negative control) was pretreated and incubated for 1 hour, and then 200ug/ml TLR2 agonist Zymosan was added to stimulate DC cells for 24 hours.
  • Day 7 After zymosan stimulates DC cells for 24 hours, collect the supernatant from each well in the 96-well plate, and detect the concentration of IL-23 by ELISA.
  • mice Seven-week-old Balb/c female mice were selected. The backs were shaved the day before the experiment, with a shaving area of 2cm ⁇ 3cm. On days 1-7 of the experiment, imiquimod (IMQ) was applied to the skin after applying the test substance for 6 hours. ) ointment (Aldara (5%)) was continuously applied to the back skin of mice for 7 days to construct a psoriasis mouse model. The control group was given the same dose of petroleum jelly ointment; the severity of skin inflammation was evaluated on days 3, 5, and 7. This includes measuring skin thickness, scabs, and erythema, and scoring each on a 5-point scale (0-5). The total score is used to assess the severity of skin inflammation. On the 7th day of the experiment, the spleen was taken and weighed, and the percentage of spleen in body weight was calculated to evaluate the degree of drug immunosuppression.
  • This experiment consists of a normal control group, a model control group, low, medium and high dose groups of compound I (0.01%, 0.03% and 0.1%), and a 0.03% reference compound A group.
  • the total score of skin inflammation is a clinical score, and the index is relatively subjective. The higher the score, the more serious the disease.
  • the degree of increase in skin thickness is an objective evaluation index. The greater the increase in thickness, the more serious the disease.
  • the proportion of spleen to body weight is an objective evaluation index. The smaller the proportion of spleen body weight, the stronger the immunosuppressive effect of the drug.

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Abstract

本公开涉及一种硼酸酯衍生物的可药用盐、其结晶形式及用途。具体而言，本公开提供了式(I)所示化合物的可药用盐、其结晶形式及用途。

Description

一种硼酸酯衍生物的可药用盐、其结晶形式及用途 技术领域
本公开属于药学领域，涉及一种硼酸酯衍生物的可药用盐、其结晶形式及用途。
背景技术
磷酸二酯酶(PDEs)为一类裂解在第二信使分子3',5'-环腺苷单磷酸(cAMP)和3',5'-环鸟苷单磷酸(cGMP)上的磷酸二酯键的细胞内酶。环核苷酸cAMP和cGMP在各种细胞途径中充当第二信使。其中，PDE4对cAMP具有高度特异性，有4种亚型：PDE4A、PDE4B、PDE4C和PDE4D。PDE4参与了促进单核细胞与巨噬细胞活化、中性粒细胞浸润、血管平滑肌的增殖、血管扩张以及心肌收缩等相关生理病理过程，对中枢神经系统功能、心血管功能、炎症/免疫系统、细胞黏附等都有影响。PDE4在促炎介质和抗炎介质的表达中起主要调节作用，PDE4抑制剂能够抑制炎症细胞释放有害介质。
近年发现许多PDE4抑制剂。例如，罗氟司特获准用于严重慢性阻塞性肺病(COPD)以减少突然发作的次数或防止COPD症状恶化；阿普司特获批用于治疗患有活动性牛皮癣性关节炎的成人。虽然PDE4抑制剂显示良好药理活性，但这些PDE抑制剂会出现副作用，诸如诱发性胃肠症状如呕吐及腹泻。仍需要开发选择性PDE4抑制剂，尤其对PDE4B和PDE4D具有亲和力的选择性PDE4抑制剂。
含硼(B)药物克立硼罗于2016年12月14日经FDA批准为一种轻度至中度特应性皮炎的局部治疗药物。该硼原子有助于皮肤渗透并与磷酸二酯酶4(PDE4)的双金属中心结合。另外，其他含硼的PDEs抑制剂小分子已有报道，诸如CN102014927A、WO2020070651。然而本公开的化合物并没有在任何文献中公开，且该类化合物展现特异性PDE4抑制效果。
PCT/CN2021/141010提供了一类新型的PDE4抑制剂，其化学名为(R)-4-(6-(8-甲氧基-2,2-二甲基苯并二氢吡喃-5-基)吡嗪-2-基)-1,2-氧杂硼杂环戊烷-2-醇，具有式I所示的结构，
作为药用活性成分的晶型往往影响到药物的化学稳定性，结晶条件及储存条件的不同有可能导致化合物的晶型结构的变化，有时还会伴随着产生其他形态的 晶型。一般来说，无定形的药物产品没有规则的晶型结构，往往具有其它缺陷，比如产物稳定性较差，析晶较细，过滤较难，易结块，流动性差等。药物的多晶型对产品储存、生产及放大有不同的要求。因此，深入研究前述化合物的晶型，改善前述化合物的各方面性质是很有必要的。
发明内容
本公开提供了一种式I所示化合物的可药用盐，
在一些实施方案中，所述可药用盐选自锂盐。
在一些实施方案中，所述式I所示化合物与碱分子或阳离子的摩尔配比为1:0.5～1:3，例如1:0.5、1:1、1:2或1:3。
在一些实施方案中，所述式I所示化合物与碱分子或阳离子的摩尔配比为1:1。
在一些实施方案中，所述式I所示化合物与酸分子或酸根的化学配比为1:0.5～1:3，例如1:0.5、1:1、1:2或1:3。本公开还提供一种上述式I所示化合物的可药用盐的制备方法，其包括将式I所示化合物与碱成盐的步骤，或者将式I所示化合物与酸成盐的步骤。
在一些实施方案中，成盐反应所用的溶剂选自乙腈、甲醇、乙醇、四氢呋喃、丙酮和二氧六环中的一种或多种。
在一些实施方案中，成盐反应所用溶剂的体积(μl)为所述式I化合物质量(mg)的1-200倍，在非限制性实施方案中为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或任意两数之间的值。
在一些实施方式中，所述的制备方法还包括离心、洗涤或干燥步骤。
本公开还提供了式I所示化合物的锂盐的晶型α，其以衍射角2θ角度表示的X-射线粉末衍射图，在13.913°、17.884°、19.894°、21.182°、33.454°处有特征峰，
在一些实施方案中，所述晶型α，以衍射角2θ角度表示的X-射线粉末衍射图，在11.855°、13.913°、14.383°、17.884°、19.894°、21.182°、22.148°、29.948°、30.310°、33.454°处有特征峰。
在一些实施方案中，所述晶型α，以衍射角2θ角度表示的X-射线粉末衍射图，在11.855°、13.913°、14.383°、17.884°、19.894°、21.182°、22.148°、23.910°、29.948°、30.310°、33.454°、36.812°处有特征峰。
在一些实施方案中，所述晶型α，以衍射角2θ角度表示的X-射线粉末衍射图，在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°、36.812°处有特征峰。
在一些实施方案中，所述晶型α，以衍射角2θ角度表示的X-射线粉末衍射谱图如图6所示。
本公开还提供了一种上述晶型α的制备方法，其包括以下步骤：(a)将式I所示化合物、氢氧化锂和溶剂混合，搅拌；(b)析晶；
在一些实施方案中，步骤(a)中的溶剂选自乙腈、甲醇、乙醇、四氢呋喃、丙酮和二氧六环中的一种或多种。
在一些实施方案中，步骤(a)中的溶剂所用体积(μl)为式I所示化合物质量(mg)的1-200倍，在非限制性实施方案中为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或任意两数之间的值。
在一些实施方案中，步骤(a)中，所述氢氧化锂的质量为式I所示化合物质量的0.5-3倍，在非限制性实施方案中为0.5、0.7、1、1.5、2、2.5、3或任意两数之间的值。
在一些实施方案中，步骤(a)中，所述搅拌的温度为10-70℃，在非限制性实施方案中为10℃、20℃、30℃、40℃、50℃、60℃、70℃或任意两数之间的值。
在一些实施方案中，步骤(a)中，所述搅拌的温度为50℃。
在一些实施方案中，步骤(b)中，所述析晶为冷却析晶，所述冷却析晶的温 度优选0℃。
在一些实施方案中，所述的析晶之后，还包括过滤、洗涤或干燥步骤。
本公开还提供了一种药物组合物，其包括上述式I所示化合物的可药用盐和任选地药学上可接受的赋形剂。
本公开还提供了一种药物组合物，其包括上述晶型α或由上述制备方法制得的晶型α，和任选地药学上可接受的赋形剂。
本公开还提供了一种上述药物组合物的制备方法，其包括将上述式I所示化合物的可药用盐或上述晶型α或由上述制备方法制得的晶型α，与药学上可接受的赋形剂混合的步骤。
本公开还提供了一种上述式I所示化合物的可药用盐或上述晶型α或由上述制备方法制得的晶型α或上述药物组合物在制备用于预防和/或治疗与PDE相关疾病的药物中的用途。
在一些实施方案中，所述与PDE相关疾病为气喘、阻塞性肺病、败血病、肾炎、糖尿病、变应性鼻炎、变应性结膜炎、溃疡性肠炎或风湿病。
本公开还提供了一种上述上述式I所示化合物的可药用盐或晶型α或由上述制备方法制得的晶型α或上述药物组合物在制备用于预防和/或治疗气喘、阻塞性肺病、败血病、肾炎、糖尿病、变应性鼻炎、变应性结膜炎、溃疡性肠炎或风湿病的药物中的用途。
本公开所述的“2θ或2θ角度”是指衍射角，θ为布拉格角，单位为°或度；每个特征峰2θ的误差范围为±0.2(包括超过1位小数的数字经过四舍五入后的情况)，可以为-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。
依据《中国药典》2015年版四部中“9103药物引湿性指导原则”中引湿性特征描述与引湿性增重的界定，
潮解：吸收足量水分形成液体；
极具引湿性：引湿增重不小于15％；
有引湿性：引湿增重小于15％但不小于2％；
略有引湿性：引湿增重小于2％但不小于0.2％；
无或几乎无引湿性：引湿增重小于0.2％。
本公开中所述的“差示扫描量热分析或DSC”是指在样品升温或恒温过程中，测量样品与参考物之间的温度差、热流差，以表征所有与热效应有关的物理变化和化学变化，得到样品的相变信息。
本公开中所述干燥温度一般为25℃～100℃，优选40℃～70℃，可以常压干燥，也可以减压干燥，压强＜-0.08MPa。
本公开中所述的“室温”一般为25℃±5℃。
本公开中所述的“赋形剂”包括但不限于任何已经被美国食品和药物管理局批准对于人类或家畜动物使用可接受的任何助剂、载体、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增香剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂或乳化剂。
本公开所述的“打浆”是指利用物质在溶剂中溶解性差，但杂质在溶剂中溶解性好的特性进行纯化的方法，打浆提纯可以去色、改变晶型或去除少量杂质。
本公开晶型制备方法中所用的起始原料可以是任意形式化合物，具体形式包括但不限于：无定形、任意晶型、水合物、溶剂合物等。
本公开中数值，例如，有关物质含量为测定计算的数据，不可避免存在一定程度的误差。一般而言，±10％均属于合理误差范围内。随其所用之处的上下文而有一定程度的误差变化，该误差变化不超过±10％，可以为±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％，优选±5％。
本公开式I化合物(R)-4-(6-(8-甲氧基-2,2-二甲基苯并二氢吡喃-5-基)吡嗪-2-基)-1,2-氧杂硼杂环戊烷-2-醇(以下简称化合物I)参照PCT/CN2021/141010中方法制备获得，本文本引用相关内容以示说明。
附图说明
图1：测试例4中各组化合物在疾病模型的临床评分比对图。
图2：测试例4中各组化合物在红斑疾病模型的临床评分比对图。
图3：测试例4中各组化合物在银屑疾病模型的临床评分比对图。
图4：测试例4中各组化合物对皮肤厚度增加的抑制作用比对图。
图5：测试例4中各组化合物对脾脏体重占比影响的比对图。
图6：晶型α的XRPD谱图。
图7：晶型α的DSC谱图。
图8：晶型α的TGA谱图。
在图1-图6中，*表示同模型组相比p<0.05，**表示同模型组相比p<0.01，***表示同模型组相比p<0.001，▼、●、◆、和#这些符号仅用于区分组别。
具体实施方式
以下将结合实施例更详细地说明本公开内容，本公开中的实施例仅用于说明本公开中的技术方案，并非限定本公开中的实质和范围。
本公开所用到的缩写的解释如下：
XRPD        X-射线粉末衍射
DSC         示差扫描量热法
TGA         热重分析
DVS          动态水分吸附
1H-NMR       液态核磁氢谱
DMF          N,N-二甲基甲酰胺
MEK          甲基乙基酮
MTBE         甲基叔丁基醚
THF          四氢呋喃
IPA          异丙醇
DCM          二氯甲烷
ACN          乙腈
本公开中实验所用仪器的测试条件：
1、X-射线粉末衍射仪(X-ray Powder Diffraction,XRPD)
仪器型号：Malver Panalytical Aeris X-射线粉末衍射仪
射线：单色Cu-Kα射线(λ＝1.54188)
扫描方式：θ/2θ，扫描范围(2θ范围)：3.5～50°
电压：40kV，电流：15mA。
2、差示扫描量热仪(Differential Scanning Calorimeter,DSC)
仪器型号：TA DSC250
吹扫气：氮气；氮气吹扫速度：50mL/min
升温速率：10℃/min
温度范围：25℃-300℃。
3、热重分析仪(Thermogravimetric Analysis，TGA)
仪器型号：TA TGA550
吹扫气：氮气；氮气吹扫速度：20ml/min
升温速率：10℃/min
温度范围：30℃-350℃。
4、动态水分吸附(DVS)
检测采用SMS Intrinsic PLUS，在25℃，湿度从50％-0％-90％，步进为10％，判断标准为每个梯度质量变化dM/dT小于0.0002％，TMAX 360min，循环两圈。
5、本发明稳定性测试中所述的高效液相色谱法(HPLC)在Agilent 1260 Infinity II上采集，HPLC条件：色谱柱：Agilen XDB C18 5μm 4.6*250mm；流动相：A-0.05％TFA(水中)，B-ACN；流速：1.0ml/min；波长：220nm。
6、化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，氘代甲醇(Methanol-d4)，内标为四甲基硅烷(TMS)。
7、化合物I制备实施例中HPLC的测定使用Agilent1100高压液相色谱仪，GAS15B DAD紫外检测器，Water Vbridge C18 150*4.6mm 5μM色谱柱。
8、MS的测定用Agilent6120三重四级杆质谱仪，G1315D DAD检测器，Waters Xbridge C18 4.6*50mm，5μM色谱柱，以正/负离子模式扫描，质量扫描范围为80～1200。
9、制备HPLC条件：水；柱：Sunfire(Prep C18 OBD 19*250mm 10μm)。
10、手性柱拆分条件：柱：Chiralpak IG 5μm 30*250mm；流动相：Hex:EtOH＝35:65，15mL/min；温度：30℃；波长:254nm。
11、薄层层析硅胶板使用烟台黄海HSGF254硅胶板，薄层色谱法(TLC)使用硅胶板采用规格是0.2mm±0.03mm，薄层层析分离纯化产品采用的规格是0.4mm-0.5mm。
12、快速柱纯化系统使用Combiflash Rf150(TELEDYNE ISCO)或者Isolara one(Biotage)。
13、正向柱层析色谱法一般使用烟台黄海硅胶200～300目或300～400目硅胶为载体，或者使用常州三泰预填预填超纯正相硅胶柱(40-63μm，60g，24g，40g，120g或其它规格)。
本公开中的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自上海泰坦科技、ABCR GmbH&Co.KG、Acros Organics、Aldrich Chemical Company、韶远化学科技(Accela ChemBio Inc)、毕得医药等公司。
实施例中无特殊说明，反应能够均在氮气氛下进行。
氮气氛是指反应瓶连接一个约1L容积的氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
氢气是由上海全浦科学仪器公司QPH-1L型氢气发生仪制得。
氮气氛或氢气氛通常抽真空，充入氮气或氢气，反复操作3次。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
实施例1：化合物I的制备
步骤1)：化合物1a(2.0g，14.6mmol)和三乙胺(1.8g，17.8mmol)溶于N,N-二甲基甲酰胺(30mL)，溶液冷却至0℃。氮气氛下，将叔丁基二苯基氯硅烷(4.0g，14.6mmol)滴加至反应体系。升至室温继续搅拌至TLC检测反应完成，将反应液倒入水中，乙酸乙酯萃取(100mL×3)。有机相用水洗(50mL×2)，无水硫酸钠干燥，浓缩，残余物经硅胶柱层析色谱法纯化(乙酸乙酯/石油醚)，得到化合物1b(5.1g)，直接用于下一步反应。
步骤2)：氮气氛下，将化合物1b(5.1g，13.6mmol)、联硼酸频那醇酯(4.2g，16.3mmol)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(512mg，0.7mmol)、乙酸钾(2.0g，20.4mmol)和二氧六环(100mL)混合物升温至80℃，并且搅拌过夜。将反应液倒入水中，乙酸乙酯萃取(100mL×2)。用水洗涤(30mL×2)，无水硫酸钠干燥，浓缩，残余物经硅胶柱层析色谱法纯化(石油醚/乙酸乙酯)，得到 化合物1c(2.0g)，LCMS：m/z 423.2(M+H)+。
步骤3)：室温下，向2000mL单口瓶中依次加入化合物a(100g,492mmol)、DMF(1000mL)、碘化钾(92.6g，837mmol)、碘化亚铜(3.13g，9.85mmol)、碳酸钾(136g,985mmol)。氮气氛下，滴加3-氯-3-甲基-1-丁炔(100mL，886mmol)。70℃搅拌16小时。冷却至室温，加入水(1000mL)，石油醚(1000mL×3)萃取。无水硫酸钠干燥，过滤。减压浓缩，残余物经柱层析色谱法(石油醚/乙酸乙酯)纯化，得到化合物b(50g)。
1H 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)。
步骤4)：室温下，向500mL单口瓶中依次加入化合物b(20.0g,74.4mmol)、正己烷(200mL)、钯碳酸钙(1.95g 18.8mmol)。氢气氛下，室温搅拌16小时。过滤，减压浓缩，得到化合物c(19g)。
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)。
步骤5)：室温下，向50mL单口瓶中依次加入化合物c(10.0g，36.9mmol)，二乙基苯胺(10mL，62.5mmol)。210℃搅拌1小时。冷却至室温，用1M HCl溶液调节pH调至中性，乙酸乙酯萃取(200mL×3)，水洗(200mL×3)。减压浓缩，得到化合物d(9.1g)。
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)。
步骤6)：室温下，向100mL单口瓶中依次加入化合物d(5.00g，18.5mmol)、甲苯(25mL)、15(5.00g，15.9mmol)。氮气氛下，100℃搅拌2小时。冷却至室温，过滤。减压浓缩，得到化合物e(3.89g)。
1H NMR(400MHz,DMSO-d6)δ7.04(d,J＝8.8Hz,1H),6.75(d,J＝8.8Hz,1H),3.71(s,3H),2.63(t,J＝6.8Hz,2H),1.78(t,J＝6.8Hz,2H),1.26(s,6H)。
步骤7)：室温下，向500mL单口瓶中依次加入化合物e(28.4g，105mmol)、二氧六环(300mL)、联硼酸频那醇酯(31.9g，126mmol)、醋酸钾(20.6g，209mmol)、[1,1’-双(二苯基膦基)二茂铁]二氯化钯(7.66g，10.5mmol)。氮气氛下，105℃搅拌16时。冷却至室温，过滤。减压浓缩，残余物经柱层析色谱法(石油醚/乙酸乙酯)纯化，得到化合物f(28.4g)。
LCMS：m/z 319(M+H)+。
步骤8)：室温下，向250mL单口瓶中依次加入化合物f(6.20g，19.5mmol)、1,4-二氧六环(80mL)、水(16mL)、2,6-二氯吡嗪(2.90g，39.0mmol)、碳酸钾(5.39g，39.0mmol)、[1,1’-双(二苯基膦基)二茂铁]二氯化钯(1.43g，1.95mmol)。氮气氛下，110℃搅拌16小时。冷却至室温，过滤。减压浓缩，残余物经柱层析色谱 法(石油醚/乙酸乙酯)纯化得到化合物g(5.7g)。
LCMS：m/z 305(M+H)+。
步骤9)：室温下，向100mL单口瓶中加入化合物g(35.0g，115mmol)、化合物1c(44.5g，149mmol)、碳酸钾(23.8g，172mmol)、醋酸钾(16.9g，172mmol)和[1,1’-双(二苯基膦基)二茂铁]二氯化钯(8.41g，11.5mmol)于1,4-二氧六环(250mL)与水(5mL)混合溶剂中。搅拌至溶解，氮气置换3次，110℃搅拌16小时。冷却至室温，加水(500mL)，乙酸乙酯(300mL×3)萃取，无水硫酸钠干燥。过滤，减压浓缩，残余物经柱层析色谱法(石油醚/乙酸乙酯)纯化，得到化合物h(37g)。
LCMS：m/z 441.1(M+H)+。
步骤10)：室温下，向250mL三口瓶中加入1,2-双(二苯基膦)乙烷(1.81g，4.54mmol)、(1,5-环辛二烯)甲氧基铱(I)二聚体(1.50g，2.27mmol)和无水1,2-二氯乙烷(100mL)。室温搅拌10分钟，加入化合物h(10.0g，22.7mmol)。升温至70℃，滴加频那醇硼烷(20.3g，159mmol)。95℃反应4小时。降温到0℃，滴加甲醇(50mL)淬灭。浓缩，残余物经柱层析色谱法(石油醚/乙酸乙酯)纯化，得到化合物i(1.4g)。
LCMS：m/z 569.3(M+H)+。
步骤11)：室温下，在50mL单口瓶中加入化合物i(9.00g，15.8mmol)和四氢呋喃(30mL)。冷却至0℃，加入2M盐酸(50mL)。50℃搅拌16小时。减压浓缩，加入乙酸乙酯(300mL×3)萃取，无水硫酸钠干燥，过滤。减压浓缩，残余物经反相柱层析色谱法(乙腈/水/三氟乙酸)纯化，得化合物I-A(3.82g)。
利用手性分离(柱：Chiralpak IG 5μm 30*250mm；流动相：Hex:EtOH＝35:65；流速：15mL/min；温度：30℃；波长：254nm)，得到化合物I。
LCMS：m/z 355.0(M+H)+。
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)。
实施例2：化合物I的锂盐的制备
将化合物I(15mg，42.3490μmol)溶解于0.2ml甲醇中，加入氢氧化锂(10.7mg)，将溶液1小时升温至50℃，50℃恒温搅拌4小时后，6小时内程序降温至0℃，0℃搅拌48小时持续反应，真空干燥，得到化合物I的锂盐。
1H 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)。
实施例3：化合物I的锂盐的晶型α的制备
将化合物I(15mg，42.3490μmol)溶解于0.2ml良溶剂(表1)中，加入氢 氧化锂(10.7mg)，将溶液1小时升温至50℃，50℃恒温搅拌4小时后，6小时内程序降温至0℃，0℃搅拌48小时，过滤，真空干燥，所得固体经XRPD检测定义为晶型α。其XRPD谱图如图6所示，其特征峰位置如表2所示，其DSC谱图如图7所示，其TGA谱图如图8所示。
表1、化合物I的锂盐的晶型α的制备用溶剂及XRPD检测结果
表2、晶型α的XRPD特征衍射峰数据
生物学评价
以下结合测试例进一步描述本公开，但这些测试例并非意味着限制本公开中的范围。
化合物A的结构为：
化合物A是采用专利申请“WO2020070651A中说明书第181页的实施例4”公开的方法制备而得的。
测试例1、体外PDE4B酶活性检测实验
1、实验材料
2、实验步骤
先在试管中以90％的DMSO(10％的水)配制浓度为10mM的化合物储备溶液，并用其制备稀释梯度为1:5的系列稀释液，终浓度从100uM开始，低至0.05nM。
将0.2ul化合物溶液转入384孔反应板中，阴性对照和阳性对照均转入0.2ul的100％DMSO。然后向孔中加入10ul的2倍浓度PDE4B1酶溶液(终浓度为0.04nM)，对于无酶活对照孔，用10ul的1倍反应缓冲液(50mM HEPES，pH 7.5,0.0015％Brij-35)替代酶溶液。1000rpm离心1min，室温孵育15分钟。接着向384孔反应板每孔中加入10ul的2倍FAM-cAMP底物溶液(底物终浓度为0.1uM)，1000rpm离心1min，25℃反应30分钟。反应结束后向384孔反应板每孔中加入60ul的反应终止液终止反应，室温下摇床600rpm振荡避光孵育60分钟。
孵育结束后读取RLU数据并计算抑制率，根据浓度和抑制率拟合曲线计算出 IC50值，其中最大值是指DMSO对照的读值，最小值是指无酶活对照的读值。
本公开的化合物I在体外对PDE4B1酶活性抑制通过以上的试验进行测定，测得的IC50值见表3。
表3
测试例2、化合物对外周血单核细胞(PBMC)促炎细胞因子的释放的抑制作用
解冻冻存的PBMC，台盼蓝染色检测细胞活率和数目。用RPMI1640完全培养基(RPMI1640+10％FBS+1％PS)清洗解冻后的PBMC，离心后弃去上清。用RPMI1640完全培养基重悬PBMC，将细胞密度调至2×106个细胞/mL。铺2×105PBMC细胞于96孔细胞培养板中，加入不同浓度的待测化合物，从化合物最大浓度100μM开始，按照1:5的比例做9个浓度的梯度稀释，双复孔检测。加入终浓度为0.1ng/mL的LPS，总体积为200μL。设置阴性和阳性对照，阴性对照孔内只加LPS和终浓度为DMSO，阳性对照孔内除细胞和LPS之外，还加入1μg/mL的地塞米松作为阳性对照。将细胞于37℃培养箱孵育24小时。孵育完成后，收集100μL细胞培养上清，用ELISA检测TNF-α的水平。向每孔剩余的细胞中加入100μL CellTiter-Glo，检测细胞活力水平。计算化合物抑制TNF-α释放的IC50值。
本公开的化合物I在体外对PBMC促炎细胞因子释放的抑制通过以上的试验进行测定，测得的IC50值见表4。
表4
测试例3、化合物体外抑制人单核细胞分化的DC细胞分泌IL-23实验
第0天：从新鲜人外周血中分离纯化单核细胞并重悬于完全的RPMI-1640培养液内，分化DC细胞时，在培养液内加入浓度为50ng/ml的IL-4和100ng/ml的GM-CSF，以1×106个细胞/mL的密度在直径为100mm的培养皿中进行分化培养，培养箱温度37℃，二氧化碳浓度为5％。第3天：将一半体积的完全RPMI培养液替换为新鲜的完全RPMI培养液，保持IL-4的浓度为50ng/ml，GM-CSF的浓度为100ng/ml。第6天：收集培养皿内未贴壁的细胞(为DC细胞)并用PBS清洗，清洗后的DC细胞用完全RPMI培养液进行重悬，重悬细胞密为1×106个细胞/mL。然后向96孔细胞培养板中每孔加入1×105个DC细胞，并加入不同浓度梯度的待 测化合物(或同等浓度的DMSO空白阴性对照)预处理孵育1小时，而后加入200ug/ml TLR2激动剂酵母聚糖(Zymosan)刺激DC细胞24小时。第7天：酵母聚糖刺激DC细胞24小时后，收集96孔板中各孔的上清，用ELISA检测IL-23的浓度。
本公开的化合物在体外对人单核细胞分化的DC细胞分泌IL-23的抑制通过以上的试验进行测定，测得的IC50值见表5。
表5
测试例4、咪喹莫特诱导的银屑病抑制实验
取适量化合物I配制成下述成分的软膏：0.1％的化合物I，9％的己二醇，78.8％的白凡士林，5％的石蜡，7％的单双硬脂酸甘油酯，0.1％的二羟基丁基甲苯，机械搅拌至软膏。
1)造模和给药
选用7周龄Balb/c雌性小鼠，实验前一天进行背部剃毛，剃毛面积为2cm×3cm；实验第1-7天在皮肤涂抹受试物6小时后，将咪喹莫特(IMQ)软膏(Aldara(5％))连续涂抹于小鼠背部皮肤7天，构建银屑病小鼠模型，对照组给予同等剂量的凡士林软膏；第3、5、7天评估皮肤炎症的严重程度，包括测量皮肤厚度，结痂，红斑情况，采用5分制(0-5)分别进行打分。总评分用于评估皮肤炎症严重程度。实验第7天取脾脏称重，计算脾脏占体重百分比用于评估药物免疫抑制作用程度。
本实验设正常对照组，模型对照组，化合物I的低、中、高剂量组(0.01％，0.03％和0.1％)，以及0.03％参比化合物A组。
2)评价指标
皮肤炎症程度的总评分为临床评分，指标相对主观，评分越高代表疾病越严重。皮肤厚度增加程度为客观评价指标，厚度增加越大代表疾病越严重。脾脏占体重比重为客观评价指标，脾脏体重占比越小代表药物免疫抑制作用越强。
3)实验结果
3.1)临床评分
化合物A以及化合物I各剂量在实验终点显著降低了临床评分。(图1-图4)。从单项评分看，化合物I各剂量组主要改善模型银屑结痂情况以及减少皮肤厚度。在皮肤厚度评分中，化合物I低高剂量组均显著抑制皮肤厚度增加，化合物A无显著作用。0.03％的化合物I对皮肤厚度增加的抑制作用显著高于相同剂量的化合物A(图4)。结果显示化合物I在改善银屑病表征上显著优于参比化合物A。各 具体评分数据见表6。
表6
3.2)脾脏体重占比
在IMQ作用下，模型组及各给药组动物体重无显著区别，而模型组脾脏占总体积比显著增加(图5)，提示脾肿大。化合物A对脾脏体重占比无显著影响，化合物I三个剂量组显著降低脾脏体重占比。提示各化合物有免疫抑制作用，且化合物I作用呈量效关系。同剂量下(0.03％)，化合物I对脾脏占体重比作用与化合物A有显著差异，结果提示化合物I免疫作用强于化合物A。具体数据见表7。
表7

Claims (14)

  1. 一种式I所示化合物的可药用盐，其中所述可药用盐选自锂盐，
  2. 根据权利要求1所述的式I所示化合物的可药用盐，其中所述式I所示化合物与碱分子或阳离子的摩尔配比为1:0.5～1:3，优选1:0.5、1:1、1:2或1:3。
  3. 一种根据权利要求1或2所述的式I所示化合物的可药用盐的制备方法，其包括将式I所示化合物与碱成盐的步骤，
  4. 根据权利要求3所述的制备方法，其中成盐反应所用的溶剂选自乙腈、甲醇、乙醇、四氢呋喃、丙酮和二氧六环中的一种或多种。
  5. 一种式I所示化合物的锂盐的晶型α，其以衍射角2θ角度表示的X-射线粉末衍射图，在13.913°、17.884°、19.894°、21.182°、33.454°处有特征峰；优选在11.855°、13.913°、14.383°、17.884°、19.894°、21.182°、22.148°、29.948°、30.310°、33.454°处有特征峰；更优选在11.855°、13.913°、14.383°、17.884°、19.894°、21.182°、22.148°、23.910°、29.948°、30.310°、33.454°、36.812°处有特征峰；进一步优选在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°、36.812°处有特征峰；更进一步优选以衍射角2θ角度表示的X-射线粉末衍射图如图6所示，
  6. 根据权利要求5所述的式I所示化合物的锂盐的晶型α，其中所述2θ值误 差范围为±0.2°。
  7. 一种根据权利要求5或6所述的式I所示化合物的锂盐的晶型α的制备方法，其包括以下步骤：
    (a)将式I所示化合物、氢氧化锂和溶剂混合，搅拌；
    (b)析晶，
  8. 根据权利要求7所述的制备方法，其中所述溶剂选自乙腈、甲醇、乙醇、四氢呋喃、丙酮和二氧六环中的一种或多种。
  9. 根据权利要求7所述的制备方法，其中，在步骤(a)中，所述搅拌的温度为10-70℃，例如10℃、20℃、30℃、40℃、50℃、60℃、70℃，优选50℃。
  10. 根据权利要求7所述的制备方法，其中，在步骤(b)中，所述析晶为冷却析晶，所述冷却析晶的温度优选0℃。
  11. 一种药物组合物，其包含根据权利要求1或2所述的式I所示化合物的可药用盐或根据权利要求5或6所述的式I所示化合物的锂盐的晶型α或由根据权利要求7至10中任一项所述的制备方法制得的式I所示化合物的锂盐的晶型α，和任选地药学上可接受的赋形剂。
  12. 一种根据权利要求11所述的药物组合物的制备方法，其包括将根据权利要求1或2所述的式I所示化合物的可药用盐或根据权利要求5或6所述的式I所示化合物的锂盐的晶型α或由根据权利要求7至10中任一项所述的制备方法制得的式I所示化合物的锂盐的晶型α，与药学上可接受的赋形剂混合的步骤。
  13. 一种根据权利要求1或2所述的式I所示化合物的可药用盐或根据权利要求5或6所述的式I所示化合物的锂盐的晶型α或由根据权利要求7至10中任一项所述的制备方法制得的式I所示化合物的锂盐的晶型α或根据权利要求11所述的药物组合物在制备用于预防和/或治疗与PDE相关疾病的药物中的用途；优选地，所述与PDE相关疾病为气喘、阻塞性肺病、败血病、肾炎、糖尿病、变应性鼻炎、变应性结膜炎、溃疡性肠炎或风湿病。
  14. 一种根据权利要求1或2所述的式I所示化合物的可药用盐或根据权利要求5或6所述的式I所示化合物的锂盐的晶型α或由根据权利要求7至10中任一项所述的制备方法制得的式I所示化合物的锂盐的晶型α或根据权利要求11所述的药物组合物在制备用于预防和/或治疗气喘、阻塞性肺病、败血病、肾炎、糖尿病、变应性鼻炎、变应性结膜炎、溃疡性肠炎或风湿病的药物中的用途。
PCT/CN2023/101654 2022-06-24 2023-06-21 一种硼酸酯衍生物的可药用盐、其结晶形式及用途 WO2023246858A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020128290A1 (en) * 1995-05-19 2002-09-12 Etsuo Ohshima Derivatives of benzofuran or benzodioxole
CN113166177A (zh) * 2018-10-05 2021-07-23 辉瑞公司 含硼的pde4抑制剂
WO2022135550A1 (zh) * 2020-12-25 2022-06-30 瑞石生物医药有限公司 一种硼酸酯衍生物及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020128290A1 (en) * 1995-05-19 2002-09-12 Etsuo Ohshima Derivatives of benzofuran or benzodioxole
CN113166177A (zh) * 2018-10-05 2021-07-23 辉瑞公司 含硼的pde4抑制剂
WO2022135550A1 (zh) * 2020-12-25 2022-06-30 瑞石生物医药有限公司 一种硼酸酯衍生物及其用途

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