WO2021185312A1 - 一种tlr8激动剂晶型 - Google Patents

一种tlr8激动剂晶型 Download PDF

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WO2021185312A1
WO2021185312A1 PCT/CN2021/081533 CN2021081533W WO2021185312A1 WO 2021185312 A1 WO2021185312 A1 WO 2021185312A1 CN 2021081533 W CN2021081533 W CN 2021081533W WO 2021185312 A1 WO2021185312 A1 WO 2021185312A1
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crystal form
compound
crystalline
crystalline form
preparation
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PCT/CN2021/081533
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English (en)
French (fr)
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蔡哲
孙飞
丁照中
陈曙辉
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正大天晴药业集团股份有限公司
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Priority to EP21771264.5A priority Critical patent/EP4122931A4/en
Priority to CN202180015947.8A priority patent/CN115175906A/zh
Priority to US17/931,456 priority patent/US20230303557A1/en
Publication of WO2021185312A1 publication Critical patent/WO2021185312A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application belongs to the field of medicinal chemistry, and relates to the crystal form of a TLR8 (Toll-like receptor 8) agonist represented by formula (I) and a preparation method thereof, and also includes the preparation of a drug for the treatment of diseases responsive to TLR8 agonism by the crystal form In the application.
  • TLR8 Toll-like receptor 8
  • TLR Toll-like receptors
  • natural immunity natural immunity
  • TLR is a single transmembrane non-catalytic protein, which is mainly expressed in a series of immune cells, such as dendritic cells, macrophages, monocytes, T cells, B cells, and NK cells.
  • Toll-like receptors can recognize molecules with conservative structures derived from microorganisms. When microorganisms break through the body's physical barriers, such as skin and mucous membranes, TLR can recognize them and activate the body to produce immune cell responses.
  • TLR1, -2, -4, -5, and -6 mainly recognize extracellular stimuli, such as bacterial lipopolysaccharides, lipopeptides, flagellin, etc.
  • TLR3, -7, -8, and -9 are in cell endosomes It plays a role in phagocytosis and envelope dissolution, binding to their ligands, and can recognize nucleic acids of microorganisms.
  • TLR8 is mainly expressed in monocytes, macrophages and myeloid dendritic cells.
  • the TLR8 signaling pathway can be activated by bacterial single-stranded RNA, small molecule agonists and microRNAs.
  • the activation of TLR8 leads to the production of Th1 polar cytokines, such as IL-12, IL-18, TNF-a and IFN- ⁇ , and various costimulatory factors such as CD80 and CD86.
  • Th1 polar cytokines such as IL-12, IL-18, TNF-a and IFN- ⁇
  • CD80 and CD86 various costimulatory factors
  • activating TLR8 on antigen-presenting cells and other immune cells in the liver can activate IL-12 and other cytokines, thereby activating specific T cells and NK cells depleted by the virus, thereby rebuilding the liver.
  • Antiviral immunity can activate IL-12 and other cytokines, thereby activating specific T cells and NK cells depleted by the virus, thereby rebuilding the liver.
  • this application provides a crystal form of the compound of formula (I),
  • crystal form A of the compound of formula (I) is provided, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 10.50 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.34 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 8.86 ⁇ 0.20°, 10.50 ⁇ 0.20°, 11.38 ⁇ 0.20°, 17.80 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.34 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 8.86 ⁇ 0.20°, 10.50 ⁇ 0.20°, 11.38 ⁇ 0.20°, 13.18 ⁇ 0.20°, 17.80 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.34 ⁇ 0.20°, 26.86 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 8.86 ⁇ 0.20°, 10.50 ⁇ 0.20°, 11.38 ⁇ 0.20°, 13.18 ⁇ 0.20°, 16.48 ⁇ 0.20°, 17.80 ⁇ 0.20°, 20.36 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.34 ⁇ 0.20°, 24.42 ⁇ 0.20°, 26.86 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 8.86 ⁇ 0.20°, 10.50 ⁇ 0.20°, 11.38 ⁇ 0.20°, 13.18 ⁇ 0.20°, 16.04 ⁇ 0.20°, 16.48 ⁇ 0.20°, 17.80 ⁇ 0.20°, 19.96 ⁇ 0.20°, 20.36 ⁇ 0.20°, 20.72 ⁇ 0.20°, 21.92 ⁇ 0.20°, 22.34 ⁇ 0.20°, 23.00 ⁇ 0.20°, 24.42 ⁇ 0.20°, 26.12 ⁇ 0.20°, 26.86 ⁇ 0.20°, 28.68 ⁇ 0.20°.
  • the XRPD pattern of the above-mentioned crystal form A is shown in FIG. 1.
  • the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A has the starting point of the endothermic peak at 228.0 ⁇ 5°C.
  • thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A has a weight loss of 0.2261% at 250.00 ⁇ 3°C.
  • the TGA pattern of the above-mentioned crystal form A is shown in FIG. 3.
  • This application also provides a method for preparing crystal form A of the compound of formula (I), including:
  • the volume ratio of acetone to water is selected from 1:1 to 10; preferably 1:1, 1:2, 1:3, 1:4, 1:5 , 1:6, 1:7, 1:8, 1:9, 1:10 or any range of values.
  • step 1) of the method for preparing crystal form A further comprises stirring at a temperature selected from 25°C to 60°C; preferably 30 to 55°C; more preferably 40 to 50°C.
  • in the preparation method 1) of the above-mentioned crystal form A further comprises stirring in a time selected from 1 hour to 72 hours; preferably 4 to 52 hours; more preferably 8 to 32 hours.
  • the weight ratio of the compound of formula (I) to the solvent is selected from 1:1-30.
  • the present application provides a crystalline composition comprising the crystalline form of the present application, wherein the crystalline form accounts for more than 50% of the weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, most It is better than 95%.
  • the present application provides a pharmaceutical composition, which includes a therapeutically effective amount of the crystal form of the present application, optionally including pharmaceutically acceptable excipients.
  • the application also provides a pharmaceutical composition, which includes a therapeutically effective amount of the crystal form composition of the crystal form of the application, and optionally includes pharmaceutically acceptable excipients.
  • the present application provides the use of the above-mentioned crystal form, crystal form composition or pharmaceutical composition thereof in the treatment of diseases responsive to TLR8 agonism.
  • the present application provides a method for stimulating TLR8, which includes administering a therapeutically effective amount of the crystal form of the present application, the crystal form composition or the pharmaceutical composition thereof to an individual in need (preferably a human).
  • the present application provides the use of the crystal form of the present application, the crystal form composition or the pharmaceutical composition thereof in the preparation of a medicine for treating diseases responsive to TLR8 agonism.
  • the disease that responds to TLR8 agonism is selected from viral infections.
  • the viral infection is selected from hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • the compound of the application has significant agonistic activity on TLR8.
  • the compounds of this application exhibit ideal TLR8 agonistic activity and specific selectivity.
  • the compound of the application exhibits ideal TLR8 pathway specific cytokine (IL-12p40, IFN- ⁇ ) inducing activity.
  • TLR8 agonists are immunomodulators. Excessive exposure may lead to excessive activation of the body's immune system, resulting in unforeseen side effects.
  • the crystal form A of the present application is easy to obtain, has good physical and chemical stability, at the same time, has good pharmaceutical properties, is suitable for use as a medicine, and therefore has high industrial application value and economic value.
  • the intermediate compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those of those skilled in the art.
  • Well-known equivalent alternatives, and preferred implementations include but are not limited to the examples of the present application.
  • Pd/C Pd/C catalyst palladium content 10w% DCM Dichloromethane THF Tetrahydrofuran Boc Tert-Butoxycarbonyl, is an amine protecting group Cbz Benzyloxycarbonyl is an amine protecting group DMF N,N-Dimethylformamide TFA Trifluoroacetate PE Petroleum ether DMSO Dimethyl sulfoxide EtOH Ethanol MeOH Methanol HOAc Acetic acid Trt Trityl
  • the powder X-ray diffraction (X-ray powder diffractometer, XRPD) of this application is detected by Dandong Haoyuan DX-2700BH X-ray diffractometer, light tube: Cu, k ⁇ ,
  • the differential thermal analysis (Differential Scanning Calorimeter, DSC) of this application uses a TA2500 differential scanning calorimeter for detection.
  • thermogravimetric Analyzer As used in this application, the thermal Gravimetric Analyzer (TGA) of this application is tested by TA TGA550 thermogravimetric analyzer.
  • the position of the peak or the relative intensity of the peak may be different due to factors such as measuring instrument, measuring method/condition and so on.
  • the measurement error of the 2 ⁇ value may be ⁇ 0.2°. Therefore, when determining each crystal type, this error should be taken into account, and the error also belongs to the scope of this application.
  • the relative intensity of the peaks in the XRPD can be calculated by peak height or peak area.
  • the relative intensity in the "Table 1 XRPD pattern analysis data of the crystal form of compound A of formula (I)" is calculated based on the peak height.
  • the position of the endothermic peak of DSC may be different due to factors such as measuring instrument, measuring method/condition and so on.
  • there may be an error in the position of the endothermic peak which can be ⁇ 5°C or ⁇ 3°C. Therefore, when determining each crystal type, this error should be taken into account, and the error also belongs to the scope of this application.
  • the location of the TGA weight loss temperature may be different due to factors such as the measuring instrument, the measuring method/condition and other factors.
  • the measuring instrument the measuring method/condition and other factors.
  • there may be an error in the position of the weight loss temperature which can be ⁇ 5°C or ⁇ 3°C. Therefore, when determining each crystal type, this error should be taken into account, and the error also belongs to the scope of this application.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues, but not Many toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipients” refer to inert substances that are administered together with the active ingredients to facilitate the administration of the active ingredients, including but not limited to those acceptable for use in humans or animals (such as those approved by the State Food and Drug Administration) Livestock) any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, Isotonic agent, solvent or emulsifier.
  • pharmaceutical composition refers to a mixture of one or more of the compounds of the application or their salts and pharmaceutically acceptable excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • the therapeutic dose of the compound of the present application or its crystalline form may be determined according to, for example, the following: the specific use of the treatment, the manner of administering the compound, the health and condition of the patient, and the judgment of the prescribing physician.
  • the ratio or concentration of the compound of the present application in the pharmaceutical composition may not be fixed, depending on various factors, including dosage, chemical properties (for example, hydrophobicity), and route of administration.
  • the dosage frequency of the crystal form described in the present application is determined by the needs of the individual patient, for example, once or twice a day, or more times a day.
  • the administration may be intermittent, for example, where the patient receives the daily dose of the crystal form during a period of several days, and then the patient does not receive the daily dose of the crystal form during a period of several days or more.
  • treatment means administering the compound or formulation described in this application to ameliorate or eliminate a disease or one or more symptoms related to the disease, and includes:
  • the term "therapeutically effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect.
  • the determination of the effective amount varies from person to person, and depends on the age and general conditions of the recipient, as well as the specific active substance. The appropriate effective amount in each case can be determined by those skilled in the art according to routine experiments.
  • Figure 1 is an XRPD spectrum of the crystal form of compound A of formula (I).
  • Figure 2 is a DSC spectrum of the crystal form of compound A of formula (I).
  • Figure 3 is a TGA spectrum of the crystal form of compound A of formula (I).
  • Step A Dissolve 1-1 (50 g, 412.54 mmol) and tetraethyl titanate (9 4.10 g, 412.54 mmol, 85.55 mL) in THF (500 mL) at 20-30 degrees Celsius, then add 2- Hexanone (41.32 g, 412.54 mmol, 51.01 ml). The reaction solution was heated to 65 degrees Celsius and stirred for 48 hours, and the obtained reaction solution was directly used in the next step.
  • Step B Cool the reaction solution of Step A to room temperature and add THF (1000 ml), then add allyl bromide (196.33 g, 1.62 mol), then slowly add zinc powder (53.06 g, 811.43 mmol) in batches, and react The liquid was stirred for 12 hours at 20 to 30 degrees Celsius under nitrogen protection.
  • the reaction solution was filtered through diatomaceous earth, and the filtrate was added with 100 ml of saturated brine, stirred and filtered through diatomaceous earth again, and the filtrate was spin-dried.
  • Step C Dissolve 1-3 (15 g, 61.12 mmol) in methanol (150 mL) and cool to 0 degrees Celsius, add hydrochloric acid dioxane solution (4M, 91.68 mL) slowly at 0 to 20 degrees Celsius, The reaction solution was stirred at 25 degrees Celsius for 2 hours. The reaction solution was directly concentrated under reduced pressure to obtain 1-4.
  • Step D Dissolve 1-4 (hydrochloride, 13 g, 73.15 mmol) and sodium bicarbonate (55.31 g, 658.36 mmol) in dioxane (90 mL) and H 2 O (60 mL), After cooling to 0 degrees Celsius, CbzCl (74.87 g, 438.91 mmol, 62.40 ml) was slowly added dropwise. The reaction solution was heated to 20-30 degrees Celsius and stirred for 2 hours, extracted twice with ethyl acetate, 100 ml each time, combined the organic phases, washed once with saturated brine (150 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure Got crude.
  • Step E Dissolve 1-5 (20.8 g, 75.53 mmol) in acetonitrile (100 mL), H 2 O (150 mL) and carbon tetrachloride (100 mL), cool to 0 degrees Celsius and slowly add periodic acid Sodium (64.62 g, 302.12 mmol), ruthenium trichloride trihydrate (394.99 mg, 1.51 mmol) was added, and the reaction solution was raised to 25 degrees Celsius and stirred for 2 hours. The reaction solution was filtered through Celite, and then extracted once with DCM (200 mL).
  • the organic phase was washed once with saturated aqueous sodium sulfite (200 mL), and then with saturated brine (200 mL), dried with anhydrous sodium sulfate, and filtered.
  • the crude product 1-6 was obtained by concentration under reduced pressure and used directly in the next step.
  • Step F Dissolve 1-6 (20 g, 68.18 mmol) and triethylamine (10.35 g, 102.26 mmol, 14.23 ml) in THF (250 ml), and add isochloroformic acid dropwise at minus 10 degrees Celsius under nitrogen protection Butyl ester (9.78 g, 71.59 mmol, 9.40 ml), stirred at minus 10 to 0 degrees Celsius for 30 minutes. Then ammonia water (63.70 g, 454.41 mmol, 70 ml, 25%) was slowly added, and the reaction was stirred at 0 to 5 degrees Celsius for 30 minutes. The reaction solution was concentrated under reduced pressure, and then extracted once with ethyl acetate (200 ml).
  • the organic phase was washed twice with saturated brine, 100 ml each time, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • Step G Stir 1-7 (15.34 g, 52.47 mmol) and N,N-dimethylformamide dimethyl acetal (134.55 g, 1.13 mol, 150 mL) at 120 degrees Celsius for 2 hours, under reduced pressure After concentration, it was dissolved in acetic acid (250 ml), and hydrazine hydrate (25.75 g, 504.09 mmol, 25 ml, 98%) was slowly added, and the mixture was stirred at 90 degrees Celsius for 2 hours under nitrogen protection.
  • reaction solution was concentrated under reduced pressure, added H 2 O (400 ml) and extracted twice with DCM, each 200 ml, the combined organic phase was washed twice with saturated brine, each 200 ml, dried with anhydrous sodium sulfate, filtered to reduce Press and concentrate to obtain crude product 1-8.
  • Step H 1-8 (15.20 g, 48.04 mmol) and DIPEA (12.42 g, 96.08 mmol, 16.74 mL) were dissolved in DCM (160 mL), and triphenylchloromethane (20.09 g, 72.06 mmol) was slowly added , The reaction solution was stirred at 25 degrees Celsius for 2 hours. Add H 2 O (100 ml) to the reaction solution and adjust the pH (7 ⁇ 8) with 2N dilute hydrochloric acid, and then extract once with 100 ml DCM. Wash the organic phase twice with saturated brine, each time 100 ml, anhydrous Dry over sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product.
  • Step I Dissolve 1-9 (12.75g, 22.82mmol) in isopropanol (300ml), add Pd/C (6g) under the protection of nitrogen, and degas the suspension under vacuum and replace it with hydrogen three times. (15psi) and stir at 25 degrees Celsius for 16 hours. The reaction solution was filtered through Celite and washed with 300 mL of DCM, and the filtrate was concentrated under reduced pressure to obtain 1-10.
  • Step A Dissolve compound 1-10 (1.91 g, 4.50 mmol) and 1-11 (900 mg, 4.50 mmol) in THF (9 mL), then add DIPEA (9.00 mmol, 1.57 mL), the mixture is in The reaction was stirred at 70 degrees Celsius for 3 hours under the protection of nitrogen. The reaction solution was concentrated under reduced pressure to obtain crude product 1-12.
  • Step B Crude product 1-12 (3.60 g, 6.12 mmol) and 2,4-dimethoxybenzylamine ((3.01 mg, 18.00 mmol, 2.71 ml) were dissolved in 1,4-dioxane (30 Under nitrogen protection, DIPEA (8.99 mmol, 1.57 ml) was added, and the mixture was stirred and reacted at 100 degrees Celsius for 12 hours.
  • the reaction solution was separated by adding water (20 ml) and ethyl acetate (50 ml), and the organic phase was saturated with Wash with brine (20 mL), dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product.
  • Step C Compound 1-13 (2.00 g, 2.78 mmol), triethylsilane (970.50 mg, 8.35 mmol, 1.33 mL) were dissolved in TFA (41.81 mL) and reacted with stirring at 28 degrees Celsius for 12 hours.
  • the reaction solution was directly concentrated under reduced pressure and then subjected to p-HPLC (column: Phenomenex luna C18 250*50mm*10 ⁇ m; fluidity: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 10%-40%, 28min, 50% min) Purification to obtain the hydrochloride salt of the compound of formula (I).
  • Experimental example 1 In vitro receptor binding activity screening experiment of human Toll-like receptor 7 (TLR7) and human Toll-like receptor 8 (TLR8)
  • the HEK-Blue TM hTLR7 (product number: hkb-htlr7) and HEK-Blue TM hTLR8 (product number: hkb-htlr8) cell lines used in this experiment were purchased from InvivoGen. These two cell lines are constructed by stably co-transfecting hTLR7 or hTLR8 with the human embryonic kidney 293 cell line and inducing the expression of the secreted alkaline phosphatase (SEAP) reporter gene, among which the secreted embryonic alkaline phosphatase (SEAP) reporter gene It is regulated by the IFN- ⁇ promoter.
  • SEAP secreted alkaline phosphatase
  • the promoter is fused with NF- ⁇ B and AP-1 binding sites, and hTLR7 or hTLR8 agonists will activate NF- ⁇ B and AP-1, and induce the expression and secretion of secreted alkaline phosphatase (SEAP).
  • SEAP secreted alkaline phosphatase
  • QUANTI-Blue TM reagent was used to detect the expression level of SEAP to identify the compound's agonistic activity on hTLR7 and hTLR8 receptors.
  • the compound was added to the cell plate in a 3-fold gradient, with a total of 10 concentrations (5000nM, 1667nM, 556nM, 185nM, 62nM, 21nM, 6.9nM, 2.3nM, 0.76nM, 0.25nM), each concentration was double-replicated. Add 1 ⁇ l DMSO to each well of the negative control well.
  • Compound activity detection Take 20 microliters of the cell supernatant from each well after induction, add it to the cell culture plate containing 180 ⁇ L QUANTI-Blue TM reagent, incubate at 37°C for 1 hour, then use the multifunctional microplate reader to detect The optical density absorption value (OD 650 ) of each hole at 650 nanometers.
  • Cell viability detection operate according to the ATPlite 1Step instruction method, and detect the luciferase signal (RLU) with a multifunctional microplate reader.
  • Test compound Human TLR8EC 50 ( ⁇ M) Human TLR7EC 50 ( ⁇ M) Hydrochloride of compound of formula (I) 0.003 33.33
  • the compound of the application exhibits ideal TLR8 agonist activity, and has the specific selectivity of TLR8 between TLR8 and TLR7.
  • TLR8 is a type of receptor for the innate immune system to sense foreign pathogens. It can recognize foreign viral single-stranded RNA and cause a series of cytokines, such as TNF- ⁇ , IL-12, and IFN- ⁇ to be released to cause resistance. Viral immune response; TLR7 is another type of receptor for the innate immune system to sense foreign pathogens. After being activated, it mainly produces antiviral cytokines such as IFN- ⁇ .
  • potential compounds of TLR8 agonists were used to stimulate human peripheral blood mononuclear cells (hPBMC), and the levels of TNF- ⁇ , IL-12p40, IFN- ⁇ and IFN- ⁇ were measured to reflect the compound's activation of TLR8 receptors. And TLR8/TLR7 selectivity.
  • CBA Flow cytometry bead microarray
  • ELISA enzyme-linked immunoassay
  • the compound of the present application has ideal TLR8 pathway specific cytokine IL-12p40, TNF- ⁇ and IFN- ⁇ inducing activity, relatively low inducing activity on TLR7 pathway specific cytokine IFN- ⁇ , showing an ideal pair The TLR8 pathway excites specifically and selectively.
  • Intravenous administration The test compound is formulated as a clear solution of 0.5 mg/ml, and the solvent is 5% DMSO/5% polyethylene glycol-15 hydroxystearate/90% water; intragastric administration: test compound Formulated into a 2 mg/ml suspension, the solvent is 0.5% sodium carboxymethyl cellulose/0.2% Tween 80/99.3% water.
  • the concentration of the test compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • LC-MS/MS high performance liquid chromatography-tandem mass spectrometry
  • the retention time of the compound and internal standard, chromatogram acquisition and chromatogram integration are processed by the software Analyst (Applied Biosystems), and the data statistics are processed by the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems).
  • mice The pharmacokinetic parameters of the hydrochloride salt of the compound of formula (I) in mice at a dose of 1 mg/Kg by intravenous injection and 5 mg/Kg by oral gavage are shown in Table 4 below.
  • Two parts are light samples (one part is a 5-day light sample, the other is a 10-day light sample); 2 light control samples (one is a 5-day light control sample, and the other is a 10-day light control sample). Place the illuminated sample into a clean watch glass, spread it into a thin layer, and cover it with a quartz glass cover.
  • the packaging method of the control sample is the same as that of the light sample, but the watch glass is covered with aluminum film.
  • Each sample is placed in an open watch glass and placed in high temperature conditions (60°C) for inspection.
  • Inner packaging double-layer medicinal low-density polyethylene bag
  • outer packaging aluminum foil bag.
  • N/A means not tested.

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Abstract

公开了式(I)所示的TLR8(Toll样受体8)激动剂的晶型及其制备方法，还包括所述晶型在制备治疗响应于TLR8激动的疾病的药物中的应用。

Description

一种TLR8激动剂晶型
相关申请的交叉引用
本申请要求于2020年03月18日向中国国家知识产权局提交的第202010193418.4号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请属于医药化学领域，涉及式(I)所示的TLR8(Toll样受体8)激动剂的晶型及其制备方法，还包括所述晶型在制备治疗响应于TLR8激动的疾病的药物中的应用。
背景技术
Toll样受体(Toll-like receptors，TLR)是参与非特异性免疫(天然免疫)的一类重要蛋白质分子，也是连接非特异性免疫和特异性免疫的桥梁。TLR是单个的跨膜非催化性蛋白质，主要表达于一系列免疫细胞，如树突细胞、巨噬细胞、单核细胞、T细胞、B细胞、NK细胞等。Toll样受体可以识别来源于微生物的具有保守结构的分子，当微生物突破机体的物理屏障，如皮肤、粘膜等时，TLR可以识别它们并激活机体产生免疫细胞应答。如TLR1，-2，-4，-5和-6主要识别胞外的刺激，如细菌的脂多糖，脂肽、鞭毛蛋白等，而TLR3，-7，-8，和-9在细胞内涵体中起作用，吞噬和包膜溶解后结合它们的配体，可识别微生物的核酸等。
在TLR不同亚型中，TLR8具有独特的功能：TLR8主要表达于单核细胞，巨噬细胞和髓样树突细胞中。TLR8的信号通路可以被细菌单链RNA，小分子激动剂和microRNAs活化。激活TLR8后导致产生Th1极性细胞因子，如IL-12，IL-18，TNF-a和IFN-γ和各种共刺激因子如CD80，CD86。这些细胞因子能激活和放大先天免疫和适应性免疫应答，并在涉及抗病毒、抗感染、自身免疫、肿瘤等疾病方面提供有益的治疗方案。例如，关于乙型肝炎，活化肝内抗原呈递细胞和其它免疫细胞上的TLR8可以激活IL-12等细胞因子，从而能激活被病毒耗尽的特异性T细胞和NK细胞，从而重建肝内的抗病毒免疫。
申请内容
一方面，本申请提供式(I)化合物的晶型，
Figure PCTCN2021081533-appb-000001
在本申请的一些方案中，提供式(I)化合物的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：10.50±0.20°、20.72±0.20°、22.34±0.20°。
在本申请的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、17.80±0.20°、20.72±0.20°、22.34±0.20°。
在本申请的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、13.18±0.20°、17.80±0.20°、20.72±0.20°、22.34±0.20°、26.86±0.20°。
在本申请的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、13.18±0.20°、16.48±0.20°、17.80±0.20°、20.36±0.20°、20.72±0.20°、22.34±0.20°、24.42±0.20°、26.86±0.20°。
在本申请的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、13.18±0.20°、16.04±0.20°、16.48±0.20°、17.80±0.20°、19.96±0.20°、20.36±0.20°、20.72±0.20°、21.92±0.20°、22.34±0.20°、23.00±0.20°、24.42±0.20°、26.12±0.20°、26.86±0.20°、28.68±0.20°。
本申请的一些方案中，上述A晶型的XRPD图谱数据如表1所示：
表1 式(I)化合物A晶型的XRPD图谱数据
编号 2θ角(±0.20°) 相对强度(％) 编号 2θ角(±0.20°) 相对强度(％)
1 8.86 22.9 14 22.34 53.6
2 10.50 46.0 15 23.00 10.2
3 11.38 27.8 16 23.80 7.9
4 13.18 17.9 17 24.42 13.7
5 15.68 8.6 18 24.64 9.2
6 16.04 13.8 19 25.28 5.4
7 16.48 17.9 20 26.12 12.8
8 17.80 35.0 21 26.86 21.8
9 19.54 9.0 22 27.38 4.1
10 19.96 18.4 23 28.68 10.5
11 20.36 30.5 24 30.16 5.6
12 20.72 100.0 25 32.46 5.5
13 21.92 11.5 / / /
。
本申请的一些方案中，上述A晶型的XRPD图谱如图1所示。
本申请的一些方案中，上述A晶型的差示扫描量热(DSC)曲线在228.0±5℃处具有吸热峰的起始点。
本申请的一些方案中，上述A晶型的DSC图谱如图2所示。
本申请的一些方案中，上述A晶型的热重分析(TGA)曲线在250.00±3℃处失重达0.2261％。
本申请的一些方案中，上述A晶型的TGA图谱如图3所示。
本申请还提供式(I)化合物的A晶型的制备方法，包括：
1)将式(I)化合物加入到丙酮与水的混合溶剂中；
2)析出固体，过滤，得到晶型A。
本申请的一些方案中，上述A晶型的制备方法，其中丙酮与水的体积比例选自1:1～10；优选1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10或任意值组成的范围。
本申请的一些方案中，上述A晶型的制备方法步骤1)中，进一步包括在选自25℃～60℃的温度下搅拌；优选30～55℃；更优选40～50℃。
本申请的一些方案中，上述A晶型的制备方法1)中，进一步包括在选自1小时～72小时的时间内搅拌；优选4～52小时；更优选8～32小时。
本申请的一些方案中，上述A晶型的制备方法，其中式(I)化合物与溶剂的重量比选自1:1～30。
本申请还有一些方案是由上述各变量任意组合而来。
另一方面，本申请提供包含本申请晶型的晶型组合物，其中，所述晶型占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上。
另一方面，本申请提供药物组合物，其包括治疗有效量的本申请的晶型，任选地包括药学上可接受的辅料。
本申请还提供药物组合物，其包括治疗有效量的本申请晶型的晶型组合物，任选地包括药学上可接受的辅料。
另一方面，本申请提供上述晶型、其晶型组合物或其药物组合物在治疗响应于TLR8激动的疾病中的用途。
另一方面，本申请提供激动TLR8的方法，包括对需要的个体(优选人类)给予治疗有效量的本申请的晶型、其晶型组合物或其药物组合物。
另一方面，本申请提供本申请的晶型、其晶型组合物或其药物组合物在制备治疗响应于TLR8激动的疾病的药物中的用途。
本申请的一些方案中，所述响应于TLR8激动的疾病选自病毒感染。
本申请的一些方案中，所述病毒感染选自乙型肝炎病毒(HBV)感染。
技术效果
本申请化合物对TLR8有显著的激动活性。本申请的化合物展示出理想的TLR8激动活性和特异选择性。本申请化合物展示出理想的TLR8通路特异性细胞因子(IL-12p40、IFN-γ)诱导活性。小鼠药代动力 学研究显示，本申请的化合物具有中等的口服生物利用度和药物曝露量，支持口服给药。TLR8激动剂属于免疫调节剂，过高的曝露量可能会导致机体免疫过度激活，从而产生不可预见的副作用。
本申请的A晶型易于获得、物理稳定性和化学稳定性均较好，同时，具有良好的药学性质，适合作为药物使用，因此具有很高的工业应用价值和经济价值。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。
本申请的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本申请，这些实施例并不意味着对本申请的任何限制。
本申请所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本申请采用下述缩略词：
Pd/C Pd/C催化剂，钯含量10w％
DCM 二氯甲烷
THF 四氢呋喃
Boc 叔丁氧羰基，是一种胺保护基团
Cbz 苄氧羰基，是一种胺保护基团
DMF N,N-二甲基甲酰胺
TFA 三氟乙酸
PE 石油醚
DMSO 二甲亚砜
EtOH 乙醇
MeOH 甲醇
HOAc 乙酸
Trt 三苯甲基
CbzCl 氯甲酸苄酯
DIPEA 二异丙基乙基胺
SiO 2 100-200目硅胶粉，用于柱层析
psi 磅力/平方英寸，压力单位
p-HPLC 制备高效液相色谱，用于化合物的纯化
如本申请所用，本申请的粉末X-射线衍射(X-ray powder diffractometer,XRPD)采用丹东浩元DX-2700BH X-射线衍射仪检测，光管：Cu,kα,
Figure PCTCN2021081533-appb-000002
如本申请所用，本申请的差热分析(Differential Scanning Calorimeter,DSC)采用TA2500差示扫描量热仪检测。
如本申请所用，本申请的热重分析(Thermal Gravimetric Analyzer,TGA)采用TA TGA550热重分析仪检测。
需要说明的是，在粉末X-射线衍射光谱中，峰的位置或峰的相对强度可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，峰的位置可能存在误差，2θ值的测定误差可以为±0.2°。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。
如本申请所用，所述XRPD中峰的相对强度可以通过峰高计算也可以通过峰面积计算。所述“表1式(I)化合物A晶型的XRPD图谱解析数据”中相对强度以峰高计算。
需要说明的是，对于同种晶型，DSC的吸热峰出现位置可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，吸热峰的位置可能存在误差，误差可以为±5℃，可以为±3℃。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。
需要说明的是，对于同种晶型，TGA的失重温度的出现位置可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，失重温度的位置可能存在误差，误差可以为±5℃，可以为±3℃。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。
所述词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。“药学上可接受的辅料”是指与活性成份一同给药的、有利于活性成份给药的惰性物质，包括但不限于国家食品药品监督管理局许可的可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对有机体给予本申请的化合物。
本申请化合物或其晶型的治疗剂量可根据例如以下而定：治疗的具体用途、给予化合物的方式、患者的健康和状态，以及签处方医师的判断。本申请化合物在药用组合物中的比例或浓度可不固定，取决于多种因素，它们包括剂量、化学特性(例如疏水性)和给药途径。
本申请所述晶型的剂量频率由患者个体的需求决定，例如，每天1次或2次，或每天更多次。给药可以是间歇性的，例如，其中在若干天的期间内，患者接受晶型的每日剂量，接着在若干天或更多天的期间，患者不接受晶型的每日剂量。
术语“治疗”意为将本申请所述化合物或制剂进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)抑制疾病或疾病状态，即遏制其发展；
(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
针对药物或药理学活性剂而言，术语“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
在本文中，除非上下文另有明确规定，否则单数术语涵盖复数指代物，反之亦然。
附图说明
图1为式(I)化合物A晶型的XRPD谱图。
图2为式(I)化合物A晶型的DSC谱图。
图3为式(I)化合物A晶型的TGA谱图。
具体实施方式
为了更好的理解本申请的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本申请的内容所做的限制。
实施例1
中间体1-10的制备：
Figure PCTCN2021081533-appb-000003
步骤A:20-30摄氏度下将1-1(50克，412.54毫尔)和钛酸四乙酯(9 4.10克，412.54毫摩尔，85.55毫升)溶于THF(500毫升)，再加入2-己酮(41.32克，412.54毫摩尔，51.01毫升)。将反应液升温至65摄氏度搅拌48小时，所得反应液直接用于下一步。
步骤B:将步骤A反应液冷却至室温补加THF(1000毫升)，再加入烯丙基溴(196.33克，1.62摩尔)，然后分批缓慢加入锌粉(53.06克，811.43毫摩尔)，反应液在氮气保护于20至30摄氏度下搅拌12小时。反应液通过硅藻土过滤，滤液加饱和食盐水100毫升，搅拌后再次通过硅藻土过滤，滤液旋干。残余物用乙酸乙酯(100毫升)溶解，分出的有机相用饱和食盐水(300毫升)洗涤一次，无水硫酸钠干燥，过滤减压浓缩得粗品。粗品经柱层析(SiO 2，PE/EtOAc＝15/1至5/1)纯化后得1-3。 1H NMR(400MHz，CDCl 3)δ5.96-5.75(m，1H)，5.23-5.08(m，2H)，3.20(s，1H)，2.39-2.20(m，2H)，1.74(br s，1H)，1.56-1.42(m，2H)，1.40-1.15(m，14H)，0.96-0.86(m，3H)。
步骤C:将1-3(15克，61.12毫摩尔)溶于甲醇(150毫升)并冷却至0摄氏度，将盐酸二氧六环溶液(4M，91.68毫升)在0至20摄氏度下缓慢加入，反应液在25摄氏度下搅拌2小时。将反应液直接减压浓缩得1-4。
步骤D:将1-4(盐酸盐，13克，73.15毫摩尔)和碳酸氢钠(55.31克，658.36毫摩尔)溶于二氧六环(90毫升)和H 2O(60毫升)，冷却至0摄氏度后缓慢滴加CbzCl(74.87克，438.91毫摩尔，62.40毫升)。反应液升温到20至30摄氏度搅拌2小时，用乙酸乙酯萃取两次，每次100毫升，合并有机相，并用饱和食盐水(150毫升)洗涤一次，无水硫酸钠干燥，过滤减压浓缩得粗品。粗品经柱层析(SiO 2，PE/EtOAc＝1/0至100/1)纯化后得1-5。 1H NMR(400MHz，CDCl 3)δ7.30-7.26(m，5H)，5.76-5.62(m，1H)，5.08-4.91(m，4H)，2.47-2.34(m，1H)，2.32-2.20(m，1H)，1.72-1.57(m，1H)，1.50-1.42(m， 1H)，1.30-1.10(m，7H)，0.76-0.76(m，1H)，0.76-0.76(m，1H)，0.82(t，J＝7.0Hz，2H)。
步骤E:将1-5(20.8克，75.53毫摩尔)溶于乙腈(100毫升)，H 2O(150毫升)和四氯化碳(100毫升)中，冷却至0摄氏度缓慢加入高碘酸钠(64.62克，302.12毫摩尔)，再加入三水合三氯化钌(394.99毫克，1.51毫摩尔)，反应液升至25摄氏度搅拌2小时。反应液通过硅藻土过滤，然后用DCM(200毫升)萃取一次，有机相用饱和亚硫酸钠水溶液(200毫升)洗涤一次，再用饱和食盐水(200毫升)洗涤一次，无水硫酸钠干燥，过滤减压浓缩得粗品1-6直接用于下一步。 1H NMR(400MHz，CDCl 3)δ7.40-7.35(m，5H)，5.19(s，1H)，5.08(s，2H)，2.89(br d，J＝14.5Hz，1H)，2.69(br d，J＝14.4Hz，1H)，1.90-1.77(m，1H)，1.74-1.62(m，1H)，1.43-1.20(m，7H)，0.90(t，J＝6.9Hz，3H)。
步骤F:将1-6(20克，68.18毫摩尔)和三乙胺(10.35克，102.26毫摩尔，14.23毫升)溶于THF(250毫升)，氮气保护下于零下10摄氏度滴加氯甲酸异丁酯(9.78克，71.59毫摩尔，9.40毫升)，零下10至0摄氏度搅拌30分钟。随后将氨水(63.70克，454.41毫摩尔，70毫升，25％)缓慢加入，反应在0至5摄氏度下搅拌30分钟。反应液减压浓缩，然后用乙酸乙酯(200毫升)萃取一次，有机相用饱和食盐水洗涤两次，每次100毫升，无水硫酸钠干燥，过滤减压浓缩得粗品。粗品经柱层析(SiO 2，PE/EtOAc＝10/1至1/1)纯化后得1-7。 1H NMR(400MHz，CDCl 3)δ7.41-7.28(m，5H)，5.62(br s，1H)，5.30-5.12(m，2H)，5.11-5.01(m，2H)，2.76(d，J＝13.2Hz，1H)，2.44(d，J＝13.3Hz，1H)，1.85-1.74(m，1H)，1.73-1.62(m，3H)，1.39-1.29(m，5H)，0.90(t，J＝7.0Hz，3H)。LCMS(ESI)m/z:293.3[M+H] +。
步骤G:将1-7(15.34克，52.47毫摩尔)和N,N-二甲基甲酰胺二甲基缩醛(134.55克，1.13摩尔，150毫升)在120摄氏度下搅拌2小时，减压浓缩后再溶于乙酸(250毫升)中，缓慢加入水合肼(25.75克，504.09毫摩尔，25毫升，98％)，氮气保护下90摄氏度搅拌2小时。反应液减压浓缩，加H 2O(400毫升)后用DCM萃取两次，每次200毫升，合并有机相用饱和食盐水洗涤两次，每次200毫升，无水硫酸钠干燥，过滤减压浓缩得粗品1-8。 1H NMR(400MHz，CDCl 3)δ7.95(s，1H)，7.43-7.29(m，5H)，5.08(s，2H)，4.90(br s，1H)，3.42(br d，J＝13.9Hz，1H)，3.12(d，J＝14.3Hz，1H)，1.87-1.77(m，1H)，1.68-1.58(m，1H)，1.41-1.17(m，7H)，0.90(br t，J＝6.5Hz，3H)。LCMS(ESI)m/z:317.2[M+H] +。
步骤H:1-8(15.20克，48.04毫摩尔)和DIPEA(12.42克，96.08毫摩尔，16.74毫升)溶于DCM(160毫升)，缓慢加入三苯基氯甲烷(20.09克，72.06毫摩尔)，反应液在25摄氏度下搅拌2小时。向反应液中加H 2O(100毫升)并用2N的稀盐酸调节pH(7～8)，再用100毫升DCM萃取一次，有机相用饱和食盐水洗涤两次，每次100毫升，无水硫酸钠干燥，过滤减压浓缩得粗品。粗品经柱层析(SiO 2，PE/EtOAc＝20/1至5/1)纯化后得1-9。 1H NMR(400MHz，CDCl 3)δ7.89(s，1H)，7.37-7.27(m，14H)，7.18-7.07(m，6H)，5.72(br s，1H)，5.16-4.93(m，2H)，3.07(d，J＝14.2Hz，1H)，2.90(d，J＝14.2Hz，1H)，1.80-1.61(m，4H)，1.33(s，3H)，0.90-0.84(m，3H)。LCMS(ESI)m/z:559.3[M+H] +。
步骤I:将1-9(12.75克，22.82毫摩尔)溶于异丙醇(300毫升)，氮气保护下加入Pd/C(6克)，悬浮液经真空脱气并用氢气置换三次，氢气氛围(15psi)下于25摄氏度搅拌16小时。反应液经硅藻土过滤，并用300毫升DCM洗涤，滤液减压浓缩所得1-10。 1H NMR(400MHz，CDCl 3)δ7.91(s，1H)，7.37-7.28(m，9H)，7.17-7.11(m，6H)，2.87(s，2H)，1.45-1.24(m，6H)，1.12(s，3H)，0.92-0.84(m，3H)。LCMS(ESI)m/z:425.2[M+H] +。
式(I)化合物盐酸盐的合成：
Figure PCTCN2021081533-appb-000004
步骤A:将化合物1-10(1.91克,4.50毫摩尔)和1-11(900毫克,4.50毫摩尔)溶于THF(9毫升)，再加入DIPEA(9.00毫摩尔,1.57毫升)，混合物在氮气保护下于70摄氏度搅拌反应3小时。反应液减压浓缩得到粗品1-12。LCMS(ESI)m/z:588.42[M+H] +。
步骤B：粗品1-12(3.60克,6.12毫摩尔)和2,4-二甲氧基苄胺((3.01毫克,18.00毫摩尔,2.71毫升)溶于1,4-二氧六环(30毫升)并氮气保护，再加入DIPEA(8.99毫摩尔,1.57毫升)，混合物在100摄氏度下搅拌反应12小时。反应液加水(20毫升)和乙酸乙酯(50毫升)分液，有机相用饱和食盐水(20毫升)洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。粗品经硅胶柱色谱(SiO 2，DCM/MeOH＝100/1至15/1)纯化后得1-13。LCMS(ESI)m/z:719.7[M+H] +。
步骤C:化合物1-13(2.00克,2.78毫摩尔)、三乙基硅烷(970.50毫克,8.35毫摩尔,1.33毫升)溶于TFA(41.81毫升)中后在28摄氏度下搅拌反应12小时。反应液直接减压浓缩后经p-HPLC(柱子:Phenomenex luna C18 250*50mm*10μm；流动性:[水(0.05％HCl)-乙腈]；乙腈％:10％-40％,28min,50％min)纯化，得到式(I)化合物的盐酸盐。 1H NMR(400MHz,CD 3OD)δ9.15(s,1H),8.90(s,1H),8.59(d,J＝5.6Hz,1H),8.26(d,J＝5.5Hz,1H),4.11(d,J＝14.8Hz,1H),3.53(d,J＝14.9Hz,1H),2.60(dt,J＝4.1,12.8Hz,1H),1.79(dt,J＝4.2,12.8Hz,1H),1.58(s,3H),1.52-1.19(m,4H),0.92(t,J＝7.2Hz,3H)。LCMS(ESI)m/z:327.1 [M+H] +。
实施例2：式(I)化合物A晶型的制备
将式(I)化合物的盐酸盐(1.4克)加入水(50毫升)中，用碳酸钠固体调节pH(9～10)，再用乙酸乙酯萃取两次，每次50毫升。将萃取液浓缩，浓缩物加入丙酮(10毫升)和水(10毫升)，将混合物在25℃搅拌1小时，减压浓缩除去丙酮(大约8mL)，室温(15～25摄氏度)搅拌约1小时，固体析出，过滤，滤饼经真空干燥箱干燥(45℃，16小时)得到白色固体即为A晶型。图1示出了该晶型的XRPD谱图，图2示出了该晶型的DSC谱图，图3示出了该晶型的TGA谱图。
实验例1：人Toll样受体7(TLR7)和人Toll样受体8(TLR8)体外受体结合活性筛选实验
本实验所用的HEK-Blue TM hTLR7(货号：hkb-htlr7)和HEK-Blue TM hTLR8(货号：hkb-htlr8)细胞株购于InvivoGen公司。这两个细胞株由人胚肾293细胞系稳定共转染hTLR7或hTLR8和诱导表达分泌型碱性磷酸酶(SEAP)报告基因所构建的，其中分泌型胚胎碱性磷酸酶(SEAP)报告基因由IFN-β启动子调控。该启动子与NF-κB和AP-1结合位点融合，hTLR7或hTLR8激动剂会激活NF-κB和AP-1，并诱导分泌型碱性磷酸酶(SEAP)的表达和分泌。用QUANTI-Blue TM试剂检测SEAP表达量，以此来鉴定化合物对hTLR7和hTLR8受体的激动活性。
实验步骤：
1.化合物按3倍梯度加入到细胞板中，共10个浓度(5000nM，1667nM，556nM，185nM，62nM，21nM，6.9nM，2.3nM，0.76nM，0.25nM)，每个浓度双复孔。阴性对照孔每孔加入1微升DMSO。
2.从CO 2培养箱取出T150培养的细胞，弃去细胞培养上清，用杜氏磷酸盐缓冲液(DPBS)清洗细胞一次，加入约10mL培养液，然后轻拍细胞培养瓶使细胞脱壁，然后用移液器将细胞团轻轻吹打均匀。细胞计数，并将细胞悬液用培养液调整到500000细胞/毫升。然后在含有化合物的96孔板中每孔加入100微升稀释后的细胞(50000细胞/孔)。
3.将化合物和细胞在37℃、5％CO 2培养箱共孵育24小时。
4.化合物活性检测：取20微升诱导后每个孔的细胞上清液，加入到含有180μL QUANTI-Blue TM试剂的细胞培养板中，37℃孵育1小时之后，用多功能酶标仪检测每孔在650纳米的光密度吸收值(OD 650)。
5.细胞活性检测：按照ATPlite 1Step说明书方法操作，荧光素酶信号(RLU)用多功能酶标仪检测。
6.数据分析：化合物活性：OD650值用GraphPad Prism软件分析，并拟合化合物剂量效应曲线，计算化合物的EC 50值(半数最大效应浓度)。
实验结果：如表2所示。
表2
受试化合物 人TLR8EC 50(μM) 人TLR7EC 50(μM)
式(I)化合物的盐酸盐 0.003 33.33
结论：本申请化合物展示出理想的TLR8激动剂活性，且在TLR8和TLR7之间的具有TLR8的特异选择性。
实验例2：外周血单个核细胞实验方案
TLR8是固有免疫系统感受外源性的病原体的一类受体，能够识别外源的病毒单链RNA，引起一系列细胞因子，如TNF-α，IL-12，IFN-γ的释放以引起抗病毒免疫反应；TLR7是另外一类固有免疫系统感受外源性的病原体的一类受体，被激活后主要产生IFN-α等抗病毒细胞因子。本实验使用TLR8激动剂的潜在化合物刺激人外周血单个核细胞(hPBMC)，通过检测上述TNF-α，IL-12p40，IFN-γ和IFN-α的水平来反映化合物对TLR8受体的活化作用和TLR8/TLR7的选择性。
实验步骤：
1.采集健康志愿者新鲜血液，EDTA-K2抗凝管(货号：BD-8516542)抗凝；
2.Ficoll密度梯度离心，分离中间云雾层的hPBMC细胞，含10％血清的RPMI1640(来源：Gibco，货号：224400-089)培养液洗2次，培养液重悬至10ml，Vi-cell细胞计数仪计数，调整细胞悬液浓度至2×10 6/mL；
3.用DMSO将化合物溶解至100mM，并分别用DMSO稀释至50mM,2mM，分别作为初始浓度，依次做3倍稀释，即取前一浓度样本5μL+10μLDMSO；稀释8个梯度；再分别用培养基稀释500倍，配成化合物工作液；
4.U底96孔板中，每孔加入hPBMC悬液100μL，以及化合物工作液100μL，终浓度依次为2000nM，666.7nM，222.2nM，74.1nM，24.7nM，8.2nM，2.7nM，0.9nM，孵育24小时，收上清-20℃冻存，待检测TNF-α，IFN-gamma，IL-12p40细胞因子；另一组化合物样本终浓度依次为50μM，16.7μM，5.6μM，1.9μM，0.6μM，0.2μM，0.1μM，0.02μM，孵育24小时，收上清-20℃冻存，待检测IFN-alpha细胞因子；
5.流式细胞小球微阵列术(CBA)检测上清中IL-12p40，TNF-α，IFN-γ；酶联免疫法(ELISA)检测细胞上清中IFN-α。
6.数据分析：化合物活性：EC 50值(半数最大效应浓度)用GraphPad Prism软件分析，并拟合化合物剂量效应曲线，计算化合物的EC 50值。
实验结果：如表3所示。
表3
受试化合物 IL-12p40 IFN-γ TNF-α IFN-α
  EC 50(nM) EC 50(nM) EC 50(nM) EC 50(nM)
式(I)化合物的盐酸盐 26 29 105 2800
结论：本申请化合物具有理想的TLR8通路特异性细胞因子IL-12p40，TNF-α和IFN-γ诱导活性，对TLR7通路特异性细胞因子IFN-α的诱导活性相对较低，展示出理想的对TLR8通路激动特异选择性。
实验例3：小鼠药物代谢动力学研究
本实验旨在评价化合物在小鼠体内单次静脉注射或灌胃给药后的药代动力学行为。静脉注射给药：受试化合物配制成0.5毫克/毫升的澄清溶液，溶媒为5％DMSO/5％聚乙二醇-15羟基硬脂酸酯/90％水；灌胃给药：受试化合物配制成2毫克/毫升的混悬液，溶媒为0.5％羧甲基纤维素钠/0.2％吐温80/99.3％水。
受试化合物在血浆中的浓度由高效液相色谱-串联质谱(LC-MS/MS)进行测定。化合物和内标的保留时间、色谱图采集和色谱图的积分采用软件Analyst(Applied Biosystems)进行处理，数据的统计采用软件Watson LIMS(Thermo Fisher Scientific)或Analyst(Applied Biosystems)进行处理。
采用WinNonlin TM Version 6.3(Pharsight，Mountain View，CA)药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数。
式(I)化合物的盐酸盐分别在静脉注射给药1mg/Kg和口服灌胃给药5mg/Kg剂量下的小鼠药代动力学相关参数如下表4所示。
表4
Figure PCTCN2021081533-appb-000005
实验例4：稳定性试验
1、影响因素试验
考查样品：
式(I)化合物A晶型
1)光稳定性试验
2份为光照样品(一份作为5天光照样品，另一份为10天光照样品)；2份光照对照样品(一份作为5天光照对照样品，另一份为10天光照对照样品)。光照样品放入干净的表面皿中，铺成薄层，加盖石英玻璃盖。对照样品的包装方式与光照样品一致，但在表面皿外面覆盖铝膜。
考察条件：光照箱。
光稳定性考察结果详见表5。
表5 光稳定性试验研究结果
Figure PCTCN2021081533-appb-000006
上述结果显示，在光照条件下，5天和10天的检测项目与初始值相比没有明显变化。说明A晶型具有良好的光照稳定性。
2)温度影响试验
每份样品放入敞口的表面皿中，放入高温条件(60℃)考察。
高温试验考察结果详见表6。
表6 高温(60℃)试验研究结果
Figure PCTCN2021081533-appb-000007
上述结果显示，在高温条件下，5天、10天和30天的检测项目与初始值相比没有明显变化。说明A晶型具有良好的高温稳定性。
3)湿度影响试验
每份样品放入敞口的扁形称量瓶中，放入高湿条件(25℃/92.5RH)考察。
高湿试验考察结果详见表7。
表7 高湿(25℃/92.5％RH)影响试验研究结果
Figure PCTCN2021081533-appb-000008
上述结果显示，在高湿条件下，5天、10天和30天的检测项目与初始值相比没有明显变化。说明A晶型具有良好的高湿稳定性。
2、加速试验
考查样品：
式(I)化合物A晶型
包装形式：
内包装：双层药用低密度聚乙烯袋；外包装：铝箔袋。
考察条件：40℃±2℃/75±5％RH
加速试验考察结果详见表8。
表8 加速试验(40℃±2℃/75±5％RH)研究结果
Figure PCTCN2021081533-appb-000009
备注：N/A表示未检测。
XRPD结果显示3个月时与初始值相比未有明显变化，说明A晶型在加速试验条件下保持稳定。

Claims (15)

  1. 式(I)化合物的晶型，
    Figure PCTCN2021081533-appb-100001
  2. 根据权利要求1所述的晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：10.50±0.20°、20.72±0.20°、22.34±0.20°；典型地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、17.80±0.20°、20.72±0.20°、22.34±0.20°；更典型地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、13.18±0.20°、17.80±0.20°、20.72±0.20°、22.34±0.20°、26.86±0.20°；进一步典型地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.86±0.20°、10.50±0.20°、11.38±0.20°、13.18±0.20°、16.48±0.20°、17.80±0.20°、20.36±0.20°、20.72±0.20°、22.34±0.20°、24.42±0.20°、26.86±0.20°。
  3. 根据权利要求1或2任一项所述的晶型，其XRPD图谱如图1所示。
  4. 根据权利要求1～3任意一项所述的晶型，其差示扫描量热曲线在228.0±5℃处具有吸热峰的起始点。
  5. 根据权利要求1～4任意一项所述的晶型，其DSC图谱如图2所示。
  6. 根据权利要求1～5任意一项所述的晶型，其热重分析曲线在250.00±3℃处失重达0.2261％。
  7. 根据权利要求1～6任意一项所述的晶型，其TGA图谱如图3所示。
  8. 权利要求2-7任一项所述的晶型的制备方法，包括：1)将式(I)化合物加入到丙酮与水的混合溶剂中；2)析出固体，过滤，得到晶型。
  9. 根据权利要求8所述的制备方法，其中丙酮与水的体积比例选自1:1～10。
  10. 根据权利要求8或9任意一项所述的制备方法，其中包括在选自25℃～60℃的温度下搅拌。
  11. 根据权利要求8-10任意一项所述的制备方法，其中包括在选自1小时～72小时的时间内搅拌。
  12. 根据权利要求8-11任意一项所述的制备方法，其中式(I)化合物与溶剂的重量比选自1:1～30。
  13. 晶型组合物，其包括权利要求1-7任意一项所述的晶型，所述晶型占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上
  14. 药物组合物，其包括治疗有效量的权利要求1-7任意一项所述的晶型或权利要求13所述的晶型组合物，任选地包括药学上可接受的辅料。
  15. 权利要求1～7任意一项所述的晶型、或权利要求13所述的晶型组合物或权利要求14所述的药物组合物在治疗响应于TLR8激动的疾病中的用途。
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