WO2019144970A1 - 一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法 - Google Patents

一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法 Download PDF

Info

Publication number
WO2019144970A1
WO2019144970A1 PCT/CN2019/073701 CN2019073701W WO2019144970A1 WO 2019144970 A1 WO2019144970 A1 WO 2019144970A1 CN 2019073701 W CN2019073701 W CN 2019073701W WO 2019144970 A1 WO2019144970 A1 WO 2019144970A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
solvent
crystalline form
water
group
Prior art date
Application number
PCT/CN2019/073701
Other languages
English (en)
French (fr)
Inventor
雷茂义
徐雨
罗云富
Original Assignee
石家庄智康弘仁新药开发有限公司
南京明德新药研发股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 石家庄智康弘仁新药开发有限公司, 南京明德新药研发股份有限公司 filed Critical 石家庄智康弘仁新药开发有限公司
Priority to EP19743686.8A priority Critical patent/EP3747884B1/en
Priority to JP2020541359A priority patent/JP7237973B2/ja
Priority to US16/965,377 priority patent/US11325907B2/en
Priority to AU2019211931A priority patent/AU2019211931B2/en
Priority to CA3089414A priority patent/CA3089414A1/en
Priority to RU2020127989A priority patent/RU2784538C2/ru
Priority to CN201980010305.1A priority patent/CN111683949B/zh
Publication of WO2019144970A1 publication Critical patent/WO2019144970A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • 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
    • 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

Definitions

  • the invention relates to a crystal form of 1H-imidazo[4,5-b]pyridine-2(3H)-one compound and a preparation method thereof, and the application of the crystal form in preparing medicine for treating diseases related to PDE4 .
  • Tumor necrosis factor is a cytokine that is released primarily by mononuclear phage cells in response to immunostimulants. TNF ⁇ promotes most processes such as cell differentiation, recruitment, proliferation, and protein degradation. At low levels, TNF ⁇ has a protective effect against infection, tumors and tissue damage. However, excessive release of TNF ⁇ can also cause diseases, such as administration of TNF ⁇ to mammals or humans, which can cause or aggravate inflammation, fever, cardiovascular effects, hemorrhage, coagulation, and acute reactions similar to acute infection and shock state.
  • TNF ⁇ production in animals or humans often suggests the following diseases: endotoxemia and/or toxic shock syndrome, cachexia, adult respiratory syndrome, cancer (such as solid tumors and hematological tumors) ), heart disease (such as congestive heart failure), viral infection, genetic disease, inflammatory disease, allergic disease or autoimmune disease.
  • Cancer is a particularly devastating disease, and an increase in the level of TNF ⁇ in the blood is predictive of a risk of spreading cancer or cancer.
  • cancer cells cannot survive in the circulatory system of healthy subjects, one of which is that the inner wall of blood vessels is a barrier to extravasation of tumor cells.
  • ELAM-1 on endothelial cells mediates the adhesion of colon cancer cells to the cytokine-treated endothelium.
  • Cyclic adenosine monophosphate plays a role in many diseases and conditions. Increased cAMP concentration in leukocytes during inflammation inhibits leukocyte activation, followed by release of inflammatory regulatory factors including TNF ⁇ and NF- ⁇ B. Increased levels of cAMP also cause relaxation of the smooth muscles of the airways.
  • PDEs cyclic nucleotide phosphodiesterases
  • the present invention provides Form A of Compound 1, the X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 theta angles: 14.10 ⁇ 0.2 °, 19.07 ⁇ 0.2 °, 21.79 ⁇ 0.2 °.
  • the X crystal powder diffraction pattern of the above Compound 1 has a characteristic diffraction peak at the following 2 ⁇ angles: 10.69 ⁇ 0.2°, 12.31 ⁇ 0.2°, 13.45 ⁇ 0.2°, 14.10 ⁇ 0.2°. , 14.62 ⁇ 0.2 °, 19.07 ⁇ 0.2 °, 20.33 ⁇ 0.2 °, 21.79 ⁇ 0.2 °.
  • the X crystal form of the above Compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 ⁇ angles: 6.25 ⁇ 0.2°, 8.93 ⁇ 0.2°, 10.69 ⁇ 0.2°, 12.31 ⁇ 0.2°. 13.45 ⁇ 0.2°, 14.10 ⁇ 0.2°, 14.62 ⁇ 0.2°, 18.16 ⁇ 0.2°, 19.07 ⁇ 0.2°, 20.33 ⁇ 0.2°, 21.79 ⁇ 0.2°.
  • the X-form analysis data of the A crystal form of the above Compound 1 is shown in Table 1.
  • the A crystalline form of Compound 1 above has a differential scanning calorimetry curve having an onset of endothermic peak at 201.70 °C ⁇ 2 °C.
  • the A crystal form of the above compound 1 has a DSC pattern as shown in FIG.
  • the A crystal form of the above compound 1 has a thermogravimetric analysis curve at a weight loss of 0.00293% at 100.00 ⁇ 2 °C.
  • the T crystal of Form A of Compound 1 above is shown in Figure 3.
  • the present invention also provides a method for preparing Form A, which comprises adding Compound 1 to an alcohol solvent, a ketone solvent, an ether solvent, a mixed solvent of an alcohol solvent and water, a mixed solvent of a ketone solvent and water, or In a mixed solvent of an ether solvent and water, it is heated and dissolved, and then cooled and crystallized to obtain.
  • the alcohol solvent is selected from the group consisting of methanol, ethanol, and isopropanol.
  • the ketone solvent is selected from the group consisting of acetone and methyl ethyl ketone.
  • the ether solvent is selected from the group consisting of ethylene glycol dimethyl ether.
  • the mixed solvent of the above alcohol solvent and water is selected from the group consisting of a mixed solvent of ethanol and water.
  • the volume ratio of the alcohol solvent to water is selected from 1:0.2 to 1.5.
  • Form A of Compound 1 above for the preparation of a disease associated with PDE4 receptors is also provided.
  • the PDE4-related condition described above refers to psoriasis, psoriatic arthritis, chronic obstructive pneumonia, ankylosing spondylitis, inflammatory bowel disease.
  • Compound A has stable crystal form properties, low hygroscopicity, and good pharmaceutical prospects.
  • the crystal form of Compound A has good stability in an alcohol solvent, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, a mixed solvent of an alcohol solvent and water, a mixed solvent of acetonitrile and water, or a mixed solvent of acetone and water.
  • the crystal form A of Compound 1 has good stability under the condition of acceleration at 40 ° C / relative humidity of 75%; Form A of Compound 1 has good stability under long-term conditions of 25 ° C / relative humidity of 60%.
  • Compound 1 exhibited excellent in vitro activity against the phosphodiesterase 4B subtype (PDE4B). Compound 1 exhibited superior in vitro activity against TNF ⁇ production in hPBMC and was superior to Apost. Compound 1 significantly improved the symptoms of collagen-induced arthritis in the three dose groups of 0.3, 1 and 3 mg/kg. In the 1 mg/kg and 3 mg/kg dose groups, there was a significant improvement in the pathological changes of arthritis. The three dose groups showed a significant dose-effect relationship in the pathological score of arthritis. The therapeutic effect of Compound 1 at 3 mg/kg (clinical score and arthritic pathology score) was better than 5 mg/kg of Apster.
  • PDE4B phosphodiesterase 4B subtype
  • intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the present invention employs the following abbreviations: DMF stands for dimethylformamide; MsOH stands for methanesulfonic acid; EtOH stands for ethanol; and NaOH stands for sodium hydroxide.
  • XRPD X-ray powder diffractometer
  • Test method Approximately 10-20 mg samples were used for XRPD detection.
  • DSC Differential Scanning Calorimeter
  • Test method The sample ( ⁇ 1 mg) was placed in a DSC aluminum pan for testing, and the sample was heated from 25 ° C to 350 ° C at a heating rate of 10 ° C / min under 50 mL / min N 2 .
  • TGA Thermal Gravimetric Analyzer
  • Test method A sample (2 to 5 mg) was placed in a TGA platinum pot for testing, and the sample was heated from room temperature to 350 ° C at a heating rate of 10 ° C/min under a condition of 25 mL/min N 2 .
  • Test conditions Samples (10-20 mg) were placed in a DVS sample pan for testing.
  • the hygroscopicity evaluation is classified as follows:
  • ⁇ W% indicates the hygroscopic weight gain of the test article at 25 ⁇ 1°C and 80 ⁇ 2%RH.
  • Figure 1 is an XRPD spectrum of Cu-K ⁇ radiation of Form A of Compound 1.
  • Figure 3 is a TGA spectrum of Form A of Compound 1.
  • the Form A of Compound 1 had a hygroscopic weight gain of 0.08% at 25 ° C and 80% RH, less than 0.2%, and no or almost no hygroscopicity.
  • Form A of Compound 1 1.5 g was weighed and placed in an open watch glass to form a thin layer. The samples placed under high temperature and high humidity conditions were placed in a desiccator for sampling on the 5th, 10th and 30th day. The test results were compared with the initial test results of 0 days; the sample cover placed under strong light conditions The transparent cover was sealed, and the parafilm was sealed. On the 5th day, the test was taken for 10 days, and the test result was compared with the initial test result of 0 days. The test results are shown in Table 3 below.
  • Compound A crystal form has good stability under long-term conditions of 25 ° C / relative humidity of 60%.
  • Test Example 1 Inhibitory activity of Compound 1 against phosphodiesterase 4B isoform (PDE4B enzyme)
  • This biological assay is based on fluorescence polarization assay to determine AMP/GMP expression, ie, tracing AMP/GMP antibody binding to indicate enzyme activity.
  • Experimental buffer solution 10 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 0.01% polyoxyethylene lauryl ether (Brij 35), 1 mM dithiothreitol (DTT) , and 1% DMSO.
  • Enzyme substrate 1 ⁇ M cAMP
  • AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer.
  • Fluorescence polarization signal was calculated according to the AMP/GMP standard curve and the % enzyme activity relative to the DMSO control by Excel software, converted to nM. Curve fitting uses GraphPad Prism (Drawing Medicine Icon).
  • Test Example 2 Evaluation of inhibition of TNF ⁇ production by human peripheral blood mononuclear cells (hPBMC) in vitro
  • Compound 1 inhibits TNF ⁇ production by lipopolysaccharide (LPS)-induced human peripheral blood mononuclear cells.
  • LPS lipopolysaccharide
  • ELISA detects the level of TNF ⁇ in the supernatant, and Graphpad Prism software fits the inhibition curve and calculates IC50.
  • n the number of tests.
  • Compound 1 exhibits excellent in vitro activity against TNF ⁇ production in hPBMC and is superior to Apost.
  • Test Example 3 In vivo CIA model
  • the collagen-induced mouse arthritis model is an animal model for evaluating the efficacy of drug treatment for psoriatic arthritis, and its pathogenesis and symptoms are significantly correlated with psoriatic arthritis diseases.
  • the model activates B cells by injecting type II collagen, the reactivity of T cells to collagen, and the activation of B cells and T cells into the joints to cause inflammation of the joints, triggering a series of human psoriatic arthritis Symptoms, such as redness and swelling of the joints, articular cartilage, joint capsule damage and other symptoms.
  • Collagen-induced mouse arthritis is often used to evaluate its effectiveness during preclinical evaluation of drug treatment of psoriatic arthritis candidate compounds.
  • the purpose of this experiment was to examine the therapeutic effect of Compound 1 on collagen-induced arthritis in mice, thereby providing preclinical pharmacodynamics information for subsequent clinical studies.
  • acetic acid was diluted to 100 mM, filtered through a 0.22 micron filter, and stored at 4 °C.
  • Bovine type II collagen solution Bovine type II collagen (CII) was dissolved in an acetic acid solution and stored at 4 ° C overnight.
  • Preparation of the emulsion The overnight stored CII solution was mixed with an equal volume of complete Freund's adjuvant and homogenized using a high speed homogenizer until the solution formed a stable emulsion.
  • LPS lipopolysaccharide
  • mice were randomly assigned to different treatment groups.
  • the first immunization day is recorded as the 0th day, and the subsequent days are marked in order.
  • the prepared collagen emulsion was subcutaneously injected into the tail.
  • mice with moderately moderate disease were selected and re-randomized according to body weight and score, with 10 mice per group.
  • Dexamethasone as a positive control drug 0.3 mg / kg dose group is a commonly used dose in the CIA model; in addition, based on the results of previous pre-experiments, the relevant dose design of Compound 1 and the control compound Apster was determined.
  • the first group was normal mice without any treatment; the second group was treated with vehicle; the third group was given dexamethasone at a dose of 0.3 mg/kg; the fourth group was given Apster at a dose of 5 Mg/kg; Compound 1, Group 1, Group 6, and Group 7, were administered at doses of 0.3, 1 and 3 mg/kg, respectively. It was administered once a day for a total of 11 days.
  • the volume of intragastric administration was 10 ml/kg.
  • mice were observed daily from 7 days before immunization to 23 days after immunization (recorded once a week). After the 23rd day, the mice were observed daily for health status, morbidity, and body weight changes (at least three times a week) until the end of the experiment.
  • mice On day 38, the mice were euthanized. The hind limbs of the mice were taken, soaked in a 10% formalin solution, decalcified with formic acid solution, embedded in paraffin, sectioned, and hematoxylin-eosin staining (HE), photographed by microscopy. The degree of joint damage was evaluated from four aspects: inflammatory cell infiltration, vasospasm formation, cartilage damage, and bone resorption, and scored according to the standard of 0-4. The various scoring criteria are shown in Table 10:
  • the experimental data were expressed as mean ⁇ standard error (Mean ⁇ SEM), body weight and clinical scores were analyzed by two-way ANOVA, pathological scores and AUC using t test, p ⁇ 0.05 was considered significant difference.
  • mice On the 25th day after the first immunization (day 2 after the second immunization), the mice began to develop clinical symptoms of arthritis. Dosing started on the 27th day. The mean clinical score of the vehicle control group gradually increased to 8.3 points on the 36th day, suggesting the successful establishment of a collagen-induced arthritis model.
  • the 5 mg/kg Aprster group also inhibited the increase in clinical scores and showed significant differences from the vehicle control group from 33 days and continued until the end of the trial.
  • the average clinical symptom score was 4.2, a decrease of 3.7 (p ⁇ 0.001) compared to the vehicle control group.
  • the area under the curve was calculated by analyzing the clinical score curve of each animal in each group, and the inhibition rate of each administration group relative to the vehicle control group was calculated by the average area under the curve between the groups.
  • the inhibition rates were 96.4% (p ⁇ 0.0001) and 41.3% (p ⁇ 0.05), respectively;
  • the area under the clinical scoring curve of arthritis animals was significantly reduced, and the inhibition rates were 43.9% (p ⁇ 0.05), 39.4% (p ⁇ 0.05) and 51.7%, respectively. p ⁇ 0.01).
  • the inhibition rate of Compound 1 in the 1 mg/kg group was comparable to that in the Apster 5 mg/kg group (p ⁇ 0.05 for both groups), while the Compound 1 3 mg/kg group was superior to the Apster 5 mg/kg group.
  • Kg group p value ⁇ 0.01 and ⁇ 0.05, respectively
  • mice The hind limbs of each group of mice were subjected to section H.E. staining, and the total scores of the two hind limbs were taken.
  • the arthritic mice in the vehicle control group had a total pathological score of 20.20 ⁇ 1.15.
  • the control compound Aptster also significantly reduced the pathological score of arthritic mice at a dose of 5 mg/kg, which was reduced to 13.90 ⁇ 1.89 (p ⁇ 0.05), and compound 1 was at 1 and
  • the pathological score of arthritis mice was significantly reduced at 3 mg/kg, which was reduced to 14.00 ⁇ 2.43 (p ⁇ 0.05) and 9.20 ⁇ 1.83 (p ⁇ 0.0001), respectively.
  • Compound 1 was comparable to the 5 mg/kg arthritis pathology score in the 1 mg/kg group (p ⁇ 0.05), and Compound 1 was superior to Apu in the 3 mg/kg group.
  • Sterling 5 mg/kg group p values ⁇ 0.0001 and ⁇ 0.05, respectively.
  • Compound 1 significantly improved the symptoms of collagen-induced arthritis in the three dose groups of 0.3, 1 and 3 mg/kg. In the 1 mg/kg and 3 mg/kg dose groups, there was a significant improvement in the pathological changes of arthritis. The three dose groups showed a significant dose-effect relationship in the pathological score of arthritis. The therapeutic effect of Compound 1 at 3 mg/kg (clinical score and arthritic pathology score) was better than 5 mg/kg of Apster.

Abstract

一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物(I)晶型及其制备方法和在制备治疗与PDE4 受体相关疾病药物中的应用。

Description

一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法
相关申请的引用
本申请主张如下优先权：
CN201810085704.1，申请日2018-01-29；
技术领域
本发明涉及一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物晶型及其制备方法，还包括所述晶型在制备治疗与PDE4相关疾病药物中的应用。
背景技术
肿瘤坏死因子(TNFα)是一种主要由单核噬菌细胞应答免疫刺激物时释放的细胞因子。TNFα能够促进细胞的分化、募集、增殖和蛋白质降解等大多数过程。在低水平下，TNFα具有防止传染物、肿瘤和组织损伤的保护作用。但TNFα释放过多也会引起疾病，如给予哺乳动物或人TNFα时，会引起或加重炎症、发烧、心血管作用、出血、凝血以及与急性感染和休克状态相类似的急性反应。动物体或人体内产生过量的或不受控制的TNFα常提示患有如下疾病：内毒素血症和/或中毒休克综合症、恶病质、成人呼吸紧张综合症、癌症(如实体瘤和血液性肿瘤)、心脏病(如充血性心力衰竭)、病毒感染、遗传疾病、炎性疾病、变应性疾病或自身免疫疾病。
癌症是具有特别破坏性的疾病，血液中TNFα水平的提高预示存在患有癌症或癌症扩散的危险。通常，癌细胞不能在健康主体的循环系统中存活，其中一个原因在于血管内壁是瘤细胞外渗的屏障。研究表明，内皮细胞上的ELAM-1能介导促进结肠癌细胞黏附在用细胞因子处理的内皮上。
环腺苷酸(cAMP)在许多疾病和病症中起作用。发炎时白细胞中cAMP浓度的升高抑制了白细胞的激活，随后释放出包括TNFα和NF-κB等炎症调控因子。cAMP水平提高也会导致呼吸道平滑肌的松弛。
cAMP失活的主要细胞机制是由于被称为环核苷酸磷酸二酯酶(PDE)的一族同工酶破坏了cAMP。已知有11个PDE家族成员。迄今，已证实抑制PDE4酶对抑制炎症介质的释放及对松弛呼吸道平滑肌特别有效，因此PDE4酶已成为热门的药物靶点之一。依据不同的基因编码，PDE-4家族可以分为4个亚型(PDE-4A、B、C、D)。其中，PDE-4A、PDE-4B和PDE-4D在炎症细胞(如B细胞、T细胞和中性粒细胞等)中的表达强于PDE-4C。抑制PDE4酶，导致cAMP水平的升高，从而调节TNFα水平，达到治疗疾病目的。
发明内容
本发明提供了化合物1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：14.10±0.2°、19.07±0.2°、21.79±0.2°。
Figure PCTCN2019073701-appb-000001
本发明的一些方案中，上述化合物1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：10.69±0.2°、12.31±0.2°、13.45±0.2°、14.10±0.2°、14.62±0.2°、19.07±0.2°、20.33±0.2°、21.79±0.2°。
本发明的一些方案中，上述化合物1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：6.25±0.2°、8.93±0.2°、10.69±0.2°、12.31±0.2°、13.45±0.2°、14.10±0.2°、14.62±0.2°、18.16±0.2°、19.07±0.2°、20.33±0.2°、21.79±0.2°。
本发明的一些方案中，上所述化合物1的A晶型，其XRPD图谱如图1所示。
本发明的一些方案中，上述化合物1的A晶型，其XRPD图谱解析数据如表1所示。
表1：化合物1的A晶型XRPD图谱解析数据
Figure PCTCN2019073701-appb-000002
本发明的一些方案中，上述化合物1的A晶型，其差示扫描量热曲线在201.70℃±2℃处具有吸热峰的起始点。
本发明的一些方案中，上述化合物1的A晶型，其DSC图谱如图2所示。
本发明的一些方案中，上述化合物1的A晶型，热重分析曲线在100.00±2℃处失重达0.02039％。
本发明的一些方案中，上述化合物1的A晶型，其TGA图谱如图3所示。
本发明还提供了A晶型的制备方法，包括将化合物1加入到醇类溶剂、酮类溶剂、醚类溶剂、醇类溶剂与水的混合溶剂中、酮类溶剂与水的混合溶剂中或醚类溶剂与水的混合溶剂中，加热溶解，然后降温析晶制得。
本发明的一些方案中，上述醇类溶剂选自甲醇、乙醇和异丙醇。
本发明的一些方案中，上述酮类溶剂选自丙酮和丁酮。
本发明的一些方案中，上述醚类溶剂选自乙二醇二甲醚。
本发明的一些方案中，上述醇类溶剂与水的混合溶剂选自乙醇和水的混合溶剂。
本发明的一些方案中，上述醇类溶剂与水的混合溶剂中，醇类溶剂和水的体积比选自1：0.2～1.5。
本发明的一些方案中，还提供了上述化合物1的A晶型在制备治疗与PDE4受体相关疾病中的应用。
本发明的一些方案中，上述PDE4相关病症是指银屑病、银屑病关节炎、慢性阻塞性肺炎、强直性脊柱炎、炎症性肠病。
技术效果：
化合物1的A晶型性质稳定，吸湿性小，成药前景良好。化合物1的A晶型在醇类溶剂，乙腈、丙酮、乙酸乙酯、四氢呋喃、醇类溶剂与水的混合溶剂、乙腈与水的混合溶剂或丙酮与水的混合溶剂中均具有良好的稳定性。化合物1的A晶型在40℃/相对湿度75％加速条件下具有良好的稳定性；化合物1的A晶型在25℃/相对湿度60％长期条件下具有良好的稳定性。
化合物1展现出优异的抑制磷酸二酯酶4B亚型(PDE4B)的体外活性。化合物1在hPBMC中展现出优异的抑制TNFα生成的体外活性，并优于阿普斯特。化合物1在0.3，1和3毫克/公斤三个剂量组对胶原诱导的关节炎的症状均有显著改善作用。并且在1毫克/公斤和3毫克/公斤剂量组对关节炎病理学病变存在显著性改善，三个剂量组在关节炎病理评分呈现明显的量效关系。化合物1在3毫克/公斤的治疗效果(临床评分与关节炎病理评分)优于5毫克/公斤的阿普斯特。
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。
本发明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发明的实施例。
本发明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发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，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发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明采用下述缩略词：DMF代表二甲基甲酰胺；MsOH代表甲磺酸；EtOH代表乙醇；NaOH代表氢氧化钠。
化合物经手工或者
Figure PCTCN2019073701-appb-000003
软件命名，市售化合物采用供应商目录名称。
本发明粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法
仪器型号：布鲁克D8advance X-射线衍射仪
测试方法：大约10～20mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2019073701-appb-000004
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
本发明差热分析(Differential Scanning Calorimeter,DSC)方法
仪器型号：TA Q2000差示扫描量热仪
测试方法：取样品(～1mg)置于DSC铝锅内进行测试，在50mL/min N 2条件下，以10℃/min的升温速率，加热样品从25℃到350℃。
本发明热重分析(Thermal Gravimetric Analyzer,TGA)方法
仪器型号：TA Q5000IR热重分析仪
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温到350℃。
本发明动态蒸汽吸附分析(Dynamic Vapor Sorption,DVS)方法
仪器型号：SEM Advantage-1动态蒸汽吸附仪
测试条件：取样品(10～20mg)置于DVS样品盘内进行测试。
详细的DVS参数如下：
温度：25℃
平衡：dm/dt＝0.01％/min(最短：10min，最长：180min)
干燥：0％RH下干燥120min
RH(％)测试梯级：10％
RH(％)测试梯级范围：0％-90％-0％
引湿性评价分类如下：
吸湿性分类 ΔW％
潮解 吸收足量水分形成液体
极具吸湿性 ΔW％≥15％
有吸湿性 15％>ΔW％≥2％
略有吸湿性 2％>ΔW％≥0.2％
无或几乎无吸湿性 ΔW％<0.2％
注：ΔW％表示受试品在25±1℃和80±2％RH下的吸湿增重。
本发明含量测定方法
仪器型号：安捷伦1260高效液相仪配置DAD检测器或岛津LC-20A高效液相仪配置PDA检测器色谱条件具体参数如下：
色谱柱：Agilent Eclipse plus C18(4.6mm×150mm，3.5μm)
柱温：40℃
流速：1.0mL/min
检测波长：230nm
进样体积：10μL
运行时间：60min
流动相A：0.04％三氟乙酸水溶液(V/V)
流动相B：乙腈
稀释剂：乙腈:纯水＝3:1(v/v)
洗针液：乙腈:纯水＝3:1(v/v)
梯度洗脱程序：
时间(min) 流动相A(％) 流动相B(％)
0.00 85 15
30.00 70 30
50.00 30 70
55.00 15 85
55.01 85 15
60.00 85 15
附图说明
图1为化合物1的A晶型的Cu-Kα辐射的XRPD谱图。
图2为化合物1的A晶型的DSC谱图。
图3为化合物1的A晶型的TGA谱图。
图4为化合物1的A晶型的DVS谱图。
具体实施方式
为了更好的理解本发明的内容，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发明的内容所做的限制。
实施例1：化合物1的A晶型的制备
Figure PCTCN2019073701-appb-000005
步骤1：化合物3的合成
室温下，将化合物b(10.00g,39.77mmol)、化合物2(9.78g,35.79mmol)和二异丙基胺(10.28g,79.53mmol,13.89mL)溶解于N,N–二甲基甲酰胺(200.00mL)中，氮气置换3次，在氮气保护下反应混合物加热至120℃并搅拌反应16小时。反应完毕后，冷却至室温，向反应混合物中加入水(400mL)，用乙酸乙酯(200mL×3)萃取。合并有机相，用饱和食盐水(100mL×3)洗涤，无水硫酸钠干燥，过滤除去干燥剂，减压浓缩，残余物经柱层析(洗脱剂：乙酸乙酯/石油醚＝1/4–1/2，体积比)，得到目标化合物3。MS–ESI m/z: 509.8[M+Na] +,511.8[M+Na+2] +. 1H NMR(400MHz,CDCl 3)δ:8.27(s,1H),7.27(d,J＝6.8Hz,1H),6.90–6.86(m,2H),6.81(d,J＝8.0Hz,1H),5.72(q,J＝6.4Hz,1H),4.04(q,J＝6.8Hz,2H),3.80(s,3H),3.68(dd,J＝6.6,14.6Hz,1H),3.40(dd,J＝6.4,14.8Hz,1H),2.52(s,3H),2.45(s,3H),1.40(t,J＝7.0Hz,3H).
步骤2：化合物4的合成
室温下，将化合物3(12.10g,24.78mmol)和邻氟苯硼酸(5.20g,37.17mmol)溶解于二氧六环(150.00mL)和水(50.00mL)中，在氮气保护下加入碳酸钾(10.27g,74.34mmol)和〔1,1’–双(二苯基磷)二茂铁〕二氯化钯二氯甲烷配合物(2.02g,2.48mmol)，在氮气保护下反应混合物加热至80℃并搅拌反应14小时。反应完毕后，冷却至室温，向反应混合物中加入水(500mL)，用乙酸乙酯(300mL×3)萃取。合并有机相，用无水硫酸钠干燥，过滤除去干燥剂，减压浓缩，残余物经柱层析(洗脱剂：乙酸乙酯/石油醚＝1/10–1/4，体积比)，得到目标化合物4。MS–ESI m/z:504.1[M+H] +. 1H NMR(400MHz,CDCl 3)δ:8.07(s,1H),7.42(d,J＝6.8Hz,1H),7.38–7.30(m,1H),7.18–7.06(m,3H),6.98–6.89(m,2H),6.83(d,J＝8.4Hz,1H),5.82(q,J＝6.6Hz,1H),4.13–4.00(m,3H),3.81(s,3H),3.43(dd,J＝6.4,14.7Hz,1H),2.55(s,3H),2.23(s,3H),1.41(t,J＝7.0Hz,3H).
步骤3：化合物5的合成
室温下，向化合物4(9.20g,18.27mmol)和氯化铵(9.77g,182.70mmol)中加甲醇(200.00mL)，在0℃下分20批加入锌粉(11.95g,182.70mmol)，反应混合物在0℃下搅拌反应16小时。反应完毕后，将反应混合物过滤除去锌粉，滤液减压浓缩得到残余物。残余物溶于二氯甲烷(200mL)中，将悬浊液过滤除去不溶物，滤液减压浓缩即可得到化合物5。MS–ESI m/z:474.0[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.55(s,1H),7.42–7.32(m,1H),7.20(d,J＝4.8Hz,2H),7.13(t,J＝9.0Hz,1H),7.08–7.03(m,2H),6.88(d,J＝8.8Hz,1H),5.70(s,1H),4.19–4.07(m,2H),4.00–3.90(m,1H),3.87(s,3H),3.58(dd,J＝6.0,14.4Hz,1H),2.78(s,3H),2.05(s,3H),1.47(t,J＝7.2Hz,3H).
步骤4：化合物1的合成
室温下，将化合物5(9.30g,19.64mmol)和三乙胺(19.87g,196.40mmol)溶解于四氢呋喃(200.00mL)中，冷却至0℃，将三光气(2.33g,7.86mmol)的四氢呋喃(50.00mL)溶液逐滴滴加到上述反应液中，滴加完毕后，在氮气保护下反应混合物在0℃搅拌反应3小时。反应完毕后，向反应混合物加入水(200mL)，用乙酸乙酯(100mL×3)萃取。合并有机相，用无水硫酸钠干燥，过滤除去干燥剂，减压浓缩，残余物经柱层析(洗脱剂：乙酸乙酯/石油醚＝1/3–2/1，体积比)，得到化合物1。 1H NMR(400MHz,CDCl 3)δ:10.13–10.01(m,1H),7.87(s,1H),7.39–7.32(m,1H),7.31(d,J＝1.6Hz,1H),7.18–7.15(m,3H),7.11(t,J＝9.2Hz,1H),6.74(d,J＝8.4Hz,1H),6.14(dd,J＝4.8,9.6Hz,1H),4.86(dd,J＝9.4,14.6Hz,1H),4.09–3.97(m,2H),3.88(dd,J＝4.4,14.8Hz,1H),3.76(s,3H),2.70(s,3H),2.16(s,3H),1.35(t,J＝7.0Hz,3H).
步骤5：化合物1的A晶型的制备
室温下，向化合物1(6.45g,12.91mmol)中加水(160.00mL)和乙醇(170.00mL)，反应混合物在90℃搅拌0.5小时，搅拌过程中反应溶液逐渐变澄清。反应混合物在搅拌中缓慢冷却至20℃，并在20℃继续搅拌16小时，搅拌期间很多白色固体析出。白色固体过滤收集，在45℃真空干燥箱中干燥18小时得化合物1的A晶型。MS–ESI m/z:500.2[M+H] +. 1H NMR(400MHz,CDCl 3)δ:9.97(s,1H),7.94(s,1H),7.46–7.39(m,1H),7.38(d,J＝1.6Hz,1H),7.26–7.22(m,3H),7.17(t,J＝9.0Hz,1H),6.81(d,J＝8.0Hz,1H),6.22(dd,J＝4.8,9.6Hz,1H),4.93(dd,J＝9.6,14.8Hz,1H),4.13–4.03(m,2H),3.95(dd,J＝4.8,14.8Hz,1H),3.83(s,3H),2.77(s,3H),2.23(s,3H),1.42(t,J＝7.0Hz,3H).
实验例1：A晶型在不同溶剂中的稳定性试验
取50mg的A晶型多份，分别加入下表中的单一或混合溶剂，40℃条件下搅拌2天后离心。收集所有样品中的固体，于真空干燥箱中(40℃)干燥过夜，XRPD检测其晶型状态。结果见表2。
表2：A晶型在不同溶剂中的稳定性实验
编号 溶剂 溶剂加入量(mL) 状态(2天后) 晶型
1 甲醇 0.4 悬浊液 A晶型
2 乙醇 0.4 悬浊液 A晶型
3 乙腈 0.4 悬浊液 A晶型
4 丙酮 0.4 悬浊液 A晶型
5 乙酸乙酯 0.4 悬浊液 A晶型
6 四氢呋喃 0.3 悬浊液 A晶型
7 甲醇：水(3:1) 0.4 悬浊液 A晶型
8 乙醇：水(3:1) 0.4 悬浊液 A晶型
9 乙腈：水(1:1) 0.4 悬浊液 A晶型
10 丙酮：水(1:2) 0.4 悬浊液 A晶型
实验结论：化合物1的A晶型在醇类溶剂，乙腈、丙酮、乙酸乙酯、四氢呋喃、醇类溶剂与水的混合溶剂、酮类溶剂与水的混合溶剂、乙腈与水的混合溶剂或丙酮与水的混合溶剂中均具有良好的稳定性。
实验例2：化合物1的A晶型的引湿性研究
实验材料：
SEM DVS Advantage-1动态蒸汽吸附仪
实验方法：
取化合物1的A晶型10～20mg置于DVS样品盘内进行测试。
实验结果：
化合物1的A晶型的DVS谱图如图4所示，△W＝0.08％。
实验结论：
化合物1的A晶型在25℃和80％RH下的吸湿增重为0.08％，小于0.2％，无或几乎无有引湿性。
实验例3：化合物1的A晶型在高温，高湿及强光条件下的固体稳定性试验
依据《原料药与制剂稳定性试验指导原则》(中国药典2015版四部通则9001)，考察化合物1的A晶型在高温(60℃，敞口)，高湿(室温/相对湿度92.5％，敞口)及强光照(4500±500lux,90μw/cm 2，封口)条件下的稳定性。
称取化合物1的A晶型1.5g，放入敞口的表面皿中，摊成薄薄一层。高温及高湿条件下放置的样品放入保干器中考察，于第5天，10天和30天取样检测，检测结果与0天的初始检测结果进行比较；强光照条件下放置的样品盖上透明的盖子，封上封口膜，于第5天，10天取样检测，检测结果与0天的初始检测结果进行比较。试验结果见下表3所示。
表3：化合物1的A晶型在高温，高湿及强光条件下的固体稳定性试验结果
Figure PCTCN2019073701-appb-000006
结论：化合物1的A晶型在高温，高湿或强光照条件下均具有良好的稳定性。
实验例4：化合物1的A晶型在加速条件下的固体稳定性试验
依据《原料药与制剂稳定性试验指导原则》(中国药典2015版四部通则9001)，考察化合物1的A晶型在高温和高湿加速条件(40℃/相对湿度75％，密封)下的稳定性。
称取化合物1的A晶型1.4g，装入双层低密度聚乙烯袋，每层低密度聚乙烯袋分别扎扣密封，再放入铝箔袋中并热封，于第1月，2月，3月和6月取样检测，检测结果与0天的初始检测结果进行比较。此试验重复三次，每次采用不同批次化合物1的A晶型，试验结果见下表4所示。
表4：化合物1的A晶型在加速条件(40℃/相对湿度75％，密封)下的固体稳定性试验结果
Figure PCTCN2019073701-appb-000007
Figure PCTCN2019073701-appb-000008
结论：化合物1的A晶型在40℃/相对湿度75％加速条件下具有良好的稳定性。
实验例5：化合物1的A晶型在长期条件下的固体稳定性试验
依据《原料药与制剂稳定性试验指导原则》(中国药典2015版四部通则9001)，考察化合物1的A晶型在长期条件(25℃/相对湿度60％，密封)下的稳定性。
称取化合物1的A晶型1.4g，装入双层低密度聚乙烯袋，每层低密度聚乙烯袋分别扎扣密封，再放入铝箔袋中并热封，于第3月，6月，9月，12月和18月取样检测，检测结果与0天的初始检测结果进行比较。此试验重复三次，每次采用不同批次化合物1的A晶型。试验结果见下表5所示。
表5：化合物1的A晶型在长期条件(25℃/相对湿度60％，密封)下的固体稳定性试验结果
Figure PCTCN2019073701-appb-000009
Figure PCTCN2019073701-appb-000010
结论：化合物1的A晶型在25℃/相对湿度60％长期条件下具有良好的稳定性。
测试例1：化合物1对磷酸二酯酶4B亚型(PDE4B酶)的抑制活性
该生物实验是根据荧光偏振测定AMP/GMP表达，即示踪AMP/GMP抗体结合来表示酶的活性。
试剂：
实验缓冲溶液：10mM三羟甲基氨基甲烷-盐酸缓冲溶液(Tris-HCl)(pH 7.5)，5mM MgCl 2，0.01％聚氧乙烯月桂醚(Brij 35)，1mM二硫苏糖醇(DTT)，和1％DMSO。
酶：重组人源PDE4B(基因登录号NM_002600；氨基酸305端)用N端GST标签在Sf9昆虫细胞中的杆状病毒来表达。MW＝78kDa。
酶作用物：1μM cAMP
检测：
Figure PCTCN2019073701-appb-000011
AMP2/GMP2抗体和AMP2/GMP2AlexaFluor633示踪。
操作步骤：
1.将重组人源PDE4B酶和酶作用物(1μM cAMP)分别溶解到新鲜制备的实验缓冲液中；
2.将上述PDE4B酶缓冲溶液转移到反应孔中；
3.通过声学技术(回声550毫微升范围)将100％DMSO溶解的化合物1加到PDE4B酶缓冲溶液反应孔中，并在室温下孵育10分钟；
4.然后，将酶作用物缓冲溶液加到上述反应孔中以启动反应；
5.在室温下孵育1小时；
6.添加检测混合物(
Figure PCTCN2019073701-appb-000012
抗体和AMP2/GMP2 AlexaFluor633示踪)以终止反应，并在缓慢混合下孵育90分钟。荧光偏振测定范围是Ex/Em＝620/688。
数据分析：荧光偏振信号根据AMP/GMP标准曲线和通过Excel软件计算相对DMSO对照的％酶活性，换算成nM。曲线拟合使用GraphPad Prism(绘制医学图标)。
表6：本发明化合物体外筛选试验结果
化合物 IC50(nM)
化合物1 0.685
结论：化合物1展现出优异的抑制磷酸二酯酶4B亚型(PDE4B)的体外活性。
测试例2：体外在人外周血单个核细胞(hPBMC)抑制TNFα生成作用的评价
化合物1对脂多糖(LPS)诱发人外周血单个核细胞的TNFα生成的抑制活性。
操作步骤：
1.采集正常人全血，EDTA-K2抗凝管抗凝；
2.Ficoll密度梯度离心分离PBMC，计数，调整细胞浓度为2x10 6/mL；
3.U底96孔板，每孔加入细胞2x10 5个细胞，加入LPS 1ng/mL,不同浓度的化合物100μM，10μM，1μM，100nM，10nM，1nM，100pM，10pM，每孔200μl体系，两复孔；
4.培养24小时，收取上清；
5.ELISA检测上清中TNFα的水平，Graphpad Prism软件拟合抑制曲线并计算IC50
表7：本发明化合物在hPBMC中对TNFα生成的抑制活性结果
化合物 IC50(nM) 化合物 IC50(nM)
阿普斯特 16.5(n＝3) 化合物1 0.56(n＝2)
n代表测试次数。
结论：化合物1在hPBMC中展现出优异的抑制TNFα生成的体外活性，并优于阿普斯特。
测试例3：体内CIA模型
实验目的：
胶原诱导的小鼠关节炎模型，是用于评价药物治疗银屑病关节炎药效的动物模型，其发病机理和症状与银屑病关节炎疾病均有比较明显的相关性。模型通过注射II型胶原蛋白从而激活B细胞，T细胞对骨胶原蛋白的反应性，被激活的B细胞和T细胞进入关节部位引起关节的炎症，从而引发一系列类似于人银屑病关节炎的症状，如关节部位红肿，关节软骨，关节囊损伤等症状。在临床前评价药物治疗银屑病关节炎候选化合物的过程中，胶原诱导的小鼠关节炎常常被用来评价其有效性。
本次实验目的是考察化合物1在胶原诱导的小鼠关节炎上的治疗效果，从而为之后的临床研究提供临床前药效学相关信息。
实验方法：
1.二型胶原/完全弗氏佐剂免疫
乙酸的配制：稀释乙酸至100mM，用0.22微米滤膜过滤后，4℃保存。
牛二型胶原溶液：将牛二型胶原(CII)溶解于乙酸溶液中，并置于4℃过夜保存。
乳剂的制备：将过夜保存的CII溶液与等体积的完全弗氏佐剂混合，使用高速匀浆机匀浆，直至溶液形成稳定的乳剂。
脂多糖(LPS)的制备：称取LPS，加入生理盐水，混匀直至形成稳定的溶液，浓度为0.3mg/kg。
2.关节炎的诱导：
将小鼠随机分配到不同的治疗组。第一次免疫当天记为第0天，随后的天数依序标注。
DBA/1小鼠经异氟烷麻醉后，在尾部皮下注射制备好的胶原乳剂。
第23天，腹腔注射100微升LPS溶液
正常组的小鼠无需免疫。
3.给药和剂量设计
第27天，当平均临床评分达到1分左右时，挑选发病程度适中的小鼠60只，按照体重和评分，重新随机分组，每组10只小鼠。
地塞米松作为阳性对照药物0.3毫克/公斤剂量组是CIA模型中普遍使用剂量；另外根据前期预实验的结果确定了化合物1和对照化合物阿普斯特的相关剂量设计。第一组为正常小鼠，不做任何处理；第二组溶媒对照组,给予溶媒；第三组给予地塞米松，剂量为0.3毫克/公斤；第四组给予阿普斯特，剂量为5毫克/公斤；第五组、第六组、第七组给予化合物1，剂量分别为0.3，1和3毫克/公斤。每天给药一次，共持续11天。灌胃给药体积为10毫升/公斤。
表8：分组及剂量设计
Figure PCTCN2019073701-appb-000013
4.关节炎发病指标测定
临床观察：从免疫前7天至免疫后第23天，每日观察DBA/1小鼠的基本健康状况及体重变化(一周记录一次)。第23天之后，每日观察小鼠健康状况，发病情况，及体重变化(一周至少记录三次)，直至实验结束。
临床评分：LPS注射后，每天观察小鼠发病情况。当小鼠开始发病之后(出现关节炎的临床症状)，根据病变的不同程度(红肿，关节变形)按照0-4分的标准进行评分，每个肢体的最高评分为4分，每只动物最高评分为16分。评分标准如表9。至少每周评分三次
表9：关节炎临床评分标准
分值 临床症状
0 无红斑和红肿
1 近跗骨附近或踝关节或跖骨出现红斑或轻度红肿，1个脚趾红肿
2 踝关节和跖骨轻微红斑和肿胀，或超过两个脚趾红肿
3 踝、腕关节和跖骨中度红斑和肿胀
4 踝、腕关节，跖骨和脚趾全部严重红肿
病理：第38天，小鼠安乐死。取小鼠两后肢，用10％福尔马林溶液浸泡，用甲酸溶液脱钙，石蜡包埋，切片，苏木精—伊红染色(hematoxylin-eosin staining，HE)，显微镜拍摄照片观察。从炎症细胞浸润、 血管翳生成、软骨损伤和骨吸收等四个方面对关节的损伤程度进行评价，并按照0-4分的标准进行评分。各项评分标准如表10：
表10：关节炎病理学评分标准
Figure PCTCN2019073701-appb-000014
5.统计学处理
实验数据应用平均数±标准误表示(Mean±SEM)，体重和临床评分用双因素方差分析(Two-way ANOVA)，病理学评分和AUC用t检验，p<0.05认为有显著性差异。
实验结果：
1.临床评分：
第一次免疫后第25天(第二次免疫后第2天)，小鼠开始出现关节炎临床症状。第27天开始给药。溶媒对照组的平均临床评分逐渐升高，至第36天达到8.3分，提示胶原诱导的关节炎模型的成功建立。
与溶媒对照组相比，化合物1在0.3，1和3毫克/公斤均可显著降低实验终点(第37天)关节炎小鼠的临床评分，三个剂量下的临床平均评分下降至3.6(p<0.0001)，4.3(p<0.001)和3.5(p<0.0001)。因此化合物1在低至0.3毫克/公斤即有效地减轻胶原诱导的关节炎。0.3毫克/公斤的地塞米松治疗可显著抑制胶原诱导的关节炎临床评分，从第30天开始起临床评分维持在0分，与溶媒对照组出现显著差异(p<0.0001)并一直持续到试验结束；5毫克/公斤的阿普斯特组也抑制了临床评分的升高，并从33天开始与溶媒对照组出现显著差异并一直持续到试验结束，至第37天时，临床症状平均评分为4.2，较溶媒对照组下降了3.7 (p<0.001)。
通过分析每组每只动物的临床评分曲线，计算曲线下面积(AUC)，通过组间曲线下面积平均值，计算各给药组相对于溶媒对照组的抑制率。与溶媒对照组相比，地塞米松组和阿普斯特组显著性降低关节炎动物的临床评分，抑制率分别为96.4％(p<0.0001)和41.3％(p<0.05)；化合物1在0.3、1和3毫克/公斤三个剂量下均能够显著性降低关节炎动物的临床评分曲线下面积，抑制率分别为43.9％(p<0.05)、39.4％(p<0.05)和51.7％(p<0.01)。化合物1的1毫克/公斤组与阿普斯特5毫克/公斤组抑制率相当(两组均p<0.05)，同时化合物1的3毫克/公斤组抑制率优于阿普斯特5毫克/公斤组(p值分别为<0.01和<0.05)
2.组织病理评分
取各组小鼠的两后肢做切片H.E.染色，取两后肢总分。溶媒对照组的关节炎小鼠，病理评分总分为20.20±1.15。与溶媒对照组相比，对照化合物阿普斯特在5毫克/公斤剂量下也可显著性降低关节炎小鼠的病理评分，可降低至13.90±1.89(p<0.05)，化合物1在1和3毫克/公斤剂量下可显著性降低关节炎小鼠的病理评分，分别降低至14.00±2.43(p<0.05)和9.20±1.83(p<0.0001)。化合物1在1毫克/公斤组与阿普斯特的5毫克/公斤关节炎病理评分效果相当(两组p<0.05)，同时化合物1在3毫克/公斤组关节炎病理评分效果优于阿普斯特5毫克/公斤组(p值分别为<0.0001和<0.05)。
3.结论
化合物1在0.3，1和3毫克/公斤三个剂量组对胶原诱导的关节炎的症状均有显著改善作用。并且在1毫克/公斤和3毫克/公斤剂量组对关节炎病理学病变存在显著性改善，三个剂量组在关节炎病理评分呈现明显的量效关系。化合物1在3毫克/公斤的治疗效果(临床评分与关节炎病理评分)优于5毫克/公斤的阿普斯特。

Claims (16)

  1. 化合物1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：14.10±0.2°、19.07±0.2°、21.79±0.2°。
    Figure PCTCN2019073701-appb-100001
  2. 根据权利要求1所述化合物1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：10.69±0.2°，12.31±0.2°，13.45±0.2°，14.10±0.2°，14.62±0.2°，19.07±0.2°，20.33±0.2°，21.79±0.2°。
  3. 根据权利要求2所述化合物1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：6.25±0.2°、8.93±0.2°、10.69±0.2°、12.31±0.2°、13.45±0.2°、14.10±0.2°、14.62±0.2°、18.16±0.2°、19.07±0.2°、20.33±0.2°、21.79±0.2°。
  4. 根据权利要求3所述化合物1的A晶型，其XRPD图谱如图1所示。
  5. 根据权利要求1～4任意一项所述化合物1的A晶型，其差示扫描量热曲线在201.70℃±2℃处具有吸热峰的起始点。
  6. 根据权利要求5所述化合物1的A晶型，其DSC图谱如图2所示。
  7. 根据权利要求1～4任意一项所述化合物1的A晶型，热重分析曲线在100.00±2℃处失重达0.02039％。
  8. 根据权利要求7所述化合物1的A晶型，其TGA图谱如图3所示。
  9. 化合物1的A晶型的制备方法，包括将化合物1加入到醇类溶剂、酮类溶剂、醚类溶剂、醇类溶剂与水的混合溶剂中、酮类溶剂与水的混合溶剂中或醚类溶剂与水的混合溶剂中，加热溶解，然后降温析晶制得。
  10. 根据权利要求9所述化合物1的A晶型的制备方法，其中，所述醇类溶剂选自甲醇、乙醇和异丙醇。
  11. 根据权利要求9所述化合物1的A晶型的制备方法，其中，所述酮类溶剂选自丙酮和丁酮。
  12. 根据权利要求9所述化合物1的A晶型的制备方法，其中，所述醚类溶剂选自乙二醇二甲醚。
  13. 根据权利要求9所述化合物1的A晶型的制备方法，其中，醇类溶剂与水的混合溶剂选自乙醇和水的混合溶剂。
  14. 根据权利要13所述化合物1的A晶型的制备方法，其中，所述醇类溶剂与水的混合溶剂中，醇类溶剂和水的体积比选自1：0.2～1.5。
  15. 根据权利要求1～8任意一项所述化合物1的A晶型在制备治疗与PDE4受体相关疾病中的应用。
  16. 根据权利要求15所述的应用，其中所述PDE4相关病症是指银屑病、银屑病关节炎、慢性阻塞性肺炎、强直性脊柱炎、炎症性肠病。
PCT/CN2019/073701 2018-01-29 2019-01-29 一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法 WO2019144970A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP19743686.8A EP3747884B1 (en) 2018-01-29 2019-01-29 Crystal form of 1h-imidazo[4,5-b]pyridine-2(3h)-one compound and preparation method therefor
JP2020541359A JP7237973B2 (ja) 2018-01-29 2019-01-29 １Ｈ－イミダゾ［４，５－ｂ］ピリジン－２（３Ｈ）－オン系化合物の結晶形及びその製造方法
US16/965,377 US11325907B2 (en) 2018-01-29 2019-01-29 Crystal form of 1H-imidazo[4,5-b]pyridine-2(3H)-one compound and preparation process therefor
AU2019211931A AU2019211931B2 (en) 2018-01-29 2019-01-29 Crystal form of 1H-imidazo[4,5-b]pyridine-2(3H)-one compound and preparation process therefor
CA3089414A CA3089414A1 (en) 2018-01-29 2019-01-29 Crystal form of 1h-imidazo[4,5-b]pyridine-2(3h)-one compound and preparation process therefor
RU2020127989A RU2784538C2 (ru) 2018-01-29 2019-01-29 Кристаллическая форма соединения 1h-имидазо[4,5-b]пиридин-2(3h)-она и способ ее получения
CN201980010305.1A CN111683949B (zh) 2018-01-29 2019-01-29 一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810085704.1 2018-01-29
CN201810085704 2018-01-29

Publications (1)

Publication Number Publication Date
WO2019144970A1 true WO2019144970A1 (zh) 2019-08-01

Family

ID=67395806

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/073701 WO2019144970A1 (zh) 2018-01-29 2019-01-29 一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法

Country Status (7)

Country Link
US (1) US11325907B2 (zh)
EP (1) EP3747884B1 (zh)
JP (1) JP7237973B2 (zh)
CN (1) CN111683949B (zh)
AU (1) AU2019211931B2 (zh)
CA (1) CA3089414A1 (zh)
WO (1) WO2019144970A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011021678A1 (ja) * 2009-08-21 2011-02-24 武田薬品工業株式会社 縮合複素環化合物
CN105407888A (zh) * 2013-06-21 2016-03-16 齐尼思表观遗传学公司 新双环溴结构域抑制剂
WO2018036469A1 (zh) * 2016-08-22 2018-03-01 南京明德新药研发股份有限公司 Pde4抑制剂
WO2018036470A1 (zh) * 2016-08-22 2018-03-01 南京明德新药研发股份有限公司 作为pde4抑制剂的并环类化合物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7244759B2 (en) 2004-07-28 2007-07-17 Celgene Corporation Isoindoline compounds and methods of making and using the same
MY165585A (en) * 2010-06-24 2018-04-05 Takeda Pharmaceuticals Co Fused heterocyclic compounds as phosphodiesterases (pdes) inhibitors
US8853175B2 (en) * 2011-01-10 2014-10-07 Celgene Corporation Phenethylsulfone isoindoline derivatives and their use
EP3142663A1 (en) * 2014-05-16 2017-03-22 Celgene Corporation Compositions and methods for the treatment of atherosclerotic cardiovascular diseases with pde4 modulators

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011021678A1 (ja) * 2009-08-21 2011-02-24 武田薬品工業株式会社 縮合複素環化合物
CN105407888A (zh) * 2013-06-21 2016-03-16 齐尼思表观遗传学公司 新双环溴结构域抑制剂
WO2018036469A1 (zh) * 2016-08-22 2018-03-01 南京明德新药研发股份有限公司 Pde4抑制剂
WO2018036470A1 (zh) * 2016-08-22 2018-03-01 南京明德新药研发股份有限公司 作为pde4抑制剂的并环类化合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Guidelines for Stability Test of APIs and Preparations", CHINESE PHARMACOPOEIA, 2015
See also references of EP3747884A4

Also Published As

Publication number Publication date
EP3747884A1 (en) 2020-12-09
RU2020127989A (ru) 2022-02-28
CN111683949A (zh) 2020-09-18
CA3089414A1 (en) 2019-08-01
CN111683949B (zh) 2023-03-21
JP7237973B2 (ja) 2023-03-13
JP2021512076A (ja) 2021-05-13
US11325907B2 (en) 2022-05-10
US20210198253A1 (en) 2021-07-01
EP3747884A4 (en) 2021-09-01
EP3747884B1 (en) 2022-11-02
AU2019211931B2 (en) 2023-08-10
AU2019211931A1 (en) 2020-08-20

Similar Documents

Publication Publication Date Title
CN104080771B (zh) 细胞凋亡信号调节激酶抑制剂
JP5765866B1 (ja) ｐ３８ＭＡＰキナーゼ阻害剤としての１−ピラゾリル−３−（４−（（２−アニリノピリミジン−４−イル）オキシ）ナフタレン−１−イル）尿素
TWI433677B (zh) 雜環化合物及其用途
US20080167314A1 (en) Condensed Imidazole Compound And Use Thereof
CN109153640A (zh) 用抗病毒剂消除乙型肝炎病毒
CN111032658B (zh) 作为C5a抑制剂的5-5稠合环类
CN108368127A (zh) 1-取代的1,2,3,4-四氢-1,7-萘啶-8-胺衍生物及其作为ep4受体拮抗剂的用途
TW200539868A (en) Novel 4-amino-thieno[3,2-c]pyridine-7-carboxylic acid amides
KR20090080998A (ko) Ｃｘｃｒ2 길항제로서의 5-술파닐메틸-[1,2,4]트리아졸[1,5-ａ]피리미딘-7-올 유도체
US11944616B2 (en) Dizocilpine derivatives as peripheral NMDA receptor antagonists
CN107667104A (zh) 化合物(s)‑3‑{4‑[5‑(2‑环戊基‑6‑甲氧基‑吡啶‑4‑基)‑[1,2,4]噁二唑‑3‑基]‑2‑乙基‑6‑甲基‑苯氧基}‑丙烷‑1,2‑二醇的结晶形式
CN112521432B (zh) 一类星状棘蛋白靶向的抗呼吸道感染病毒的双功能化合物及其盐类的制备方法和应用
WO2019144970A1 (zh) 一种1H-咪唑并[4,5-b]吡啶-2(3H)-酮类化合物的晶型及其制备方法
Li et al. Novel O-benzylcinnamic acid derivative L26 treats acute lung injury in mice by MD-2
RU2784538C2 (ru) Кристаллическая форма соединения 1h-имидазо[4,5-b]пиридин-2(3h)-она и способ ее получения
CN115038701A (zh) Jak抑制剂的晶型及其应用
CN114072382B (zh) 一种苯并咪唑-2-酮类化合物的晶型、溶剂化物、溶剂化物的晶型及它们的制备方法
WO2023006057A1 (zh) 氨基吡唑并嘧啶化合物的晶体
EP3468969B1 (en) Novel dizocilpine derivatives as peripheral nmda receptor antagonists
KR101192063B1 (ko) 염증성 질환의 예방 및 치료용 약제학적 조성물
CN113330013A (zh) 杂环化合物盐及其应用
CN110655516A (zh) 一种抗凝血药物的晶型

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19743686

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3089414

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2020541359

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2019211931

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019211931

Country of ref document: AU

Date of ref document: 20190129

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019743686

Country of ref document: EP

Effective date: 20200831