WO2020249139A1 - 一种5型磷酸二酯酶抑制剂的钾盐晶型b及其制备方法和应用 - Google Patents

一种5型磷酸二酯酶抑制剂的钾盐晶型b及其制备方法和应用 Download PDF

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WO2020249139A1
WO2020249139A1 PCT/CN2020/107811 CN2020107811W WO2020249139A1 WO 2020249139 A1 WO2020249139 A1 WO 2020249139A1 CN 2020107811 W CN2020107811 W CN 2020107811W WO 2020249139 A1 WO2020249139 A1 WO 2020249139A1
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crystal form
potassium salt
salt crystal
compound
group
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PCT/CN2020/107811
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French (fr)
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赵子建
穆云萍
李芳红
赵正刚
朱惠丹
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广州华真医药科技有限公司
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Priority to US17/617,655 priority Critical patent/US20220251093A1/en
Priority to GB2117860.3A priority patent/GB2599528B/en
Priority to AU2020293524A priority patent/AU2020293524B2/en
Publication of WO2020249139A1 publication Critical patent/WO2020249139A1/zh
Priority to ZA2021/10585A priority patent/ZA202110585B/en

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    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—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

Definitions

  • the invention relates to the field of chemical pharmacy, in particular to a potassium salt crystal form B of a type 5 phosphodiesterase inhibitor, and a preparation method and application thereof.
  • Phosphodiesterase inhibitors are drugs that inhibit the activity of phosphodiesterase (PDEs), and have broad application prospects in diseases such as heart failure, asthma, and erectile dysfunction.
  • Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are two important second messengers in cells, which activate protein kinase A (protein kinase C, PKA) and protein kinase G (protein kinase C, PKG) pathways Participating in a variety of metabolic activities of the body, the regulation of its intracellular concentration is mainly determined by the balance between the synthesis of adenylate cyclase and the hydrolysis of PDE.
  • PDE can specifically catalyze the hydrolysis of cGMP and cAMP in cells to produce corresponding inactive 5'-nucleotides.
  • phosphodiesterase type 5 PDE5
  • PDE-5i can regulate the contractility of the corpus cavernosum and the vascular smooth muscle of the lung, and participate in the conduction of the NO-cGMP signal pathway in the brain, as well as the growth and apoptosis of breast cancer cancer cells.
  • the therapeutic effect of PDE-5i on IPF has not been reported yet, and further research is needed.
  • Sildenafil is a selective inhibitor of cGMP-specific PDE5. It is a drug for erectile dysfunction (ED) that was accidentally invented during the development of drugs for the treatment of cardiovascular diseases. Sildenafil selectively inhibits PDE5, can prevent the degradation of cGMP and increase the level of cGMP in cells, thereby improving the effect of nitric oxide in relaxing vascular smooth muscle and reducing vascular resistance. It was the first drug candidate to enter clinical research as a drug for the treatment of cardiovascular diseases.
  • PDE5i can increase pulmonary artery vasodilation and inhibit pulmonary vascular remodeling, thereby reducing pulmonary artery pressure and pulmonary vascular resistance, significantly increasing the survival rate of patients and improving their quality of life.
  • Sildenafil has the disadvantages of large dosage (100mg/day), short biological half-life, low bioavailability (about 41%), and patients need to take the drug multiple times a day, which greatly increases the adverse drug reactions and treatment costs.
  • the selective effect of Sildenafil on PDE5 is only 10 times that of PDE6.
  • PDE6 is an enzyme that exists in the retina. Therefore, high doses or high blood concentration of Sildenafil may cause color vision abnormalities such as photophobia and blurred vision.
  • Tadalafil's biological half-life is significantly prolonged (about 17.5 hours), and the daily therapeutic dose required is also reduced (40mg/day), which greatly reduces the patient's treatment costs.
  • Tadalafil also has a certain inhibitory effect on other PDEs, adverse reactions are also more significant, such as headache, stomach discomfort or pain, diarrhea, back pain, and muscle pain.
  • Chinese Patent Document (CN102020645A) discloses a pyrazolopyrimidinone derivative with small side effects and can be used to prepare and treat male impotence, pulmonary hypertension, lower urinary tract syndrome, and benign prostate Therapeutic drugs for enlargement, chronic heart failure, stroke, coronary artery disease and neuropathic pituitary disease.
  • the Chinese patent document also specifically discloses a compound WYQ: 5-(2-H-5(N-butoxycarbonylaminosulfonyl)phenyl)-1-methyl-3-propyl-1,6-di Hydrogen-7H-pyrazole[4,3-d]pyrimidin-7-one has a structural formula as shown in formula (I).
  • Compound WYQ is a derivative of Sildenafil, which has the same curative effect as Sildenafil and Tadalafil. Compared with Sildenafil, compound WYQ has the characteristics of low dose and high efficacy. Since the efficacy of compound WYQ is dependent on the therapeutic dose, and more importantly, the solubility of compound WYQ in water is very low, which increases the daily usage of compound WYQ, and it mainly depends on liver and kidney metabolism. If liver and kidney dysfunction is incomplete When the patient is given high-dose long-term treatment, it will increase the burden on the liver and kidney, cause serious adverse reactions, and significantly increase the treatment cost of the patient.
  • the present invention aims to provide a potassium salt crystal form of compound WYQ with good water solubility and stable physical and chemical properties.
  • the present invention provides a potassium salt crystal form B of a compound, the structural formula of the compound is shown in formula (I),
  • the X-ray powder diffraction (XRPD) pattern of the potassium salt crystal form B has characteristic peaks at the following 2 ⁇ angles: 5.71° ⁇ 0.2°, 8.23° ⁇ 0.2°, 11.37° ⁇ 0.2°, 13.22° ⁇ 0.2°, 17.09 ° ⁇ 0.2°, 21.56° ⁇ 0.2°, 23.99° ⁇ 0.2°, 25.85° ⁇ 0.2°.
  • the X-ray powder diffraction (XRPD) pattern of the potassium salt crystal form B has characteristic peaks at the following 2 ⁇ angles: 15.88° ⁇ 0.2°, 16.35° ⁇ 0.2°, 18.47° ⁇ 0.2°, 19.70° ⁇ 0.2°, 22.90° ⁇ 0.2°, 23.64° ⁇ 0.2°, 31.92° ⁇ 0.2°.
  • X-ray powder diffraction (XRPD) pattern of the potassium salt crystal form B has characteristic peaks at the following 2 ⁇ angles: 25.04° ⁇ 0.2°, 26.54° ⁇ 0.2°, 28.36° ⁇ 0.2°, 29.94° ⁇ 0.2°, 35.48° ⁇ 0.2°, 37.83° ⁇ 0.2°.
  • the potassium salt crystal form B has an X-ray powder diffraction (XRPD) pattern as shown in FIG. 1.
  • XRPD X-ray powder diffraction
  • the differential scanning calorimetry (DSC) spectrum of the potassium salt crystal form B has characteristic melting absorption peaks at peaks of 191.3°C and 217.9°C.
  • the potassium salt crystal form B has a TG-DSC chart as shown in FIG. 2.
  • the molecular formula of the potassium salt crystal form B is: C 20 H 24 KN 5 O 5 S.
  • the present invention also provides a method for preparing the potassium salt crystal form B of the compound represented by formula (I), including:
  • Vacuum filtration was performed to separate the solid material and vacuum dry to obtain the potassium salt crystal form B of the compound.
  • the solvent is selected from acetone, tetrahydrofuran, ethyl acetate or ethanol.
  • the mass-volume ratio of the compound represented by formula I and the solvent is 25-30 mg/mL, and the mass ratio of the potassium hydroxide and the compound represented by formula I is 1:(7-10 ).
  • the stirring and crystallization is as follows: the suspension 2 is stirred at 45-50°C for 5-10 min, at 20-30°C for 20-24 h, and at 45-50°C for 8-10 h.
  • the preparation method of the potassium salt crystal form B of the compound includes:
  • Potassium hydroxide is added to the suspension 1 to aid dissolution by ultrasound to form a suspension 2, wherein the mass ratio of potassium hydroxide and the compound represented by formula I is 1:8;
  • Vacuum filtration was performed to separate the solid material and vacuum dry to obtain the potassium salt crystal form B of the compound.
  • the present invention also provides a pharmaceutical composition, comprising the potassium salt crystal form B of the compound represented by the above formula (I) and a pharmaceutically acceptable carrier.
  • the present invention also provides the application of the potassium salt crystal form B of the compound represented by the above formula (I) in the preparation of a medicine for treating pulmonary hypertension.
  • the present invention also provides the application of the potassium salt crystal form B of the compound represented by the above formula (I) in preparing a medicine for treating idiopathic pulmonary fibrosis.
  • the present invention also provides the application of the potassium salt crystal form B of the compound represented by the above formula (I) in preparing a medicine for treating renal fibrosis.
  • the present invention also provides the application of the potassium salt crystal form B of the compound represented by the above formula (I) in preparing a medicine for treating myocardial hypertrophy.
  • the present invention also provides the application of the potassium salt crystal form B of the compound represented by formula (I) in the preparation of a medicine for treating erectile dysfunction.
  • the present invention also provides a method for treating pulmonary hypertension, which comprises administering to a subject the potassium salt crystal form B of the compound represented by formula (I); preferably, the oral administration dose of the potassium salt crystal form B of the compound It is 0.324-3.24 mg.kg -1 .d -1 , and the preferred administration time is once a day, preferably for 14 consecutive days; preferably, the injection dosage of the potassium salt crystal form B of the compound is 0.097-1.296 mg.kg -1 .d -1 , the preferred administration time is once a day, preferably for 14 consecutive days.
  • the present invention also provides a method for treating idiopathic pulmonary fibrosis, comprising administering to a subject the potassium salt crystal form B of the compound represented by formula (I); preferably, the potassium salt crystal form B of the compound
  • the oral administration dose of the compound is 0.405-1.62 mg.kg -1 .d -1
  • the preferred administration time is once a day, preferably for 23 consecutive days
  • the injection administration dose of the potassium salt crystal form B of the compound is 0.122- 0.648 mg.kg -1 .d -1
  • the preferred administration time is once a day, preferably 23 consecutive days.
  • the present invention also provides a method for treating renal fibrosis, comprising administering to a subject the potassium salt crystal form B of the compound represented by formula (I); preferably, oral administration of the potassium salt crystal form B of the compound
  • the dosage is 0.405 mg.kg -1 .d -1
  • the preferred administration time is once a day, preferably 10 consecutive days, more preferably 7 consecutive days;
  • the injection dosage of the potassium salt crystal form B of the compound is 0.1215 -0.162 mg.kg -1 .d -1
  • the administration time is once a day, preferably 10 consecutive days, more preferably 7 consecutive days.
  • the present invention also provides a method for treating myocardial hypertrophy, comprising administering to a subject the potassium salt crystal form B of the compound represented by formula (I); preferably, the oral administration dose of the potassium salt crystal form B of the compound It is 0.324 mg.kg -1 .d -1 , and the preferred administration time is once a day, preferably for 7 consecutive days; preferably, the injection dosage of the potassium salt crystal form B of the compound is 0.097-0.1296 mg.kg -1 .d -1 , the preferred administration time is once a day, preferably 7 consecutive days.
  • the present invention also provides a method for treating erectile dysfunction, which comprises administering to a subject the potassium salt crystal form B of the compound represented by formula (I); preferably, oral administration of the potassium salt crystal form B of the compound
  • the dosage is 0.324-3.24mg.kg -1 .d -1
  • the preferred administration time is once a day, preferably for 14 consecutive days; preferably, the injection dosage of the potassium salt crystal form B of the compound is 0.097-1.296 mg. kg -1 .d -1
  • the preferred administration time is once a day, preferably for 14 consecutive days.
  • the present invention obtains a potassium salt crystal form B of compound WYQ by screening the salt or co-crystal form of compound WYQ, which has better water solubility and stable physical and chemical properties;
  • the potassium salt crystal form B has also been found to have therapeutic effects on idiopathic pulmonary fibrosis, renal fibrosis and myocardial hypertrophy. It has the characteristics of low dose and high efficacy; the preparation method is simple, easy to operate, and has good stability, and has the potential to be developed as a drug for the treatment of the above diseases.
  • Fig. 1 is an X-ray powder diffraction pattern of potassium salt crystal form B prepared in Example 1 of the present application;
  • Example 4 is a temperature-variable X-ray powder diffraction test diagram of potassium salt crystal form B prepared in Example 1 of the present application;
  • FIG. 6 is a comparison diagram of XRPD before and after DVS test of potassium salt crystal form B prepared in Example 1 of the present application;
  • Figure 7 is a graph of the dynamic solubility of potassium salt crystal form B prepared in Example 1 in water at 37°C;
  • Figure 8 is a graph of the dynamic solubility of potassium salt crystal form B prepared in Example 1 in SGF at 37°C;
  • Figure 9 is a graph of the dynamic solubility of potassium salt crystal form B prepared in Example 1 in FaSSIF at 37°C;
  • Figure 10 is a graph of the dynamic solubility of potassium salt crystal form B prepared in Example 1 in FeSSIF at 37°C;
  • Figure 11 is a comparison diagram of XRPD results of solids obtained from a sample of compound WYQ in water and SGF at 37°C;
  • Figure 12 is a comparison diagram of XRPD results of solids obtained from a sample of compound WYQ in FaSSIF and FeSSIF at 37°C;
  • FIG. 13 is a comparison diagram of the XRPD results of the potassium salt crystal form B prepared in Example 1 at 37°C in water;
  • 16 is a comparison diagram of the XRPD results of the potassium salt crystal form B prepared in Example 1 in FeSSIF at 37°C;
  • Figure 17 is a comparison diagram of XRPD results of compound WYQ samples placed under solid-state stability test conditions
  • Figure 19 is a graph showing the average blood concentration-time curve of the potassium salt crystal form B prepared in Example 1 at 2.0 mg ⁇ kg -1 in rats;
  • Figure 20 is a graph showing the effects of different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil on the RVSP of PAH rats after 21 days of treatment;
  • Figure 21 is a graph showing the effects of different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil on the RVMI of PAH rats after 21 days of treatment;
  • Figure 22 is a standard picture of H&E staining of pulmonary arterioles in different groups of rats with different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil for 21 days after treatment of PAH rats;
  • Figure 23 is a statistical diagram of the pulmonary arteriole-media thickness of different groups of rats in different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil for 21 days after treating PAH rats;
  • Figure 24 is a statistical graph showing the percentage of pulmonary arteriole-media thickness (WT%) in different groups of rats in different groups after treating PAH rats with different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil for 21 days;
  • Figure 25 shows the effect of WYQ potassium salt crystal form B micro-dose treatment (Mini-dose) on erectile function in rats;
  • Figure 26 shows the effect of WYQ potassium salt crystal form B low-dose treatment (Low-dose) on erectile function in rats.
  • Figure 27 shows the effect of WYQ potassium salt crystal form B medium-dose treatment (Medium-dose) on erectile function in rats;
  • Figure 28 shows the effect of WYQ potassium salt crystal form B high-dose treatment (High-dose) on erectile function in rats;
  • Figure 29 shows the effect of sildenafil treatment on erectile function in rats
  • Figure 30 shows the effect of tadalafil treatment on erectile function in rats
  • Figure 31 is a graph showing the effect of different doses of WYQ potassium salt crystal form B and the current clinically approved positive control drug nintedanib (BIBF) on the weight of the left lung of IPF rats after 23 days;
  • Figure 32 is a graph showing the effect of different doses of WYQ potassium salt crystal form B and BIBF on the left lung volume of rats after 23 days of treatment of IPF rats.
  • Figure 33 is a comparison chart (H&E staining) of left lung pulmonary fibrosis lesions and lesion area changes after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats.
  • A is the model group and B is the BIBF-50mg/ kg group, C is CPD-1-2.5mg/kg group, D is CPD-1-10mg/kg group;
  • Figure 34 shows the comparison of changes in left lung fibrosis lesions and lesions in IPF rats with different doses of WYQ potassium salt crystal form B and BIBF for 23 days (Masson Trichrom staining), where A is the model group and B is the BIBF-50mg /kg group, C is CPD-1-2.5mg/kg group, D is CPD-1-10mg/kg group;
  • Figure 35 shows the comparison of histological changes of bronchiole and pulmonary arterioles in left lung pulmonary fibrosis lesions in IPF rats with different doses of WYQ potassium salt crystal form B and BIBF for 23 days (HE staining, image magnification: ⁇ 200) ,
  • A is the contralateral lung tissue of the model group
  • B is the model group
  • C is the BIBF-50mg/kg group
  • D is the CPD-1-2.5mg/kg group
  • E is the CPD-1-10mg/kg group
  • Figure 36 is a comparison diagram of histological changes of bronchiole and pulmonary arterioles at the edge of left lung pulmonary fibrosis lesions after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (HE staining, image magnification: ⁇ 200) ,
  • A is the contralateral lung tissue of the model group
  • B is the model group
  • C is the BIBF-50mg/kg group
  • D is the CPD-1-2.5mg/kg group
  • E is the CPD-1-10mg/kg group;
  • Figure 37 is a comparison diagram of changes in alveolar tissue structure in the left lung pulmonary fibrosis lesions after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (HE staining, image magnification: ⁇ 200), where A is the model Group contralateral lung tissue, B is the model group, C is the BIBF-50mg/kg group, D is the CPD-1-2.5mg/kg group, and E is the CPD-1-10mg/kg group;
  • Figure 38 is a comparison diagram of changes in the histological structure of the left lung pulmonary fibrosis lesion alveolar after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (Masson Trichrom staining, image magnification: ⁇ 200), where A is The contralateral lung tissue of the model group, B is the model group, C is the BIBF-50mg/kg group, D is the CPD-1-2.5mg/kg group, and E is the CPD-1-10mg/kg group;
  • Figure 39 is a comparison diagram of changes in the area of left lung pulmonary fibrosis after treatment with different doses of WYQ potassium salt crystal form B and BIBF for 23 days in IPF rats;
  • Figure 40 is the standard of pulmonary fibrosis pathology score (Masson Trichrome staining), and Figures A-I are the standard pictures of Masson Trichrome staining of fibrosis grade 0-8 in Ashcroft scoring system;
  • Figure 41 is a comparison diagram of changes in left lung pulmonary fibrosis lesion scores after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats;
  • Fig. 42 is a comparison chart of changes in the score percentage of left lung pulmonary fibrosis lesions after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats.
  • Figure 43 shows that WYQ potassium salt crystal form B can improve renal fibrosis in UIRI model mice in vivo, where A is WYQ potassium salt crystal form B inhibits FN, Collagen I, PAI-1 and ⁇ -SMA in renal fibrotic lesions Western blot standard graph of protein expression, the number (1, 2, 3) represents each animal in each group, BE is the statistical graph of relative content determination of FN, Collagen I, PAI-1 and ⁇ -SMA;
  • Figure 44 shows the comparison of the changes in the structure of the glomeruli and tubules in the left renal fibrosis lesions (HE staining) after treatment with WYQ potassium salt crystal form B in UIRI model mice for ten days.
  • AC represents the sham operation group, UIRI model group, and WYQ potassium salt crystal form B treatment group;
  • Figure 45 shows the comparison of the changes in the structure of the glomeruli and tubules in the left renal fibrosis lesions in the left renal fibrosis lesions after WYQ potassium salt crystal form B treatment of UIRI model mice for ten days (Masson Trichrome staining).
  • AC represents the sham operation group and the UIRI model group, respectively , WYQ potassium salt crystal form B treatment group;
  • Figure 46 shows the changes in the expression of FN and ⁇ -SMA in the renal fibrotic lesions of UIRI model mice after treatment with WYQ potassium salt crystal form B for ten days (immunohistochemistry).
  • AC represents the sham operation group, UIRI model group, and WYQ potassium salt, respectively Crystal Form B treatment group;
  • Figure 47 is a comparison diagram of histological changes (HE staining) of glomeruli and renal tubules in the left kidney fibrosis lesion after treatment with WYQ potassium salt crystal form B in UUO model mice for 7 days, where A is the normal control group and B is the model Group C is the WYQ potassium salt crystal form B treatment group;
  • Figure 48 shows the changes of FN expression in renal fibrotic lesions in UUO mice treated with WYQ potassium salt crystal form B for 7 days (immunohistochemistry), where A is the normal control group, B is the model group, and C is the WYQ potassium salt Form B group;
  • Figure 49 is a statistical diagram of the fibrosis area in the left kidney fibrosis lesion after treatment of UUO mice with WYQ potassium salt crystal form B for 7 days;
  • Figure 50 shows that WYQ potassium salt crystal form B improves the cardiac function parameters of rats with isoprenaline-induced myocardial hypertrophy after 7 days of treatment.
  • AC is the normal control group
  • DF is a statistical graph of left ventricular pressure, Max dp/dt and Min dp/dt of rats in each group;
  • Figure 51 shows WYQ potassium salt crystal form B treatment for 7 days, the reduction of isoproterenol-induced cardiac hypertrophy in rats with cardiac obesity index, where A is the normal control group, model group and WYQ potassium salt crystal form B treatment group rats HW-Tibia ratio statistical graph, B is the statistical graph of left ventricle-tibia length ratio of rats in each group;
  • Figure 52 shows that WYQ potassium salt crystal form B reduced the expression of hypertrophic factor ANP in the cardiac tissue of rats with myocardial hypertrophy induced by isoproterenol after 7 days of treatment.
  • a and B are normal control group, model group and WYQ potassium salt crystal
  • C is the statistical graph of ANP gene expression in rats in each group.
  • Figure 53 shows that WYQ potassium salt crystal form B reduced the expression of hypertrophic factor BNP in the cardiac tissue of rats with myocardial hypertrophy induced by isoproterenol after 7 days of treatment.
  • Potassium hydroxide was purchased from China National Pharmaceutical Group Corporation;
  • Vacuum filtration was performed to separate the above solid material, and vacuum-dried it at 25° C. for 24 h to obtain 290.1 mg of crystalline solid (mass yield: 72.5%).
  • X-ray powder diffraction (XRPD) chart is shown in Figure 1; TG-DSC chart is shown in Figure 2; HPLC purity 98.7area%; IC test (Thermo ICS1100) results show that the stoichiometric ratio of compound WYQ to potassium ion is 1:7 ⁇ 1 :10;
  • the polarized light microscope (PLM, Axio Lab.A1) image is shown in Figure 3, indicating that the prepared potassium salt crystal form B is composed of small particles with agglomeration.
  • Vacuum filtration was performed to separate the above solid material, and vacuum-dried it at room temperature for 12 hours to obtain 290.1 mg of crystalline solid (mass yield: 72.5%).
  • X-ray powder diffraction (XRPD) pattern is basically consistent with Figure 1; TG-DSC pattern is basically consistent with Figure 2; HPLC purity is 99area%.
  • Vacuum filtration was performed to separate the above solid material and vacuum-dried it at room temperature for 20 h to obtain 290.1 mg of crystalline solid (mass yield: 72.5%).
  • X-ray powder diffraction (XRPD) pattern is basically consistent with Figure 1; TG-DSC pattern is basically consistent with Figure 2; HPLC purity is 99area%.
  • the present invention provides the potassium salt crystal form B of compound WYQ, and uses various methods and instruments to study the properties of the potassium salt crystal form B of compound WYQ prepared in Example 1.
  • the present invention adopts (X'Pert 3 ) model X-ray powder diffractometer, at a scanning speed of 2° per minute, using a copper-chromium target to obtain the XRPD pattern of potassium salt crystal form B prepared in Example 1 as shown in Figure 1. Show.
  • the XRPD test parameter settings are shown in Table 1, and the detailed X-ray powder diffraction parameters in Figure 1 are shown in Table 2.
  • the potassium salt crystal form B prepared in Example 1 has a weight loss of 0.9% before 150°C, and has absorption at peaks 191.3°C (starting temperature 188.5°C) and 217.9°C (starting temperature 208.7°C) Hot peak. Combining that the potassium salt crystal form B has a small weight loss before melting and decomposition, it is speculated that the potassium salt crystal form B prepared in Example 1 is an anhydrous crystal.
  • the temperature-variable X-ray powder diffraction (VT-XRPD) test was carried out.
  • the test conditions were no nitrogen protection at 30°C, nitrogen purging at 30°C for 30 minutes, heating to 150°C under nitrogen protection, and cooling down to 30°C under nitrogen protection.
  • the test results are shown in Figure 4.
  • the potassium salt crystal form B sample prepared in Example 1 was purged with nitrogen at 30°C for 30 minutes, the crystal form did not change.
  • partial diffraction peaks were observed After shifting and cooling to 30°C, the crystal form is consistent with the potassium salt crystal form B, confirming that the potassium salt crystal form B of the compound WYQ is an anhydrous crystal.
  • a DVS Intrinsic instrument of SMS was used to conduct a dynamic vapor adsorption experiment at 25° C. to conduct a moisture absorption test on the potassium salt crystal-free type B prepared in Example 1.
  • the test sample is pre-dried under the condition of 0% relative humidity (RH) to remove the adsorbed solvent or water and then the test is started.
  • the target relative humidity is 0-95%.
  • the mass change (moisture adsorption capacity) of the potassium salt crystal form B sample under the condition of 25°C/80%RH is 0.6%, indicating that it is slightly hygroscopic.
  • the XRPD comparison chart shows that the crystal form of the test sample has not changed before and after the DVS test.
  • DVS test parameter settings are shown in Table 4.
  • the potassium salt crystal form B sample prepared in Example 1 was used in four solvents: water, artificial gastric juice (SGF), fasting artificial intestinal fluid (FaSSIF), and fed artificial intestinal fluid (FeSSIF)
  • SGF artificial gastric juice
  • FaSSIF fasting artificial intestinal fluid
  • FeSSIF fed artificial intestinal fluid
  • the dynamic solubility in WYQ was tested, and the dynamic solubility data of the WYQ sample under the same conditions were collected.
  • the solid-state stability of the potassium salt crystal form B prepared in Example 1 was tested, and 5.22 mg of the potassium salt crystal form B sample was weighed and placed in an open at 25°C/60% RH, and 5.19 mg of the potassium salt crystal form was weighed Sample B was placed in the open under the condition of 40°C/75%RH. After one week, XRPD characterization (see Table 1 for XRPD detection parameter settings) and HPLC purity test (Agilent 1100) were performed on all samples to detect crystal form and purity changes. HPLC The purity test parameters are shown in Table 6.
  • the potassium salt crystal form B sample of compound WYQ prepared in Example 1 of the present invention has better moisture absorption, solubility, and physical and chemical stability than that of compound WYQ.
  • the pharmaceutical use of the potassium salt crystal form B prepared in the embodiment of the present invention will be specifically described below in conjunction with specific experiments.
  • the potassium salt crystal form B of compound WYQ was prepared in Example 1; sodium carboxymethyl cellulose, physiological saline, and sodium pentobarbital were purchased from China National Pharmaceutical Group Co., Ltd.; acetonitrile, formic acid, and methanol were purchased from China Bailingwei Chemical Reagent Co., Ltd.
  • mice Male SPF SD rats, weighing 180 ⁇ 200g, were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd., experimental animal license number: SCXK ( ⁇ )-2002-0011, raised in the experimental animal room of Nanjing Medical University, all large The rats were placed in a temperature (22 ⁇ 2) °C, humidity (60 ⁇ 5) °C, 12h alternating light and dark environment.
  • Example 2 6 healthy male SD rats, fasted and freely drinking water for 12 hours, the potassium salt crystal form B prepared in Example 1 was administered by gavage at 2 mg.kg -1 , respectively 0.2h, 0.5h, 1h, Blood was collected from the ocular venous plexus at 1.5h, 2h, 4h, 6h and 8h, and acetonitrile: water (1:1) was added to centrifuge and the supernatant was centrifuged to perform chromatographic analysis.
  • the potassium salt crystal form B of compound WYQ was prepared in Example 1; Sildenafil and Tadalafil were synthesized by WuXi AppTec New Drug Development Co., Ltd. (batch numbers: EW10443-180-P1, EW10443-228-P1); monocrotaline (monocrotaline, MCT) and sodium carboxymethyl cellulose are the products of American Sigma reagent company; heparin, urethane solution, xylene, and paraffin were purchased from China National Pharmaceutical Group Co., Ltd.; hematoxylin-eosin and neutral gum were purchased from China Bi Yuntian Biological Technology Co., Ltd.
  • SD rats were randomly divided into 8 groups, 15 per group, namely: normal control group (Vehicle); PAH model group (MCT); PAH+WYQ potassium salt crystal form B micro-dose group (Mini-dose, hereinafter referred to as micro-dose) Group); PAH+WYQ potassium salt crystal form B low-dose group (Low-dose, hereinafter referred to as low-dose group); PAH+WYQ potassium salt crystal form B medium-dose group (Medium-dose, hereinafter referred to as medium-dose group); PAH +WYQ potassium salt crystal form B high-dose group (High-dose, hereinafter referred to as high-dose group); PAH+Sildenafil control group (hereinafter referred to as sildenafil group); PAH+Tadalafil control group (hereinafter referred to as tadalafil group) ).
  • normal control group Vehicle
  • MCT PAH model group
  • PAH+WYQ potassium salt crystal form B micro-dose group Mini
  • the normal control group was injected with saline (50mg/kg) under the abdominal cavity, and the other groups were injected with MCT (50mg/kg) under the abdominal cavity at one time.
  • PAH model group no treatment
  • Micro-dose group WYQ potassium salt crystal form B is mixed with normal saline to make 0.4mg/ml medicinal solution (currently used), administered by gavage, and the dosage is 2mg.kg -1 .d -1 ;
  • WYQ potassium salt crystal form B is made into 1mg/ml medicinal solution with physiological saline (currently used), administered by gavage, and the dosage is 5mg.kg -1 .d -1 ;
  • Middle-dose group WYQ potassium salt crystal form B is made into 2mg/ml medicinal solution with physiological saline (currently used), administered by gavage, and the dosage is 10mg.kg -1 .d -1 ;
  • High-dose group WYQ potassium salt crystal form B is mixed with physiological saline to make 4mg/ml medicinal solution (currently used now), administered by gavage, and the dosage is 20mg.kg -1 .d -1 ;
  • Sildenafil group Sildenafil was prepared with 0.5% sodium carboxymethyl cellulose into a 5mg/ml liquid (currently used now), administered by gavage, and the dosage was 25mg.kg -1 .d -1 ;
  • Tadalafil group Tadalafil was formulated with 0.5% sodium carboxymethyl cellulose into a 2 mg/ml liquid (currently prepared), administered by gavage, and the dosage was 10 mg.kg -1 .d -1 .
  • the day of injection was the starting day of the experiment, and the administration was started 7 days after the model was built, once a day for 14 consecutive days.
  • the conversion factor for rats and humans is 0.162. Therefore, according to the gavage dosage of rats in the examples, the population taking WYQ potassium salt crystal form B is estimated to be 0.324, 0.81, 1.62 and 3.24 mg.kg -1 .d -1 respectively. , The application time is once a day for 14 consecutive days.
  • the ratio of intramuscular and intraperitoneal injection to oral dose is about 0.3-0.4. From this, it is calculated that the WYQ potassium salt crystal form B human injection medicine is micro, The low, medium and high doses are 0.097-0.129, 0.243-0.324, 0.486-0.648 and 0.972-1.296 mg.kg -1 .d -1 respectively .
  • the administration time is once a day for 14 consecutive days (drug concentration: 1mg/ml ).
  • RVSP right ventricular systolic blood pressure
  • RVMI right ventricle
  • LV+S left ventricular wall plus septum
  • the rats were treated with heparin (50IU/100g, intraperitoneal injection) for 5 minutes, and the animals were anesthetized with 20% urethane solution (0.5ml/100g), and blood pressure indicators were detected and recorded;
  • the lung was dissected, the right lung was ligated, and the left lung lobe was perfused with fixative (4% paraformaldehyde) and then fixed in the fixative.
  • fixative 4% paraformaldehyde
  • Different slices were taken to randomly observe the changes in the thickness and thickness of the arteriolar media in 6 fields of view, and compared with the control group to determine the lung tissue of each group Learning changes.
  • RVSP Right ventricular systolic blood pressure
  • Figure 20 is a graph showing the effect of different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil on the RVSP of PAH rats after 21 days of treatment.
  • the results showed that compared with the normal control group (22.9 ⁇ 0.5mmHg), the RVSP (72.5 ⁇ 4.5mmHg) of rats in the PAH model group was significantly increased ( ** P ⁇ 0.01); micro-dose group, low-dose group, middle-dose group The RVSP of the high-dose group and the high-dose group were significantly decreased and improved (micro-dose group: 56.2 ⁇ 0.5mmHg, # P ⁇ 0.05; low-dose group: 47.6 ⁇ 1.9mmHg, ## P ⁇ 0.01; medium-dose group: 40.7 ⁇ 1.5mmHg , ## P ⁇ 0.01; high-dose group: 29.8 ⁇ 1.6mmHg, ## P ⁇ 0.01), and the inhibitory effect on RVSP was dose-dependent.
  • Figure 21 is a graph showing the effects of different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil on the RVMI of PAH rats after 21 days of treatment.
  • the results showed that: compared with the normal control group (24.2 ⁇ 0.7%), the RVMI (51.7 ⁇ 2.3%) of the PAH model group was significantly increased ( ** P ⁇ 0.01); the WYQ potassium salt crystal form B administration was micro
  • the RVSP of the dose group, low dose group, middle dose group and high dose group were significantly decreased and improved (micro dose group: 42.8 ⁇ 2.1%, # P ⁇ 0.05; low dose group: 40.6 ⁇ 2.7%, ## P ⁇ 0.01 ; Middle dose group: 38.7 ⁇ 2.1%, ## P ⁇ 0.01; high dose group: 34.6 ⁇ 19%, ## P ⁇ 0.01), different from the inhibitory effect on RVSP, WYQ potassium salt crystal form B inhibits RVMI The effect is not dose-dependent; compared with the PAH model group, the RVMI of the tadalafil group was
  • Figure 22 is a standard picture of H&E staining of pulmonary arterioles in different groups of rats after 21 days of treatment with different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil for PAH rats.
  • Figure 25-30 shows the comparison of the penile erection of rats in different doses of WYQ potassium salt crystal form B and sildenafil and tadalafil treatment groups after intragastric administration for 1h/40min/30min.
  • Observation results 1 hour after oral administration of WYQ potassium salt crystal form B micro-dose group, 30% of rats developed penile erections; 1 hour after oral administration of the low-dose group, 50% rats developed penile erections; After 1 hour, 80% of the rats developed penile erections; 40 minutes after intragastric administration in the high-dose group, 80% of the rats developed penile erections.
  • the drug dose increased, the erect penis became thicker and more red.
  • Bleomycin hydrochloride is a product of Nippon Kayaku Co., Ltd.; HPLC grade water, BIBF is provided by Case Air Biotechnology (Suzhou) Co., Ltd.; Sodium carboxymethyl cellulose is a product of Sigma Reagent Company; the potassium salt crystal of compound WYQ Form B was prepared in Example 1; isoflurane, pentobarbital sodium anesthetic, and formalin were purchased from China National Pharmaceutical Group Corporation.
  • 32 male SD rats were randomly divided into 4 groups according to their body weight, 8 rats/group: model group, nintedanib 50mg/kg group (BIBF-50mg/kg), WYQ potassium salt crystal form B 2.5mg/kg group (CPD-1-2.5mg/kg), WYQ potassium salt crystal form B 10mg/kg group (CPD-1-10mg/kg).
  • Model group Administer physiological saline by gavage according to body weight, the dose is 1ml.100g -1 .d -1 ;
  • Trinidad Technip 50mg / kg group (BIBF-50mg / kg): BIBF with 0.5% sodium carboxymethylcellulose formulated 10mg / ml solution, administered orally, the dose 50mg.kg -1 .d - 1 ;
  • WYQ potassium salt crystal form B 2.5mg/kg group (CPD-1-2.5mg/kg): WYQ potassium salt crystal form B is diluted with normal saline to a solution of 0.5mg/ml, administered by gavage, the dosage is 2.5mg .kg -1 .d -1 ;
  • WYQ potassium salt crystal form B10mg/kg group (CPD-1-10mg/kg): WYQ potassium salt crystal form B is diluted with physiological saline into a 2mg/ml solution, and administered by gavage, the dosage is 10mg.kg -1 . d -1 ;
  • Rats in each group were given intragastric administration on the day of modeling, once a day, for a total of 23 days.
  • the conversion factor for rats and humans is 0.162. Therefore, according to the gavage dosage of rats in the examples, the population dosage of WYQ potassium salt crystal form B is estimated to be 0.405 and 1.62 mg.kg -1 .d -1 respectively, and the administration time is once a day for 23 consecutive days .
  • the ratio of intramuscular and intraperitoneal injection to oral dose is about 0.3-0.4. From this, the human injection dosage of WYQ potassium salt crystal form B is calculated. It is 0.122-0.162, 0.486-0.648 mg.kg -1 .d -1 , and the administration time is once a day for 23 consecutive days (drug concentration: 1 mg/ml).
  • mice in each group were given intraperitoneal injection of pentobarbital sodium anesthetic (100mg/kg) to euthanize all animals in accordance with the KCI standard operating procedures for animal euthanasia.
  • pentobarbital sodium anesthetic 100mg/kg
  • the animals were perfused systemically with low-temperature PBS and then systemic formalin perfusion Fix, take the left lung, perfuse the lung with an equal amount of formalin solution, perform weighing and follow-up lung pathology related tests.
  • the gross pathological examination of the left lung After the left lung is perfused with an equal amount of formalin, the wet weight of the left lung after the perfusion is weighed and recorded with a microbalance; the left lung volume after the perfusion is measured and recorded with a toe volume meter.
  • the left lung was dehydrated according to the KCI pathological standard SOP.
  • the left lung was made by paraffin block, and the left lung was paraffin sectioned with a thickness of 3-4 ⁇ m; followed the KCI pathological standard staining SOP for HE staining and Masson Trichrome staining, and passed Hamamatsu NanoZoomer Digital Pathology( S210) Slice scanner to perform panoramic slice scanning; Masson Trichrome staining slices to calculate lung lesion area, left lung fibrosis area (%) is the percentage of fibrosis area to left lung area; 10 area sizes are randomly selected in the lesion area With a field of 1 mm 2 , the pathologist performs semi-quantitative scoring under double-blind conditions according to the Ashcroft scoring system (shown in Table 8 and Figure 40).
  • FIG. 31 shows the weight of the left lung of the IPF rats after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF.
  • Figure 32 shows the effect of different doses of WYQ potassium salt crystal form B and BIBF on the left lung volume of rats after 23 days of treatment of IPF rats.
  • Model group 8 1.56 ⁇ 0.11 1739 ⁇ 116.23 BIBF-50mg/kg 8 1.58 ⁇ 0.12 1736.9 ⁇ 136.10 CPD-1-2.5mg/kg 8 1.59 ⁇ 0.10 1779.9 ⁇ 105.76 CPD-1-10mg/kg 8 1.47 ⁇ 0.16 1604.5 ⁇ 153.72
  • Figures 33-34 show significant lung injury with clear lung tissue boundaries.
  • Two different lung histological stains H&E and Masson Trichrom staining
  • H&E and Masson Trichrom staining clearly show uniform fibrotic lesions and lesion distribution in the left lung.
  • WYQ potassium salt crystal form B and BIBF After 23 days of treatment of IPF rats with different doses of WYQ potassium salt crystal form B and BIBF, compared with the model group, there was no significant difference in left lung pulmonary fibrosis lesions and lesion range.
  • bronchiole, terminal bronchiole, and alveolar duct epithelial cells were hyperplasia in varying degrees, part of the epithelium and even full-thickness epithelium were gobletized, and varying amounts of mucus tissue were seen in the lumen.
  • Figure 35 shows the comparison of histological changes (HE staining) of bronchioles and pulmonary arterioles in the left lung pulmonary fibrosis lesions after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats.
  • Figure 36 shows different doses of WYQ Comparison of histological changes of bronchiole and pulmonary arterioles at the edge of the left lung pulmonary fibrosis lesion after 23 days of treatment with potassium salt crystal form B and BIBF in IPF rats.
  • Alveolar tissue in the left lung fibrosis lesion of IPF rats is damaged to varying degrees, manifested as alveolar epithelial shedding and regeneration, alveolar wall thickening, and fibrosis; varying degrees of fibrous tissue deposition in the alveolar cavity, inflammatory exudation, and inflammatory cell infiltration
  • the flaky alveolar structure in the fibrotic lesion is damaged and disappeared, and it is filled with a large number of exuding inflammatory cells and hyperplastic connective tissue. Inflammatory exudate and hyperplastic connective tissue can be seen in the remaining alveolar cavity.
  • Figure 37 is a comparison diagram of changes in alveolar tissue structure in the left lung pulmonary fibrosis lesion after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (HE staining, picture magnification: ⁇ 200).
  • Figure 38 is a comparison diagram of changes in the histological structure of the left lung pulmonary fibrosis lesion alveolar after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (Masson Trichrom staining, image magnification: ⁇ 200).
  • Figure 39 is a comparison diagram of changes in left lung lung fibrosis area after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (the normal control group in the figure refers to the untreated lung in the model group). Show: The lesion area is basically the same between the model group and each administration group, suggesting the stability and uniformity of the model.
  • Figure 40 shows the pathology score of pulmonary fibrosis (Masson Trichrome staining) standard. Table 10 shows the results of the Ashcraft score and score percentage statistics of pulmonary fibrosis.
  • Figure 41 shows the comparison of the scores of left lung pulmonary fibrosis lesions after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (normal control in the figure) The group refers to the untreated lung in the model group);
  • Figure 42 shows the comparison of the percentage changes in left lung pulmonary fibrosis lesion scores after 23 days of treatment with different doses of WYQ potassium salt crystal form B and BIBF in IPF rats (normal in the figure)
  • the control group refers to the untreated lung in the model group).
  • Table 10 shows the pulmonary fibrosis Ashcraft score and score percentage statistical results
  • Pulmonary fibrosis Ashcraft score results showed that the positive control drug BIBF significantly improved the degree of left lung fibrosis in rats compared with the model group at a dose of 50mg/kg (p ⁇ 0.05); WYQ potassium salt crystal form B was at 10mg/kg Under the dose, oral administration once a day for 23 consecutive days can significantly inhibit pulmonary fibrosis, which is significantly different from the model group (p ⁇ 0.05).
  • the percentage of pulmonary fibrosis levels below 3 (including 3 points) or above 4 (including 4 points) is calculated.
  • the results show that nearly 51% of the lesion areas in the model group are scored at 4 points Or more than 4 points.
  • the score of the lesion area of each drug treatment group animal is above 4 points, and the area is between 25-50%.
  • the statistical results showed that the percentage of pulmonary fibrosis after treatment with the positive drug BIBF was significantly reduced compared with the model group (p ⁇ 0.05); WYQ potassium salt crystal form B at a dose of 10 mg/kg, the treatment group and the model group compared lung fibers Compared with the model group, the percentage of the degree of transformation was significantly reduced (p ⁇ 0.05).
  • Mouse anti- ⁇ -SMA antibody, mouse anti- ⁇ -tubulin antibody, rabbit anti-Fibronectin antibody, and sodium carboxymethylcellulose are the products of Sigma Reagent Company; rabbit anti-Collagen-I antibody, rabbit anti-Kim-1 antibody, purchased from Millipore, USA Company; anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson, the United States; the potassium salt crystal form B of compound WYQ was prepared in Example 1; isoflurane, pentobarbital sodium anesthetic, and formalin were purchased from China National Medicines Group Co., Ltd.
  • Tissue dehydration machine HistoCore Pearl, Leica; Embedding machine: HistoCore Arcadia, Leica; Microtome: RM2235, Leica; Automatic staining machine: LEICA Autostainer ST5020; Section scanner: Hamamatsu NanoZoomer Digital Pathology (S210); Analytical balance: German Precia Company; Weight scale: Changshu Shuangjie Test Instrument Factory, T1000; Surgical microscope: Luckbird XTS-4A; Gel imaging system: American Bio-Rad; Electrophoresis tank and electrophoresis instrument: American Bio-Rad; pH meter: Switzerland ETTLER company.
  • SPF male BALB/c mice weigh about 20g. Purchased from the Laboratory Animal Center of Southern Medical University, license number: SCXK (Guangdong)-2011-0015. The experimental process strictly followed the "Guiding Opinions on the Good Treatment of Laboratory Animals” issued in 2006 to dispose of animals.
  • mice Fifteen male BALB/c mice were randomly divided into: 5 in the Sham group (sham operation group) and 10 in the surgical model. Operation method: After anesthesia, an incision was made on the left abdomen of the mouse to expose the kidney and bluntly separate the renal pedicle. In the sham operation group, only the renal pedicle was exposed and not clipped; the model used a non-invasive micro-arterial clip to clamp the left renal renal pedicle for 30 minutes. During the clipping, the mice were placed on a 37°C constant temperature plate to maintain a constant body temperature, and the surgical incision was covered Gauze soaked with saline to prevent dehydration of kidney tissue.
  • the model mice were randomly divided into 2 groups: UIRI group (model group), WYQ potassium salt crystal form B treatment group (5 mg/kg), 5 mice in each group.
  • the first gavage was started 2 hours after the operation.
  • 1Sham group and UIRI model group Administer normal saline by gavage according to body weight, the dose is 0.1ml.10g -1 .d -1 ;
  • WYQ potassium salt crystal form B treatment group WYQ potassium salt crystal form B was diluted with normal saline to a 1mg/ml solution, and administered by gavage, the dosage was 5mg.kg -1 .d-1;
  • the conversion factor for mice and humans is 0.081. Therefore, according to the mouse gavage dose in the examples, the population dose of WYQ potassium salt crystal form B is estimated to be 0.405 mg.kg -1 .d -1 , and the administration time is once a day for 10 consecutive days.
  • the ratio of intramuscular and intraperitoneal injection to oral dose is about 0.3-0.4. From this, the human injection dosage of WYQ potassium salt crystal form B is calculated. It is 0.1215-0.162 mg.kg -1 .d -1 , and the administration time is once a day for 10 consecutive days (drug concentration: 1 mg/ml).
  • mice were anesthetized in the same way, the right back was incised, the right kidney was exposed, and the kidney pedicle was ligated and then the right kidney was removed. In the sham operation group, only the renal capsule was removed, and the right kidney was not removed.
  • the abdominal cavity was opened, and the left kidney tissue of the mice was stripped. Care should be taken to maintain the integrity of the kidney. After the kidney is taken out, it is quickly transferred to pre-cooled PBS, and the kidney is cut for different tests. Divide the kidney into four parts with a scalpel.
  • kidney tissue is transferred Store in the refrigerator at -80°C.
  • the dorsal side of the kidney was fixed in 4% paraformaldehyde for making paraffin sections.
  • HE staining and Masson staining were used to observe the morphological changes of the kidney tissue, and immunohistochemistry was used to observe the changes in the expression of fibrosis marker protein in the kidney tissue.
  • mice Fifteen male C57BL/6 mice were adaptively fed for one week and randomly divided into two groups: 5 in the Sham group (sham operation group), and 10 in the surgical model. Operation method: The mice in the sham operation group were under anesthesia and the left ureter was opened without ligation, and the abdomen was closed and sutured; the mice in the model group were under anesthesia with the left ureter ligation. Model mice were randomly divided into 2 groups: UUO group (model group), WYQ potassium salt crystal form B treatment group (5mg/kg), 5 mice in each group.
  • the first gavage was started 2 hours after the operation.
  • 1Sham group and UUO model group Administer physiological saline by gavage according to body weight, the dose is 0.1ml.10g -1 .d -1 ;
  • WYQ potassium salt crystal form B treatment group WYQ potassium salt crystal form B was diluted with physiological saline to a 1mg/ml solution, and administered by gavage, the dosage was 5mg.kg -1 .d -1 ;
  • the conversion factor for mice and humans is 0.081. Therefore, according to the gavage dose of rats in the examples, the population dose of WYQ potassium salt crystal form B is estimated to be 0.405 mg.kg -1 .d -1 , and the administration time is once a day for 7 consecutive days.
  • the ratio of intramuscular and intraperitoneal injection to oral dose is about 0.3-0.4
  • the human injection dose of WYQ potassium salt crystal form B is calculated as 0.1215-0.162 mg.kg -1 .d -1
  • the administration time is once a day for 7 consecutive days (drug concentration: 1 mg/ml).
  • mice All the mice were sacrificed 7 days after the administration, the abdominal cavity was opened, and the left and right kidney tissues of the mice were stripped. Care should be taken to maintain the integrity of the kidney. After the kidney is taken out, it is quickly transferred to pre-cooled PBS, and the kidney is cut for different tests. Divide the kidney into four parts with a scalpel. The back of the kidney is fixed in 4% paraformaldehyde for making paraffin sections. HE staining and Masson staining are used to observe the morphological changes of the kidney tissue, and immunohistochemistry to observe the fibers in the kidney tissue Changes in expression of chemical marker proteins.
  • Isoproterenol, mouse anti- ⁇ -SMA antibody, and sodium carboxymethylcellulose are the products of Sigma Reagent Company; rabbit anti-Collagen-I antibody was purchased from Millipore Company; mouse anti-BNP antibody and rabbit anti-ANP antibody were purchased from abcam company; anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson, USA; the potassium salt crystal form B of compound WYQ was prepared in Example 1; heparin, uratan solution, and formalin were purchased from China National Pharmaceutical Group Company limited by shares.
  • the rat ANP and BNP primers were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.
  • RM6240E multi-channel physiological signal recorder (Chengdu Instrument Factory); analytical balance: German Precia Company; weight scale: Changshu Shuangjie Test Instrument Factory, T1000; operating microscope: Luckbird XTS-4A; gel imaging system: American Bio-Rad Company; electrophoresis tank, electrophoresis instrument: American Bio-Rad Company; pH instrument: Switzerland ETTLER Company.
  • SPF male SD rats purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002. Raised in the Experimental Animal Center of South China University of Technology, the animal center license number: SYXK (Guangdong) 2017-0178, weight: 250 ⁇ 10 grams.
  • SD rats were randomly divided into 3 groups, 10 rats/group, namely: normal control group (CON); isoproterenol-induced cardiac hypertrophy model group (Iso); Iso+WYQ potassium salt crystal form B treatment group (hereinafter referred to as treatment group).
  • CON normal control group
  • Iso isoproterenol-induced cardiac hypertrophy model group
  • Iso+WYQ potassium salt crystal form B treatment group hereinafter referred to as treatment group.
  • Isoproterenol is prepared with double-distilled water to make a 4mg/ml medicinal solution, which is now prepared for immediate use.
  • the normal control group was injected with normal saline (5mg/kg) into the abdominal cavity, and the other groups were injected with isoproterenol (5mg/kg) into the abdominal cavity once a day for 7 days.
  • Iso model group no drug treatment
  • WYQ potassium salt crystal form B was prepared with normal saline to make 0.4mg/ml medicinal solution (prepared for current use), administered by gavage, and the dosage was 2mg.kg -1 .d -1 ;
  • the day of injection was the starting day of the experiment, and the administration was started 2 hours after the model was built, once a day for 7 consecutive days.
  • the conversion factor for rats and humans is 0.162. Therefore, according to the gavage dose of rats in the examples, the population dose of WYQ potassium salt crystal form B is estimated to be 0.324 mg.kg -1 .d -1 , and the administration time is once a day for 7 consecutive days.
  • the ratio of intramuscular and intraperitoneal injection to oral dose is about 0.3-0.4
  • the human injection dose of WYQ potassium salt crystal form B is calculated as 0.0972-0.1296 mg.kg -1 .d -1
  • the administration time is once a day for 7 consecutive days (drug concentration: 1 mg/ml).
  • the left ventricle was cannulated retrogradely through the right common carotid artery, and hemodynamic index data of each group of rats were collected on the RM-6240E multi-channel physiological recorder, including: left ventricular pressure (LVP) and The rate of change of ventricular pressure (dp/dt).
  • LVP left ventricular pressure
  • dp/dt The rate of change of ventricular pressure
  • the animals were anesthetized and the hemodynamic test (RM-6240E multi-channel physiological recorder) was performed. After the pressure measurement, the animals were sacrificed. The heart tissues were immediately dissected and the blood was rinsed in frozen normal saline. , Weigh the heart quickly, cut the left and right atria along the atrio-ventricular junction with ophthalmological scissors, and separate the left ventricle. After the filter paper absorbs water and weighs, it is put into liquid nitrogen for protein and mRNA extraction, and detection of hypertrophy-related factor genes. The expression level changes. After 2 hours, the left ventricular tissue was transferred to -80°C freezer for freezing.
  • Left ventricular pressure reflects the change of left ventricular pressure in rats, and the rate of change of ventricular pressure (dp/dt) reflects the rate of change of left ventricular pressure during cardiac diastole and contraction, where Max dp/dt represents left ventricular pressure during isovolumic systole The maximum rate of rise reflects the systolic function of the heart; Min dp/dt represents the maximum rate of the left ventricular pressure drop during isovolumic diastole, which reflects the diastolic function of the heart.
  • the cardiac hypertrophy index of rats can be expressed by the heart-weight ratio.
  • the heart-tibia length (HW-Tibia) is measured to express the rat myocardial hypertrophy index.
  • the experimental results showed that: (1) Compared with CON, the HW-Tibia ratio of the rats in the Iso model group was significantly increased; and after the intervention of WYQ potassium salt crystal form B, compared with the model group, the HW-Tibia ratio of the rats in the treatment group Significantly reduced (Figure 51A).
  • the expression of atrial natriuretic peptide (ANP) in cardiomyocytes can be used as a measure of cardiac hypertrophy.
  • the experimental results showed that: (1) Compared with CON, the expression of ANP gene and protein in the left heart tissue of rats in the Iso model group increased significantly; and after the intervention of WYQ potassium salt crystal form B, compared with the model group, the ANP gene in the treatment group And protein expression was significantly reduced (Figure 52). (2) Compared with the CON, the expression of BNP gene and protein in the heart tissue of the Iso model group was significantly increased; and after the intervention of WYQ potassium salt crystal form B, compared with the model group, the expression of BNP gene in the treatment group was significantly reduced (Figure 53 ). It shows that WYQ potassium salt crystal form B has a significant therapeutic effect on myocardial hypertrophy caused by isoproterenol.
  • the present invention provides a PDE5i potassium salt crystal form B, which has the advantages of good water solubility, low hygroscopicity, and stable physical and chemical properties, and the present invention proves that WYQ potassium salt crystal form B can be used
  • Treatment of PAH, IPF and erectile dysfunction can effectively reduce RVSP, relieve right ventricular hypertrophy, improve pulmonary arteriole hyperplasia, reduce the degree of fibrosis in pulmonary fibrosis lesions, and relieve alveolar structure damage and the proliferation of small bronchi and small pulmonary arteries .
  • the potassium salt crystal form B provided by the invention can also be used to treat renal fibrosis and myocardial hypertrophy.
  • the new crystal form of PDE5i has significantly improved water solubility, which can reduce its treatment dosage, reduce liver and kidney pressure, and reduce the economic cost of treating diseases.

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Abstract

本发明公开了一种5型磷酸二酯酶抑制剂的钾盐晶型B及其制备方法和应用。所述5型磷酸二酯酶抑制剂的结构式如式(I)所示，所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角有特征峰：5.71°±0.2°，8.23°±0.2°，11.37°±0.2°，13.22°±0.2°，17.09°±0.2°，21.56°±0.2°，23.99°±0.2°，25.85°±0.2°。本发明还公开了该钾盐晶型在制备在治疗肺动脉高压、特发性肺纤维化、肾脏纤维化、心肌肥大或勃起功能障碍的药物中的应用。

Description

一种5型磷酸二酯酶抑制剂的钾盐晶型B及其制备方法和应用 技术领域
本发明涉及化学制药领域，具体涉及一种5型磷酸二酯酶抑制剂的钾盐晶型B及其制备方法和应用。
背景技术
磷酸二酯酶抑制剂(phosphodiesterase inhibitors，PDEi)是一种抑制磷酸二酯酶(phosphodiesterase，PDEs)活性的药物，在心衰、哮喘、勃起功能障碍等疾病中具有广泛的应用前景。环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)是细胞内两种重要的第二信使，通过激活蛋白激酶A(protein kinase C，PKA)和蛋白激酶G(protein kinase C，PKG)途径参与机体的多种新陈代谢活动，其细胞内浓度的调节主要由腺(鸟)苷酸环化酶的合成和PDE的水解作用之间的平衡决定。PDE能特异性地催化细胞内的cGMP和cAMP水解生成相应的无活性的5’-核苷酸，其中，5型磷酸二酯酶(PDE5)可特异性水解cGMP，主要分布于肺、胰腺、大脑、阴茎海绵体、血管平滑肌细胞、血小板、骨骼肌细胞以及心肌细胞中。已有研究表明，PDE-5i可以调节阴茎海绵体和肺部血管平滑肌的收缩力，同时参与脑部NO-cGMP信号途径的传导，以及乳腺癌癌细胞的生长和凋亡过程。但是，PDE-5i对IPF的治疗作用尚未见报道，还有待进一步研究。
西地那非(Sildenafil)为cGMP特异性PDE5的选择性抑制药，是一种研发治疗心血管疾病药物时意外发明出的治疗男性勃起功能障碍(erectile dysfunction，ED)药物。Sildenafil通过选择性抑制PDE5，能阻止cGMP的降解，升高细胞内cGMP水平，从而提高一氧化氮舒张血管平滑肌、降低血管阻力的作用，是最早作为治疗心血管疾病的候选药物而进入临床研究。2005年，美国FDA批准了Sildenafil用于治疗肺动脉高压(PAH)，2009年又批准了另外一种PDE5i他达拉非(Tadalafil)，目前均作为PAH的一线治疗用药。PDE5i能够增加肺动脉血管扩张，抑制肺血管重塑，从而降低肺动脉压和肺血管阻力，显著提高患者的存活率并改善其生存质量。但Sildenafil存在使用量大(100mg/天)、生物半衰期短的缺点，生物利用度低(约41％)，患者需要每天多次服药，大大增加了药物的不良反应以及治疗成本。此外，Sildenafil对PDE5的选择性作用仅是对PDE6的10倍，PDE6是存在于视网膜中的一种酶，因此，高剂量或者高血药浓度的Sildenafil可能会引起畏光和视力模糊等色觉异常。与Sildenafil相比，Tadalafil生物半衰期显著延长(约17.5小时)，每天所需治疗剂量也降低(40mg/天)，大大减少了患者的治疗费用。然而，由于Tadalafil对其他PDE也有一定的抑制作用，所以不良反应也比较显著，如头痛、胃部不适或疼痛、腹泻、背痛和肌肉疼痛等。
为了克服Sildenafil和Tadalafil临床表现出的副作用，中国专利文献(CN102020645A)公开了一种吡唑并嘧啶酮衍生物，副作用小，可用于制备治疗男性阳痿、肺动脉高压、下尿路综合征、良性前列腺肿大、慢性心衰、中风、冠脉疾病和神经性垂体功能疾病等的治疗药物。该中国专利文献还具体公开了一种化合物WYQ：5-(2-H-5(N-丁氧羰基氨基磺酰基)苯基)-1-甲基-3-丙基-1,6-二氢-7H-吡唑[4,3-d]嘧啶-7-酮，具有如式(I)所示的结构 式，与具有如式(II)所示结构式的Sildenafil相比，化合物WYQ保留了Sildenafil上与环磷酸鸟苷(cGMP)分子结构高度相似的母环，保证了衍生物活性，同时去除了侧链苯环上可能被分解的乙氧基，并将环状的甲基哌嗪替换成链状的正丁氧甲酰胺以降低张力，从而提高药物在生物体内的稳定性。
Figure PCTCN2020107811-appb-000001
化合物WYQ是Sildenafil的一种衍生物，具有与Sildenafil和Tadalafil相同的疗效，且相比Sildenafil，化合物WYQ具有低剂量高药效的特点。由于化合物WYQ的疗效与治疗剂量存在依赖关系，更重要的是化合物WYQ在水中的溶解度非常低，这更加大了化合物WYQ的每日使用量，且其主要依靠肝肾代谢，如果肝肾功能不全者给予大剂量的长期治疗时，会加重其肝肾的负担，引起严重的不良反应，也显著增加患者的治疗费用。鉴于现有技术尚存不足，本领域仍需要开发具有良好的水溶性和理化稳定性的PDE5i晶型，以满足临床用药对于活性物质的形态、水溶性及纯度等理化性质的严格要求。目前尚未有化合物WYQ的钾盐晶型的相关报道。
发明内容
因此，本发明旨在提供一种水溶性良好、理化性质稳定的化合物WYQ的钾盐晶型。
本发明提供一种化合物的钾盐晶型B，所述化合物的结构式如式(I)所示，
Figure PCTCN2020107811-appb-000002
Figure PCTCN2020107811-appb-000003
所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角有特征峰：5.71°±0.2°，8.23°±0.2°，11.37°±0.2°，13.22°±0.2°，17.09°±0.2°，21.56°±0.2°，23.99°±0.2°，25.85°±0.2°。
进一步地，所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角还有特征峰：15.88°±0.2°，16.35°±0.2°，18.47°±0.2°，19.70°±0.2°，22.90°±0.2°，23.64°±0.2°，31.92°±0.2°。
进一步地，所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角还有特征峰：25.04°±0.2°，26.54°±0.2°，28.36°±0.2°，29.94°±0.2°，35.48°±0.2°，37.83°±0.2°。
进一步地，所述钾盐晶型B具有如图1所示的X-射线粉末衍射(XRPD)图。
进一步地，所述钾盐晶型B的差示扫描量热法图谱(DSC)在峰值191.3℃和217.9℃处具有特征熔融吸收峰。
进一步地，所述钾盐晶型B具有如图2所示的TG-DSC图谱。
进一步地，所述钾盐晶型B的分子式为：C 20H 24KN 5O 5S。
进一步地，所述钾盐晶型B的结构式为：
Figure PCTCN2020107811-appb-000004
本发明还提供一种上述式(I)所示化合物的钾盐晶型B的制备方法，包括：
将如式I所示化合物和溶剂混合形成悬浊液1；
向所述悬浊液1中加入氢氧化钾溶解，形成悬浊液2，搅拌结晶，析出固体料；
真空抽滤，分离得到所述固体料并真空干燥，得到所述化合物的钾盐晶型B。
进一步地，所述溶剂选用丙酮、四氢呋喃、乙酸乙酯或乙醇。
进一步地，所述如式I所示的化合物和溶剂的质量体积比为25-30mg/mL，所述氢氧化钾和所述如式I所示的化合物的质量比为1:(7-10)。
进一步地，所述搅拌结晶为：将悬浊液2依次在45-50℃下搅拌5-10min，在20-30℃下搅拌20-24h，在45-50℃下搅拌8-10h。
进一步地，所述的化合物的钾盐晶型B的制备方法包括：
将如式I所示化合物和丙酮按质量体积比28mg/mL混合形成悬浊液1；
向所述悬浊液1中加入氢氧化钾，超声助溶，形成悬浊液2，其中，氢氧化钾和如式I所示的化合物的质量比为1:8；
将悬浊液2依次在50℃下搅拌5min，在25℃下搅拌24h，在50℃下搅拌9h，析出固体料；
真空抽滤，分离得到所述固体料并真空干燥，得到所述化合物的钾盐晶型B。
本发明还提供一种药物组合物，包括上述式(I)所示化合物的钾盐晶型B和药学上可接受的载体。
本发明还提供了上述式(I)所示化合物的钾盐晶型B在制备治疗肺动脉高压的药物中的应用。
本发明还提供了上述式(I)所示化合物的钾盐晶型B在制备治疗特发性肺纤维化的药物中的应用。
本发明还提供了上述式(I)所示化合物的钾盐晶型B在制备治疗肾脏纤维化的药物中的应用。
本发明还提供了上述式(I)所示化合物的钾盐晶型B在制备治疗心肌肥大的药物中的应用。
本发明还提供了上述式(I)所示化合物的钾盐晶型B在制备治疗勃起功能障碍的药物中的应用。
本发明还提供了一种治疗肺动脉高压的方法，包括向受试者施用上述式(I)所示化合物的钾盐晶型B；优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324-3.24mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天；优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-1.296mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天。
本发明还提供了一种治疗特发性肺纤维化的方法，包括向受试者施用上述式(I)所示化合物的钾盐晶型B；优选地，所述化合物的钾盐晶型B的口服施用剂量为0.405-1.62mg.kg -1.d -1，优选施用时间为每天1次，优选连续23天；优选地，所述化合物的钾盐晶型B的注射施用剂量为0.122-0.648mg.kg -1.d -1，优选施用时间为每天1次，优选连续23天。
本发明还提供了一种治疗肾脏纤维化的方法，包括向受试者施用上述式(I)所示化合物的钾盐晶型B；优选地，所述化合物的钾盐晶型B的口服施用剂量为0.405mg.kg -1.d -1，优选施用时间为每天1次，优选连续10天，更优选连续7天；优选地，所述化合物的钾盐晶型B的注射施用剂量为0.1215-0.162mg.kg -1.d -1，优选施用时间为每天1次，优选连续10天，更优选连续7天。
本发明还提供了一种治疗心肌肥大的方法，包括向受试者施用上述式(I)所示化合物的钾盐晶型B；优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324mg.kg -1.d -1，优选施用时间为每天1次，优选连续7天；优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-0.1296mg.kg -1.d -1，优选施用时间为每天1次，优选连续7天。
本发明还提供了一种治疗勃起功能障碍的方法，包括向受试者施用上述式(I)所示化合物的钾盐晶型B；优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324-3.24mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天；优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-1.296mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天。
本发明技术方案，具有如下优点：本发明通过对化合物WYQ成盐或共晶所成晶型筛选，得到了一种化合物WYQ的钾盐晶型B，水溶性较优、理化性质稳定；在肺动脉高压和勃起功能障碍等疾病的治疗中具有低剂量、高药效的特点，尤其是还发现了该钾盐晶型B对特发性肺纤维化、肾脏纤维化和心肌肥大的治疗作用，同样具有低剂量、高药效的特 点；制备方法简单、易于操作、稳定性好，具有被开发为治疗以上疾病的药物的潜力。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请实施例1制备的钾盐晶型B的X-射线粉末衍射图；
图2是本申请实施例1制备的钾盐晶型B的TG-DSC图谱；
图3是本申请实施例1制备的钾盐晶型B的偏光显微镜图；
图4是本申请实施例1制备的钾盐晶型B的变温X-射线粉末衍射测试图；
图5是本申请实施例1制备的钾盐晶型B的DVS测试图；
图6是本申请实施例1制备的钾盐晶型B的DVS测试前后的XRPD对比图；
图7是实施例1制备的钾盐晶型B在水中37℃条件下的动态溶解度图；
图8是实施例1制备的钾盐晶型B在SGF中37℃条件下的动态溶解度图；
图9是实施例1制备的钾盐晶型B在FaSSIF中37℃条件下的动态溶解度图；
图10是实施例1制备的钾盐晶型B在FeSSIF中37℃条件下的动态溶解度图；
图11是化合物WYQ样品在水和SGF中37℃条件下所得固体的XRPD结果对比图；
图12是化合物WYQ样品在FaSSIF和FeSSIF中37℃条件下所得固体的XRPD结果对比图；
图13是实施例1制备的钾盐晶型B在水中37℃条件下所得固体的XRPD结果对比图；
图14是实施例1制备的钾盐晶型B在SGF中37℃条件下所得固体的XRPD结果对比图；
图15是实施例1制备的钾盐晶型B在FaSSIF中37℃条件下所得固体的XRPD结果对比图；
图16是实施例1制备的钾盐晶型B在FeSSIF中37℃条件下所得固体的XRPD结果对比图；
图17是化合物WYQ样品在固态稳定性测试条件下放置后的XRPD结果对比图；
图18是实施例1制备的钾盐晶型B在固态稳定性测试条件下放置后的XRPD结果对比图；
图19是2.0mg·kg -1实施例1制备的钾盐晶型B在大鼠体内的平均血药浓度-时间曲线图；
图20为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，对大鼠RVSP的影响图；
图21为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，对大鼠RVMI的影响图；
图22为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，放大200倍的不同组大鼠肺小动脉H&E染色标准图；
图23为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，不同组大鼠肺小动脉中膜厚度统计图；
图24为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，不 同组大鼠肺小动脉中膜厚度百分比(WT％)统计图；
图25为WYQ钾盐晶型B微剂量治疗(Mini-dose)对大鼠阴茎勃起功能的影响；
图26为WYQ钾盐晶型B低剂量治疗(Low-dose)对大鼠阴茎勃起功能的影响。
图27为WYQ钾盐晶型B中剂量治疗(Medium-dose)对大鼠阴茎勃起功能的影响；
图28为WYQ钾盐晶型B高剂量治疗(High-dose)对大鼠阴茎勃起功能的影响；
图29为sildenafil治疗对大鼠阴茎勃起功能的影响；
图30为tadalafil治疗对大鼠阴茎勃起功能的影响；
图31为不同剂量WYQ钾盐晶型B和目前临床批准的阳性对照药物尼达尼布(BIBF)治疗IPF大鼠23天后，对大鼠左肺重量的影响图；
图32为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，对大鼠左肺体积的影响图。
图33为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶和病灶范围变化对比图(H&E染色)，其中，A为模型组、B为BIBF-50mg/kg组、C为CPD-1-2.5mg/kg组、D为CPD-1-10mg/kg组；
图34为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶和病灶范围变化对比图(Masson Trichrom染色)，其中，A为模型组、B为BIBF-50mg/kg组、C为CPD-1-2.5mg/kg组、D为CPD-1-10mg/kg组；
图35为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶内细支气管和肺细小动脉的组织学变化对比图(HE染色，图片放大倍率：×200)，其中，A为模型组对侧肺组织、B为模型组、C为BIBF-50mg/kg组、D为CPD-1-2.5mg/kg组、E为CPD-1-10mg/kg组；
图36为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶边缘细支气管和肺细小动脉的组织学变化对比图(HE染色，图片放大倍率：×200)，其中，A为模型组对侧肺组织、B为模型组、C为BIBF-50mg/kg组、D为CPD-1-2.5mg/kg组、E为CPD-1-10mg/kg组；
图37为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶内肺泡组织结构变化对比图(HE染色，图片放大倍率：×200)，其中，A为模型组对侧肺组织、B为模型组、C为BIBF-50mg/kg组、D为CPD-1-2.5mg/kg组、E为CPD-1-10mg/kg组；
图38为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶肺泡的组织结构变化对比图(Masson Trichrom染色，图片放大倍率：×200)，其中，A为模型组对侧肺组织、B为模型组、C为BIBF-50mg/kg组、D为CPD-1-2.5mg/kg组、E为CPD-1-10mg/kg组；
图39为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化面积的变化对比图；
图40为肺纤维化病理评分(Masson Trichrome染色)标准，图A-I依次为Ashcroft评分系统中纤维化分级0-8级的Masson Trichrome染色标准图片；
图41为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶评分的变化对比图；
图42为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病 灶评分百分比的变化对比图。
图43为WYQ钾盐晶型B在体内可改善UIRI模型小鼠的肾脏纤维化，其中，A为WYQ钾盐晶型B抑制肾纤维化病灶中FN、Collagen Ⅰ、PAI-1和α-SMA蛋白表达的免疫印迹标准图，数字(1，2，3)表示每组中的每只动物，B-E为FN、Collagen Ⅰ、PAI-1和α-SMA相对含量测定统计图；
图44为WYQ钾盐晶型B治疗UIRI模型小鼠十天后，左肾纤维化病灶内肾小球、肾小管组织结构变化对比图(HE染色)，A-C分别表示假手术组、UIRI模型组、WYQ钾盐晶型B治疗组；
图45为WYQ钾盐晶型B治疗UIRI模型小鼠十天后，左肾纤维化病灶内肾小球、肾小管组织结构变化对比图(Masson Trichrome染色)，A-C分别表示假手术组、UIRI模型组、WYQ钾盐晶型B治疗组；
图46为WYQ钾盐晶型B治疗UIRI模型小鼠十天后肾脏纤维化病灶内FN和α-SMA表达量的变化(免疫组化)，A-C分别表示假手术组、UIRI模型组、WYQ钾盐晶型B治疗组；
图47为WYQ钾盐晶型B治疗UUO模型小鼠7天后，左肾纤维化病灶内肾小球和肾小管组织学变化对比图(HE染色)，其中，A为正常对照组、B为模型组、C为WYQ钾盐晶型B治疗组；
图48为WYQ钾盐晶型B治疗UUO小鼠7天后，肾脏纤维化病灶内FN表达量的变化(免疫组化)，其中，A为正常对照组、B为模型组、C为WYQ钾盐晶型B组；
图49为WYQ钾盐晶型B治疗UUO小鼠7天后，左肾纤维化病灶内纤维化面积计算统计图；
图50为WYQ钾盐晶型B治疗7天后，改善异丙肾上腺素诱导的心肌肥大大鼠心脏功能各参数，其中，A-C为正常对照组、模型组和WYQ钾盐晶型B治疗组大鼠左心室压标准曲线，D-F为各组大鼠左心室压、Max dp/dt和Min dp/dt统计图；
图51为WYQ钾盐晶型B治疗7天后，减小异丙肾上腺素诱导的心肌肥大大鼠心肥指数，其中，A为正常对照组、模型组和WYQ钾盐晶型B治疗组大鼠HW-Tibia比统计图，B为各组大鼠左心室-胫骨长度比统计图；
图52为WYQ钾盐晶型B治疗7天后，降低异丙肾上腺素诱导的心肌肥大大鼠心脏组织中肥大因子ANP的表达，其中，A、B为正常对照组、模型组和WYQ钾盐晶型B治疗组大鼠左心组织中ANP蛋白表达的免疫印迹标准图和统计图，C为各组大鼠ANP基因表达的统计图。
图53为WYQ钾盐晶型B治疗7天后，降低异丙肾上腺素诱导的心肌肥大大鼠心脏组织中肥大因子BNP的表达。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
试剂来源
氢氧化钾购自中国国药集团股份有限公司；
化合物WYQ由药明康德新药开发有限公司合成(批号：ET2951-2-P1)，具体合成路线如下：
(1)将化合物1(50.0g，274mmol，1.00eq)和化合物1A(41.5g，274mmol，1.00eq)在二氧杂环乙烷(350ml)中混合成悬浊液，然后通过薄层色谱分析(TLC)形成亚胺后，在80℃下将混合物搅拌3小时，在O 2条件下添加CuCl 2(36.9g，274mmol，1.00eq)，同样在O 2(15psi)条件下将混合物在80℃搅拌2小时，TLC(石油醚/乙酸乙酯＝3/1，Rf＝0.43)显示反应完成，将反应混合物倒入冰水(500ml)中搅拌20分钟，过滤并减压浓缩，得到残渣，用甲基叔丁基醚(MTBE，500ml)洗涤反应混合物，过滤，减压浓缩过滤，得到白色固体化合物2(50.0g，160mmol，58.2％产率)：
Figure PCTCN2020107811-appb-000005
(2)在N 2条件下将化合物2(50.0g，160mmol，1.00eq)溶于甲醇(MeOH，250ml)和四氢呋喃(THF，250ml)中，添加钯碳催化剂(Pd/C，5.00g，10％纯度)，真空条件下对悬浮液脱气并用H 2吹扫数次，在50℃下将混合物在H 2(50psi)下搅拌5小时，TLC(石油醚/乙酸乙酯＝0/1，Rf＝0.33)表明反应完全，过滤反应混合物并浓缩滤液，用MTBE(200ml×3)洗涤粗产物，得到白色固体化合物3(40.0g，118.6mmol，74.3％产率，84.0％纯度)：
Figure PCTCN2020107811-appb-000006
(3)化合物3(40.0g，141mmol，1.00eq)溶于乙腈(MeCN，500mL)，添加BF 3.HF(18.6g，212mmol，1.50eq)和第三丁基亚硝酸盐(21.8g，212mmol，1.50eq)，然后在0℃下在N 2条件下将混合物搅拌0.5小时，在MeCN(100ml)中添加SO 2(90.4g，1.41mol，10.0eq)和CuCl(20.9g，212mmol，1.50eq)并用N 2吹扫3次，然后在25℃下在N 2条件下搅拌2.5小时，TLC(石油醚/乙酸乙酯＝0/1，Rf＝0.36)表明反应完全，将反应混合物添加到冰水(500ml)中，将混合物在0℃搅拌0.5小时，过滤后，滤饼在低压下浓缩，得到残渣，粗产物用MTBE(200ml)洗涤，过滤，滤饼减压浓缩，得到白色固体化合物4(40.0g，109mmol，77.2％产率)：
Figure PCTCN2020107811-appb-000007
(4)化合物4(40.0g，109mmol，1.00eq)溶于NH 3.H 2O(300ml，25％纯度)中形成混合物，用N 2吹扫3次，然后在30℃下N 2条件将混合物搅拌12小时，TLC(石油醚/乙酸乙酯＝0/1，Rf＝0.24)表明反应完全，反应混合物是浓缩过滤，减压浓缩滤饼，得到残渣，用THF(300ml)稀释残余物，经Na 2SO 4干燥，过滤并减压浓缩，得到呈蓝色固体的化合物5(30.0g，86.4mmol，79.2％产率)：
Figure PCTCN2020107811-appb-000008
(5)化合物5(15.0g，43.2mmol，1.00eq)溶于N,N-二甲基甲酰胺(DMF，75.0ml)和二氯甲烷(DCM，75.0ml)中形成混合物，添加4-二甲氨基吡啶(DMAP，2.64g，21.6mmol，0.50eq)和三乙基胺(TEA，8.74g，86.4mmol，2.00eq)，0℃条件下添加化合物5A(11.8g，86.4mmol，2.00eq)，用N 2吹扫3次，25℃下在N 2条件下搅拌12小时，TLC(二氯甲烷/甲醇＝10/1，Rf＝0.31)表明反应完全，使反应混合物在乙酸乙酯(EtOAc，200ml)和盐水(300ml)中分离，有机相分离出来，用盐水(200ml×3)洗涤，经Na 2SO 4干燥，过滤，减压浓缩，得到残渣，通过柱层析法(SiO 2，二氯甲烷-甲醇101)纯化残余物，然后通过制备高效液相色谱法(TFA条件，柱：Phenomene×LunaC18250×50mm×10um，流动相：[水0.1％乙腈]，B％：35％65％，20分钟后得到化合物WYQ(10.2g，22.5mmol，52.2％产率，98.8％纯度)白色固体：
Figure PCTCN2020107811-appb-000009
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1
化合物WYQ的钾盐晶型B的制备方法：
称取400.1mg化合物WYQ于20mL小瓶中，加入14.4mL的丙酮(丙酮:水＝19:1，v/v)混合形成悬浊液1；
称取50.5mg氢氧化钾，加入悬浊液1中，超声助溶30s，形成悬浊液2；
将悬浊液2依次在50℃下搅拌5min，在25℃下搅拌24h，在50℃下搅拌9h，析出固体料；
真空抽滤，分离上述固体料，将其在25℃下真空干燥24h，得到290.1mg结晶固体(质量收率：72.5％)。
X-射线粉末衍射(XRPD)图见图1；TG-DSC图谱见图2；HPLC纯度98.7area％；IC测试(Thermo ICS1100)结果表明化合物WYQ与钾离子的化学计量比为1:7～1:10；偏光显微镜(PLM，Axio Lab.A1)图如图3所示，表明制备的钾盐晶型B由小颗粒组成，且伴有团聚。
实施例2
化合物WYQ的钾盐晶型B的制备方法：
称取480mg化合物WYQ于40mL小瓶中，加入20mL的四氢呋喃(四氢呋喃:水＝19:1，v/v)混合形成悬浊液1；
称取69mg氢氧化钾，加入悬浊液1中，超声助溶60s，形成悬浊液2；
将悬浊液2依次在45℃下搅拌10min，在20℃下搅拌24h，在45℃下搅拌10h，析出固体料；
真空抽滤，分离上述固体料，将其在室温下真空干燥12h，得到290.1mg结晶固体(质量收率：72.5％)。
X-射线粉末衍射(XRPD)图与图1基本一致；TG-DSC图谱与图2基本一致；HPLC纯度99area％。
实施例3
化合物WYQ的钾盐晶型B的制备方法：
称取600mg化合物WYQ于40mL小瓶中，加入20mL的乙酸乙酯(乙酸乙酯:水＝19:1，v/v)混合形成悬浊液1；
称取60mg氢氧化钾，加入悬浊液1中，超声助溶40s，形成悬浊液2；
将悬浊液2依次在50℃下搅拌8min，在30℃下搅拌20h，在50℃下搅拌8h，析出固体料；
真空抽滤，分离上述固体料，将其在室温下真空干燥20h，得到290.1mg结晶固体(质量收率：72.5％)。
X-射线粉末衍射(XRPD)图与图1基本一致；TG-DSC图谱与图2基本一致；HPLC纯度99area％。
本发明提供了化合物WYQ的钾盐晶型B，并采用多种方式和仪器对实施例1制备得到的化合物WYQ的钾盐晶型B的性质进行了研究。
一、X-射线粉末衍射(XRPD)
本发明采用(X’Pert 3)型号的X-射线粉末衍射仪，以2°每分钟的扫描速度，采用铜铬射靶获取实施例1制备的钾盐晶型B的XRPD图谱如图1所示。XRPD测试参数设置见表1，图1中各详细X-射线粉末衍射参数见表2。
表1 XRPD测试参数
Figure PCTCN2020107811-appb-000010
表2实施例1制备得到的钾盐晶型B的X-射线粉末衍射参数
Figure PCTCN2020107811-appb-000011
二、热分析和变温XRPD测试
采用(美国TA公司Discovery系列DSC2500)差示扫描量热分析仪，获得实施例1制备的钾盐晶型B的差示扫描量热扫描图谱(DSC曲线)；采用(美国TA公司Q系列Q500/5000)热重分析仪，获得实施例1制备的钾盐晶型B的热重曲线(TG曲线)，得到如图2所示的实施例1制备的钾盐晶型B的TG-DSC图谱。DSC和TGA测试参 数设置见表3。
表3 DSC和TGA测试参数
参数 TGA DSC
方法 线性升温 线性升温
样品盘 铝盘，敞开 铝盘，压盖
温度范围 室温-目标温度 25℃-目标温度
扫描速率(℃/min) 10 10
保护气体 氮气 氮气
如图2所示，实施例1制备的钾盐晶型B在150℃前存在0.9％的失重，且在在峰值191.3℃(起点温度188.5℃)和217.9℃(起点温度208.7℃)处具有吸热峰。结合该钾盐晶型B在熔化分解前具有较小的失重，推测实施例1制备的钾盐晶型B为无水晶型。
进一步进行变温X-射线粉末衍射(VT-XRPD)测试，测试条件分别为30℃下无氮气保护、30℃下氮气吹扫30分钟、氮气保护下升温至150℃、氮气保护下降温至30℃，得到测试结果如图4所示，将实施例1制备的钾盐晶型B样品在30℃氮气吹扫30分钟后，晶型无变化，氮气保护下升温至150℃后观察到部分衍射峰偏移，降温至30℃后，晶型与钾盐晶型B一致，证实化合物WYQ的钾盐晶型B为无水晶型。
三、动态蒸汽吸附(DVS)实验
采用SMS(Surface Measurement Systems)的DVS Intrinsic仪器在25℃条件下进行动态蒸汽吸附实验，以对实施例1制得的钾盐无水晶型B进行引湿性测试。将测试样品预先在0％相对湿度(RH)条件下干燥去除吸附的溶剂或水后开始测试，目标相对湿度为0-95％。如图5所示，钾盐晶型B样品在25℃/80％RH条件下质量变化(水分吸附量)为0.6％，表明其略有引湿性。如图6所示，XRPD对比图显示DVS测试前后测试样品的晶型未发生改变。DVS测试参数设置见表4。
表4 DVS测试参数
Figure PCTCN2020107811-appb-000012
四、动态溶解度测试
在37℃/60％RH的条件下对实施例1制备的钾盐晶型B样品在水、人工胃液(SGF)、禁食态人工肠液(FaSSIF)、喂食态人工肠液(FeSSIF)四种溶媒中的动态溶解度进行测试，并收集化合物WYQ样品相同条件下的动态溶解度数据。在动态溶解度测试实验中，分别称取约16mg钾盐晶型B样品/化合物WYQ样品与3.2mL溶媒在4mL离心管中混合，并将离心管密封固定在转速为25r/min的旋转盘上，在37℃条件下旋转混合1h、4h和24h后取样，检测各取样点的溶解度(S，mg/mL)、pH值和经XRPD检测后的晶型变化(FC，其中“是”代表晶型部分或全部变成了化合物WYQ)，检测 结果如表5所示。钾盐晶型B样品在水、SGF、FaSSIF、FeSSIF中的溶解度曲线图如图7-10所示，化合物WYQ样品在不同溶媒中1h和24h取样点的XRPD结果对比如图11-12所示(XRPD检测参数设置见表1)，钾盐晶型B样品在不同溶媒中1h和24h取样点的XRPD结果对比如图13-16所示。
表5各取样点的溶解度、pH值和经XRPD检测后的晶型变化
Figure PCTCN2020107811-appb-000013
以上结果均显示，实施例1制备的钾盐晶型B在37℃条件下在水、SGF、FaSSIF、FeSSIF中的溶解度均高于化合物WYQ样品，且在水中溶解度最高，可达12.9mg/mL。
五、固态稳定性测试
对实施例1制备的钾盐晶型B的固态稳定性进行测试，称取5.22mg钾盐晶型B样品在25℃/60％RH条件下敞口放置，并称取5.19mg钾盐晶型B样品在40℃/75％RH条件下敞口放置，一周后对所有样品进行XRPD表征(XRPD检测参数设置见表1)和HPLC纯度测试(Agilent 1100)，以检测晶型和纯度变化，HPLC纯度测试参数见表6。同时，称取4.59mg化合物WYQ样品在25℃/60％RH条件下敞口放置，并称取5.11mg化合物WYQ样品在40℃/75％RH条件下敞口放置，在相同条件下进行了固态稳定性测试，用于比较参考。
表6 HPLC纯度测试参数
Figure PCTCN2020107811-appb-000014
Figure PCTCN2020107811-appb-000015
测试结果结果汇总于表7，XRPD测试结果如图17及图18所示。结果表明，实施例1制备的化合物WYQ的钾盐晶型B的HPLC纯度为98.71area％，钾盐晶型B样品放置一周后，晶型不变且未观察到明显的纯度变化，在两种测试条件下具有良好的理化稳定性；化合物WYQ样品在25℃/60％RH条件下放置一周后纯度无明显下降，在40℃/75％RH条件下放置一周后，检测到约1％的HPLC纯度(area％)降低。
表7实施例1制备的钾盐晶型B的固态稳定性测试结果
Figure PCTCN2020107811-appb-000016
综上所述，本发明实施例1制备得到的化合物WYQ的钾盐晶型B样品与化合物WYQ样品相比，具有更优异的引湿性、溶解度和理化稳定性。以下将结合具体实验对本发明实施例制备的钾盐晶型B的药物用途进行具体说明。
实验例实施例1制备的钾盐晶型B的药物活性检测实验
一、实施例1制备的钾盐晶型B的药代动力学实验
1.实验材料
1.1试剂
化合物WYQ的钾盐晶型B为实施例1中制备得到；羧甲基纤维素钠、生理盐水、戊巴比妥钠购自中国国药集团股份有限公司；乙腈、甲酸、甲醇购自中国百灵威化学试剂有限公司。
1.2仪器
美国Ag-ilentSeries1200液相色谱及Agilent6410质谱仪。
1.3实验动物
雄性SPF级SD大鼠，体重180～200g，购于北京维通利华实验动物有限公司，实验动物许可证号:SCXK(京)-2002-0011，饲养于南京医科大学实验动物房，所有大鼠置于温度(22±2)℃、湿度(60±5)℃，12h明暗交替环境中。
2.实验方法
健康雄性SD大鼠6只，禁食自由饮水12h后，将实施例1制备的钾盐晶型B按2mg.kg -1灌胃给药，分别于给药后0.2h、0.5h、1h、1.5h、2h、4h、6h和8h眼球静脉丛取血，加入乙腈:水(1:1)离心取上清液，进行色谱分析。色谱条件：色谱柱Thermo  Fisher Hypersil gold column(3μm，20mm×2.1mm)，进样量10μL，流速为0.3mL/min，柱温40℃，流动相水(1％甲酸)和甲醇，梯度洗脱：甲醇10％(0min)；10％(1min)；90％(4min)；90％(8min)；10％(9min)。质谱条件：多反应监测(MRM)，电喷雾离子化(ESI)，驻留时间(Dwell Time)200ms，检测离子m/z448.0-347.8，血药浓度数据用Winnonlin6.3软件进行分析，得到药代动力学参数，形成平均血药浓度-时间曲线图如图19所示。
3.实验结果
化合物WYQ的钾盐晶型B在雄性SD大鼠体内的口服半衰期(t 1/2)＝(2.30±0.27)h，给药(0.63±0.12)h后药物浓度达到峰值，最大药物浓度(C max)＝(333.8±23.7)ng.mL -1，给药后8h内的平均血药浓度-时间曲线下面积AUC (0－t)＝(638.8±72.4)h.ng.mL -1，平均血药浓度-时间曲线下总面积AUC (0－∞)＝(650.2±70.8)h.ng.mL -1。
二、肺动脉高压(PAH)大鼠实验
1.实验材料
1.1试剂
化合物WYQ的钾盐晶型B为实施例1中制备得到；Sildenafil、Tadalafil由药明康德新药开发有限公司合成(批号:EW10443-180-P1、EW10443-228-P1)；野百合碱(monocrotaline，MCT)、羧甲基纤维素钠为美国Sigma试剂公司产品；肝素(heparin)、乌拉坦溶液、二甲苯、石蜡购自中国国药集团股份有限公司；苏木素-伊红、中性树胶购自中国碧云天生物科技有限公司。
1.2仪器
日本Nikon光学显微镜摄像系统；包埋机：HistoCore Arcadia，Leica；切片机：RM2235，Leica；自动染色机：LEICA Autostainer ST5020；切片扫描仪：Hamamatsu NanoZoomer Digital Pathology(S210)；分析天平：METTLERToledo，ALT104；体重秤：常熟市双杰测试仪器厂，T1000；手术显微镜：Luckbird XTS-4A；RM6240E生理记录仪(成都仪器厂)。
1.3实验动物
SPF级雄性SD大鼠，由山东济南朋悦实验动物繁育有限公司提供，许可证号：SCXK(鲁)20140007，批号No.37009200014851，体重：150±5克。
广州呼吸疾病研究所动物房，实验单位使用许可证编号：SYXK(粤)2013-0093，动物房温度20-25℃，湿度55％-65％，12h明暗交替，自由摄食饮水。
2.实验方法
2.1实验分组
SD大鼠随机分8组，15只/组，分别为：正常对照组(Vehicle)；PAH模型组(MCT)；PAH+WYQ钾盐晶型B微剂量组(Mini-dose，下称微剂量组)；PAH+WYQ钾盐晶型B低剂量组(Low-dose，下称低剂量组)；PAH+WYQ钾盐晶型B中剂量组(Medium-dose，下称中剂量组)；PAH+WYQ钾盐晶型B高剂量组(High-dose，下称高剂量组)；PAH+Sildenafil对照组(下称西地那非组)；PAH+Tadalafil对照组(下称他达拉非组)。
2.2动物造模
MCT用1M稀HCl充分溶解后，加双蒸水稀释，再用3M的NaOH调至pH＝7.2， 配成终浓度为20mg/ml的MCT药液备用。
正常对照组于腹腔下注射生理盐水(50mg/kg)，其余各组于腹腔下一次性注射MCT(50mg/kg)。
2.3给药量及给药方式
正常对照组：不给药处理；
PAH模型组：不给药处理；
微剂量组：WYQ钾盐晶型B用生理盐水配成0.4mg/ml的药液(现用现配)，灌胃给药，给药量2mg.kg -1.d -1；
低剂量组：WYQ钾盐晶型B用生理盐水配成1mg/ml的药液(现用现配)，灌胃给药，给药量5mg.kg -1.d -1；
中剂量组：WYQ钾盐晶型B用生理盐水配成2mg/ml的药液(现用现配)，灌胃给药，给药量10mg.kg -1.d -1；
高剂量组：WYQ钾盐晶型B用生理盐水配成4mg/ml的药液(现用现配)，灌胃给药，给药量20mg.kg -1.d -1；
西地那非组：Sildenafil用0.5％羧甲基纤维素钠配成5mg/ml的药液(现用现配)，灌胃给药，给药量25mg.kg -1.d -1；
他达拉非组：Tadalafil用0.5％羧甲基纤维素钠配成2mg/ml的药液(现用现配)，灌胃给药，给药量10mg.kg -1.d -1。
注射当天为实验起始日，造模7天后开始给药，每天1次，连续14天。
根据美国FDA目前所采用的种属间剂量换算方法，大鼠和人换算系数为0.162。因此，根据实施例中的大鼠灌胃用药剂量推算WYQ钾盐晶型B的人口服用药微、低、中和高剂量分别为0.324、0.81、1.62和3.24mg.kg -1.d -1，施用时间为每天1次，连续14天。
根据《药理实验方法学》中不同给药途径间的剂量换算，肌肉注射和腹腔注射与口服的剂量之比约为0.3-0.4，由此推算出WYQ钾盐晶型B的人注射用药微、低、中和高剂量分别为0.097-0.129、0.243-0.324、0.486-0.648和0.972-1.296mg.kg -1.d -1，施用时间为每天1次，连续14天(药物浓度：1mg/ml)。
2.4试验指标及检测方法
2.4.1 PAH大鼠右心室收缩压(RVSP)
实验起始日起3周后，在RM-6240E多导生理记录仪上采集各组大鼠RVSP数据。
2.4.2 PAH大鼠右心室肥大指数(RVMI)
实验起始日起3周后，麻醉动物，进行血流动力学检测(RM-6240E多导生理记录仪)，测压完处死动物，立即剖取心肺组织，用眼科剪沿房室交界处剪去左右心房及大血管，分离出右心室(RV)及左心室+室间隔(left ventricular wall plus septum，LV+S)，滤纸吸去水分后分别称质量，并计算RV/(LV+S)即RVMI。
2.4.3 PAH大鼠肺小动脉病理学观察及定量分析
实验起始日起3周后，大鼠肝素化(heparin，50IU/100g，腹腔注射)处理5min，注射20％乌拉坦溶液(0.5ml/100g)麻醉动物，检测并记录血压各项指标；
剖取心脏，分离右心室壁，分离左心室和室间隔，滤纸吸干后分别称重，计算右心室肥大指数；
剖取肺，结扎右肺，用固定液(4％多聚甲醛)灌注左肺叶后将其置于固定液内固定，经乙醇梯度脱水、二甲苯透明、浸蜡、石蜡包埋、切片(4μm)、苏木素-伊红(HE)染色，中性树胶封片后显微镜下观察，取不同切片随机观察6个视野中小动脉中膜厚度和厚度的变化情况，与对照组对比确定各组动物肺组织学变化。
应用图像采集分析系统测量肺小动脉(直径为50～150μM)中膜厚度、外径，然后计算肺小动脉中膜厚度及厚度百分比(WT％)，WT％＝(2×中膜厚度)/外径×100％。
2.4.4 PAH大鼠阴茎勃起功能观察
灌胃处理后，观察动物阴茎勃起状况，记录各组灌胃后出现阴茎勃起现象的时间。
3.实验结果
3.1 PAH大鼠右心室收缩压(RVSP)
图20为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，对大鼠RVSP的影响图。结果显示：与正常对照组(22.9±0.5mmHg)相比，PAH模型组大鼠的RVSP(72.5±4.5mmHg)显著升高( **P<0.01)；微剂量组、低剂量组、中剂量组、高剂量组的RVSP均明显下降和改善(微剂量组：56.2±0.5mmHg， #P<0.05；低剂量组：47.6±1.9mmHg， ##P<0.01；中剂量组:40.7±1.5mmHg， ##P<0.01；高剂量组：29.8±1.6mmHg， ##P<0.01)，且对RVSP的抑制作用呈剂量依赖性，高剂量组的RVSP已接近正常水平；他达拉非组与PAH模型组相比，RVSP也明显下降(52.5±2.4mmHg， ##P<0.01)，下降的幅度相当于低剂量组；西地那非组与PAH模型组相比，RVSP也有所下降，但下降的幅度不如WYQ钾盐晶型B各剂量给药组和他达拉非组，与PAH模型组相比无显著统计学意义(60.4±5.0mmHg，P＝0.089)；与微剂量组相比，低剂量组的RVSP显著下降( $P＜0.05)，与低剂量组相比，中剂量组的RVSP显著下降( $P＜0.05)，与中剂量组相比，高剂量组的RVSP显著下降( $$P＜0.01)。
3.2 PAH大鼠右心室肥大指数(RVMI)
图21为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，对大鼠RVMI的影响图。结果显示：与正常对照组(24.2±0.7％)相比，PAH模型组大鼠的RVMI(51.7±2.3％)显著升高( **P<0.01)；WYQ钾盐晶型B给药的微剂量组、低剂量组、中剂量组、高剂量组的RVSP均明显下降和改善(微剂量组：42.8±2.1％， #P<0.05；低剂量组：40.6±2.7％， ##P<0.01；中剂量组：38.7±2.1％， ##P<0.01；高剂量组：34.6±19％， ##P<0.01)，与对RVSP的抑制作用不同，WYQ钾盐晶型B对RVMI的抑制作用不呈剂量依赖性；他达拉非组与PAH模型组相比，RVMI也明显下降(41.8±1.9％， ##P<0.01)，下降幅度相当于WYQ钾盐晶型B低剂量组；西地那非组与PAH模型组相比，RVMI也有所下降，但下降的幅度不如WYQ钾盐晶型B各剂量组和他达拉非组，与PAH模型组相比无显著统计学意义(48.4±1.0％，P＝0.468)；与微剂量组相比，高剂量组的RVMI显著下降( $$P＜0.01)。
3.3 PAH大鼠肺小动脉病理学观察及定量分析
对各组大鼠肺小动脉中膜厚度和WT％数据进行统计。图22为不同剂量WYQ钾盐晶型B及sildenafil和tadalafil治疗PAH大鼠21天后，放大200倍的不同组大鼠肺小动脉H&E染色标准图。图23和图24结果显示：与正常对照组相比，PAH模型组大鼠肺小动脉中膜厚度和WT％均显著增加(P<0.05)；WYQ钾盐晶型B给药的低剂量组、中剂量组、高剂量组的中膜厚度和WT％均明显下降和改善；他达拉非组与PAH 模型组相比，肺小动脉中膜厚度及WT％也明显下降；西地那非组与PAH模型组相比，中膜厚度有所下降，但下降的幅度不如WYQ钾盐晶型B各剂量组和他达拉非组，且WT％与PAH模型组相比无显著统计学意义(P＝0.468)。
3.4 PAH大鼠阴茎勃起功能观察
图25-30显示了不同剂量WYQ钾盐晶型B和sildenafil、tadalafil治疗组大鼠灌胃1h/40min/30min后的阴茎勃起情况对比。观察结果：WYQ钾盐晶型B微剂量组灌胃后1小时，有30％大鼠出现阴茎勃起；低剂量组灌胃后1小时，有50％大鼠出现阴茎勃起；中剂量组灌胃后1小时，有80％大鼠出现阴茎勃起；高剂量组灌胃后40分钟，有80％大鼠出现阴茎勃起，随着药物剂量增大，勃起后的阴茎尺寸更粗、颜色更红。他达拉非组灌胃后30分钟，有80％大鼠出现阴茎勃起，同样，西地那非组灌胃后30分钟，也有80％大鼠出现阴茎勃起，且与WYQ钾盐晶型B相比，Sildenafil和Tadalafil灌胃后出现阴茎勃起现象的时间更短、效果更显著。
三、特发性肺纤维化(IPF)大鼠实验
1.实验材料
1.1试剂
盐酸博莱霉素为日本化药株式会社产品；HPLC级水，BIBF由凯斯艾生物科技(苏州)有限公司提供；羧甲基纤维素钠为美国Sigma试剂公司产品；化合物WYQ的钾盐晶型B为实施例1中制备得到；异氟烷、戊巴比妥钠麻醉剂、福尔马林购自中国国药集团股份有限公司。
1.2仪器
日本Nikon光学显微镜摄像系统；动物呼吸机(HX-300S)、呼吸麻醉机(R580)：深圳市瑞沃德生命科技有限公司；组织脱水机：HistoCore Pearl，Leica；包埋机：HistoCore Arcadia，Leica；切片机：RM2235，Leica；自动染色机：LEICA Autostainer ST5020；切片扫描仪：Hamamatsu NanoZoomer Digital Pathology(S210)；分析天平：METTLERToledo，ALT104；体重秤：常熟市双杰测试仪器厂，T1000；电热毯：Jwilch，China；手术显微镜：Luckbird XTS-4A；足趾容量测量仪：(上海欣软信息科技有限公司)。
1.3实验动物
SPF级雄性SD大鼠。动物饲养于凯斯艾生物科技(苏州)有限公司动物中心SPF级屏障系统内，实验单位使用许可证号：SYXK(苏)2017-0041，遵循国际标准温、湿、光控制系统。本实验动物操作方案经由凯斯艾生物科技(苏州)有限公司IACUC委员会联合审批确认。严格遵循凯斯艾生物科技(苏州)有限公司(KCI)的相关标准操作规程(SOP)实行一切操作和管理。
2.实验方法
2.1实验分组
雄性SD大鼠32只，根据体重随机分为4组，8只/组：模型组、尼达尼布50mg/kg组(BIBF-50mg/kg)、WYQ钾盐晶型B 2.5mg/kg组(CPD-1-2.5mg/kg)、WYQ钾盐晶型B 10mg/kg组(CPD-1-10mg/kg)。
2.2动物造模
在KCI动物实验操作SOP指导原则下实施本实验所涉及到的一切操作。动物购入 后，适应性饲养3-7天后开始建模。动物称重后采用异氟烷吸入麻醉，确认动物麻醉之后，消毒颈部，剪开颈部皮肤，顿性分离肌肉暴露主气管，沿气管环之间切开一小口，插入PE-20管至左侧主支气管，直接注入博莱霉素(剂量：3mg/kg，体积：1.0ml/kg)，缝合气管以及皮肤。手术完毕后，将动物置于37℃电热毯保温至动物完全苏醒，确认能够自由采食和饮水后将动物返回饲养笼正常饲养。
2.3给药量及给药方式
模型组：按体重给予生理盐水灌胃，剂量为1ml.100g -1.d -1；
尼达尼布50mg/kg组(BIBF-50mg/kg)：BIBF用0.5％羧甲基纤维素钠配成10mg/ml的溶液，灌胃给药，给药量50mg.kg -1.d -1；
WYQ钾盐晶型B 2.5mg/kg组(CPD-1-2.5mg/kg)：WYQ钾盐晶型B用生理盐水稀释成0.5mg/ml的溶液，灌胃给药，给药量2.5mg.kg -1.d -1；
WYQ钾盐晶型B10mg/kg组(CPD-1-10mg/kg)：WYQ钾盐晶型B用生理盐水稀释成2mg/ml的溶液，灌胃给药，给药量10mg.kg -1.d -1；
各组大鼠于造模当天开始灌胃给药，每天一次，共计给药23天。
根据美国FDA目前所采用的种属间剂量换算方法，大鼠和人换算系数为0.162。因此，根据实施例中的大鼠灌胃用药剂量推算WYQ钾盐晶型B的人口服用药剂量分别为0.405、1.62mg.kg -1.d -1，施用时间为每天1次，连续23天。
根据《药理实验方法学》中不同给药途径间的剂量换算，肌肉注射和腹腔注射与口服的剂量之比约为0.3-0.4，由此推算出WYQ钾盐晶型B的人注射用药剂量分别为0.122-0.162、0.486-0.648mg.kg -1.d -1，施用时间为每天1次，连续23天(药物浓度：1mg/ml)。
2.4试验指标及检测方法
2.4.1 IPF大鼠左肺重量和体积
连续给药23天后，各组动物按照KCI动物安乐死标准操作规程，给予动物腹腔注射戊巴比妥钠麻醉剂(100mg/kg)安乐死全部动物，经低温PBS全身灌注，再进行全身福尔马林灌注固定，取左肺，进行肺内等量福尔马林液灌注，进行称重及后续肺病理相关检测。
左肺大体病理检测：左肺等量福尔马林液灌注后采用微量天平分别称取并记录灌注后左肺湿重；采用足趾容量测量仪测量并记录灌注后左肺体积。
2.4.2 IPF大鼠肺组织病理学检测
按照KCI病理标准SOP进行左肺整肺脱水，石蜡块制作，左肺整肺石蜡切片，片厚3-4μm；遵循KCI病理标准染色SOP进行HE染色、Masson Trichrome染色，并通过Hamamatsu NanoZoomer Digital Pathology(S210)切片扫描仪进行切片全景扫描；通过Masson Trichrome染色切片进行肺病变面积计算，左肺纤维化面积(％)为纤维化面积占左肺面积的百分比；在病变区域内随机选择10个面积大小为1mm 2的视野，病理学家根据Ashcroft评分系统(如表8和图40所示)在双盲条件下进行半定量评分。
表8 Ashcroft评分标准
Figure PCTCN2020107811-appb-000017
Figure PCTCN2020107811-appb-000018
3.实验结果
3.1 IPF大鼠左肺重量和体积
连续给药23天后各组IPF大鼠左肺重量和体积的统计结果如表9所示，图31为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，对大鼠左肺重量的影响图；图32为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，对大鼠左肺体积的影响图。
表9连续给药23天后各组IPF大鼠左肺重量和体积的统计结果
组别 动物数(只) 左肺重量(g) 左肺体积(mm 3)
模型组 8 1.56±0.11 1739±116.23
BIBF-50mg/kg 8 1.58±0.12 1736.9±136.10
CPD-1-2.5mg/kg 8 1.59±0.10 1779.9±105.76
CPD-1-10mg/kg 8 1.47±0.16 1604.5±153.72
结果显示：各给药组和模型组间左肺重量和体积在统计学上无显著性差异。
3.2 IPF大鼠肺组织病理学检测
3.2.1左肺肺纤维化病灶和病灶范围
由图33-34可见肺部组织界限较为清晰的显著性肺损伤，两种不同的肺组织学染色(H&E和Masson Trichrom染色)清晰可见左肺均匀一致的纤维化病灶以及病灶分布范围。不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，与模型组相比，左肺肺纤维化病灶和病灶范围无显著性差异。
3.2.2 IPF大鼠左肺肺纤维化病灶内细支气管和肺细小动脉的组织学变化
观察表现为细支气管、终末细支气管、肺泡管上皮细胞不同程度的增生，部分上皮乃至全层上皮杯状细胞化，管腔内可见不等量粘液组织。肺细小动脉管壁不同程度的炎细胞浸润，部分管壁增厚，平滑肌增生以及管壁外模膜肉芽组织增生。图35为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶内细支气管和肺细小动脉的组织学变化对比图(HE染色)，图36为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶边缘细支气管和肺细小动脉的组织学变化对比图。由图35-36可见治疗后左肺肺纤维化病灶内细支气管和肺细小动脉，病灶边缘细支气管和肺细小动脉平滑肌增生及炎细胞浸润状况得到改善。与模型组相比，CPD-1-10mg/kg治疗组效果更明显。
3.2.3 IPF大鼠左肺肺纤维化病灶内肺泡组织损伤
IPF大鼠左肺肺纤维化病灶内肺泡组织不同程度损伤，表现为肺泡上皮脱落与再生，肺泡壁增厚，纤维化；肺泡腔内不同程度的纤维组织沉积，炎性渗出，炎细胞浸润；纤维化病灶内片状肺泡结构损伤、消失，由大量渗出的炎细胞和增生的结缔组织充填，残存肺泡腔内可见炎性渗出物和增生的结缔组织。
图37为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶内肺泡组织结构变化对比图(HE染色，图片放大倍率：×200)。结果显示：BIBF-50mg/kg治疗组，纤维化病灶内部分肺泡结构仍有损伤，残存肺泡壁增厚，壁内炎细胞浸润；CPD-1-2.5mg/kg治疗组，纤维化病灶内部分肺泡结构损伤，残存肺泡壁增厚，壁内有炎细胞浸润；CPD-1-10mg/kg治疗组，纤维化病灶内肺泡结构保存，肺泡壁增厚，壁内炎细胞浸润。
图38为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶肺泡的组织结构变化对比图(Masson Trichrom染色，图片放大倍率：×200)。结果显示：BIBF-50mg/kg治疗组，纤维化病灶内部分肺泡结构消失，残存肺泡壁增厚；CPD-1-2.5mg/kg治疗组，纤维化病灶内部分肺泡结构保存，肺泡壁增厚，肺泡壁损伤区域可见炎性渗出物和增生的结缔组织；CPD-1-10mg/kg治疗组，纤维化病灶内大部分肺泡结构保存，肺泡壁增厚，部分修复的肺泡壁结构，肺泡腔内可见少量炎性渗出物。
3.2.4 IPF大鼠左肺肺纤维化病灶面积
图39为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化面积的变化对比图(图中正常对照组是指模型组未给药的一侧肺)，结果显示：模型组和各给药组之间病灶面积基本相同，提示本模型的稳定和均匀一致性。
3.2.5 IPF大鼠左肺肺纤维化病灶评分和评分百分比
图40为肺纤维化病理评分(Masson Trichrome染色)标准。表10为肺纤维化Ashcraft评分和评分百分比统计结果，图41为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶评分的变化对比图(图中正常对照组是指模型组未给药的一侧肺)；图42为不同剂量WYQ钾盐晶型B和BIBF治疗IPF大鼠23天后，左肺肺纤维化病灶评分百分比的变化对比图(图中正常对照组是指模型组未给药的一侧肺)。
表10为肺纤维化Ashcraft评分和评分百分比统计结果
Figure PCTCN2020107811-appb-000019
注：One-way ANOVA: *p<0.05vs模型组； **p<0.01vs.模型组； ***p<0.001vs.模型组； ###p<0.001vs.正常对照组。
肺纤维化Ashcraft评分结果显示：阳性对照药品BIBF在50mg/kg剂量下与模型组相比，显著改善大鼠左肺纤维化的程度(p<0.05)；WYQ钾盐晶型B在10mg/kg剂量下，每天一次口服给药，连续23天，可显著抑制肺纤维化，与模型组比较具有显著性差异(p<0.05)。
以Ashcraft评分3分为界，计算3分以下(包含3分)或4分以上(包含4分)的肺纤维化程度的百分比，结果显示：模型组接近51％以上的病灶区域评分在4分或4分以上，经过药物治疗后各药物治疗组动物的病灶区评分在4分以上区域在25-50％之间。统计学结果显示，阳性药BIBF治疗后肺纤维化程度的百分比与模型组相比显著减少(p<0.05)；WYQ钾盐晶型B在10mg/kg剂量下，治疗组与模型组比较肺纤维化程度的百分比与模型组相比均显著减少(p<0.05)。
四、肾脏纤维化动物实验
1.实验材料
1.1试剂
鼠抗α-SMA抗体、鼠抗α-tubulin抗体、兔抗Fibronectin抗体、羧甲基纤维素钠为美国Sigma试剂公司产品；兔抗Collagen-Ⅰ抗体、兔抗Kim-1抗体、购自美国Millipore公司；抗兔、抗鼠二抗购自美国Jackson公司；化合物WYQ的钾盐晶型B为实施例1中制备得到；异氟烷、戊巴比妥钠麻醉剂、福尔马林购自中国国药集团股份有限公司。
1.2仪器
组织脱水机：HistoCore Pearl，Leica；包埋机：HistoCore Arcadia，Leica；切片机：RM2235，Leica；自动染色机：LEICA Autostainer ST5020；切片扫描仪：Hamamatsu NanoZoomer Digital Pathology(S210)；分析天平：德国Precia公司；体重秤：常熟市双杰测试仪器厂，T1000；手术显微镜：Luckbird XTS-4A；凝胶成像系统：美国Bio-Rad公司；电泳槽、电泳仪：美国Bio-Rad公司；pH仪：瑞士ETTLER公司。
1.3实验动物
SPF级雄性BALB/c小鼠，体重20g左右。购自南方医科大学实验动物中心，许可证号：SCXK(粤)-2011-0015。实验过程严格参照2006年发布的《关于善待实验动物的指导意见》处置动物。
2实验方法
2.1单侧肾缺血再灌注小鼠模型(UIRI模型)
2.1.1造模与分组
雄性BALB/c小鼠15只，随机分为：Sham组(假手术组)5只，手术造模组10只。手术方法：小鼠麻醉后于左侧腹部切口，暴露肾脏并钝性分离肾蒂。假手术组仅暴露肾蒂而不夹闭；造模组用无损伤微型动脉夹夹闭左肾肾蒂30min，夹闭期间小鼠置于37℃恒温板上以维持体温恒定，手术切口处覆盖生理盐水浸润的纱布以防肾组织脱水。30min后取掉动脉夹，观察肾脏在1min内由紫黑色逐渐转变为鲜红色，表示血流再灌注成功。将肾脏归位，缝合伤口。造模组小鼠再随机分为2组：UIRI组(模型组)、WYQ钾盐晶型B治疗组(5mg/kg)，每组5只。
2.1.2给药
术后2小时开始第一次灌胃给药。
①Sham组和UIRI模型组：按体重给予生理盐水灌胃，剂量为0.1ml.10g -1.d -1；
②WYQ钾盐晶型B治疗组：WYQ钾盐晶型B用生理盐水稀释成1mg/ml的溶液，灌胃给药，给药量5mg.kg -1.d-1；
每天一次，共计给药10天。
根据美国FDA目前所采用的种属间剂量换算方法，小鼠和人换算系数为0.081。因此，根据实施例中的小鼠灌胃用药剂量推算WYQ钾盐晶型B的人口服用药剂量为0.405mg.kg -1.d -1，施用时间为每天1次，连续10天。
根据《药理实验方法学》中不同给药途径间的剂量换算，肌肉注射和腹腔注射与口服的剂量之比约为0.3-0.4，由此推算出WYQ钾盐晶型B的人注射用药剂量分别为0.1215-0.162mg.kg -1.d -1，施用时间为每天1次，连续10天(药物浓度：1mg/ml)。
2.1.3标本采集与处理
术后第10天，同样方法麻醉小鼠，切开右背部，暴露右肾，结扎肾蒂后切除右肾。假手术组小鼠仅剥离肾包膜，不切除右肾。术后第11天将小鼠全部处死，打开腹腔，剥离小鼠左肾脏组织，需要注意保持肾脏的完整性。肾脏取出后迅速转移到预冷的PBS中，切割肾脏，以做不同的检测。用手术刀将肾脏分为四部分，肾脏腹侧上极和下极放入液氮用于蛋白质和mRNA的提取，检测纤维化相关因子蛋白和基因的表达量变化，2小时后将肾脏组织转入-80℃冰箱冻存。肾脏背侧放入4％多聚甲醛中固定，用于制作石蜡切片，HE染色和Masson染色观察肾脏组织的形态学变化，免疫组化观察肾脏组织中纤维化标志蛋白表达量的变化。
2.2小鼠单侧输尿管梗阻模型(UUO模型)
2.2.1造模与分组
将15只雄性C57BL/6小鼠适应性喂养一周，随机分为两组：Sham组(假手术组)5只，手术造模组10只。手术方法：假手术组小鼠在麻醉状态下打开腹腔游离左侧输尿管，不结扎，关腹缝合；造模组小鼠在麻醉状态下行左侧输尿管结扎术。造模组小鼠再随机分为2组：UUO组(模型组)、WYQ钾盐晶型B治疗组(5mg/kg)，每组5只。
2.2.2给药
术后2小时开始第一次灌胃给药。
①Sham组和UUO模型组：按体重给予生理盐水灌胃，剂量为0.1ml.10g -1.d -1；
②WYQ钾盐晶型B治疗组：WYQ钾盐晶型B用生理盐水稀释成1mg/ml的溶液，灌胃给药，给药量5mg.kg -1.d -1；
每天一次，共计给药7天。
根据美国FDA目前所采用的种属间剂量换算方法，小鼠和人换算系数为0.081。因此，根据实施例中的大鼠灌胃用药剂量推算WYQ钾盐晶型B的人口服用药剂量为0.405mg.kg -1.d -1，施用时间为每天1次，连续7天。
根据《药理实验方法学》中不同给药途径间的剂量换算，肌肉注射和腹腔注射与口服的剂量之比约为0.3-0.4，由此推算出WYQ钾盐晶型B的人注射用药剂量为0.1215-0.162mg.kg -1.d -1，施用时间为每天1次，连续7天(药物浓度：1mg/ml)。2.2.3标本采集与处理
给药7天后将小鼠全部处死，打开腹腔，剥离小鼠左右肾脏组织，需要注意保持肾脏的完整性。肾脏取出后迅速转移到预冷的PBS中，切割肾脏，以做不同的检测。用手术刀将肾脏分为四部分，肾脏背侧放入4％多聚甲醛中固定，用于制作石蜡切片，HE染色和Masson染色观察肾脏组织的形态学变化，免疫组化观察肾脏组织中纤维化标志蛋白表达量的变化。
3实验结果
3.1 WYQ钾盐晶型B对UIRI模型小鼠的治疗作用
3.1.1 WYQ钾盐晶型B显著降低UIRI小鼠肾脏组织中FN1、Collagen Ⅰ、PAI-1及α-SMA的表达
Western Blot结果显示(图43)，小鼠肾脏组织中，纤维化标志性因子FN1、Collagen Ⅰ、PAI-1及α-SMA蛋白在基础状态下的表达很低，UIRI模型小鼠肾脏组织中其表达量均显著增高，而在WYQ钾盐晶型B预防用药组中，这些蛋白的表达水平均显著降低。表明，WYQ钾盐晶型B能够抑制缺血再灌注诱导的肾脏纤维化的形成。
3.1.2 WYQ钾盐晶型B有效缓解UIRI小鼠肾脏纤维化病变情况
HE染色(图44)和Masson染色(图45)结果显示：假手术组小鼠肾脏组织结构正常，肾小球无萎缩，未见肾小管扩张、炎细胞浸润及间质纤维组织增生等病理改变。与假手术组小鼠相比，UIRI手术组小鼠的肾组织、细胞结构发生不可逆损伤，肾小管萎缩或消失，肾小球硬化，胶原纤维在肾间质显著增多，胶原沉积显著，间质炎症细胞浸润发生纤维化。免疫组化(图46)结果显示肌成纤维细胞分泌的FN1以及α-SMA蛋白在UIRI小鼠肾脏纤维化病灶中显著增加。WYQ钾盐晶型B治疗后，肾脏中炎症细胞浸润明显减少，细胞外基质沉积减少，FN1以及α-SMA蛋白表达水平也显著降低。
3.2 WYQ钾盐晶型B对UUO模型小鼠的治疗作用
3.2.1 WYQ钾盐晶型B有效缓解UUO小鼠肾脏纤维化病变情况
HE染色(图47)结果显示：假手术组小鼠肾脏组织结构正常，未见肾小管萎缩或扩张、肾小球病变、炎细胞浸润及间质纤维组织增生等病理改变。与假手术组小鼠相比，UUO手术组小鼠的肾脏肾盂肾盏扩张明显，肾间质中有大量炎症细胞浸润，肾小管刷状缘的完整性破坏，有不同程度的萎缩、坏死，肾小球基底膜变厚、小球玻璃样改变，肾脏间质中胶原沉积明显，纤维化面积显著增加。免疫组化(图48)结果显示肌成纤维细胞分泌的FN1蛋白在UUO小鼠肾脏组织中显著增加。WYQ钾盐晶型B 治疗后，肾脏中炎症细胞浸润明显减少，肾间质胶原纤维增生减少，FN1蛋白表达水平也显著降低，与UUO模型组相比，WYQ钾盐晶型B治疗组中，肾纤维化病灶中纤维化面积显著减少(图49)。
五、心肌肥大动物实验
1.实验材料
1.1试剂
异丙肾上腺素、鼠抗α-SMA抗体、羧甲基纤维素钠为美国Sigma试剂公司产品；兔抗Collagen-Ⅰ抗体购自美国Millipore公司；鼠抗-BNP抗体、兔抗-ANP抗体购自abcam公司；抗兔、抗鼠二抗购自美国Jackson公司；化合物WYQ的钾盐晶型B为实施例1中制备得到；肝素(heparin)、乌拉坦溶液、福尔马林购自中国国药集团股份有限公司。大鼠ANP、BNP引物由生工生物工程(上海)有限公司合成。
1.2仪器
RM6240E多道生理信号记录仪(成都仪器厂)；分析天平：德国Precia公司；体重秤：常熟市双杰测试仪器厂，T1000；手术显微镜：Luckbird XTS-4A；凝胶成像系统：美国Bio-Rad公司；电泳槽、电泳仪：美国Bio-Rad公司；pH仪：瑞士ETTLER公司。
1.3实验动物
SPF级雄性SD大鼠，购自湖南斯莱克景达实验动物有限公司，生产许可号：SCXK(湘)2016-0002。饲养于华南理工大学实验动物中心，动物中心使用许可证号：SYXK(粤)2017-0178，体重：250±10克。
2.实验方法
2.1实验分组
SD大鼠随机分3组，10只/组，分别为：正常对照组(CON)；异丙肾上腺素致心肌肥大模型组(Iso)；Iso+WYQ钾盐晶型B治疗组(下称治疗组)。
2.2动物造模
异丙肾上腺素用双蒸水配成4mg/ml的药液，现配现用。正常对照组于腹腔下注射生理盐水(5mg/kg)，其余各组于腹腔下注射异丙肾上腺素(5mg/kg)，每天一次，持续注射7天。
2.3给药量及给药方式
CON组：不给药处理；
Iso模型组：不给药处理；
治疗组：WYQ钾盐晶型B用生理盐水配成0.4mg/ml的药液(现用现配)，灌胃给药，给药量2mg.kg -1.d -1；
注射当天为实验起始日，造模2小时后开始给药，每天1次，连续7天。
根据美国FDA目前所采用的种属间剂量换算方法，大鼠和人换算系数为0.162。因此，根据实施例中的大鼠灌胃用药剂量推算WYQ钾盐晶型B的人口服用药剂量为0.324mg.kg -1.d -1，施用时间为每天1次，连续7天。
根据《药理实验方法学》中不同给药途径间的剂量换算，肌肉注射和腹腔注射与口服的剂量之比约为0.3-0.4，由此推算出WYQ钾盐晶型B的人注射用药剂量为0.0972-0.1296mg.kg -1.d -1，施用时间为每天1次，连续7天(药物浓度：1mg/ml)。
2.4实验指标及检测方法
2.4.1心肌肥大大鼠血流动力学指标
实验起始日起7天后，经右颈总动脉逆行左心室插管，在RM-6240E多导生理记录仪上采集各组大鼠血流动力学指标数据，包括：左心室压(LVP)和心室压力变化速率(dp/dt)。
2.4.2心肌肥大大鼠心肥指数的测量及组织分离
实验起始日起1周后，麻醉动物，进行血流动力学检测(RM-6240E多导生理记录仪)，测压完处死动物，立即剖取心脏组织，于冰冻的生理盐水中冲洗干净血液，迅速将心脏称重，用眼科剪沿房室交界处剪去左右心房，分离出左心室，滤纸吸去水分称重后放入液氮用于蛋白质和mRNA的提取，检测肥大相关因子基因的表达量变化，2小时后将左心室组织转入-80℃冰箱冻存。完成以上操作后，用手术剪剪开大鼠腿部毛皮及肌肉，充分暴露胫骨，测量胫骨长度。大鼠的心肌肥大指数通过心脏-胫骨长度(HW-Tibia)表示。
3.实验结果
3.1 WYQ钾盐晶型B对异丙肾上腺素诱导的心肌肥大大鼠心脏功能各参数的影响
左心室压(LVP)反映大鼠左心室压力的变化，心室压力变化速率(dp/dt)反映心脏舒缩过程中左心室压力变化的速率，其中Max dp/dt表示等容收缩期左心室压力上升的最大速率，反映心脏收缩功能；Min dp/dt表示等容舒张期左心室压力下降的最大速率，反映心脏舒张功能。实验结果显示：(1)与CON相比，Iso模型组大鼠的左心室压显著降低；而WYQ钾盐晶型B干预后，与模型组相比，治疗组大鼠的左心室压明显升高(图50A-D)。(2)与CON相比，Iso模型组大鼠的Max dp/dt显著降低；WYQ钾盐晶型B干预后，与模型组相比，治疗组大鼠Max dp/dt明显升高(图50E)。(3)与CON相比，Iso模型组大鼠的Min dp/dt显著降低；WYQ钾盐晶型B干预后，与模型组相比，治疗组大鼠Min dp/dt明显升高(图50F)。表明，WYQ钾盐晶型B能够很好的恢复异丙肾上腺素诱导的心肌肥大大鼠左心功能，具有潜在的药物开发价值。
3.2 WYQ钾盐晶型B对异丙肾上腺素诱导的大鼠心肌肥大指数的影响
大鼠的心肌肥大指数可以通过心脏-体重比表示。鉴于大鼠个体体重增长差异性较大，而胫骨长度随月龄增长变化相对一致，因此通过测定心脏-胫骨长度(HW-Tibia)表示大鼠心肌肥大指数。实验结果显示：(1)与CON相比，Iso模型组大鼠的HW-Tibia比显著增加；而WYQ钾盐晶型B干预后，与模型组相比，治疗组大鼠的HW-Tibia比明显减少(图51A)。(2)与CON相比，Iso模型组大鼠的左心室-胫骨长度比显著增加；而WYQ钾盐晶型B干预后，与模型组相比，治疗组大鼠的左心室-胫骨长度比明显减少(图51B)。表明，WYQ钾盐晶型B可显著减轻由异丙肾上腺素诱导的心肌肥大。
3.3 WYQ钾盐晶型B对异丙肾上腺素诱导的心肌肥大大鼠心脏组织中肥大因子表达量的影响
心肌细胞心房钠尿肽(ANP)的表达量可作为心肌肥大的衡量标志。实验结果显示：(1)与CON相比，Iso模型组大鼠左心组织中ANP基因与蛋白表达量显著增加；而WYQ钾盐晶型B干预后，与模型组相比，治疗组ANP基因与蛋白表达量明显减少(图52)。(2)与CON相比，Iso模型组心组织中BNP基因与蛋白表达量显著增加； 而WYQ钾盐晶型B干预后，与模型组相比，治疗组BNP基因表达量明显减少(图53)。表明，WYQ钾盐晶型B对于异丙肾上腺素造成的心肌肥大有明显的治疗作用。
以上实验结果证明，本发明提供了一种PDE5i钾盐晶型B，该钾盐晶型具有水溶性好、吸湿性小、理化性质稳定的优势，且本发明证实WYQ钾盐晶型B可用于治疗PAH、IPF及勃起功能障碍，能够有效降低RVSP，缓解右心室肥厚，改善肺小动脉增生，减轻肺纤维化病灶内纤维化程度，使肺泡结构损伤和和细小支气管、肺小动脉增生得到缓解。本发明提供的钾盐晶型B还可用于治疗肾脏纤维化、心肌肥大。此外，PDE5i新晶型水溶性显著提高，从而可以减少其治疗用量，减轻肝肾压力，降低治疗疾病的经济费用。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (23)

  1. 一种化合物的钾盐晶型B，其特征在于，所述化合物的结构式如式(I)所示，
    Figure PCTCN2020107811-appb-100001
    所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角有特征峰：5.71°±0.2°，8.23°±0.2°，11.37°±0.2°，13.22°±0.2°，17.09°±0.2°，21.56°±0.2°，23.99°±0.2°，25.85°±0.2°。
  2. 根据权利要求1所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角还有特征峰：15.88°±0.2°，16.35°±0.2°，18.47°±0.2°，19.70°±0.2°，22.90°±0.2°，23.64°±0.2°，31.92°±0.2°。
  3. 根据权利要求2所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B的X-射线粉末衍射(XRPD)图在下述2θ角还有特征峰：25.04°±0.2°，26.54°±0.2°，28.36°±0.2°，29.94°±0.2°，35.48°±0.2°，37.83°±0.2°。
  4. 根据权利要求1-3任一所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B具有如图1所示的X-射线粉末衍射(XRPD)图。
  5. 根据权利要求1-4任一所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B的差示扫描量热法图谱(DSC)在峰值191.3℃和217.9℃处具有吸热峰。
  6. 根据权利要求1-5任一所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B具有如图2所示的TG-DSC图谱。
  7. 根据权利要求1-6任一所述的化合物的钾盐晶型B，其特征在于，所述钾盐晶型B的结构式为：
    Figure PCTCN2020107811-appb-100002
  8. 一种权利要求1-7任一所述的化合物的钾盐晶型B的制备方法，其特征在于，包括：
    将如式I所示化合物和溶剂混合形成悬浊液1；
    向所述悬浊液1中加入氢氧化钾溶解，形成悬浊液2，搅拌结晶，析出固体料；
    真空抽滤，分离得到所述固体料并真空干燥，得到所述化合物的钾盐晶型B。
  9. 根据权利要求1-8任一所述的化合物的钾盐晶型B的制备方法，其特征在于，所述溶剂选用丙酮、四氢呋喃、乙酸乙酯或乙醇。
  10. 根据权利要求1-9任一所述的化合物的钾盐晶型B的制备方法，其特征在于，所述如式I所示的化合物和溶剂的质量体积比为25-30mg/mL，所述氢氧化钾和所述如式I所示的化合物的质量比为1:(7-10)。
  11. 根据权利要求1-10任一所述的化合物的钾盐晶型B的制备方法，其特征在于，所述搅拌结晶为：将悬浊液2依次在45-50℃下搅拌5-10min，在20-30℃下搅拌20-24h，在45-50℃下搅拌8-10h。
  12. 根据权利要求1-11任一所述的化合物的钾盐晶型B的制备方法，其特征在于，包括：
    将如式I所示化合物和丙酮按质量体积比28mg/mL混合形成悬浊液1；
    向所述悬浊液1中加入氢氧化钾，超声助溶，形成悬浊液2，其中，氢氧化钾和如式I所示的化合物的质量比为1:8；
    将悬浊液2依次在50℃下搅拌5min，在25℃下搅拌24h，在50℃下搅拌9h，析出固体料；
    真空抽滤，分离得到所述固体料并真空干燥，得到所述化合物的钾盐晶型B。
  13. 一种药物组合物，其特征在于，包括权利要求1-7任一所述的化合物的钾盐晶型B和药学上可接受的载体。
  14. 权利要求1-7任一所述的化合物的钾盐晶型B在制备治疗肺动脉高压的药物中的应用。
  15. 权利要求1-7任一所述的化合物的钾盐晶型B在制备治疗特发性肺纤维化的药物中的应用。
  16. 权利要求1-7任一所述的化合物的钾盐晶型B在制备治疗肾脏纤维化的药物中的应用。
  17. 权利要求1-7任一所述的化合物的钾盐晶型B在制备治疗心肌肥大的药物中的应用。
  18. 权利要求1-7任一所述的化合物的钾盐晶型B在制备治疗勃起功能障碍的药物中的应用。
  19. 一种治疗肺动脉高压的方法，其特征在于，包括向受试者施用权利要求1-7任一所述的化合物的钾盐晶型B；
    优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324-3.24mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天；
    优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-1.296mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天。
  20. 一种治疗特发性肺纤维化的方法，其特征在于，包括向受试者施用权利要求1-7任一所述的化合物的钾盐晶型B；
    优选地，所述化合物的钾盐晶型B的口服施用剂量为0.405-1.62mg.kg -1.d -1，优选施用时间为每天1次，优选连续23天；
    优选地，所述化合物的钾盐晶型B的注射施用剂量为0.122-0.648mg.kg -1.d -1，优选施用时间为每天1次，优选连续23天。
  21. 一种治疗肾脏纤维化的方法，其特征在于，包括向受试者施用权利要求1-7任一所述的化合物的钾盐晶型B；
    优选地，所述化合物的钾盐晶型B的口服施用剂量为0.405mg.kg -1.d -1，优选施用时间为每天1次，优选连续10天，更优选连续7天；
    优选地，所述化合物的钾盐晶型B的注射施用剂量为0.1215-0.162mg.kg -1.d -1，优选施用时间为每天1次，优选连续10天，更优选连续7天。
  22. 一种治疗心肌肥大的方法，其特征在于，包括向受试者施用权利要求1-7任一所述的化合物的钾盐晶型B；
    优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324mg.kg -1.d -1，优选施用时间为每天1次，优选连续7天；
    优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-0.1296mg.kg -1.d -1，优选施用时间为每天1次，优选连续7天。
  23. 一种治疗勃起功能障碍的方法，其特征在于，包括向受试者施用权利要求1-7任一所述的化合物的钾盐晶型B；
    优选地，所述化合物的钾盐晶型B的口服施用剂量为0.324-3.24mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天；
    优选地，所述化合物的钾盐晶型B的注射施用剂量为0.097-1.296mg.kg -1.d -1，优选施用时间为每天1次，优选连续14天。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6225315B1 (en) * 1998-11-30 2001-05-01 Pfizer Inc Method of treating nitrate-induced tolerance
WO2007056955A1 (fr) * 2005-11-17 2007-05-24 Topharman Shanghai Co., Ltd. Derives de la pirazolopyrimidinone, leur preparation et leur utilisation
CN102020645A (zh) * 2010-09-30 2011-04-20 中山大学 吡唑并嘧啶酮衍生物及其可药用盐、其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6225315B1 (en) * 1998-11-30 2001-05-01 Pfizer Inc Method of treating nitrate-induced tolerance
WO2007056955A1 (fr) * 2005-11-17 2007-05-24 Topharman Shanghai Co., Ltd. Derives de la pirazolopyrimidinone, leur preparation et leur utilisation
CN102020645A (zh) * 2010-09-30 2011-04-20 中山大学 吡唑并嘧啶酮衍生物及其可药用盐、其制备方法和应用

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