WO2023169557A1 - 一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用 - Google Patents

一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用 Download PDF

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WO2023169557A1
WO2023169557A1 PCT/CN2023/080746 CN2023080746W WO2023169557A1 WO 2023169557 A1 WO2023169557 A1 WO 2023169557A1 CN 2023080746 W CN2023080746 W CN 2023080746W WO 2023169557 A1 WO2023169557 A1 WO 2023169557A1
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pulmonary fibrosis
compound
hypoxanthine
group
caused
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French (fr)
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黄文�
李为民
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Sichuan Yiasuo Pharmaceutical Technology Co Ltd
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Sichuan Yiasuo Pharmaceutical Technology Co Ltd
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Priority to JP2024554175A priority Critical patent/JP2025507180A/ja
Priority to US18/845,212 priority patent/US20250188082A1/en
Priority to EP23766134.3A priority patent/EP4491183A4/en
Priority to CN202380023408.8A priority patent/CN118742310B/zh
Publication of WO2023169557A1 publication Critical patent/WO2023169557A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/28—Oxygen atom
    • C07D473/30—Oxygen atom attached in position 6, e.g. hypoxanthine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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

Definitions

  • the present invention relates to the technical field of biomedicine, and in particular to the application of a hypoxanthine derivative compound in the preparation of drugs for treating pulmonary fibrosis.
  • the causative factors of pulmonary fibrosis are diverse, and the clinical pathological manifestations are generally fibroblast proliferation, massive extracellular matrix accumulation, inflammatory response, and destruction of lung tissue structure.
  • the mortality rate of pulmonary fibrosis is extremely high, even higher than the mortality rate of most tumors.
  • the average survival time after diagnosis of pulmonary fibrosis is 2.8 years.
  • pulmonary fibrosis will eventually form. They are different manifestations of the same disease at different stages of development. Interstitial pneumonia or other worsening manifestations of pneumonia are pulmonary fibrosis. Therefore, it is crucial to find a drug that can prevent and treat pneumonia and pulmonary fibrosis, reduce the pulmonary inflammatory response, and block the transformation of mild pneumonia into severe pneumonia pulmonary fibrosis.
  • Hypoxanthine also known as "6-hydroxypurine”
  • hypoxanthine compounds can block the development of pulmonary fibrosis and reverse pathological damage.
  • the purpose of the present invention is to propose the application of a hypoxanthine derivative compound in the preparation of drugs for the treatment of pulmonary fibrosis, so as to accelerate the development of new drugs for the treatment of pulmonary fibrosis.
  • the many technical effects that can be produced by the preferred technical solutions of the present invention are described in detail below.
  • the first aspect of the present invention relates to the application of a hypoxanthine derivative compound in the preparation of a drug for treating pulmonary fibrosis.
  • the hypoxanthine derivative compound has the activity of treating pulmonary fibrosis, and the hypoxanthine derivative compound has the following structure: one:
  • R 2 , R 3 , and R 4 are optionally H, C 1 -C 18 alkyl, halogen-substituted C 1 -C 18 alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid group, or R 2 _ _ _ _ , R 3 and R 4 are optionally C 1 -C 18 alkyl or fatty acid groups substituted by oxygen atoms, sulfur atoms or nitrogen atoms; when R 3 and R 4 are individually substituted, the double bond is connected at the unsubstituted N position, and all are replaced by There are no double bonds when substituting.
  • a second aspect of the invention relates to compounds having the following structure:
  • R 2 , R 3 , and R 4 are optionally H, C 1 -C 18 alkyl, halogen-substituted C 1 -C 18 alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid group, or R 2 _ _ _ _ , R 3 and R 4 are optionally C 1 -C 18 alkyl or fatty acid groups substituted by oxygen atoms, sulfur atoms or nitrogen atoms; when R 3 and R 4 are individually substituted, the double bond is connected at the unsubstituted N position, and all are replaced by There are no double bonds when substituting.
  • the compound is selected from the following group:
  • a third aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition further contains pharmaceutically acceptable excipients or auxiliary ingredients.
  • the pharmaceutical composition is an oral preparation, an injection preparation or a nasal mucosal administration preparation.
  • a fourth aspect of the present invention relates to a method of treating pulmonary fibrosis by administering an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention to an individual in need thereof.
  • the drugs for treating pulmonary fibrosis include drugs that have the effect of preventing and treating pulmonary fibrosis and its complications.
  • the drug for treating pulmonary fibrosis is a preparation prepared by using a hypoxanthine derivative compound or a salt thereof as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.
  • the preparation is an oral preparation, an injection preparation or a nasal mucosal administration preparation.
  • the pulmonary fibrosis includes one or more of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis and interstitial pneumonia. kind.
  • the pulmonary fibrosis includes one of bacterial pulmonary fibrosis, viral pulmonary fibrosis, mycoplasmal pulmonary fibrosis, chlamydial pulmonary fibrosis, immune pulmonary fibrosis and fungal pulmonary fibrosis. or more.
  • the pulmonary fibrosis includes pulmonary fibrosis caused by Streptococcus pneumoniae, pulmonary fibrosis caused by influenza A virus, pulmonary fibrosis caused by influenza B virus, pulmonary fibrosis caused by coronavirus, and One or more of the pulmonary fibrosis caused by the new coronavirus.
  • the pulmonary fibrosis also includes pulmonary fibrosis caused by Klebsiella spp., pulmonary fibrosis caused by Streptococcus fibrosus, pulmonary fibrosis caused by resistant enterococci, and drug-resistant Staphylococcus aureus.
  • pulmonary fibrosis caused by Acinetobacter baumannii and pulmonary fibrosis caused by Acinetobacter baumannii are examples of pulmonary fibrosis caused by Klebsiella spp.
  • the compounds and derivatives provided by the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.
  • alkyl is a linear or branched saturated hydrocarbon radical.
  • Examples of C1 to C3 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3) and isopropyl (C3).
  • pharmaceutically acceptable means a carrier, carrier, diluent, excipient, and/or salt formed that is generally chemically or physically compatible with the other ingredients constituting a pharmaceutical dosage form and is physiologically compatible Compatible with receptors.
  • salts refers to acidic and/or basic salts of the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, For example, alkylammonium salts.
  • These salts can be obtained directly from the final isolation and purification of the compounds. It can also be obtained by appropriately mixing the above compound with a certain amount of acid or base (for example, equivalent amounts). These salts may form a precipitate in the solution and be collected by filtration, or may be recovered after evaporation of the solvent, or may be obtained by reacting in an aqueous medium and then freeze-drying.
  • the salt mentioned in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, butylene salt of the compound. salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • the administration mode of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative administration modes include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or solubilizers, for example, starch , lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, such as , glycerin; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarder, such as paraffin; (f) absorption Accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compounds can also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils,
  • compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions may contain, in addition to the active compound, suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.
  • compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays and inhalants, for example nasal mucosal formulations.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.
  • the pharmaceutically acceptable excipients mentioned in the present invention refer to substances other than active ingredients contained in the dosage form.
  • the pharmaceutically acceptable auxiliary component of the present invention has certain physiological activity, but the addition of this component will not change the dominant position of the above-mentioned pharmaceutical composition in the process of disease treatment, but only exerts auxiliary effects. These auxiliary effects are only It is the utilization of the known activity of this ingredient and is a commonly used auxiliary treatment method in the medical field. If the above auxiliary ingredients are used in conjunction with the pharmaceutical composition of the present invention, they should still fall within the scope of protection of the present invention.
  • hypoxanthine derivative compound provided by the present invention in the preparation of anti-pulmonary fibrosis drugs, through the detection of the anti-pulmonary fibrosis pharmacological activity of the structurally modified and transformed hypoxanthine derivative compound, in a variety of animal disease models
  • the pharmacological activity of this type of compound was tested, and data on the activity in preventing and treating different types of pulmonary fibrosis were provided. It was confirmed that they all have good activity, and the effect is significantly better than that of nintedanib, a commonly used positive drug in clinical practice, and the effect is also significantly better than that of subxanthinib.
  • hypoxanthine derivative compound provided by the present invention in the preparation of anti-pulmonary fibrosis drugs can provide a new framework for the screening of new compounds for the preparation of anti-pulmonary fibrosis drugs and lay a theoretical foundation for the development of new lead compounds.
  • hypoxanthine derivative compound provided by the present invention in the preparation of anti-pulmonary fibrosis drugs can significantly improve pulmonary fibrosis caused by viruses and bacteria. More importantly, the hypoxanthin provided by the invention Derivative compounds can improve early-stage pulmonary fibrosis symptoms and reverse late-stage pulmonary fibrosis symptoms.
  • hypoxanthine derivative compound provided by the present invention in the preparation of anti-pulmonary fibrosis drugs, through the detection of the anti-pulmonary fibrosis pharmacological activity of the structurally modified and transformed hypoxanthine derivative compound, in a variety of animal disease models The pharmacological activity of this type of compound was tested, and data on the activity in preventing and treating different types of pulmonary fibrosis were provided.
  • hypoxanthine derivative compound provided by the present invention in the preparation of anti-pulmonary fibrosis drugs can provide a new framework for the screening of new compounds for the preparation of anti-pulmonary fibrosis drugs and lay a theoretical foundation for the development of new lead compounds.
  • hypoxanthine analogs A, B, C, and D are as follows:
  • Hypoxanthine analogues A, B, C, and D were prepared as follows for the preparation of compound 2, compound 4, and/or compound 6.
  • hypoxanthine derivative compound provided by the present invention in preparing anti-pulmonary fibrosis drugs will be described in detail below with reference to Examples 1 to 8.
  • This example describes the preparation method of Compound 1 to Compound 12 in detail.
  • Compound 1 to Compound 12 are prepared by alkylation reaction of hypoxanthine.
  • This example provides methods for preparing 12 compounds.
  • the structures of all the compounds prepared were determined by nuclear magnetic resonance spectroscopy and mass spectrometry.
  • This example is used to detect the anti-extensive pulmonary fibrosis activity of Compound 1 to Compound 12 prepared in Example 1.
  • mice SPF grade C57BL/6 mice (weighing about 22-25g) were randomly divided into several groups, including a blank control group, a model group, and nintedanib. Positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, and compound group 1, compound group 2... compound group 12, 9 animals in each group, at SPF level The animals were kept in the animal center for 7 days. The day before the experiment, the mice were fasted for 12 hours, and then anesthetized with sodium pentobarbital.
  • mice in the blank control group were given In addition to sterile PBS, the model group, nintedanib positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, compound group 1, compound group 2... Mice in compound 12 group were given the same volume of 5 mg/kg bleomycin.
  • each group was administered intragastric administration.
  • the mice in the blank control group and the model group were given the same volume of normal saline as the drug group according to their body weight.
  • the nintedanib positive control group was given an appropriate volume of saline according to their body weight. Nintedanib (120 mg/kg), hypoxanthine analog group A, hypoxanthine analog group B, and hypoxanthine analog group C were given hypoxanthine analog A (120 mg/kg/d), hypoxanthine analog group Xanthine analog B (120 mg/kg/d), hypoxanthine analog C (120 mg/kg/d), compound group 1, compound group 2... compound group 12 were given compound 1 (prepared in Example 1) respectively. 120mg/kg/d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d). It was administered twice a day.
  • mice After 14 days of administration, the mice were anesthetized with 0.8% sodium pentobarbital solution (10 mL/kg) on the 15th day. Blood was taken from the abdominal aorta to detect changes in blood routine and evaluate the lung tissue. To determine the severity of fibrosis, perform HE staining on paraffin sections of lung tissue and perform Ashcroft scoring. The results are shown in Table 2-1 and Table 2-2 below.
  • mice SPF grade C57BL/6 mice (weighing about 22-25g) were randomly divided into several groups, including a blank control group, a model group, and nintedanib. Positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, and compound group 1, compound group 2... compound group 12, 9 animals in each group, at SPF level The animals were kept in the animal center for 7 days. The day before the experiment, the mice were fasted for 12 hours, and then anesthetized with sodium pentobarbital.
  • mice in the blank control group were given In addition to sterile PBS, the model group, nintedanib positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, compound group 1, compound group 2... Mice in compound 12 group were given the same volume of 5 mg/kg bleomycin. Three weeks after the establishment of the model, each group was administered intragastric administration. The mice in the blank control group and the model group were given the same volume of normal saline as the drug group according to their body weight.
  • the nintedanib positive control group was given an appropriate volume of saline according to their body weight. Nintedanib (120 mg/kg), hypoxanthine analog group A, hypoxanthine analog group B, and hypoxanthine analog group C were given hypoxanthine respectively.
  • Analogue A 120mg/kg/d
  • hypoxanthine analogue B 120mg/kg/d
  • hypoxanthine analogue C 120mg/kg/d
  • compound group 1 compound group 2... compound 12
  • Each group was administered Compound 1 (120 mg/kg/d), Compound 2 (120 mg/kg/d), and Compound 12 (120 mg/kg/d) prepared in Example 1 respectively. It was administered twice a day.
  • mice After 14 days of administration, the mice were anesthetized with 0.8% sodium pentobarbital solution (10 mL/kg) on the 15th day. Blood was taken from the abdominal aorta to detect changes in blood routine and evaluate the lung tissue. To determine the severity of fibrosis, perform HE staining on paraffin sections of lung tissue and perform Ashcroft scoring. The results are shown in Tables 2-3 and 2-4 below.
  • Compounds 1 to 12 prepared in Example 1 have significant inhibitory effects on inflammatory factors such as leukocytes, neutrophils, and lymphocytes in the blood of advanced pulmonary fibrosis, while nintedanib
  • the anti-inflammatory effects of tablets and hypoxanthine analog A, hypoxanthine analog B, and hypoxanthine analog C were significantly lower than those of compounds 1 to 12.
  • hypoxanthine analogues A, B, C, D and E1-12 are as follows:
  • Hypoxanthine analogs A, B, C, D and E-1 to E-12 were prepared as follows for preparing compound 2, compound 4 and/or compound 6.
  • mice SPF grade C57BL/6 mice (weighing about 22-25g) were randomly divided into several groups, including a blank control group, a model group, and nintedanib. Positive control group, hypoxanthine analogue group A, hypoxanthine analogue group B, hypoxanthine analogue group C, hypoxanthine analogue D, compound group E-1 to E-12, compound group 1, compound Group 2... Compound 12 groups, 9 animals in each group, were raised in an SPF grade animal center for 7 days. The day before the experiment, the mice were fasted for 12 hours, and then anesthetized with sodium pentobarbital.
  • mice in the blank control group were given In addition to sterile PBS, model group, nintedanib positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, hypoxanthine analog D, compound E- Mice in groups 1 to E-12 and compound group 1, compound 2, and compound 12 were given the same volume of 5 mg/kg bleomycin. Two weeks after the establishment of the model, each group was administered intragastric administration. The mice in the blank control group and the model group were given the same volume of normal saline as the drug group according to their body weight.
  • the nintedanib positive control group was given an appropriate volume of saline according to their body weight.
  • Nintedanib 120 mg/kg
  • hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, and hypoxanthine analog group D were given hypoxanthine analog A ( 120mg/kg/d), hypoxanthine analog B (120mg/kg/d), hypoxanthine analog C (120mg/kg/d), hypoxanthine analog D (120mg/kg/d); compound 1 Group, Compound 2 Group... Compound 12 Group were respectively administered Compound 1 (120 mg/kg/d), Compound 2 (120 mg/kg/d)...
  • Compound 12 120 mg/kg/d prepared in Example 1; Group E-1, compound E-2 group...compound E-12 group were respectively given compound E-1 (120mg/kg/d), compound E-2 (120mg/kg/d)...compound E-12 (120mg /kg/d). It was administered twice a day. After 14 days of administration, the mice were anesthetized with 0.8% sodium pentobarbital solution (10 mL/kg) on the 15th day. The alveolar lavage fluid and lung tissue were collected, and the alveolar lavage fluid and lung tissue were detected. The levels of hydroxyproline and TGF- ⁇ , the gold indicators of pulmonary fibrosis in tissues, were used to evaluate the anti-pulmonary fibrosis level of the compound of this patent application. The results are shown in Table 2-5 below.
  • the hydroxyproline levels in alveolar lavage fluid of the compound 1-12 intervention group were significantly lower than those of the hypoxanthine analog AD and the compound E1-12 control group (the P values of each group's comparison statistical analysis were less than 0.05 , with significant difference); the levels of hydroxyproline in the lung tissue of the compound 1-12 intervention group were significantly lower than those of the hypoxanthine analog AD and the compound E1-12 control group (the P values of the comparative statistical analysis of each group were less than 0.001, with significant differences); the TGF- ⁇ levels in alveolar lavage fluid of the compound 1-12 intervention group were significantly lower than those of the hypoxanthine analogue AD and compound E1-12 control groups (the P values of the comparative statistical analysis of each group were less than 0.001, There is a significant difference); the TGF- ⁇ levels in the lung tissue of the compound 1-12 intervention group were significantly lower than those of the hypoxanthine analogue AD and the compound E1-12 control group (the P values of the comparative statistical analysis
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against pulmonary fibrosis caused by Streptococcus pneumoniae.
  • mice SPF grade C57BL/6 mice (weighing about 22-25g) were randomly divided into several groups, including a blank control group, a model group, and nintedanidine. Positive control group, hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, compound group 1, compound group 2... compound group 12, 9 animals in each group, at SPF Level animal center for 7 days. On the first day, the mice were lightly anesthetized with ether inhalation. The mice in the blank control group were instilled with physiological saline through the nasal cavity.
  • mice in the other groups were inhaled through the trachea by spontaneous breathing of 0.5 mL/kg Streptococcus pneumoniae liquid ( The concentration is 1.0 ⁇ 10 9 CFU/mL). The next day, 5 mg/kg of bleomycin was administered via the trachea to induce pulmonary fibrosis.
  • each group was administered intragastric administration.
  • the mice in the blank control group and the model group were given the same volume of normal saline as the drug group according to their body weight.
  • the nintedanib positive control group was given an appropriate volume of saline according to their body weight.
  • hypoxanthine analog group A (120 mg/kg)
  • hypoxanthine analog group B (120 mg/kg/d)
  • hypoxanthine analog group C (120 mg/kg/d)
  • compound group 1 compound group 2...
  • compound group 12 were given compound 1 (prepared in Example 1) respectively.
  • 120mg/kg/d compound 2 (120mg/kg/d).
  • compound 12 (120mg/kg/d). It was administered twice a day. After 14 days of administration, the mice were anesthetized with 0.8% sodium pentobarbital solution (10 mL/kg) on the 15th day.
  • Compound 1 to Compound 12 prepared in Example 1 have a significant inhibitory effect on inflammatory factors such as leukocytes, neutrophils and lymphocytes in the blood of pulmonary fibrosis caused by Streptococcus pneumoniae.
  • inflammatory factors such as leukocytes, neutrophils and lymphocytes in the blood of pulmonary fibrosis caused by Streptococcus pneumoniae.
  • hypoxanthine analog A, hypoxanthine analog B, and hypoxanthine analog C were significantly lower than those of compounds 1 to 12.
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against pulmonary fibrosis induced by influenza A virus.
  • mice C57BL/6 mice (22-25g) were randomly divided into several groups, namely blank control group, model group, nintedanib positive control group, Hypoxanthine analog group A, hypoxanthine analog group B, hypoxanthine analog group C, and compound group 1, compound group 2... compound group 12, 9 animals in each group.
  • the mice in the blank control group were intranasally instilled with normal saline, and the mice in the other groups were infected with the FM1 strain of influenza A H1N1 virus (30 ⁇ L) through intranasal instillation.
  • mice in the blank control group and the model group were given the same dose of normal saline in the drug group, and the positive control group was given nintedanib.
  • Nibu 120 mg/kg
  • hypoxanthine analog group A hypoxanthine analog group B
  • hypoxanthine analog group C were given hypoxanthine analog A (120 mg/kg/d) and hypoxanthine respectively.
  • Analogue B 120 mg/kg/d
  • hypoxanthine analog C 120 mg/kg/d
  • compound group 1 compound group 2...
  • compound group 12 were respectively administered with compound 1 (120 mg/ kg/d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d). Administration was continued for 14 days, and the mice were observed daily and their body weight and death were recorded. After the administration, blood was taken from the eyeballs, and the expression levels of NF- ⁇ B, TNF- ⁇ , IL-1, and IL-6 in the serum were immediately detected, and lung tissue was taken for HE detection and Ashcroft score. The results are shown in Table 4 below. 1 and Table 4-2.
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against pulmonary fibrosis induced by influenza B virus.
  • mice C57BL/6 mice (22-25g) were randomly divided into several groups, namely blank control group, model group, nintedanib positive control group, Hypoxanthine analogue group A, hypoxanthine analogue group B, hypoxanthine analogue group C, and compounds There are 1 group of objects, 2 groups of compounds...12 groups of compounds, 9 animals in each group.
  • the mice in the blank control group were intranasally instilled with normal saline, and the mice in the other groups were all infected with type B H7N9 influenza virus strain (30 ⁇ L) through intranasal instillation.
  • mice in the blank control group and the model group were given the same dose of normal saline in the drug group, and the positive control group was given nintedanib.
  • Nibu 120 mg/kg
  • hypoxanthine analog group A hypoxanthine analog group B
  • hypoxanthine analog group C were given hypoxanthine analog A (120 mg/kg/d) and hypoxanthine respectively.
  • Analogue B 120 mg/kg/d
  • hypoxanthine analog C 120 mg/kg/d
  • compound group 1 compound group 2...
  • compound group 12 were respectively administered with compound 1 (120 mg/ kg/d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d). Administration was continued for 14 days, and the mice were observed daily and their body weight and death were recorded. After the administration, blood was taken from the eyeballs, and the expression levels of NF- ⁇ B, TNF- ⁇ , IL-1, and IL-6 in the serum were immediately detected, and lung tissue was taken for HE detection and Ashcroft score. The results are shown in Table 5 below. 1 and Table 5-2.
  • Compound 1 to Compound 12 prepared in Example 1 can inhibit IL-1 ⁇ , IL-6, TNF- ⁇ , and NF- ⁇ B in the serum of mice with pulmonary fibrosis caused by influenza B virus.
  • the levels were significantly reduced, indicating that compound 1 to compound 12 have significant activity against pulmonary fibrosis caused by influenza B virus, and the effect is better than that of hypoxanthine analog A, hypoxanthine analog B, and hypoxanthine analog Object C and nintedanib, and it was found that no fibrotic lesions appeared in the lungs of mice in the compound 1-compound intervention group.
  • Compound 1 to Compound 12 prepared in Example 1 have a significant effect on reducing the degree of pulmonary fibrosis in mice with pulmonary fibrosis model caused by influenza B virus.
  • Compound 1 to Compound 12 12 has a significant effect on improving pulmonary fibrosis caused by influenza B virus, and the effect is better than that of hypoxanthine analog compound A, hypoxanthine analog B, hypoxanthine analog C and nintedanib tablets.
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against coronavirus-induced pulmonary fibrosis.
  • mice C57BL/6 mice (22-25g) were randomly divided into several groups, namely the blank control group, the model group, the nintedanib positive control group, and the hypoxanthide group.
  • the mice in the blank control group were intranasally instilled with normal saline, and the mice in the other groups were infected with HcoV-OC43 coronavirus strain (30 ⁇ L) through intranasal instillation.
  • mice in the blank control group and the model group were given the same dose of normal saline in the drug group, and the positive control group was given nintedanib.
  • Nibu 120 mg/kg
  • hypoxanthine analog group A hypoxanthine analog group B
  • hypoxanthine analog group C were given hypoxanthine analog A (120 mg/kg/d) and hypoxanthine respectively.
  • Analogue B 120 mg/kg/d
  • hypoxanthine analog C 120 mg/kg/d
  • compound group 1 compound group 2...
  • compound group 12 were respectively administered with compound 1 (120 mg/ kg/d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d). Administration was continued for 14 days, and the mice were observed daily and their body weight and death were recorded. After the administration, blood was taken from the eyeballs, and the expression levels of NF- ⁇ B, TNF- ⁇ , IL-1, and IL-6 in the serum were immediately detected, and lung tissue was taken for HE detection and Ashcroft score. The results are shown in Table 6 below- 1 and Table 6-2.
  • Compounds 1 to 12 prepared in Example 1 all significantly reduced the degree of pulmonary fibrosis in the pulmonary fibrosis model mice caused by coronavirus, indicating that Compounds 1 to 12 have significant anti-coronavirus-induced pulmonary fibrosis effects.
  • the effect of fibrosis is better than that of nintedanib, and the effect is better than that of hypoxanthine analog A, hypoxanthine analog B and hypoxanthine analog C.
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against pulmonary fibrosis caused by novel coronavirus.
  • mice C57BL/6 mice (22-25g) were randomly divided into several groups, namely the blank control group, the model group, the nintedanib positive control group, and the subgroup.
  • the mice in the blank control group were intranasally instilled with normal saline, and the mice in the other groups were infected with the COVID-19 novel coronavirus strain (30 ⁇ L) through intranasal instillation.
  • mice in the blank control group and the model group were given the same dose of normal saline in the drug group, and the nintedanib positive control group was given nintedanib.
  • hypoxanthine analog group A, hypoxanthine analog group B, and hypoxanthine analog group C were given hypoxanthine analog A (120 mg/kg/d) and hypoxanthine analog group respectively.
  • B 120 mg/kg/d
  • hypoxanthine analog C 120 mg/kg/d
  • compound group 1 compound group 2...
  • compound group 12 were respectively administered with compound 1 prepared in Example 1 (120 mg/kg/ d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d). Administration was continued for 14 days, and the mice were observed daily and their body weight and death were recorded. After the administration, blood was taken from the eyeballs, and the expression levels of NF- ⁇ B, TNF- ⁇ , IL-1, and IL-6 in the serum were immediately detected, and lung tissue was taken for HE detection and Ashcroft score. The results are shown in Table 7 below. 1 and Table 7-2.
  • Compound 1 to Compound 12 prepared in Example 1 all significantly reduce the risk of novel coronavirus infection.
  • the degree of pulmonary fibrosis in the virus-induced pulmonary fibrosis model mice shows that Compound 1 to Compound 12 have significant anti-pulmonary fibrosis effects caused by the new coronavirus, and the effect is better than that of nintedanib and hypoxanthine.
  • This example is used to detect the activity of Compound 1 to Compound 12 prepared in Example 1 against mycoplasma-induced pulmonary fibrosis.
  • mice C57BL/6 mice (22-25g) were randomly divided into several groups, namely blank control group, model group, nintedanib positive control group, and hypoxanthine Analog group A, hypoxanthine analog group B, hypoxanthine analog group C, compound group 1, compound group 2... compound group 12, 9 animals in each group. Before modeling, the mice were anesthetized with ether. Except for the mice in the blank control group, which were instilled with 100 ⁇ L of physiological saline into the nasal cavity, the other groups were slowly instilled with the same volume of MPFH strain solution (containing 1 ⁇ 10 7 mL -1 ) into the nasal cavity.
  • MPFH strain solution containing 1 ⁇ 10 7 mL -1
  • mice in the blank control group and the model group were given the same dose of normal saline in the drug group, and the positive control group was given nintedanib.
  • Nibu 120 mg/kg
  • hypoxanthine analog group A hypoxanthine analog group B
  • hypoxanthine analog group C hypoxanthine analog A (120 mg/kg/d) and hypoxanthine respectively.
  • Analogue B 120 mg/kg/d
  • hypoxanthine analog C 120 mg/kg/d
  • compound group 1 compound group 2... compound group 12 were respectively administered with compound 1 (120 mg/ kg/d), compound 2 (120mg/kg/d)... compound 12 (120mg/kg/d).
  • continuous administration for 14 days. The mice were observed daily and their body weight and mortality were recorded. On the last day of administration, the mice were sacrificed, and blood was collected from the eyeballs, and the blood routine indicators to be detected were stored at -80°C.
  • Compound 1 to Compound 12 prepared in Example 1 all have the effect of reducing the levels of neutrophils, lymphocytes and leukocytes in the blood of mycoplasma-infected mice, indicating that Compound 1 to Compound 12 have It has anti-mycoplasma infection effect and has anti-pulmonary fibrosis activity, and the effect is better than hypoxanthine analog A, hypoxanthine analog B, hypoxanthine analog C and nintedanib. And it was found that no fibrotic lesions appeared in the lungs of mice in the intervention group of Compound 1 to Compound 12.
  • Compound 1 to Compound 12 prepared in Example 1 have a significant reducing effect on the degree of pulmonary fibrosis in mice with pulmonary fibrosis model caused by mycoplasma infection.
  • Compound 1 to Compound 12 show that Compound 1 to Compound 12 It has a significant effect on improving pulmonary fibrosis caused by mycoplasma, and the effect is better than that of hypoxanthine analog A, hypoxanthine analog B, hypoxanthine analog C and nintedanib tablets.

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Abstract

一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用，通过对结构修饰与改造后的次黄碱衍化合物进行治疗肺纤维化药理活性的检测，在多种动物疾病模型中测试了该类化合物的药理活性，并提供了防治不同类型肺纤维化活性的数据，证实其均有良好活性，效果明显优于临床常用的阳性药物尼达尼布，效果也明显优于次黄嘌类似物A、B、C、D和现有技术中的其他次黄嘌类似物。

Description

一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用 技术领域
本发明涉及生物医药技术领域，尤其涉及一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用。
背景技术
肺纤维化的致病因素多种多样，临床的病理表现一般为成纤维细胞增殖、大量细胞外基质聚集、炎症反应以及肺组织结构破坏等。肺纤维化的致死率极高，甚至高于大多数肿瘤的致死率，肺纤维化确诊后的平均生存期为2.8年。肺炎经过不断发展最终会形成肺纤维化，它们是同一类疾病在不同发展时期的不同表现形式，间质性肺炎或其它肺炎恶化的表现则是肺纤维化。因此，寻找一种可以防治肺炎、肺纤维化，减少肺部炎症反应，阻断轻症肺炎向重症肺炎肺纤维化转化的药物对肺炎及肺纤维的防治至关重要。
次黄嘌呤(次黄碱衍，Hypoxanthine)，也称“6-羟基嘌呤”，是一种天然存在的嘌呤类化合物，它是核酸的嘌呤核苷酸的合成前体。目前尚无次黄嘌类化合物可阻断肺纤维化发展，并逆转病理损伤的报道。
发明内容
本发明的目的是提出一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用，以加快研发新的治疗肺纤维化药物的进程。本发明优选技术方案所能产生的诸多技术效果详见下文阐述。
为实现上述目的，本发明提供了以下技术方案：
本发明第一方面涉及次黄碱衍化合物在制备治疗肺纤维化药物中的应用，所述的次黄碱衍化合物具有治疗肺纤维化活性，并且所述的次黄碱衍化合物具有如下结构之一：
其中：
R1任选为O、N、C、S或者＝O；
R2、R3、R4任选为H、C1-C18烷基、卤素取代的C1-C18烷基、三氟甲基、磺酰基、磺酰胺、亚磺酰基、氨基酸基、2-[双(新戊酰氧基)甲氧基]膦酰甲氧基乙基、C1-C18脂肪酸基、C3-C12杂环基、C1-C18脂肪酸；或者R2、R3、R4任选为氧原子、硫原子或氮原子取代的C1-C18的烷基或脂肪酸基；R3、R4单个取代时双键连接在非取代N位置，全被取代时无双键。
本发明第二方面涉及具有如下结构的化合物：
其中：
R1任选为O、N、C、S或者＝O；
R2、R3、R4任选为H、C1-C18烷基、卤素取代的C1-C18烷基、三氟甲基、磺酰基、磺酰胺、亚磺酰基、氨基酸基、2-[双(新戊酰氧基)甲氧基]膦酰甲氧基乙基、C1-C18脂肪酸基、C3-C12杂环基、C1-C18脂肪酸；或者R2、R3、R4任选为氧原子、硫原子或氮原子取代的C1-C18的烷基或脂肪酸基；R3、R4单个取代时双键连接在非取代N位置，全被取代时无双键。
根据一个优选实施方式，所述的化合物选自下组：
本发明第三方面涉及药物组合物，其包含本发明的化合物或其药学上可接受的盐。
根据一个优选实施方式，所述的药物组合物进一步包含药学上可接受的辅料或者辅助性成分。
根据一个优选实施方式，所述的药物组合物为口服制剂、注射制剂或鼻腔黏膜给药制剂。
本发明第四方面涉及治疗肺纤维化的方法，向有需要的个体施用有效量的本发明的化合物或其药学上可接受的盐，或本发明的药物组合物。
根据一个优选实施方式，所述的治疗肺纤维化药物包括具有防治肺纤维化及其并发症功效的药物。
根据一个优选实施方式，所述的治疗肺纤维化药物是以次黄碱衍化合物或其盐为活性成分，加入药学上可接受的辅料或者辅助性成分制备而成的制剂。
根据一个优选实施方式，所述的制剂为口服制剂、注射制剂或鼻腔黏膜给药制剂。
根据一个优选实施方式，所述的肺纤维化包括原发性肺纤维化、继发性肺纤维化、特发性肺纤维化、肺间质纤维化和间质性肺炎中的一种或多种。
根据一个优选实施方式，所述的肺纤维化包括细菌性肺纤维化、病毒性肺纤维化、支原体肺纤维化、衣原体肺纤维化、免疫性肺纤维化和真菌性肺纤维化中的一种或多种。
根据一个优选实施方式，所述的肺纤维化包括肺炎链球菌引发的肺纤维化、甲型流感病毒引发的肺纤维化、乙型流感病毒引发的肺纤维化、冠状病毒引发的肺纤维化和新冠状病毒引发的肺纤维化中的一种或多种。
根据一个优选实施方式，所述的肺纤维化还包括克雷伯菌引发的肺纤维化、肺纤维化链球菌引发的肺纤维化、耐万古肠球菌引发的肺纤维化、耐药金葡菌引发的肺纤维化和鲍曼不动杆菌引发的肺纤维化中的一种或多种。
术语定义：
本发明提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或CAS(化学文摘服务社，Columbus，OH)命名系统命名。
术语“烷基”是直链或支链的饱和烃基的基团。C1～C3烷基的实例包括甲基(C1)、乙基(C2)、正丙基(C3)和异丙基(C3)。
术语“药学上可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
术语“药学上可接受的盐”是指本发明化合物与无机和/或有机酸和碱形成的酸式和/或碱式盐，也包括两性离子盐(内盐)，还包括季铵盐，例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集，或在溶剂蒸发后回收而得到，或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增溶剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1，3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混 合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂，例如鼻腔黏膜给药制剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明所述药学上可接受的辅料，是指除活性成分以外包含在剂型中的物质。
本发明所述药学上可接受的辅助性成分，它具有一定生理活性，但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位，而仅仅发挥辅助功效，这些辅助功效仅仅是对该成分已知活性的利用，是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明药物组合物配合使用，仍然应属于本发明保护的范围。
本发明提供的次黄碱衍化合物至少具有如下有益技术效果：
本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用，通过对结构修饰与改造后的次黄碱衍化合物进行抗肺纤维化药理活性的检测，在多种动物疾病模型中测试了该类化合物的药理活性，并提供了防治不同类型肺纤维化活性的数据，证实其均有良好活性，效果明显优于临床常用的阳性药物尼达尼布，效果也明显优于次黄嘌类似物A、B、C、D和现有技术中的其他次黄嘌类似物。即本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用，可为制备抗肺纤维化药物的新化合物的筛选提供新型骨架，为开发新型先导化合物奠定理论基础。
具体的，本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用，可显著改善病毒、细菌引发的肺纤维化。更为重要的是，本发明提供的次黄碱 衍化合物可改善早期肺纤维化病症，并可逆转晚期肺纤维化的病症。
具体实施方式
为使本发明的目的、优点和技术方案更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，包括对本发明的次黄碱衍化合物治疗肺纤维化的延伸研究，都属于本发明所保护的范围。
通过对天然产物药物化学和化学研究，已经开发出许多植物内源性化合物及衍生物。对天然产物结构进行修饰和改造，得到了许多具有优良药理药化活性的衍生物。本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用，通过对结构修饰与改造后的次黄碱衍化合物进行抗肺纤维化药理活性的检测，在多种动物疾病模型中测试了该类化合物的药理活性，并提供了防治不同类型肺纤维化活性的数据，证实其均有良好活性，效果明显优于临床常用的阳性药物尼达尼布，效果也明显优于次黄嘌类似物A、B、C、D和现有技术中的其他次黄嘌类似物。即本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用，可为制备抗肺纤维化药物的新化合物的筛选提供新型骨架，为开发新型先导化合物奠定理论基础。
次黄嘌类似物A、B、C、D的结构分别如下所示：
次黄嘌类似物A、B、C、D按如下制备化合物2、化合物4和/或化合物6的方法制备。
下面结合实施例1～8对本发明提供的次黄碱衍化合物在制备抗肺纤维化药物中的应用进行详细说明。
实施例1
本实施例对化合物1～化合物12的制备方法进行详细说明。
根据一个优选实施方式，化合物1～化合物12是通过次黄嘌呤的烷基化反应制得的。
本实施例提供了12种化合物的制备方法，所有制得的化合物均通过核磁共振谱和质谱确定其结构。
化合物1和化合物5的制备
化合物1和化合物5的合成路线如下所示：
反应：(1)在0℃条件下，向250mL四口瓶中加入3.45g NaH(4.5eq)，抽换气3次后N2气氛下缓慢加入40mL(8V)无水THF；(2)往体系中缓慢滴加12.3mL异丙醇(4.5eq)，反应30min；(3)随后将5g化合物1(1.0eq)的100mL(20V)异丙醇混合物缓慢滴加至反应体系；(4)升温至80℃，体系反应10h。
后处理：(1)反应完全后加水淬灭，加入醋酸中和至pH＝8～10；(2)乙酸乙酯萃取5次，合并有机相；(3)有机相使用无水硫酸钠干燥，浓缩后快速硅胶柱层析分离得到淡黄色固体；(4)TLC监测：展开剂：二氯甲烷/甲醇＝10：1Rf(化合物1)＝0.4。
化合物1和化合物5的相关谱图数据如下：
化合物1：1H NMR(400MHz，DMSO-d6)δ13.33(s，1H)，8.44(s，1H)，8.32(s，1H)，5.55(hept，J＝6.1Hz，1H)，1.37(d，J＝6.2Hz，6H).HRMS(ESI-TOF)calc’d for C8H10N4OH+[M+H+]：179.09；found 179.10。
化合物5：11H NMR(400MHz，DMSO-d6)δ13.37(s，1H)，8.47(s，1H)，8.35(s，1H)，5.57(hept，J＝6.2Hz，1H)，1.39(d，J＝6.2Hz，6H).HRMS(ESI-TOF)[M+H+]：179.09；found 179.00。
化合物2、化合物4和化合物6的制备
化合物2、化合物4和化合物6的合成路线如下所示：
化合物2、化合物4和化合物6的相关谱图数据如下：
化合物2：1H NMR(400MHz，Chloroform-d)δ13.19(s，1H)，8.24(s，1H)，7.89(s，1H)，4.80(hept，J＝6.8Hz，1H)，1.60(d，J＝6.8Hz，6H).HRMS(ESI-TOF)[M+H+]：179.19；found 179.30。
化合物4：1H NMR(400MHz，Chloroform-d)δ8.58(s，1H)，8.07(s，1H)，5.63(hept，J＝6.2Hz，1H)，4.90(hept，J＝6.7Hz，1H)，1.59(d，J＝6.8Hz，6H)，1.44(d，J＝6.2Hz，6H).HRMS(ESI-TOF)[M+H+]：221.27；found 221.20。
化合物6：1H NMR(400MHz，Chloroform-d)δ13.21(s，1H)，8.27(s，1H)，7.82(s，1H)，4.70(hept，J＝6.8Hz，1H)，1.65(d，J＝6.6Hz，6H).HRMS(ESI-TOF)[M+H+]：179.19；found 179.30。
化合物3的制备
化合物3的合成路线如下所示：
化合物3的相关谱图数据如下：
化合物3：1H NMR(400MHz，DMSO-d6)δ8.43(s，1H)，8.19(s，1H)，5.08(hept，J＝6.9Hz，1H)，4.71(hept，J＝6.8Hz，1H)，1.52(d，J＝6.8Hz，6H)，1.41(d，J＝6.9Hz，6H).HRMS(ESI-TOF)[M+H+]：221.27；found 221.30。
化合物7～化合物12的制备
化合物7～化合物12按如上制备化合物1～化合物6中的一个化合物的方法制备。
化合物7～化合物12的相关谱图数据如下：
化合物7：1H NMR(500MHz，Chloroform-d)δ8.58(s，1H)，7.99(d，J＝0.9Hz，1H)，4.89(heptd，J＝5.6，0.5Hz，1H)，4.18(s，3H)，1.63(s，6H).HRMS(ESI-TOF)[M+H+]：193.10；found 193.10。
化合物8：1H NMR(500MHz，Chloroform-d)δ8.55(s，1H)，7.95(d，J＝0.9Hz，1H)，4.85(heptd，J＝5.2，0.5Hz，1H)，4.62(q，J＝6.5Hz，2H)，1.57(s，3H)，1.50(t，J＝6.5Hz，3H).HRMS(ESI-TOF)[M+H+]：207.12；found 207.10。
化合物9：1H NMR(500MHz，Chloroform-d)δ8.51(s，1H)，7.57(d，J＝0.3Hz，1H)，4.57(heptd，J＝5.0，0.7Hz，1H)，4.61(t，J＝5.4Hz，2H)，1.90(qt，J＝7.3，5.9Hz，2H)，1.63(s，6H)，1.15(t，J＝7.4Hz，3H).HRMS(ESI-TOF)[M+H+]：221.13；found 221.10。
化合物10：1H NMR(500MHz，Chloroform-d)δ8.37(s，1H)，5.33-5.27(m，3H)，4.74-4.64(m，2H)，1.25(d，J＝6.2Hz，6H)，1.17(dd，J＝6.7，5.2Hz，12H).HRMS(ESI-TOF)[M+H+]：265.20；found 265.20。
化合物11：1H NMR(500MHz，Chloroform-d)δ7.99-7.88(m，2H)，4.83(pd，J＝5.6，0.8Hz，1H)，2.32(s，3H)，1.53(s，6H).HRMS(ESI-TOF)[M+H+]：221.10；found 221.10。
化合物12：1H NMR(500MHz，Chloroform-d)δ7.99-7.90(m，2H)，4.85(heptd，J＝5.2，0.5Hz，1H)，2.54(q，J＝7.7Hz，2H)，1.55(s，3H)，1.19-1.11(m，3H).HRMS(ESI-TOF)[M+H+]：235.10；found 235.10。
实施例2
本实施例用来检测实施例1制得的化合物1～化合物12抗广泛性肺纤维化的活性。
(1)化合物1～化合物12抗早期肺纤维化的活性。
实验方法：建立体内博来霉素引发的肺纤维化模型：将SPF级的C57BL/6小鼠(重约22～25g)随机平均分成若干组，包括空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只，在SPF级动物中心饲养7天。实验前一天禁食12小时，然后用戊巴比妥钠进行麻醉，通过气管利用小鼠自主呼吸吸入博来霉素(5mg/kg)至肺部诱导肺纤维化，除空白对照组小鼠给无菌PBS外，模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组的小鼠分别给同体积5mg/kg博来霉素。模型构建1周后，对各组进行灌胃给药干预，空白对照组和模型组小鼠按体重给予和药物组同等体重体积的生理盐水，尼达尼布阳性对照组按体重给予适量体积的尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。每天给药两次，给药14d后，第15天将小鼠用0.8％戊巴比妥钠溶液(10mL/kg)麻醉，在腹主动脉处取血并检测血常规变化，评价肺组织肺纤维化的严重程度，对肺组织石蜡切片进行HE染色，并进行Ashcroft评分，结果见下表2-1和表2-2。
表2-1早期肺纤维化炎性检测结果表

表2-2早期肺纤维化程度Ashcroft评分表

通过表2-1数据可以看出，实施例1制得的化合物1～化合物12对早期肺纤维化血液中的白细胞、中性粒细胞和淋巴细胞等炎性因子有显著的抑制作用，而尼达尼布片和次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C的抗炎效果明显低于化合物1～化合物12。
通过表2-2数据明显看出肺纤维化模型小鼠的肺纤维程度在实施例1制得的化合物1～化合物12作用下得到了显著的改善，表明实施例1制得的化合物1～化合物12具有显著的抗肺纤维化作用，且改善效果优于尼达尼布片以及次黄嘌呤类似物A、次黄嘌呤类似物B和次黄嘌呤类似物C。
(2)化合物1～化合物12抗晚期肺纤维化的活性。
实验方法：建立体内博来霉素引发的肺纤维化模型：将SPF级的C57BL/6小鼠(重约22～25g)随机平均分成若干组，包括空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只，在SPF级动物中心饲养7天。实验前一天禁食12小时，然后用戊巴比妥钠进行麻醉，通过气管利用小鼠自主呼吸吸入博来霉素(5mg/kg)至肺部诱导肺纤维化，除空白对照组小鼠给无菌PBS外，模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组的小鼠分别给同体积5mg/kg博来霉素。模型构建3周后，对各组进行灌胃给药干预，空白对照组和模型组小鼠按体重给予和药物组同等体重体积的生理盐水，尼达尼布阳性对照组按体重给予适量体积的尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤 类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。每天给药两次，给药14d后，第15天将小鼠用0.8％戊巴比妥钠溶液(10mL/kg)麻醉，在腹主动脉处取血并检测血常规变化，评价肺组织肺纤维化的严重程度，对肺组织石蜡切片进行HE染色，并进行Ashcroft评分，结果见下表2-3和表2-4。
表2-3晚期肺纤维化炎性检测结果表
表2-4晚期肺纤维化程度Ashcroft评分表

从表2-3可知，实施例1制得的化合物1～化合物12对晚期肺纤维化血液中的白细胞、中性粒细胞和淋巴细胞等炎性因子有显著的抑制作用，而尼达尼布片和次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C的抗炎效果明显低于化合物1～化合物12。
从表2-4明显看出肺纤维化模型小鼠的肺纤维程度在实施例1制得的化合物1～化合物12作用下得到了显著的改善，表明实施例1制得的化合物1～化合物12具有显著的抗肺纤维化作用，且改善效果优于尼达尼布片以及次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C。
(3)化合物1～化合物12与对比药物抗肺纤维化的活性的比较。
对比药物次黄嘌类似物A、B、C、D及E1-12化合物结构如下所示：
次黄嘌类似物A、B、C、D及E-1～E-12按如下制备化合物2、化合物4和/或化合物6的方法制备。
实验方法：建立体内博来霉素引发的肺纤维化模型：将SPF级的C57BL/6小鼠(重约22～25g)随机平均分成若干组，包括空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、次黄嘌类似物D、化合物E-1～E-12组以及化合物1组、化合物2组……化合物12组，每组9只，在SPF级动物中心饲养7天。实验前一天禁食12小时，然后用戊巴比妥钠进行麻醉，通过气管利用小鼠自主呼吸吸入博来霉素(5mg/kg)至肺部诱导肺纤维化，除空白对照组小鼠给无菌PBS外，模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、次黄嘌类似物D、化合物E-1～E-12组以及化合物1组、化合物2组……化合物12组的小鼠分别给同体积5mg/kg博来霉素。模型构建2周后，对各组进行灌胃给药干预，空白对照组和模型组小鼠按体重给予和药物组同等体重体积的生理盐水，尼达尼布阳性对照组按体重给予适量体积的尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、次黄嘌类似物D组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)、次黄嘌类似物D(120mg/kg/d)；化合物1 组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)；化合物E-1组、化合物E-2组……化合物E-12组分别给予化合物E-1(120mg/kg/d)、化合物E-2(120mg/kg/d)……化合物E-12(120mg/kg/d)。每天给药两次，给药14d后，第15天将小鼠用0.8％戊巴比妥钠溶液(10mL/kg)麻醉，取肺泡灌洗液、肺组织，并检测肺泡灌洗液、肺组织中的肺纤维化金指标羟脯氨酸和TGF-β水平，评价本专利申请化合物的抗肺纤维化水平，结果见下表2-5。
表2-5.肺泡灌洗液及肺组织羟脯氨酸检测结果

通过表2-5数据可以看出，通过one-way ANOVA数据分析，化合物1-12干预组的肺组织羟脯氨酸水平均显著低于模型对照组(P＜0.001)；化合物1-12 干预组的肺泡灌洗液羟脯氨酸水平均显著低于模型对照组(P＜0.05)；化合物1-12干预组的肺组织中TGF-β水平均显著低于模型对照组(P＜0.05)；化合物1-12干预组的肺泡灌洗液TGF-β水平均显著低于模型对照组(P＜0.001)。重要是，我们还发现化合物1-12干预组的肺泡灌洗液羟脯氨酸水平均显著低于次黄嘌类似物A-D及化合物E1-12对照组(各组对比统计分析P值均小于0.05，具有显著性差异)；化合物1-12干预组的肺组织羟脯氨酸水平均显著低于次黄嘌类似物A-D及化合物E1-12对照组(各组对比统计分析P值均小于0.001，具有显著性差异)；化合物1-12干预组的肺泡灌洗液TGF-β水平均显著低于次黄嘌类似物A-D及化合物E1-12对照组(各组对比统计分析P值均小于0.001，具有显著性差异)；化合物1-12干预组的肺组织TGF-β水平均显著低于次黄嘌类似物A-D及化合物E1-12对照组(各组对比统计分析P值均小于0.05，具有显著性差异)。这些结果表明实施例1制得的化合物1～化合物12对肺纤维化模型小鼠肺泡灌洗液和肺组织中的羟脯氨酸、TGF-β具有显著的抑制作用，并且阳性对照尼达尼布和次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C、次黄嘌呤类似物D及化合物E1-12的抗肺纤维化效果明显低于化合物1～化合物12。
通过表2-5数据明显看出肺纤维化模型小鼠的肺纤维程度在实施例1制得的化合物1～化合物12作用下得到了显著的改善，表明实施例1制得的化合物1～化合物12具有显著的抗肺纤维化作用，且改善效果显著优于尼达尼布以及次黄嘌呤类似物A-D及化合物E1-12。
实施例3
本实施例用来检测实施例1制得的化合物1～化合物12抗肺炎链球菌引发的肺纤维化的活性。
实验方法：建立体内肺炎链球菌引发的肺纤维化模型：将SPF级的C57BL/6小鼠(重约22～25g)随机平均分成若干组，包括空白对照组、模型组、尼达尼 布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只，在SPF级动物中心饲养7天。第1天，先用乙醚吸入的方式将小鼠轻度麻醉，空白对照组小鼠经鼻腔滴注生理盐水，其它各组通过气管利用小鼠自主呼吸吸入0.5mL/kg肺炎链球菌菌液(浓度为1.0×109CFU/mL)。第二天，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。模型构建1周后，对各组进行灌胃给药干预，空白对照组和模型组小鼠按体重给予和药物组同等体重体积的生理盐水，尼达尼布阳性对照组按体重给予适量体积的尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。每天给药两次，给药14d后，第15天将小鼠用0.8％戊巴比妥钠溶液(10mL/kg)麻醉，在腹主动脉处取血并检测血常规变化，评价肺组织肺纤维化的严重程度，对肺组织石蜡切片进行HE染色，并进行Ashcroft评分，结果见下表3-1和表3-2。
表3-1肺炎链球菌引发肺纤维化炎性检测结果表

表3-2肺炎链球菌引发肺纤维化程度Ashcroft评分表
从表3-1可知，实施例1制得的化合物1～化合物12对肺炎链球菌引发肺纤维化血液中的白细胞、中性粒细胞和淋巴细胞等炎性因子有显著的抑制作用， 而尼达尼布片和次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C的抗炎效果明显低于化合物1～化合物12。
从表3-2明显看出肺炎链球菌引发的肺纤维程度在实施例1制得的化合物1～化合物12作用下得到了显著的改善，肺纤维化程度显著降低，表明实施例1制得的化合物1～化合物12具有显著的抗肺炎链球菌引发的肺纤维化的作用，且改善效果优于尼达尼布片以及次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C。
实施例4
本实施例用来检测实施例1制得的化合物1～化合物12抗甲型流感病毒引发肺纤维化的活性。
实验方法：建立体内甲型流感病毒引发的肺纤维化模型：随机将C57BL/6小鼠(22～25g)平分成若干组，分别为空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只。第1天，空白对照组小鼠经鼻腔滴注生理盐水，其它各组小鼠均经滴鼻感染甲型H1N1流感病毒FM1株(30μL)。第二天，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。造模两周后，对各组进行灌胃连续给药干预14天，空白对照组和模型组小鼠均给予药物组同等剂量的生理盐水灌胃服用，尼达尼布阳性对照组给予尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。连续给药14天，每日观察小鼠并记录其体重和死亡情况。给药结束后，眼球取血，立即检测血清中的NF-κB、TNF-α、IL-1、和IL-6表达水平，并取肺组织进行HE检测及Ashcroft评分，结果见下表4-1和表4-2。
表4-1甲型流感病毒引发肺纤维化炎性指标检测结果表
表4-2甲型流感病毒引发肺纤维化程度Ashcroft评分表

通过表4-1数据可以看出，实施例1制得的化合物1～化合物12对血清中的IL-1β、IL-6、TNF-α、NF-κB水平均有显著降低的效果，表明化合物1～化合物12抗甲型流感病毒引发肺纤维化的活性很显著，且效果优于次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C和尼达尼布，并且发现化合物1～化合物12干预组小鼠肺部未出现纤维化病变。
通过表4-2数据可以看出，实施例1制得的化合物1～化合物12对甲型流感病毒感染引发的肺纤维化模型小鼠肺纤维化程度分有显著的降低效果，说明合物1～化合物12对抗甲型流感病毒引发肺纤维化的改善作用显著，且效果优于次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C和尼达尼布片。
实施例5
本实施例用来检测实施例1制得的化合物1～化合物12抗乙型流感病毒引发肺纤维化的活性。
实验方法：建立体内乙型流感病毒引发的肺纤维化模型：随机将C57BL/6小鼠(22～25g)平分成若干组，分别为空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合 物1组、化合物2组……化合物12组，每组9只。第1天，空白对照组小鼠经鼻腔滴注生理盐水，其它各组小鼠均经滴鼻感染乙型H7N9流感病毒株(30μL)。第二天，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。造模两周后，对各组进行灌胃连续给药干预14天，空白对照组和模型组小鼠均给予药物组同等剂量的生理盐水灌胃服用，尼达尼布阳性对照组给予尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。连续给药14天，每日观察小鼠并记录其体重和死亡情况。给药结束后，眼球取血，立即检测血清中的NF-κB、TNF-α、IL-1、和IL-6表达水平，并取肺组织进行HE检测及Ashcroft评分，结果见下表5-1和表5-2。
表5-1乙型流感病毒引发肺纤维化炎性指标检测结果表

表5-2乙型流感病毒引发肺纤维化程度Ashcroft评分表
通过表5-1数据可以看出，实施例1制得的化合物1～化合物12对乙型流感病毒引发肺纤维化小鼠血清中的IL-1β、IL-6、TNF-α、NF-κB水平均有显著降低的效果，表明化合物1～化合物12抗乙型流感病毒引发肺纤维化的活性很显著，且效果优于次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C 和尼达尼布，并且发现化合物1～化合物干预组小鼠肺部未出现纤维化病变。
通过表5-2数据可以看出，实施例1制得的化合物1～化合物12对乙型流感病毒引发的肺纤维化模型小鼠肺纤维化程度有显著的降低效果，说明合物1～化合物12对乙型流感病毒引发的肺纤维化的改善作用显著，且效果优于次黄嘌呤类似物化合物A、次黄嘌呤类似物B、次黄嘌呤类似物C和尼达尼布片。
实施例6
本实施例用来检测实施例1制得的化合物1～化合物12抗冠状病毒引发肺纤维化的活性。
实验方法：建立体内冠状病毒引发的肺纤维化模型：随机将C57BL/6小鼠(22～25g)平分成若干组，分别为空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只。第1天，空白对照组小鼠经鼻腔滴注生理盐水，其它各组小鼠均经滴鼻感染HcoV-OC43冠状病毒株(30μL)。第二天，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。造模两周后，对各组进行灌胃连续给药干预14天，空白对照组和模型组小鼠均给予药物组同等剂量的生理盐水灌胃服用，尼达尼布阳性对照组给予尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。连续给药14天，每日观察小鼠并记录其体重和死亡情况。给药结束后，眼球取血，立即检测血清中的NF-κB、TNF-α、IL-1、和IL-6表达水平，并取肺组织进行HE检测及Ashcroft评分，结果见下表6-1和表6-2。
表6-1冠状病毒引发肺纤维化炎性指标检测结果表

表6-2冠状病毒引发肺纤维化程度Ashcroft评分表

从表6-1可知，实施例1制得的化合物1～12均显著降低血清中的NF-κB、TNF-α、IL-1β和IL-6水平，表明化合物1～化合物12具有显著的抗冠状病毒(HcoV-OC43)引发的肺纤维化的活性，且效果优于尼达尼布，效果也优于次黄嘌呤类似物A、次黄嘌呤类似物B和次黄嘌呤类似物C。并且发现化合物1～化合物12干预组小鼠肺部未出现纤维化病变。
从表6-2可知，实施例1制得的化合物1～化合物12均显著降低冠状病毒引发的肺纤维化模型小鼠肺纤维化程度，表明化合物1～化合物12具有显著的抗冠状病毒引发肺纤维化的作用，且效果优于尼达尼布，效果也优于次黄嘌呤类似物A、次黄嘌呤类似物B和次黄嘌呤类似物C。
实施例7
本实施例用来检测实施例1制得的化合物1～化合物12抗新型冠状病毒引发肺纤维化的活性。
实验方法：建立体内新型冠状病毒引发的肺纤维化模型：随机将C57BL/6小鼠(22～25g)平分成若干组，分别为空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只。第1天，空白对照组小鼠经鼻腔滴注生理盐水，其它各组小鼠均经滴鼻感染COVID-19新型冠状病毒株(30μL)。第二天，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。造模 两周后，对各组进行灌胃连续给药干预14天，空白对照组和模型组小鼠均给予药物组同等剂量的生理盐水灌胃服用，尼达尼布阳性对照组给予尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。连续给药14天，每日观察小鼠并记录其体重和死亡情况。给药结束后，眼球取血，立即检测血清中的NF-κB、TNF-α、IL-1、和IL-6表达水平，并取肺组织进行HE检测及Ashcroft评分，结果见下表7-1和表7-2。
表7-1新型冠状病毒引发肺纤维化炎性指标检测结果表

表7-2新型冠状病毒引发肺纤维化程度Ashcroft评分表
从表7-1可知，实施例1制得的化合物1～化合物12均显著降低血清中的NF-κB、TNF-α、IL-1β和IL-6水平，表明化合物1～化合物12具有显著的抗新型冠状病毒引发的肺纤维化的活性，且效果优于尼达尼布，效果也优于次黄嘌呤类似物A、次黄嘌呤类似物B和次黄嘌呤类似物C。并且发现化合物1～化合物12干预组小鼠肺部未出现纤维化病变。
从表7-2可知，实施例1制得的化合物1～化合物12均显著降低新型冠状 病毒引起的肺纤维化模型小鼠肺纤维化程度，表明化合物1～化合物12具有显著的抗新型冠状病毒引发肺纤维化的作用，且效果优于尼达尼布，效果也优于次黄嘌呤类似物A、次黄嘌呤类似物B和次黄嘌呤类似物C。
实施例8
本实施例用来检测实施例1制得的化合物1～化合物12抗支原体引发肺纤维化的活性。
实验方法：建立体内支原体引发的肺纤维化模型：随机将C57BL/6小鼠(22～25g)平分成若干组，分别为空白对照组、模型组、尼达尼布阳性对照组、次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组、以及化合物1组、化合物2组……化合物12组，每组9只。造模前用乙醚将小鼠麻醉，除空白对照组小鼠鼻腔滴注100μL生理盐水外，其余各组缓慢滴注同体积的MPFH菌株溶液(含1×107mL-1)至鼻腔，使其吸入进支气管，连续滴注3天。经气管滴注支原体后，通过气管给予5mg/kg的博来霉素引发其发生肺纤维化。造模两周后，对各组进行灌胃连续给药干预14天，空白对照组和模型组小鼠均给予药物组同等剂量的生理盐水灌胃服用，尼达尼布阳性对照组给予尼达尼布(120mg/kg)，次黄嘌类似物A组、次黄嘌类似物B组、次黄嘌类似物C组分别给予次黄嘌呤类似物A(120mg/kg/d)、次黄嘌类似物B(120mg/kg/d)、次黄嘌类似物C(120mg/kg/d)，化合物1组、化合物2组……化合物12组分别给予实施例1制得的化合物1(120mg/kg/d)、化合物2(120mg/kg/d)……化合物12(120mg/kg/d)。每日1次，连续给药治疗14天。每日观察小鼠并记录其体重和死亡情况。给药最后一天，将小鼠进行处死，采用眼球取血的方式，-80℃保存待检测血常规指标。同时，用生理盐水将肺部进行灌洗，分离收集灌洗液，检测白细胞计数及分类，并取肺组织进行HE染色以及纤维化程度Ashcroft评分，结果见下表8-1和8-2。
表8-1支原体引发肺纤维化炎性检测结果表

表8-2支原体引发肺纤维化程度Ashcroft评分表

通过表8-1数据可以看出，实施例1制得的化合物1～化合物12均对支原体感染小鼠血液中中性粒细胞、淋巴细胞和白细胞水平有降低效果，表明化合物1～化合物12具有抗支原体感染的效果，并具有抗肺纤维化的活性，且效果优于次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C和尼达尼布。并且发现化合物1～化合物12干预组小鼠肺部未出现纤维化病变。
通过表8-2数据可以看出，实施例1制得的化合物1～化合物12对支原体感染引发的肺纤维化模型小鼠的肺纤维化程度有显著的降低效果，说明合物1～化合物12对支原体引发肺纤维化的改善作用显著，且效果优于次黄嘌呤类似物A、次黄嘌呤类似物B、次黄嘌呤类似物C和尼达尼布片。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (19)

  1. 一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的次黄碱衍化合物具有治疗肺纤维化活性，并且所述的次黄碱衍化合物具有如下结构之一：
    其中：
    R1任选为O、N、C、S或者＝O；
    R2、R3、R4任选为H、C1-C18烷基、卤素取代的C1-C18烷基、三氟甲基、磺酰基、磺酰胺、亚磺酰基、氨基酸基、2-[双(新戊酰氧基)甲氧基]膦酰甲氧基乙基、C1-C18脂肪酸基、C3-C12杂环基、C1-C18脂肪酸；或者R2、R3、R4任选为氧原子、硫原子或氮原子取代的C1-C18的烷基或脂肪酸基；R3、R4单个取代时双键连接在非取代N位置，全被取代时无双键。
  2. 根据权利要求1所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的次黄碱衍化合物为以下化合物中的一种或多种：
  3. 根据权利要求1所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的治疗肺纤维化药物包括具有防治肺纤维化及其并发症功效的药物。
  4. 根据权利要求1所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的治疗肺纤维化药物是以次黄碱衍化合物或其盐为活性成分，加入药学上可接受的辅料或者辅助性成分制备而成的制剂。
  5. 根据权利要求4所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的制剂为口服制剂、注射制剂或鼻腔黏膜给药制剂。
  6. 根据权利要求1所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的肺纤维化包括原发性肺纤维化、继发性肺纤维化、特发性肺纤维化、肺间质纤维化和间质性肺炎中的一种或多种。
  7. 根据权利要求6所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的肺纤维化包括细菌性肺纤维化、病毒性肺纤维化、支原体肺纤维化、衣原体肺纤维化、免疫性肺纤维化和真菌性肺纤维化中的一种或多种。
  8. 根据权利要求7所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的肺纤维化包括肺炎链球菌引发的肺纤维化、甲型流感病毒引发的肺纤维化、乙型流感病毒引发的肺纤维化、冠状病毒引发的肺纤维化和新冠状病毒引发的肺纤维化中的一种或多种。
  9. 根据权利要求8所述的次黄碱衍化合物在制备治疗肺纤维化药物中的应用，其特征在于，所述的肺纤维化还包括克雷伯菌引发的肺纤维化、肺纤维化链球菌引发的肺纤维化、耐万古肠球菌引发的肺纤维化、耐药金葡菌引发的肺纤维化和鲍曼不动杆菌引发的肺纤维化中的一种或多种。
  10. 具有如下结构的化合物：
    其中：
    R1任选为O、N、C、S或者＝O；
    R2、R3、R4任选为H、C1-C18烷基、卤素取代的C1-C18烷基、三氟甲基、磺酰基、磺酰胺、亚磺酰基、氨基酸基、2-[双(新戊酰氧基)甲氧基]膦酰甲氧基乙基、C1-C18脂肪酸基、C3-C12杂环基、C1-C18脂肪酸；或者R2、R3、R4任选为氧原子、硫原子或氮原子取代的C1-C18的烷基或脂肪酸基；R3、R4单个取代时双键连接在非取代N位置，全被取代时无双键。
  11. 根据权利要求10所述的化合物，选自下组：
  12. 药物组合物，其包含权利要求10或11的化合物或其药学上可接受的盐。
  13. 根据权利要求12所述的药物组合物，其进一步包含药学上可接受的辅料或者辅助性成分。
  14. 根据权利要求12或13所述的药物组合物，其为口服制剂、注射制剂或鼻腔黏膜给药制剂。
  15. 一种治疗肺纤维化的方法，其特征在于，向有需要的个体施用有效量的权利要求10或11的化合物或其药学上可接受的盐，或权利要求12-14的药物组合物。
  16. 根据权利要求15所述的方法，其特征在于，所述的肺纤维化包括原发性肺纤维化、继发性肺纤维化、特发性肺纤维化、肺间质纤维化和间质性肺炎中的一种或多种。
  17. 根据权利要求15所述的方法，其特征在于，所述的肺纤维化包括细菌性肺纤维化、病毒性肺纤维化、支原体肺纤维化、衣原体肺纤维化、免疫性肺纤维化和真菌性肺纤维化中的一种或多种。
  18. 根据权利要求15所述的方法，其特征在于，所述的肺纤维化包括肺炎链球菌引发的肺纤维化、甲型流感病毒引发的肺纤维化、乙型流感病毒引发的肺纤维化、冠状病毒引发的肺纤维化和新冠状病毒引发的肺纤维化中的一种或多种。
  19. 根据权利要求15所述的方法，其特征在于，所述的肺纤维化包括克雷伯菌引发的肺纤维化、肺纤维化链球菌引发的肺纤维化、耐万古肠球菌引发的肺纤维化、耐药金葡菌引发的肺纤维化和鲍曼不动杆菌引发的肺纤维化中的一种或多种。
PCT/CN2023/080746 2022-03-11 2023-03-10 一种次黄碱衍化合物在制备治疗肺纤维化药物中的应用 Ceased WO2023169557A1 (zh)

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