WO2007104242A1 - Compounds capable of inhibiting zinc ion metalloproteinases - Google Patents

Compounds capable of inhibiting zinc ion metalloproteinases Download PDF

Info

Publication number
WO2007104242A1
WO2007104242A1 PCT/CN2007/000742 CN2007000742W WO2007104242A1 WO 2007104242 A1 WO2007104242 A1 WO 2007104242A1 CN 2007000742 W CN2007000742 W CN 2007000742W WO 2007104242 A1 WO2007104242 A1 WO 2007104242A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
substituted
unsubstituted
alkyl
aryl
Prior art date
Application number
PCT/CN2007/000742
Other languages
French (fr)
Chinese (zh)
Inventor
Xuexun Fang
Original Assignee
Xuexun Fang
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xuexun Fang filed Critical Xuexun Fang
Publication of WO2007104242A1 publication Critical patent/WO2007104242A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/84—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring
    • C07C69/88—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring with esterified carboxyl groups
    • 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
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C243/00—Compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
    • C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
    • C07C39/10—Polyhydroxy benzenes; Alkylated derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/82—Ketones containing a keto group bound to a six-membered aromatic ring containing hydroxy groups
    • C07C49/825—Ketones containing a keto group bound to a six-membered aromatic ring containing hydroxy groups all hydroxy groups bound to the ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/16—Quinones the quinoid structure being part of a condensed ring system containing three rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones

Definitions

  • the present invention relates to a class of compounds capable of inhibiting zinc ion metalloproteinases, uses thereof, and pharmaceutical compositions containing the same.
  • Matrix metalloproteinases are a general term for the family of Zn 2+ metal endopeptidases secreted by cells and are involved in the degradation of extracellular matrix (ECM).
  • ECM extracellular matrix
  • MMPs are a family of Zn 2+ -dependent proteases, which are mainly synthesized and secreted by fibroblasts, endothelial cells, macrophages, neutrophils, etc., usually under neutral conditions and with the participation of Ca 2+ . Found 23 species of human Ps.
  • Various Ps should generally have the following characteristics: 1 The cDNA sequences of different P. s have homology.
  • Enzyme activity depends on the Zn 2+ and Ca 2+ of the active center of the enzyme. 4 Enzyme activity is inhibited by specific matrix metalloproteinase tissue inhibitors (TIMPs) and can also be inhibited by chelators.
  • TIMPs matrix metalloproteinase tissue inhibitors
  • Extracellular matrix not only acts as a mechanical support and connection between cells and cells, but also serves as a bridge between cells and cells.
  • MMPs are ECM proteolytic enzymes that regulate the dynamic balance between degradation and recombination by breaking down ECM components to eliminate certain specific signals, reveal certain hidden signals, and even release existing biologically active substances in the matrix. Or the purpose of activation, and thus participate in various morphological and functional changes in cells and tissues. These changes include the growth, proliferation, and differentiation of various tissues and cells; the cyclical changes of the endometrium during reproduction, physiological changes such as ovulation, blastocyst implantation, and the infiltration and metastasis of tumor cells, the occurrence and spread of inflammation. And other pathological changes.
  • MMPs In addition to the degradation of extracellular matrix (ECM), the biological functions of MMPs play an important role in the regulation of the synthesis and secretion of cytokines, growth factors, hormones and cell adhesion molecule receptors, and thus participate in apoptosis and morphology. A series of pathological and physiological processes such as occurrence, proliferation and development. The key role of ⁇ Ps in tumorigenesis, growth, invasion, metastasis, tumor angiogenesis and apoptosis has been established. The development of Ps inhibitors has broad prospects for important diseases including cancer, neurodegenerative diseases such as Alzheimer's disease, cardiovascular disease and various inflammatory drug treatments.
  • ADAMs a disintegrin and metalloproteinase proteolytic enzymes.
  • ADAMs also known as MDC (metalloproteinase/disintergrin/cystein-rich)
  • MDC metaloproteinase/disintergrin/cystein-rich
  • ADAMs are widely distributed in organisms, in multicellular In animals, such as humans, pigs, rats, Xenopus, Drosophila, C. elegans, etc., such as brain, testis, testis, ovary, breast, placenta, small intestine, colon, heart, liver, lung, bone , muscles, etc.
  • members of the family although they all contain fairly conserved structural domains, their tissue distribution, activity, and biological roles vary. They may play an important role in cellular activities such as hydrolysis of extracellular matrices, cell-cell and cell-matrix adhesion, cell fusion, and signaling.
  • the family plays an important role in multiple physiological processes such as sperm-egg binding, neurodevelopment, myotube formation, cell proliferation and differentiation, extracellular matrix remodeling, and angiogenesis.
  • AMMS also plays a key role in many pathological processes such as tumorigenesis, invasion and metastasis, Alzheimer's disease, arthritis and cardiovascular disease.
  • ADAM-TS (a disintegrin and metalloprotease with thrombospondin motif) is a newly discovered class of zinc ion metalloproteinases. More than 20 species have been discovered. ADAM-TS is expressed in tissues of mammals, fruit flies, and nematodes. ADAM-TS shares some structural features with the ADAMs family. For example, the amino terminus of a protein contains a signal peptide, a leader peptide, a metalloproteinase region, a de-integrin region, and a cysteine-rich region.
  • ADAM-TS does not contain Epidermal growth factor-like repeat domain and transmembrane domain, replaced by different numbers of type I thrombin sensitive protein (thrombospondin 1 ) repeat structure. It is mainly involved in the degradation of extracellular matrix, cell-matrix adhesion and tissue organ remodeling, and plays an important role in physiological and pathological processes such as ovulation, embryonic development, connective tissue diseases, and tumors.
  • the catalytically active sites contain a conserved sequence of Zn 2+ -bound HEXXH, which is similar to the structure of the active center of MMPs. Therefore, inhibitors of ⁇ Ps should have a similar effect on ADAMs and ADAM-TS.
  • MMP inhibitor bursts are an important branch of academic and industrial research.
  • many pharmaceutical companies around the world showed strong interest in the screening of ⁇ Ps and its inhibitors, and several broad-spectrum, small-molecular-weight MMP inhibitors for several types of tumors.
  • MMP inhibitors can effectively inhibit the activity of Luo P and effectively prevent the growth and metastasis of tumors in preclinical animal experiments, the results of clinical treatment of cancer are not satisfactory, eventually leading to late clinical practice. The failure of the test. This is mainly because the complexity of the MMP family is not fully understood.
  • the specificity of these inhibitors is not strong, and the normal physiological functions of other types of MMPs have also been affected to varying degrees.
  • MMP as a therapeutic target for disease is complex because MMPs are essential for maintaining a normal physiological environment, and their functions are diverse, with reproducible and context-dependent expression and activity.
  • MMPs are essential for maintaining a normal physiological environment, and their functions are diverse, with reproducible and context-dependent expression and activity.
  • MMPs are closely related to many diseases, these ⁇ Pis are very likely to be used for the treatment of cardiovascular diseases in the future; arthritis; periodontal disease; multiple hard Inflammation; pain; corneal ulcer; bacterial meningitis; diabetes syndrome; wound healing; nephropathy; neurodegenerative diseases; AIDS; herpes; allergies; endometriosis; osteoporosis; For organ transplantation; control of fertilization process and regeneration capacity; these inhibitors can also be used for anti-aging; antibacterial; as an additive for the industrial production of extracellular matrix/collagen products, cosmetics, and beauty products.
  • 1,2,3-trihydroxybenzene and its derivatives have an inhibitory effect on zinc ion metalloproteinases.
  • the inhibitory effect of 1,2,3-trihydroxybenzene and its derivatives on zinc ion metalloproteinases is universal and specific. Three of the adjacent hydroxyl groups are the key sites for inhibition, and the three adjacent hydroxyl groups must be on the same plane in the benzene ring structure.
  • the mechanism by which 1,2,3-trihydroxybenzene inhibits matrix metalloproteinases is competitive inhibition or competitive advantage inhibition.
  • the invention relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting a zinc ion metalloproteinase, said compound of formula I
  • R 1 , R 2 and R 3 are independently a hydrogen atom, a substituted or unsubstituted ( ⁇ -C 18 alkyl group, a substituted or unsubstituted C 2 -C 18 alkane interrupted by one or several discrete 0 atoms) Base, or by -CO-, - C00-, - 0C0-, - 0C00-, - C0-N (R 4 ) -, _N (R 4 ) - CO -, - N (R 4 ) -C0-N ( R 4) -, - [N (R 4)] 2 - C0-, - N (R 4) - substituted or unsubstituted spaced COO- C 2 - C 18 alkyl, a substituent independently selected from _0R 5 , - 0C0_R 5 , - COO- R 5 , _N (R 4 ) - R 5 , - N (R 4 ) Base,
  • C 2 aryl group and CrC 2 containing 0, S or N heteroaryl oxygen Base -C0-0R 4 or -CO- N (R 4 ) 2 ; or R 1 and R 2 , or R 2 and R 3 together form a substituted or unsubstituted C e -C 2 .
  • Drugs that inhibit zinc ion metalloproteinases are preferably used to modulate the physiological and pathological processes involved in matrix metalloproteinases (Ps), ADAMs, or ADAM-TS.
  • the physiological and pathological processes involved in matrix metalloproteinases (MMPs), ADAMs or ADAM-TS are preferably angiogenesis, wound healing, organ transplantation, control of fertilization and regeneration, bone remodeling or pain.
  • the drug for inhibiting zinc ion metalloproteinase is preferably used for the treatment and prevention of cancer, cardiovascular disease, arthritis, periodontal disease, multiple sclerosis, inflammation, corneal ulcer, bacterial meningitis, diabetes syndrome, kidney disease, sputum regression Sexual diseases, AIDS, herpes, allergies, endometriosis, osteoporosis, asthma, anti-aging or antibacterial. More preferably used for the treatment and prevention of cancer, cardiovascular disease or arthritis.
  • the present invention provides the use of a compound of the formula I or a salt thereof as an additive in a health food, a cosmetic or a conventional product for inhibiting zinc ion metalloproteinase.
  • the compound of formula I is preferably pyrophoric acid, propyl gallate, tannic acid, myricetin, baicalin, terminal acid, quercetin, scutellarin, scutellin, locust pavilion, valerian Kiosk, myricetin, lauryl gallate, gallic tea, delphinidin or benserazide. More preferred is pyrophoric acid. Pyroallic acid (Pyrogal l ic acid)
  • Another aspect of the invention provides a pharmaceutical composition for use as a zinc ion metalloproteinase inhibitor comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is preferably used to modulate the physiological and pathological processes involved in matrix metalloproteinases (as Ps;), ADAMs or ADAM-TS.
  • the physiological and pathological processes involved in matrix metalloproteinases (MMPs), ADAMs or ADAM-TS are preferably angiogenesis, wound healing, organ transplantation, control of fertilization and regeneration, bone remodeling or pain.
  • the above pharmaceutical composition is preferably used for the treatment and prevention of cancer, cardiovascular disease, arthritis, periodontal disease, multiple sclerosis, inflammation, corneal ulcer, bacterial meningitis, diabetes syndrome, nephropathy, neurodegenerative diseases, AIDS, blister Rash, allergies, endometriosis, osteoporosis, asthma, anti-aging or antibacterial. More preferably used for the treatment and prevention of cancer, cardiovascular disease or arthritis.
  • a health food, cosmetic or daily use composition having an action of inhibiting zinc ion metalloproteinase, comprising a compound of the formula I or a salt thereof as an additive.
  • Figure 1 The abscissa from left to right represents the positive control group, the three different doses of the drug-administered group, and the negative control group.
  • the positive control group 100 ⁇ 13 was significantly larger than the three drug-administered groups (72 soil, 10, 69 ⁇ /12, 35 ⁇ 5; P ⁇ 0.001) and the negative control group (25 ⁇ /3, P ⁇ 0.001).
  • Example 1 1, 2, 3-trihydroxybenzene and its derivatives inhibit zinc ion metalloproteinase
  • the buffer system used was 50 mM HEPES (pH 7.5) with 0.2 M NaCl, lOraM CaCl 2 , and 0.05% Brij-35, and the substrates used were DQ-Gelatin and P126; the total reaction volume was determined at room temperature. 100 1.
  • the enzyme and inhibitor should be incubated in the buffer system for 30 minutes and then added to the substrate for determination. All instruments tested were FLX800 fluorescence microplate reader (Bio-Tek), for DQ-Gelatin excitation wavelength 495 nm, emission wavelength 515, and for P126, excitation wavelength 328, emission wavelength 393 nm.
  • the reaction time was determined to be 8 minutes, and the slope of the line composed of the points at which the fluorescence values were taken was the velocity, and the uninhibited matter was recorded as V. , plus inhibitors, to Vi / V. Indicates the degree of inhibition of the inhibitor; then the concentration of the added inhibitor is plotted on the abscissa, Vi/V. The value of the ordinate is plotted against the ordinate; in Vi/V. The concentration of the inhibitor corresponding to 0.5 is IC50.
  • the cells were counted, and then introduced into a 24-well plate at a cell density of 10 V, and a specified amount of pyrophoric acid was added to make the final concentration of each drug. as followed
  • the cells were counted, and then introduced into a 24-well plate at a cell density of 107 cells, and a specified amount of pyrophoric acid was added to make the final drug concentration per well. In order of 0
  • the adhesion and extension of tumor cells are related to MMPs.
  • the experimental results show that different concentrations of 1,2,3-trihydroxybenzene have inhibitory effects on HT1080 tumor cell adhesion and extension.
  • the HT1080 cells were cultured at a cell density of 107 cells in a 24-well plate. When the cells were grown to about 80% of the bottom wall, the plate was removed, and the supernatant was aspirated, using a 200 ⁇ L pipette tip. A diagonal line of the same width is drawn in each well of the culture plate, and then a specified amount of pyrophoric acid is added, so that the final concentration of the drug in each well is 0 ⁇ ⁇ , 2 ⁇ ⁇ , 5 ⁇ ⁇ , 10 ⁇ ⁇ , 20 ⁇ ⁇ , and the final volume of the medium in each well was 1 mL, and then placed in a 37 ⁇ incubator. After 12 h, observe the filling of the scribe line in each well of the plate. The migration of tumor cells is associated with ⁇ Ps. Different concentrations of 1,2,3-trihydroxybenzene inhibited the migration of ⁇ 080 tumor cells. '
  • one 24-well culture plate, several Transwell chambers, Matrigel, 200uL gun heads should be placed in the 4°C refrigerator for more than 5 hours, and transferred to the ultra-clean table with the ice box.
  • the cold 200 ⁇ L tip plus 70 ⁇ L/well Matrigel is added to the Transwell. It is usually added in two portions.
  • the medium is configured with a final concentration of 0 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M cell suspension for use (approximately 10 6 HT1080 cells per suspension).
  • the proliferation of tumor cells is associated with MMPs.
  • the MTT method was used to determine the degree of killing of HT1080 cells by different levels of non-phagic acid.
  • the experimental methods were as follows: The vigorous growth of HT1080 cells was suspended in 10% FBS DMEM medium at a final concentration of 10 4 /well. The cells were added to a 96-well plate and cultured for one day. After the cells adhered to 70% of the bottom wall, they were cultured for one day in a medium of 2% serum (FBS). Each well was then administered at a concentration of 0 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M. Incubate overnight, and 20 ⁇ l of each well was added to each well for 4 h. Carefully aspirate the supernatant, add 150 ⁇ l of DMS0 to each well, gently shake to dissolve the crystal violet formed in the well, and then read the 0D value of each well at 490 nm with a microplate reader.
  • the ascites cells of sputum 22 were inoculated for 8 days, and the cell concentration was IX 107 ml.
  • Each mouse was subcutaneously inoculated with 0.1 ml in the left hind limb. The next day, the rats were randomly divided into groups, 12 rats in each group. 1 time (dose by dose). Continuous administration for 10 days, weighing once every 3 days, the mice were killed by dislocation on the next day after the last administration, and the tumor weight was called, and the tumor inhibition was calculated according to the following formula.
  • Table 2 Inhibitory effect of pyrogallic acid on the growth of mouse H 22 tumor
  • Example 3 is specific for the treatment of atherosclerosis with the drug of P.
  • Atherosclerosis is the pathological basis of clinical syndromes such as acute myocardial infarction and unstable angina.
  • Matrix metalloproteinase Ps plays an important role in cardiovascular remodeling by degrading extracellular matrix. Expression of MMPs is associated with both intimal thickening, plaque instability, and formation of restenosis lesions. MMPs that have been upregulated in atherosclerosis have been detected to include MMP-1, -2, -7, -9, -12. Many Ps are associated with matrix remodeling of arteriosclerotic lesions, such as ⁇ P-1, -2, -3, -7, -9, -12, -13, -14. Therefore, the development of therapeutic drugs specific for MMP will help prevent the development of atherosclerosis, plaque rupture and restenosis.
  • Atherosclerotic intimal thickening Atherosclerosis can be initiated by chemical or mechanical damage of the endothelium, and then monocytes in the blood enter the inner membrane through transendothelial osmosis, where they activate or synthesize many cytokines and growth. factor.
  • monocytes in the blood enter the inner membrane through transendothelial osmosis, where they activate or synthesize many cytokines and growth. factor.
  • vascular smooth muscle cells migrated from the media to the intima and experienced a proliferative process. Matrix degradation is essential for the migration of monocytes and vascular smooth muscle cells, as cells have to cross extracellular disorders, including the subendothelial and basement membrane surrounding each smooth muscle cell.
  • plaque stability unstable plaque easily lead to coronary thrombosis and occlusion, triggering acute coronary syndrome (ACS).
  • ACS acute coronary syndrome
  • PCI coronary intervention
  • Rats are usually fed with high fat, high fat for at least three months, 5% egg yolk (rich in cholesterol, crushed), 10% lard, 0.2% methylthiouracil, 1% cholesterol; 3 months After the abdominal aorta forms obvious plaques. Vascular remodeling and restenosis were studied using balloon injury methods.
  • the effect of MMP inhibitor on the disease was investigated by using the smectin P inhibitor in the balloon injury model of rats. This experiment is based on the carotid artery of rats, and immediately after modeling. The sputum sputum was injected into the abdominal cavity 300 mg/kg for 70 days.
  • the relevant testing indicators are as follows:
  • the degree of remodeling is determined by the ratio of the total vessel wall area (EEL AREA) of the balloon lesion to the wall area of the standard portion of the joint without damage (EEL AREA), ie the remodeling ratio (remodeling) Ratio, RR).
  • the carotid artery of the rat was taken for zymography analysis 3 days after the balloon injury, and the relative density of the MMP-2 band was analyzed by a density analyzer.
  • the positive control group was significantly larger than the three drug-administered groups and the negative control group (Fig. 1). Explain that medication can reduce the activity of MMP.
  • Endometrial translocation occurs after the endometrial glands infiltrate normal tissues. Endometriosis is a process of infiltration. Matrix metalloproteinases are essential for the remodeling of extracellular matrices. Matrix metalloproteinases are involved in normal tissue metabolic turnover, including intimal rupture during the menstrual cycle. There is sufficient evidence that metalloproteinases and their tissue inhibitors are involved in the pathological process of endometrial translocation and are associated with the development of endometrial translocation. The metalloproteinases and their tissue inhibitors currently associated with endometrial translocation include MMP-1, MMP-2, MMP-3, Sa P-9, TACE and TIMP-1, TIMP-3.
  • EMs endometriosis
  • the exogenous matrix metalloproteinase inhibitor is delphinidin.
  • B is the exogenous inhibitor Feiyancaoyuan treatment group: 100mg/kg, intraperitoneal injection, once a day, for a total of four weeks.
  • C is the exogenous inhibitor Feiyancaoyuan treatment group: 150mg/kg, intraperitoneal injection, once a day, for a total of four weeks.
  • Control group D was a positive control group: gonadotropin-releasing hormone agonist (LHRH-A) - nafarelin 0. lmg/kg, subcutaneous injection, once a day for four weeks.
  • E is a negative control group: 0. 9% normal saline 0. 5ml / kg, intraperitoneal injection, once a day, a total of four weeks.
  • exogenous inhibitor treatment group ectopic lesions Compared with the western medicine control group, there was no statistically significant difference (P>0.05). It can be seen that exogenous matrix metal inhibitor and narfarrein treated EMs, and the ectopic endometrial growth was significantly inhibited. And the exogenous matrix metal inhibitors are similar to the inhibitory effects of nafarelin.
  • the length, width and height of the ectopic endometrium at the uterus bifurcation, ovary and abdominal wall were measured before administration (4 weeks after surgery) and after drug treatment (4 weeks after administration).
  • the total volume (Vl, V2) was calculated, and the inhibition rate of the ectopic endometrium of each group of drugs was calculated.
  • the rats in the model group had extensive adhesions in the peritoneal cavity.
  • the grafts were visible in 10 rats, ranging from 1 to 5 mm 3 , and some were cystic vesicles containing bloody fluids. It is a cystic vesicle containing transparent colorless liquid, and some of them are cystic.
  • the connective tissue was formed on the surface of the graft, and one ectopic lesion was shrunk and atrophied.
  • the model group showed that the intima of the transplant survived, and the epithelial cells proliferated in a columnar, cubic or flat shape, and some of the vacuoles and the top paddle were secreted, showing a cyclical change close to the eutopic endometrium.
  • the interstitial cells are small and sparse, the growth is not active, the number of glands is reduced, some glands are changed in shape, and there are gaps. Some glandular cavities are extremely enlarged, there are papillary processes, and some hemosiderin can be seen in the glandular cavity. Cells and foam cells, some glands disappear, blood vessels are rich.
  • the exogenous inhibitor treatment group had morphological changes in the lining of the endometrial tissue, but the gland structure was intact, the glandular epithelium was neatly arranged, and the interstitial was dense; the ectopic endometrial tissue was slit-like, the surface epithelium was short cubic, epithelium The cells are arranged disorderly, the endometrial glands are reduced, the glandular epithelium is atrophied, the surface vessels are reduced, and the interstitial cells are small and sparse.
  • a sputum inhibitor was used to demonstrate that the intraperitoneal injection of the bismuth inhibitor, delphinidin, has a therapeutic and palliative effect on this disease.
  • Osteoarthritis is a chronic joint disease characterized by degeneration, destruction and bone hyperplasia of articular cartilage. It is the most common joint disease in China and one of the main causes of pain and disability in the elderly. Its pathogenesis is still unclear, but the mechanism of its cartilage degradation has been described in many ways.
  • the basic purpose of OA treatment is to relieve symptoms, improve function, delay progression and correct deformity.
  • OA should focus on early diagnosis, early treatment, long course of treatment, that is, comprehensive treatment should be used when patients have mild symptoms.
  • a variety of Ps degrades the extracellular matrix of articular cartilage, changes the structure of the articular cartilage matrix, and reduces the resistance to external forces, eventually leading to the progressive destruction of articular cartilage. Therefore, the use of MMP inhibitors in the treatment of osteoarthritis is an important means to achieve cartilage protection to delay the development of the disease.
  • the first week 24 large white rabbits underwent ACL surgery.
  • the third week and the fifth week Animals were randomly divided into three groups, group A (intra-articular injection of normal saline), group B (joint cavity injection of 100 mg/ml wild scutella solution), group C (joint injection of lOOmg/tnl pentoxide) Flavonoid solution).
  • the rabbits were anesthetized with 3% sodium pentobarbital (30 mg/kg) in the ear vein, fixed on the operating table, localized in the right knee joint, prepared for skin, disinfected, draped, and strictly aseptic. Take a longitudinal incision about 4 cm long inside the knee joint, and explore the medial collateral ligament after no primary lesion in the joint cavity, further remove the medial meniscus and anterior and posterior cruciate ligaments, be careful not to damage the articular cartilage surface, completely stop bleeding, flush the joint cavity, After the drawer test is positive, the joint capsule is sutured layer by layer, subcutaneous, skin, and sterile dressing are used to dress the wound and will not be fixed.
  • Grade I No abnormal pain response
  • Grade II Contraction of the affected limb
  • Grade III Contraction of the affected limb, convulsion, mild systemic reaction, such as trembling around the body, turning back, etc.
  • W severe contraction of the affected limb, paralysis, whole body Trembling, snarling, struggling.
  • Gait changes According to the walking gait of the affected limb, the gait is divided into 4 levels.
  • Grade I The affected limbs are innocent, running normally, and powerful; Grade II: Mild limp while running on the affected limb, strong and powerful; Grade III: The affected limb is involved in walking, but the movement is obvious; Grade IV: The affected limb cannot participate Walking, can't touch the ground, squatting.
  • Class I 90° or more; Class II: 45° to 90°; Class III: 15° to 45° Class IV: 15°.
  • Group C 2 4 2 0 3 4 1 0 4 2 1 1 4 3 1
  • group A compared with group B
  • group C p ⁇ 0.01.
  • the scores were scored according to the following principles: 0 points, articular surface smoothness, color As usual; 1 point, the joint surface is rough, there are small cracks and the color is gray; 2 points, the articular surface is erosive, the cartilage defect is deep to the middle layer of the cartilage; 3 points, the articular surface ulcer is formed, the defect is deep to the deep cartilage; 4 points, cartilage Exfoliation, subchondral bone exposure.
  • the 10% formalin-fixed knee joint was decalcified in the decalcifying agent for approximately one week. After the decalcification is complete, the knee joint traverses in the leading plane, so the intermediate and lateral circulation is maintained, and the two halves are embedded in the paraffin.
  • the number of cartilage destruction is determined by 1/3, 2/3 or 3/3 of the histological surface, and the above scores are multiplied by 1, 2 or 3, respectively, to reflect the extent of the participating tibial plates.
  • the size of the epiphysis measured with an eyepiece micrometer: 1, 2, or 3 are mild, moderate, or severe, respectively.
  • the data in the above table is significantly different from the ⁇ group and the C group at the p ⁇ 0.05 level.
  • MMP inhibitors were used to demonstrate the treatment and alleviation of the disease by intra-articular injection of MMP inhibitors of scutellarin and quercetin solution.

Abstract

The invention has disclosed 1,2,3-trihydroxy benzene and its derivatives or pharmaceutically acceptable salts of formula I for zinc ion metal-protease inhibition. These compounds are used for selective depressants of zinc ion metal-proteases such as MT1-MMP,gelatinase A, B and collagenase, matrilysins, metal-elastase or stromelysin-1. These depressants can be used to adjust many physiological or pathology courses which are concerned with MMPs, ADAMs, ADAM-TS such as vessel rebirth, wound healing, organ transplantation, fecundation course and reactivation capability, bone rebuilding and ache. They are used for treating cancer, cardiovascular diseases, arthritis, periodontal disease, multiple sclerosis, inflammation, adenomyosis, cornea ulcer, bacterial meningitis, diabetic syndrome, kidney diseases, nerve retrogression diseases, AIDS, bleb, anaphylaxis, adenomyosis, osteoporosis, asthma and so on. These blocking agents can be used for antisenescence, antibiosis, commercial manufacture addition agents of cell epimatrix, collagen product, cosmetics and make-up preparation. They can used for animals and other living bodies.

Description

一类能够抑制锌离子金属蛋白酶的化合物  a class of compounds capable of inhibiting zinc ion metalloproteinases
技术领域 Technical field
本发明涉及一类能够抑制锌离子金属蛋白酶的化合物， 其用途以及含有该化合物的药物 组合物。  The present invention relates to a class of compounds capable of inhibiting zinc ion metalloproteinases, uses thereof, and pharmaceutical compositions containing the same.
背景技术 Background technique
基质金属蛋白酶即 Matrix Metalloproteinases, 简称顧 Ps， 是由细胞分泌的 Zn2+金属 内肽酶家族的总称， 参与细胞外基质 (ECM)的降解。 MMPs是一个 Zn2+依赖性蛋白酶家族， 主要 由成纤维细胞、 内皮细胞、 巨噬细胞、 中性粒细胞等合成与分泌， 通常在中性条件下及 Ca2+ 参与下发挥作用， 目前已发现 23种人源疆 Ps。各种應 Ps普遍具有如下特征： ①不同的丽 Ps 的 cDNA序列具有同源性。 ②均以酶原形式分泌,受蛋白酶或其它因子激活， 活化后能降解一 种或多种 ECM。 ③酶活性依赖酶活性中心的 Zn2+及 Ca2+。 ④酶活性受特异性基质金属蛋白酶组 织抑制剂 (TIMPs)抑制， 亦可受螯合剂抑制。 Matrix metalloproteinases, referred to as Gu Ps, are a general term for the family of Zn 2+ metal endopeptidases secreted by cells and are involved in the degradation of extracellular matrix (ECM). MMPs are a family of Zn 2+ -dependent proteases, which are mainly synthesized and secreted by fibroblasts, endothelial cells, macrophages, neutrophils, etc., usually under neutral conditions and with the participation of Ca 2+ . Found 23 species of human Ps. Various Ps should generally have the following characteristics: 1 The cDNA sequences of different P. s have homology. Both are secreted in the form of zymogens, activated by proteases or other factors, and can degrade one or more ECMs upon activation. 3 Enzyme activity depends on the Zn 2+ and Ca 2+ of the active center of the enzyme. 4 Enzyme activity is inhibited by specific matrix metalloproteinase tissue inhibitors (TIMPs) and can also be inhibited by chelators.
细胞外基质（ECM)不仅起细胞与细胞之间机械支持和连接作用，也是细胞和细胞之间信 号传递的桥梁。 MMPs是 ECM蛋白水解酶， 可以通过分解 ECM成分来调节其降解和重组之间的 动态平衡， 以达到消除某些特殊信号、 显现某些隐含信号、 甚至使基质中已存在的生物活性 物质释放或激活的目的， 进而参与细胞和组织器官的各种形态和功能变化。 这些变化既包括 各组织和细胞的生长、 增殖、 分化； 生殖过程中子宫内膜的周期性变化、 ***、 胚泡着床等 生理变化， 也包括肿瘤细胞的浸润和转移， 炎症的发生及扩散等病理变化。 MMPs的生物功能 除对细胞外基质（ECM)的降解外， 它们在调控细胞因子、 生长因子、 激素和细胞粘附分子受 体等的合成和分泌中发挥重要作用， 进而参与细胞凋亡、 形态发生、 增殖和发育等一系列病 理和生理过程。 而匪 Ps在肿瘤的发生、 发展过程中生长、 浸润、 转移、 肿瘤血管新生和细胞 凋亡中的关键作用已被确立。画 Ps抑制剂的开发对于重要疾病包括癌症、 神经退行性疾病如 老年痴呆症、 心血管疾病和各类炎症药物治疗具有广阔的前景。  Extracellular matrix (ECM) not only acts as a mechanical support and connection between cells and cells, but also serves as a bridge between cells and cells. MMPs are ECM proteolytic enzymes that regulate the dynamic balance between degradation and recombination by breaking down ECM components to eliminate certain specific signals, reveal certain hidden signals, and even release existing biologically active substances in the matrix. Or the purpose of activation, and thus participate in various morphological and functional changes in cells and tissues. These changes include the growth, proliferation, and differentiation of various tissues and cells; the cyclical changes of the endometrium during reproduction, physiological changes such as ovulation, blastocyst implantation, and the infiltration and metastasis of tumor cells, the occurrence and spread of inflammation. And other pathological changes. In addition to the degradation of extracellular matrix (ECM), the biological functions of MMPs play an important role in the regulation of the synthesis and secretion of cytokines, growth factors, hormones and cell adhesion molecule receptors, and thus participate in apoptosis and morphology. A series of pathological and physiological processes such as occurrence, proliferation and development. The key role of 匪Ps in tumorigenesis, growth, invasion, metastasis, tumor angiogenesis and apoptosis has been established. The development of Ps inhibitors has broad prospects for important diseases including cancer, neurodegenerative diseases such as Alzheimer's disease, cardiovascular disease and various inflammatory drug treatments.
锌离子金属蛋白酶还包括 ADAMs (a disintegrin and metalloproteinase)蛋白水解 酶。 ADAMs又被称为 MDC (metalloproteinase/disintergrin/cystein-rich) ， 是近年新发 现的一类跨膜糖蛋白，目前人们已发现三十余种 ADAMs。 ADAMs在生物中分布很广， 在多细胞 动物中， 如人、 猪、 大鼠、 爪蟾、 果蝇、 线虫等的细胞、 组织和器官， 如脑、 睾丸、 睾、 卵 巢、 乳腺、 胎盘、 小肠、 结肠、 心、 肝、 肺、 骨、 肌肉等处。 尽管该家族成员尽管都含有相 当保守的结构功能域， 但是它们的组织分布、 活性功能以及发挥的生物学作用各不相同。 它 们在在细胞外基质的水解、 细胞一细胞和细胞一基质的粘连、 细胞融合及信号传导等细胞活 动中可能具有重要作用。 该家族在精卵结合、 神经发育、 肌管形成、 细胞的增殖与分化、 胞 外基质重建以及血管新生等多个生理过程中的重要作用。 同时， AMMS 在肿瘤的发生、 浸润 和转移， 老年痴呆症， 关节炎以及心血管疾病等多个病理过程中也有关键的作用。 Zinc ion metalloproteinases also include ADAMs (a disintegrin and metalloproteinase) proteolytic enzymes. ADAMs, also known as MDC (metalloproteinase/disintergrin/cystein-rich), are a new class of transmembrane glycoproteins discovered in recent years. More than 30 kinds of ADAMs have been discovered. ADAMs are widely distributed in organisms, in multicellular In animals, such as humans, pigs, rats, Xenopus, Drosophila, C. elegans, etc., such as brain, testis, testis, ovary, breast, placenta, small intestine, colon, heart, liver, lung, bone , muscles, etc. Although members of the family, although they all contain fairly conserved structural domains, their tissue distribution, activity, and biological roles vary. They may play an important role in cellular activities such as hydrolysis of extracellular matrices, cell-cell and cell-matrix adhesion, cell fusion, and signaling. The family plays an important role in multiple physiological processes such as sperm-egg binding, neurodevelopment, myotube formation, cell proliferation and differentiation, extracellular matrix remodeling, and angiogenesis. At the same time, AMMS also plays a key role in many pathological processes such as tumorigenesis, invasion and metastasis, Alzheimer's disease, arthritis and cardiovascular disease.
ADAM-TS (a disintegrin and metalloprotease with thrombospondin motif) 是最近 新发现的一类锌离子金属蛋白酶现人们已发现二十余种。 ADAM- TS在哺乳动物、 果蝇、 线虫 等多种动物的组织都有中表达。 ADAM- TS与 ADAMs家族具有一些共同的结构特征， 如蛋白质 的氨基端依次含有信号肽、前导肽、金属蛋白酶区、去整合蛋白区及富含半胱氨酸区， 但是， ADAM-TS 不含表皮生长因子样重复域和跨膜域， 代之以不同数目的 I 型凝血酶敏感蛋白 (thrombospondin 1 ) 重复结构。 主要参与细胞外基质的降解、 细胞与基质的粘附和组织器 官的重构， 在***、 胚胎发育及***疾病、 肿瘤等生理和病理过程中起着重要的作用。  ADAM-TS (a disintegrin and metalloprotease with thrombospondin motif) is a newly discovered class of zinc ion metalloproteinases. More than 20 species have been discovered. ADAM-TS is expressed in tissues of mammals, fruit flies, and nematodes. ADAM-TS shares some structural features with the ADAMs family. For example, the amino terminus of a protein contains a signal peptide, a leader peptide, a metalloproteinase region, a de-integrin region, and a cysteine-rich region. However, ADAM-TS does not contain Epidermal growth factor-like repeat domain and transmembrane domain, replaced by different numbers of type I thrombin sensitive protein (thrombospondin 1 ) repeat structure. It is mainly involved in the degradation of extracellular matrix, cell-matrix adhesion and tissue organ remodeling, and plays an important role in physiological and pathological processes such as ovulation, embryonic development, connective tissue diseases, and tumors.
对于已发现的 ADAMs和 ADAM-TS而言，其催化活性部位都包含 Zn2+结合的 HEXXH保守序 列， 这与 MMPs的活性中心的结构相似。 因此， 匪 Ps的抑制剂， 对于 ADAMs和 ADAM- TS也应 有类似的作用。 For the ADAMs and ADAM-TS that have been discovered, the catalytically active sites contain a conserved sequence of Zn 2+ -bound HEXXH, which is similar to the structure of the active center of MMPs. Therefore, inhibitors of 匪Ps should have a similar effect on ADAMs and ADAM-TS.
MMP抑制剂幵发是学术和工业研究的一个重要分支。在二十世纪九十年代末期， 全球多 家医药公司都对匪 Ps及其抑制剂的筛选表现出浓厚的兴趣，并且几种广谱的，小分子量的用 于数种类型肿瘤的 MMP抑制剂已被临床评估。 虽然这些小分子药物在临床前的动物实验中， 能高效的抑制羅 P的活性， 并有效阻止肿瘤的生长和转移， 但是在临床上来治疗癌症的结果 并不令人满意， 最终导致了后期临床检验的失败。 这主要是因为 MMP家族的复杂性并没有被 完全了解， 这些抑制剂的特异性不强， 对于其他种类的 MMP的正常生理功能也起了不同程度 的影响。以 MMP为疾病的治疗靶点的概念是复杂的， 因为 MMP是维持正常生理环境所必需的， 并且他们的功能多样化， 具有重复性和背景依赖的表达与活性。 尽管有着 40年的研究历史， 合成并测试了成千种化合物， 只有一种胶原酶抑制剂 （Periostat, doxycycline hyclate) 通过美国 FDA认证， 并作为治疗牙周疾病药物。 因此， 寻求高效的、 特异性强的 MMP抑制剂 是这个领域当前工作的重点， 也是国内外开发抗肿瘤药物的热点。 同时， 由于 MMPs和许多疾 病密切相关， 这些匪 Pis今后极有可能用于治疗心血管疾病； 关节炎； 牙周疾病； 多发性硬 化症； 炎症； 疼痛； 角膜溃疡； 细菌性脑膜炎； 糖尿病综合症； 伤口愈合； 肾病； 神经退行 性疾病； AIDS; 疱疹； 过敏症； 子宫内膜异位症； 骨质疏松； 哮喘； 可以用于器官移植； 对 受精过程和再生能力的控制；这些抑制剂还可以用于抗衰老； 抗菌； 作为细胞外基质 /胶原产 物， 化妆品， 美容产品的工业生产的添加剂。 MMP inhibitor bursts are an important branch of academic and industrial research. In the late 1990s, many pharmaceutical companies around the world showed strong interest in the screening of 匪Ps and its inhibitors, and several broad-spectrum, small-molecular-weight MMP inhibitors for several types of tumors. Has been clinically evaluated. Although these small molecule drugs can effectively inhibit the activity of Luo P and effectively prevent the growth and metastasis of tumors in preclinical animal experiments, the results of clinical treatment of cancer are not satisfactory, eventually leading to late clinical practice. The failure of the test. This is mainly because the complexity of the MMP family is not fully understood. The specificity of these inhibitors is not strong, and the normal physiological functions of other types of MMPs have also been affected to varying degrees. The concept of MMP as a therapeutic target for disease is complex because MMPs are essential for maintaining a normal physiological environment, and their functions are diverse, with reproducible and context-dependent expression and activity. Despite 40 years of research history, thousands of compounds have been synthesized and tested, and only one collagenase inhibitor (Periostat, doxycycline hyclate) has been approved by the US FDA as a drug for the treatment of periodontal disease. Therefore, the search for efficient and specific MMP inhibitors is the focus of current work in this field, and it is also a hot spot for the development of anti-tumor drugs at home and abroad. At the same time, because MMPs are closely related to many diseases, these 匪Pis are very likely to be used for the treatment of cardiovascular diseases in the future; arthritis; periodontal disease; multiple hard Inflammation; pain; corneal ulcer; bacterial meningitis; diabetes syndrome; wound healing; nephropathy; neurodegenerative diseases; AIDS; herpes; allergies; endometriosis; osteoporosis; For organ transplantation; control of fertilization process and regeneration capacity; these inhibitors can also be used for anti-aging; antibacterial; as an additive for the industrial production of extracellular matrix/collagen products, cosmetics, and beauty products.
发明内容 Summary of the invention
我们发现并证明了 1, 2， 3-三羟基苯及其衍生物对锌离子金属蛋白酶有抑制作用。 1，2，3- 三羟基苯及其衍生物对锌离子金属蛋白酶的抑制作用具有普遍性和特异性。 其中三个相邻的 羟基是抑制的关键部位， 并且这三个相邻的羟基必须是在苯环结构上， 处于同一平面。 我们 进一步证明了 1， 2， 3-三羟基苯对基质金属蛋白酶抑制的机理是竞争性抑制或竞争性优势抑制 作用，  We have found and demonstrated that 1,2,3-trihydroxybenzene and its derivatives have an inhibitory effect on zinc ion metalloproteinases. The inhibitory effect of 1,2,3-trihydroxybenzene and its derivatives on zinc ion metalloproteinases is universal and specific. Three of the adjacent hydroxyl groups are the key sites for inhibition, and the three adjacent hydroxyl groups must be on the same plane in the benzene ring structure. We further demonstrated that the mechanism by which 1,2,3-trihydroxybenzene inhibits matrix metalloproteinases is competitive inhibition or competitive advantage inhibition.
一方面，本发明涉及式 I化合物或其药学上可接受的盐在制备用于抑制锌离子金属蛋白 酶的药物中的应用， 所述式 I化合物是  In one aspect, the invention relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting a zinc ion metalloproteinase, said compound of formula I
Figure imgf000005_0001
Figure imgf000005_0001
其中 R1, R2和 R3独立的是氢原子， 取代或未取代的(^- C18烷基， 被一个或几个不连续 0原子间隔 的取代或未取代的 C2- C18烷基， 或被- CO-、 - C00-、 - 0C0-、 - 0C00-、 - C0-N (R4) -、 _N (R4) - CO -、 - N (R4) -C0-N (R4) -、 - [N (R4) ]2- C0-、 - N (R4) - COO-间隔的取代或未取代的 C2- C18烷基， 取代基独 立的选自 _0R5、 - 0C0_R5、 - COO- R5、 _N (R4) - R5、 - N (R4) - CO- R5和- CO- N (R4) - R5 ; C2- C12链烯基或被 一个或几个不连续 0原子间隔的 C2- C12链烯基； 取代或未取代的 C6- C2。的芳基， 或是取代或未取 代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤素、 0、 S、 d- C8烷基、 Cr ( 8垸 硫基、 d- C8烷氧基、 - 的芳基、 C4-C2。的含 0、 S或 N的杂芳基、 Ce- C2。的芳基氧基和 CrC2。的含 0、 S或 N的杂芳基氧基； -C0-0R4或 - CO- N (R4) 2；或者 R1和 R2,或 R2和 R3共同形成取代或未取代的 Ce-C2。的芳基， 或是取代或未取代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤 素、 0、 S、 d- C8烧基、 d- ( 8烷硫基、 C,- C8烷氧基、 CerC2。的芳基、 C4- Ca)的含 0、 S或 N的杂芳基、 ( 6-(]2。的芳基氧基和(4-( 2。的含0、 S或 N的杂芳基氧基； 和 R5相互独立的是氢， C「 C18烷基， 被 一个或几个不连续 Q原子间隔的 ( Cw烷基， Ο ( ^环垸基， -^链烯基， 取代或未取代的 Q G,, 的芳基， 或是取代或未取代的 C.,- „的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤素、 0、 S、 d- C«垸基、 d- (：„烷硫基、 (：«垸氧基、 C。的芳基、 d- C 的含 0、 S或 N的杂芳基、 C«- 的芳基氧基和 C.,- 的含 0、 S或 N的杂芳基氧基。 Wherein R 1 , R 2 and R 3 are independently a hydrogen atom, a substituted or unsubstituted (^-C 18 alkyl group, a substituted or unsubstituted C 2 -C 18 alkane interrupted by one or several discrete 0 atoms) Base, or by -CO-, - C00-, - 0C0-, - 0C00-, - C0-N (R 4 ) -, _N (R 4 ) - CO -, - N (R 4 ) -C0-N ( R 4) -, - [N (R 4)] 2 - C0-, - N (R 4) - substituted or unsubstituted spaced COO- C 2 - C 18 alkyl, a substituent independently selected from _0R 5 , - 0C0_R 5 , - COO- R 5 , _N (R 4 ) - R 5 , - N (R 4 ) - CO- R 5 and - CO- N (R 4 ) - R 5 ; C 2 - C 12 Alkenyl or C 2 -C 12 alkenyl separated by one or several discrete 0 atoms; substituted or unsubstituted C 6 -C 2 aryl, or substituted or unsubstituted C 4 - C 2 containing 0, S or N heteroaryl substituents independently selected from hydroxy, halo, 0, S, d- C 8 alkyl, Cr (8 embankment alkylthio, d- C 8 alkoxy, - aryl, C 4 -C 2 containing 0, S or N heteroaryl, C e -.. C 2 aryl group and CrC 2 containing 0, S or N heteroaryl oxygen Base; -C0-0R 4 or -CO- N (R 4 ) 2 ; or R 1 and R 2 , or R 2 and R 3 together form a substituted or unsubstituted C e -C 2 . aryl group, or a substituted or unsubstituted C 4 - C 2 , a heteroaryl group containing 0, S or N, substituents independently selected from hydroxy, halo, 0, S, d- C 8 burn-yl, d- (8 alkylthio, C, -. C 8 alkoxy, CerC 2 aryl, C 4 - C a .) containing 0, S or N heteroaryl, (6 - (] 2 aryl group, and (4 - (2-containing 0, S or N heteroaryl group; and R 5 each Independent of hydrogen, C "C 18 alkyl, was One or more discontinuous Q atoms spaced apart (Cw alkyl, Ο (cyclohexyl), -^ alkenyl, substituted or unsubstituted QG, aryl, or substituted or unsubstituted C., - a heteroaryl group containing 0, S or N, the substituent being independently selected from the group consisting of hydroxy, halogen, 0, S, d-C« fluorenyl, d- (: thiol, (: « methoxy) An aryl group of C, a heteroaryl group containing 0, S or N of d-C, an aryloxy group of C«- and a heteroaryloxy group containing 0, S or N of C.,-.
抑制锌离子金属蛋白酶的药物优选用于调节基质金属蛋白酶（删 Ps )、 ADAMs或 ADAM - TS 参与的生理及病理过程。 其中基质金属蛋白酶 （MMPs )、 ADAMs或 ADAM-TS参与的生理及病理 过程优选血管新生， 伤口愈合， 器官移植， 对受精过程和再生能力的控制， 骨的重建或疼痛。  Drugs that inhibit zinc ion metalloproteinases are preferably used to modulate the physiological and pathological processes involved in matrix metalloproteinases (Ps), ADAMs, or ADAM-TS. The physiological and pathological processes involved in matrix metalloproteinases (MMPs), ADAMs or ADAM-TS are preferably angiogenesis, wound healing, organ transplantation, control of fertilization and regeneration, bone remodeling or pain.
抑制锌离子金属蛋白酶的药物优选用于治疗和预防癌症， 心血管疾病， 关节炎， 牙周疾 病， 多发性硬化症， 炎症， 角膜溃疡， 细菌性脑膜炎， 糖尿病综合症， 肾病， 祌经退行性疾 病， AIDS , 疱疹， 过敏症， 子宫内膜异位症， 骨质疏松、 哮喘、 抗衰老或抗菌。 更优选用于 治疗和预防癌症、 心血管疾病或关节炎。  The drug for inhibiting zinc ion metalloproteinase is preferably used for the treatment and prevention of cancer, cardiovascular disease, arthritis, periodontal disease, multiple sclerosis, inflammation, corneal ulcer, bacterial meningitis, diabetes syndrome, kidney disease, sputum regression Sexual diseases, AIDS, herpes, allergies, endometriosis, osteoporosis, asthma, anti-aging or antibacterial. More preferably used for the treatment and prevention of cancer, cardiovascular disease or arthritis.
另一方面， 本发明还提供了式 I化合物或其盐在保健食品、 化妆品或 常用品中作为添 加剂用于抑制锌离子金属蛋白酶的应用。  In another aspect, the present invention provides the use of a compound of the formula I or a salt thereof as an additive in a health food, a cosmetic or a conventional product for inhibiting zinc ion metalloproteinase.
式 I化合物优选焦性末食子酸、 末食子酸丙酯、 单宁酸、 杨梅黄酮、 黄芩苷、末食子酸、 五羟黄酮、 野黄芩素、 小木麻黄素、 刺槐亭、 栎草亭、 杨梅苷、 没食子酸月桂酯、 没食子儿 茶精、 飞燕草甙元或苄丝肼。 更优选焦性末食子酸。 焦性没食子酸 （Pyrogal l ic acid)  The compound of formula I is preferably pyrophoric acid, propyl gallate, tannic acid, myricetin, baicalin, terminal acid, quercetin, scutellarin, scutellin, locust pavilion, valerian Kiosk, myricetin, lauryl gallate, gallic tea, delphinidin or benserazide. More preferred is pyrophoric acid. Pyroallic acid (Pyrogal l ic acid)
Figure imgf000006_0001
Figure imgf000006_0001
3, 4, 5—三羟基苯甲酸丙酯 ( Propyl gal l ic acid )
Figure imgf000007_0001
Propyl gal l ic acid 3, 4, 5-trihydroxybenzoic acid
Figure imgf000007_0001
没食子酸 （Gallic acid)
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0004
野黄芩素 （Scutellarein)
Figure imgf000008_0001
Gallic acid
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0004
Scutellarein
Figure imgf000008_0001
栎草亭 (Quercetagetin)
Figure imgf000008_0002
栎草亭(Quercetagetin)
Figure imgf000008_0002
杨梅苷 (Myricitrin) Myricitrin
Figure imgf000009_0001
Figure imgf000009_0001
没食子酸月桂酯 （Lauryl gallate)
Figure imgf000009_0002
Lauryl gallate
Figure imgf000009_0002
没食子儿茶精 （Gallocatechol)
Figure imgf000009_0003
Gallocatechol
Figure imgf000009_0003
丝肼 (Benserazide) Benserazide
Figure imgf000009_0004
Figure imgf000009_0004
Figure imgf000010_0001
Figure imgf000010_0001
Ruf igal lol Ruf igal lol
Figure imgf000010_0002
Figure imgf000010_0002
Tr icet i n Tr icet i n
Figure imgf000010_0003
Figure imgf000010_0003
本发明的另一方面提供了用作锌离子金属蛋白酶抑制剂的药物组合物， 包含式 I化合物 或其药学上可接受的盐以及药学上可接受的载体、 稀释剂或赋形剂。 Another aspect of the invention provides a pharmaceutical composition for use as a zinc ion metalloproteinase inhibitor comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
该药物组合物优选用于调节基质金属蛋白酶（隨 Ps；)、 ADAMs或 ADAM-TS参与的生理及病 理过程。 基质金属蛋白酶 （MMPs )、 ADAMs或 ADAM- TS参与的生理及病理过程优选血管新生， 伤口愈合， 器官移植， 对受精过程和再生能力的控制， 骨的重建或疼痛。  The pharmaceutical composition is preferably used to modulate the physiological and pathological processes involved in matrix metalloproteinases (as Ps;), ADAMs or ADAM-TS. The physiological and pathological processes involved in matrix metalloproteinases (MMPs), ADAMs or ADAM-TS are preferably angiogenesis, wound healing, organ transplantation, control of fertilization and regeneration, bone remodeling or pain.
上述药物组合物优选用于治疗和预防癌症， 心血管疾病， 关节炎， 牙周疾病， 多发性硬 化症， 炎症， 角膜溃疡.， 细菌性脑膜炎， 糖尿病综合症， 肾病， 神经退行性疾病， AIDS , 疱 疹， 过敏症， 子宫内膜异位症， 骨质疏松、 哮喘、 抗衰老或抗菌。 更优选用于治疗和预防癌 症， 心血管疾病或关节炎。 The above pharmaceutical composition is preferably used for the treatment and prevention of cancer, cardiovascular disease, arthritis, periodontal disease, multiple sclerosis, inflammation, corneal ulcer, bacterial meningitis, diabetes syndrome, nephropathy, neurodegenerative diseases, AIDS, blister Rash, allergies, endometriosis, osteoporosis, asthma, anti-aging or antibacterial. More preferably used for the treatment and prevention of cancer, cardiovascular disease or arthritis.
本发明再一方面提供了具有抑制锌离子金属蛋白酶作用的保健食品、 化妆品或日常用品 组合物， 包含式 I化合物或其盐作为添加剂。  According to still another aspect of the present invention, there is provided a health food, cosmetic or daily use composition having an action of inhibiting zinc ion metalloproteinase, comprising a compound of the formula I or a salt thereof as an additive.
附图说明 DRAWINGS
图 1 横坐标从左到右依次表示阳性对照组， 三个不同剂量的给药组， 以及阴性对照组。 阳性对照组 （100±13) 明显比 3个给药组 (72土 10， 69±/12， 35±5； P<0.001)和阴性对 照组 (25±/3， P〈0.001)大。  Figure 1 The abscissa from left to right represents the positive control group, the three different doses of the drug-administered group, and the negative control group. The positive control group (100±13) was significantly larger than the three drug-administered groups (72 soil, 10, 69±/12, 35±5; P<0.001) and the negative control group (25±/3, P<0.001).
具体实施方式 Detailed ways
下列实施例更详细地说明本发明， 但是并不是要将本发明限定为实施例。  The following examples illustrate the invention in more detail, but are not intended to limit the invention to the examples.
实施例 1 1, 2， 3-三羟基苯及其衍生物对锌离子金属蛋白酶有抑制作用 Example 1 1, 2, 3-trihydroxybenzene and its derivatives inhibit zinc ion metalloproteinase
酶学实验步骤： Enzymatic experimental steps:
所用缓冲液体系为 50mM HEPES (pH7.5) , with 0.2M NaCl, lOraM CaCl2, and 0.05 % Brij-35, 所用的底物为 DQ-Gelatin和 P126;在室温条件下测定， 反应总体积为 100 1。 在 加有抑制剂时， 酶与抑制剂应在缓冲液体系中孵育 30分钟， 然后再加底物测定。 所有检测 的仪器为 FLX800荧光酶标仪（Bio- Tek)，对于 DQ- Gelatin激发波长 495nm，发射波长 515訓， 而对于 P126， 激发波长 328 ， 发射波长 393nm。 The buffer system used was 50 mM HEPES (pH 7.5) with 0.2 M NaCl, lOraM CaCl 2 , and 0.05% Brij-35, and the substrates used were DQ-Gelatin and P126; the total reaction volume was determined at room temperature. 100 1. When an inhibitor is added, the enzyme and inhibitor should be incubated in the buffer system for 30 minutes and then added to the substrate for determination. All instruments tested were FLX800 fluorescence microplate reader (Bio-Tek), for DQ-Gelatin excitation wavelength 495 nm, emission wavelength 515, and for P126, excitation wavelength 328, emission wavelength 393 nm.
测定反应时间为 8分钟， 取荧光值的点所组成的线的斜率为速度， 其中未加抑制剂的记 为 V。，加抑制剂的为 ， 以 Vi/V。表示抑制剂的抑制程度；然后以所加抑制剂的浓度为横坐标， 以 Vi/V。的值为纵坐标， 做出相应曲线； 在 Vi/V。为 0.5处所对应的抑制剂的浓度为 IC50。  The reaction time was determined to be 8 minutes, and the slope of the line composed of the points at which the fluorescence values were taken was the velocity, and the uninhibited matter was recorded as V. , plus inhibitors, to Vi / V. Indicates the degree of inhibition of the inhibitor; then the concentration of the added inhibitor is plotted on the abscissa, Vi/V. The value of the ordinate is plotted against the ordinate; in Vi/V. The concentration of the inhibitor corresponding to 0.5 is IC50.
我们利用 Dixon plot的方法对 1， 2， 3—三羟基苯对 MMP-2的抑制机理进行了检测： 以 1, 2， 3—三羟基苯的浓度为横坐标， 1/V为纵坐标， 取三个不同的底物浓度， 可作出三条直 线； 这三条直线的交点在 Y轴正半轴和 X轴负半轴之间， 这说明其机理是竞争性或是以竞争 性为主的抑制机理。 我们进一步证明了 1， 2， 3-三轻基苯对基质金属蛋白酶抑制的机理是竞争 性抑制或竞争性优势抑制作用， 见表 1。 表 1 1, 2, 3-三羟基苯及其几种衍生物对锌离子金属蛋白酶抑制的 IC50 ( μ Μ)  We used the Dixon plot method to detect the inhibition mechanism of 1,2,3-trihydroxybenzene on MMP-2: the concentration of 1,2,3-trihydroxybenzene as the abscissa and 1/V as the ordinate. Taking three different substrate concentrations, three straight lines can be made; the intersection of the three straight lines is between the positive half-axis of the Y-axis and the negative half-axis of the X-axis, indicating that the mechanism is competitive or competitive-based suppression. mechanism. We further demonstrated that the mechanism by which 1,2,3-trisylbenzene inhibits matrix metalloproteinases is competitive inhibition or competitive superiority inhibition, as shown in Table 1. Table 1 IC50 (μ Μ) for inhibition of zinc ion metalloproteinase by 1, 2, 3-trihydroxybenzene and its derivatives
画 Ρ- ΜΜΡ-2 醒 Ρ-3 ΜΜΡ-7 ΜΜΡ-9 隨 Ρ- 1 MP-1 Ρ-2 Painting Ρ- ΜΜΡ-2 醒 Ρ-3 ΜΜΡ-7 ΜΜΡ-9 随Ρ - 1 MP-1 Ρ-2
1 4 6 6 1 4 6 6
1， 2, 3-三羟基 2.51 4.67 12.77 0.84 4.11 35.09 33.22 34.91 苯 /焦性末食子 ±0. ±0.1 ±0.3 ±0.0 ±0.5 ±0.8 ±5.6 ±4.5 酸 PYR0GALLIC 17 7 6 4 4 4 9 9 ACID (uM) 1, 2, 3-trihydroxy 2.51 4.67 12.77 0.84 4.11 35.09 33.22 34.91 Benzene / agonistic eater ±0. ±0.1 ±0.3 ±0.0 ±0.5 ±0.8 ±5.6 ±4.5 Acid PYR0GALLIC 17 7 6 4 4 4 9 9 ACID (uM)
末食子酸丙酯 ND >500 ND >500 394.2 296.4 22.69 approPropyl galactate ND >500 ND >500 394.2 296.4 22.69 appro
PROPYL GALLIC 4 1 ximatPROPYL GALLIC 4 1 ximat
ACID (μΜ) e ACID (μΜ) e
150 单宁酸 TANNIC 0.25 0.779 10.40 0.017 0.145 0. Ill 0.129 51.2 150 tannic acid TANNIC 0.25 0.779 10.40 0.017 0.145 0. Ill 0.129 51.2
ACID (μΜ) 8±0 ±0.0 土 ±0.0 ±0.0 ±0.0 ±0.0 ACID (μΜ) 8±0 ±0.0 soil ±0.0 ±0.0 ±0.0 ±0.0
.008 86 1.30 00 01 02 09  .008 86 1.30 00 01 02 09
杨梅黄酮 1.05 0.23Myricel Flavonoids 1.05 0.23
MYRICETIN ±0. 1.69 4.26 0.46 0.59 0.75 0.72 ±0.0MYRICETIN ±0. 1.69 4.26 0.46 0.59 0.75 0.72 ±0.0
(μΜ) 07 ±0.1 ±0.6 ±0.0 ±0.0 ±0.0 ±0.1 4 (μΜ) 07 ±0.1 ±0.6 ±0.0 ±0.0 ±0.0 ±0.1 4
4 3 04 2 2 7  4 3 04 2 2 7
黄芩苷 ND 13.85 ND 5.70 7.53 44.93 5.69 35.1Baicalin ND 13.85 ND 5.70 7.53 44.93 5.69 35.1
BAICALEIN ±0.3 ±0.1 ±1.2 ±6.9 ±1.5 BAICALEIN ±0.3 ±0.1 ±1.2 ±6.9 ±1.5
(μΜ) 3 1 9 2 3  (μΜ) 3 1 9 2 3
EGCG (μΜ) ND 6 ND 1.5 0.8 81.67 0.9 ND 末 食 子 酸 ND 51.6 ND 147 80 >200 ND 161.9 gallic acid  EGCG (μΜ) ND 6 ND 1.5 0.8 81.67 0.9 ND End Acid Acid ND 51.6 ND 147 80 >200 ND 161.9 gallic acid
(uM)  (uM)
五 羟 黄 酮 ND 15.2 ND 8.3 18.5 ND 21.6 NDPentahydroxyketone ND 15.2 ND 8.3 18.5 ND 21.6 ND
Tricetin Tricetin
(μΜ)  (μΜ)
野 黄 芩 素 ND 5.4 ND 4.2 2.5 ND 30.8 NDWild scorpion ND 5.4 ND 4.2 2.5 ND 30.8 ND
Scutellarein Scutellarein
( Μ)  ( Μ)
小木麻黄素 ND 0.6 ND 0.23 1.1 ND 5.2 NDSmall casuetin ND 0.6 ND 0.23 1.1 ND 5.2 ND
Strictinin Strictinin
(μΜ)  (μΜ)
Rufigallol ND 22.8 ND ND 54.1 ND ND ND Rufigallol ND 22.8 ND ND 54.1 ND ND ND
(μΜ) (μΜ)
剌 槐 亭 ND 9.7 ND ND 2.6 ND 14.5 ND剌 亭 Pavilion ND 9.7 ND ND 2.6 ND 14.5 ND
Rob i net in Rob i net in
(μΜ)  (μΜ)
栎草亭 ND 6.6 ND ND 15.2 ND 22.3 ND栎草亭 ND 6.6 ND ND 15.2 ND 22.3 ND
Quercetagetin Quercetagetin
(uM)  (uM)
杨 梅 苷 ND 8.7 ND ND 32.1 ND 18.3 NDMyosin ND 8.7 ND ND 32.1 ND 18.3 ND
Myricitrin Myricitrin
(μΜ)  (μΜ)
Methanone ND 15.6 ND ND 18.6 ND 23.4 ND Methanone ND 15.6 ND ND 18.6 ND 23.4 ND
( Μ) ( Μ)
没食子酸月桂 ND 18.2 ND ND 3.6 ND 10.2 ND 酯 Lauryl Gallic acid Laurel ND 18.2 ND ND 3.6 ND 10.2 ND ester Lauryl
gal late ( μ M) 没食子儿茶精 ND 12. 2 ND ND 8. 7 ND 23. 2 ND Gal late ( μ M) Innocent tea ND 12. 2 ND ND 8. 7 ND 23. 2 ND
Gallocatechol  Gallocatechol
( μ Μ)  ( μ Μ)
飞燕草甙元 ND 2. 1 ND ND 0. 7 ND 1. 5 ND  Feiyancaoyuan ND 2. 1 ND ND 0. 7 ND 1. 5 ND
Delphinidin  Delphinidin
( Μ)  ( Μ)
苄丝肼 ND 12. 1 ND ND 1. 1 ND 14. 8 ND  Benserazide ND 12. 1 ND ND 1. 1 ND 14. 8 ND
Benserazide  Benserazide
( μ Μ) 实施例 2 焦性没食子酸的抗肿瘤实验  ( μ Μ) Example 2 Anti-tumor experiment of pyrogallic acid
1. 焦性没食子酸对 HT1080肿瘤细胞粘附和伸展的抑制  1. Inhibition of HT1080 tumor cell adhesion and extension by pyrogalllic acid
黏附实验： Adhesion experiment:
将处于对数生长期的 HT1080细胞经消化重悬以后，进行细胞计数,然后以 10V孔的细胞密 度传入 24孔板中，加入指定量的焦性没食指酸，使每孔的药物终浓度依次为  After resuspending the HT1080 cells in the logarithmic growth phase, the cells were counted, and then introduced into a 24-well plate at a cell density of 10 V, and a specified amount of pyrophoric acid was added to make the final concentration of each drug. as followed
0 μ M， 2 μ M, 5 μ M, 10 μ M, 20 μ M，然后每孔加入 lml的 10%FBS的 DMEM培养基混匀后，放入 37°C培 养箱培养 30rain左右后取出，用孔板离心机 1000转离心 5min，然后吸去上清,在倒置显微镜下观 察各孔里边黏附在孔板底部的细胞数量. 0 μ M, 2 μ M, 5 μ M, 10 μ M, 20 μ M, then add 1 ml of 10% FBS DMEM medium to each well, mix it, and place it in a 37 ° C incubator for about 30 min. The cells were centrifuged at 1000 rpm for 5 min, then the supernatant was aspirated, and the number of cells adhering to the bottom of each well was observed under an inverted microscope.
伸展试验： Stretch test:
将处于对数生长期的 HT1080细胞经消化重悬以后,进行细胞计数,然后以 107孔的细胞密 度传入 24孔板中，加入指定量的焦性没食指酸,使每孔的药物终浓度依次为 0  After resuspending the HT1080 cells in the logarithmic growth phase, the cells were counted, and then introduced into a 24-well plate at a cell density of 107 cells, and a specified amount of pyrophoric acid was added to make the final drug concentration per well. In order of 0
μ M, 2 μ M， 5 μ M, 10 μ M， 20 μ Μ, 然后每孔加入 lml的 10%FBS的 DMEM培养基混匀,放入 37°C培养 箱培养,在细胞培养时间分别为 30min, 45min和 60min的时候,利用倒置显微镜观察细胞的形态 及生长情况。 μ M, 2 μ M, 5 μ M, 10 μ M, 20 μ Μ, then add 1 ml of 10% FBS DMEM medium to each well and mix in a 37 ° C incubator at the cell culture time. At 30 min, 45 min and 60 min, the morphology and growth of the cells were observed using an inverted microscope.
肿瘤细胞的黏附和伸展与 MMPs有关， 实验结果证明： 不同浓度的 1, 2， 3-三羟基苯对 HT1080肿瘤细胞粘附和伸展有抑制作用。  The adhesion and extension of tumor cells are related to MMPs. The experimental results show that different concentrations of 1,2,3-trihydroxybenzene have inhibitory effects on HT1080 tumor cell adhesion and extension.
2. 焦性没食子酸对 HT1080肿瘤细胞迁移的抑制  2. Inhibition of HT1080 tumor cell migration by pyrogalllic acid
先在 24孔板中以 107孔的细胞密度培养 HT1080细胞，待细胞生长到占底壁的 80%左右的时 候，将培养板取出，吸去上清液,用 200 μ L的移液枪头在各培养板孔中划一条宽度相同的斜线， 然后加入指定量的焦性没食指酸，使每孔的药物终浓度依次为 0 μ Μ, 2 μ Μ, 5 μ Μ, 10 μ Μ, 20 μ Μ， 并使每孔的培养基终体积为 lmL，然后放入 37Ό的培养箱中， 12h后观察培养板各孔中划线部分 的填平情况. 肿瘤细胞的迁移与匪 Ps有关。不同浓度的 1， 2， 3-三羟基苯对 ΗΠ080肿瘤细胞的迁移有抑 制作用。 ' The HT1080 cells were cultured at a cell density of 107 cells in a 24-well plate. When the cells were grown to about 80% of the bottom wall, the plate was removed, and the supernatant was aspirated, using a 200 μL pipette tip. A diagonal line of the same width is drawn in each well of the culture plate, and then a specified amount of pyrophoric acid is added, so that the final concentration of the drug in each well is 0 μ Μ, 2 μ Μ, 5 μ Μ, 10 μ Μ, 20 μ Μ, and the final volume of the medium in each well was 1 mL, and then placed in a 37 培养 incubator. After 12 h, observe the filling of the scribe line in each well of the plate. The migration of tumor cells is associated with 匪Ps. Different concentrations of 1,2,3-trihydroxybenzene inhibited the migration of ΗΠ080 tumor cells. '
3. 焦性没食子酸对 HT1080肿瘤细胞浸润的抑制  3. Inhibition of HT1080 tumor cell infiltration by pyrogallic acid
实验前需要将 24孔培养板 1个， Transwell小室数个， Matrigel， 200uL枪头数个放于 4°C 冰箱中 5小时以上,并用冰盒转移到超净台里面.实验时，先用预冷过的 200 μ L的枪头加 70 ϋ L/ 孔的 Matrigel入 Transwell中，一般分两次加入.将 24孔板封好后，放入 37°C培养箱 20min左右 并用 10%FBS的 DMEM培养基配置好终浓度为 0 μ M， 2 μ M, 5 μ M, 10 μ M, 20 μ Μ的细胞悬液以备用 (每份悬液中含大约 106个 HT1080细胞）.将凝胶后的 24孔板取出，下部加入 150 y L/孔的 ΝΙΗ 3Τ3 细胞上清作为趋化液，上部的 Matrigel上加入已配好的细胞悬液.然后放入 37°C的培养箱中 培养 2- 3天，取出孔板除去 Transwell小室上的 Matrigel,用 Giemsa染液将透膜细胞染色以后在 倒置显微镜下观察各孔中透膜细胞数量的多少. Before the experiment, one 24-well culture plate, several Transwell chambers, Matrigel, 200uL gun heads should be placed in the 4°C refrigerator for more than 5 hours, and transferred to the ultra-clean table with the ice box. The cold 200 μL tip plus 70 ϋ L/well Matrigel is added to the Transwell. It is usually added in two portions. After the 24-well plate is sealed, it is placed in a 37 °C incubator for about 20 min and 10% FBS in DMEM. The medium is configured with a final concentration of 0 μM, 2 μM, 5 μM, 10 μM, 20 μM cell suspension for use (approximately 10 6 HT1080 cells per suspension). After the 24-well plate was taken out, 150 μL/well of ΝΙΗ 3Τ3 cell supernatant was added as a chemotaxis solution, and the prepared cell suspension was added to the upper Matrigel. Then, it was cultured in a 37 ° C incubator. - 3 days, remove the well plate to remove Matrigel on the Transwell chamber, stain the transmembrane cells with Giemsa stain, and observe the number of transmembrane cells in each well under an inverted microscope.
肿瘤细胞的浸润与醒 Ps有关。不同浓度的 1， 2, 3-三羟基苯对 HT1080肿瘤细胞的浸润有抑 制作用。  Infiltration of tumor cells is associated with waking Ps. Different concentrations of 1,2,3-trihydroxybenzene inhibited the infiltration of HT1080 tumor cells.
4. 焦性没食子酸对 Hela、 HT1080肿瘤细胞增殖的影响  4. Effect of pyrogallic acid on proliferation of Hela and HT1080 tumor cells
肿瘤细胞的增殖与 MMPs有关。 我们用 MTT法测定不同浓度下的没食指酸对 HT1080细胞的 杀伤程度， 实验方法如下： 将生长旺盛期的 HT1080细胞,用 10%FBS的 DMEM培养基悬浊以后以 104/孔的终浓度加入 96孔板中， 培养一天， 待细胞贴壁生长达到底壁的 70%， 再换成 2 %血清 (FBS)的培养基培养一天。 然后将各孔按 0 μ M， 2 μ M, 5 μ M， 10 μ M， 20 μ Μ的浓度给药.培养过 夜， 每孔加入 20 μ 1 ΜΤΤ后继续培养 4 h。 小心吸去上清， 每孔加入 150 μ 1的 DMS0, 轻轻振荡， 使孔内形成的结晶紫充分溶解后， 用酶标仪读出各孔在 490nm下的 0D值. The proliferation of tumor cells is associated with MMPs. We used the MTT method to determine the degree of killing of HT1080 cells by different levels of non-phagic acid. The experimental methods were as follows: The vigorous growth of HT1080 cells was suspended in 10% FBS DMEM medium at a final concentration of 10 4 /well. The cells were added to a 96-well plate and cultured for one day. After the cells adhered to 70% of the bottom wall, they were cultured for one day in a medium of 2% serum (FBS). Each well was then administered at a concentration of 0 μM, 2 μM, 5 μM, 10 μM, 20 μM. Incubate overnight, and 20 μl of each well was added to each well for 4 h. Carefully aspirate the supernatant, add 150 μl of DMS0 to each well, gently shake to dissolve the crystal violet formed in the well, and then read the 0D value of each well at 490 nm with a microplate reader.
不同浓度的 1, 2，3-三羟基苯对 1£1、 HT1080肿瘤细胞增殖有抑制作用。 1， 2, 3-三羟基苯 XiHela, ΗΠ080肿瘤细胞增殖的半数抑制浓度 IC50分别为 41. 7 μ Μ和 56. 8 μ Μ。  Different concentrations of 1,2,3-trihydroxybenzene inhibited the proliferation of tumor cells of 1£1 and HT1080. 1,2,3-Trihydroxybenzene XiHela, ΗΠ080 The half-inhibitory concentration of tumor cell proliferation IC50 was 41. 7 μ Μ and 56. 8 μ Μ, respectively.
5. 焦性没食子酸体内抗肿瘤实验  5. In vivo anti-tumor experiment of pyrogallic acid
体内抗肿瘤实验： In vivo anti-tumor experiments:
在无菌条件下， 抽取接种 8d的 Η22肝癌腹水细胞， 调细胞浓度为 I X 107ml， 每只小鼠 左后肢皮下接种 0. 1 ml, 接种次日随机分组， 每组 12只， 每天灌胃给药 1次（按剂量给药） 。 连续给药 10天， 每 3天称重 1次， 末次给药后次日脱臼处死小鼠， 称瘤重， 并按下式计算抑瘤 表 2焦性没食子酸对小鼠 H22荷瘤生长的抑制作用 Under aseptic conditions, the ascites cells of sputum 22 were inoculated for 8 days, and the cell concentration was IX 107 ml. Each mouse was subcutaneously inoculated with 0.1 ml in the left hind limb. The next day, the rats were randomly divided into groups, 12 rats in each group. 1 time (dose by dose). Continuous administration for 10 days, weighing once every 3 days, the mice were killed by dislocation on the next day after the last administration, and the tumor weight was called, and the tumor inhibition was calculated according to the following formula. Table 2 Inhibitory effect of pyrogallic acid on the growth of mouse H 22 tumor
组别 剂量 动物数（只） 舰 (g) 抑瘤率 )  Group dose number of animals (only) ship (g) tumor inhibition rate)
(mg/kg)  (mg/kg)
对照组 12/12 1. 57±0. 31  Control group 12/12 1. 57±0. 31
焦性没食子酸 100 12/12 0. 745±0. 18 53  Pyrogal gallic acid 100 12/12 0. 745±0. 18 53
5-Fu 20 12/12 0. 503 ±0. 43. 68  5-Fu 20 12/12 0. 503 ±0. 43. 68
焦性没食子酸在 lOOmg/kg时对荷瘤小鼠 ¾2肿瘤的抑制率为 53%，说明焦性没食子酸具有体内 抗癌活性。 用药组和对照组瘤重有显著差异 （P (0. 05〉 实施例 3特异性对抗謹 P的药物治疗动脉粥样硬化 The inhibitory rate of pyrogalllic acid on tumor-bearing mice 3⁄4 2 tumor at 100 mg/kg was 53%, indicating that pyrogallic acid has anticancer activity in vivo. There was a significant difference in tumor weight between the drug group and the control group (P (0.05) Example 3 is specific for the treatment of atherosclerosis with the drug of P.
一、动脉粥样硬化与 MMPs 1. Atherosclerosis and MMPs
动脉粥样硬化是急性心肌梗死和不稳定型心绞痛等临床综合征的病理基础，基质金属蛋 白酶醒 Ps通过降解细胞外基质在心血管重塑中起到重要作用。 MMPs表达既与内膜增厚、 斑块 的不稳定有关， 也与再狭窄病变的形成有关。 已检测出的动脉粥样硬化中表达上调的 MMPs包 括 MMP-1， -2， -7， -9， -12。很多丽 Ps同动脉硬化损伤的基质重塑有关，例如丽 P- 1， -2， - 3， -7， -9, -12， -13， -14。 因此特异性对抗 MMP的治疗药物的发展将有助于预防动脉粥样硬化 发展、 斑块破裂和再狭窄的形成。  Atherosclerosis is the pathological basis of clinical syndromes such as acute myocardial infarction and unstable angina. Matrix metalloproteinase Ps plays an important role in cardiovascular remodeling by degrading extracellular matrix. Expression of MMPs is associated with both intimal thickening, plaque instability, and formation of restenosis lesions. MMPs that have been upregulated in atherosclerosis have been detected to include MMP-1, -2, -7, -9, -12. Many Ps are associated with matrix remodeling of arteriosclerotic lesions, such as 丽 P-1, -2, -3, -7, -9, -12, -13, -14. Therefore, the development of therapeutic drugs specific for MMP will help prevent the development of atherosclerosis, plaque rupture and restenosis.
1、 动脉粥样硬化内膜增厚： 动脉粥样硬化可由内皮的化学或机械损伤启动， 随即血中 单核细胞通过转内皮渗透作用进入内膜， 在那里他们激活或合成许多细胞因子和生长因子。 相应于这些刺激， 血管平滑肌细胞从中膜迁至内膜经历了增殖过程。 基质降解是单核细胞与 血管平滑肌细胞迁移的必要条件， 因为细胞不得不穿越细胞外障碍， 包括内皮下和环绕每个 平滑肌细胞的基底膜。 这些观察说明 MMPs参与了内膜增厚的作用， 特别是血管平滑肌细胞的 迁移。  1. Atherosclerotic intimal thickening: Atherosclerosis can be initiated by chemical or mechanical damage of the endothelium, and then monocytes in the blood enter the inner membrane through transendothelial osmosis, where they activate or synthesize many cytokines and growth. factor. Corresponding to these stimuli, vascular smooth muscle cells migrated from the media to the intima and experienced a proliferative process. Matrix degradation is essential for the migration of monocytes and vascular smooth muscle cells, as cells have to cross extracellular disorders, including the subendothelial and basement membrane surrounding each smooth muscle cell. These observations suggest that MMPs are involved in the enhancement of intimal hyperplasia, particularly the migration of vascular smooth muscle cells.
2、斑块稳定性：不稳定斑块易导致冠脉内血栓形成和闭塞，触发急性冠脉综合征（ACS)。 在动脉粥样硬化斑块中可检测到增强的匪 Ps表达，而 MMPs可通过降解 ECM导致斑块纤维帽的削 弱， 因此画 P家族的活动与斑块破裂有关。  2, plaque stability: unstable plaque easily lead to coronary thrombosis and occlusion, triggering acute coronary syndrome (ACS). Enhanced 匪 Ps expression was detected in atherosclerotic plaques, whereas MMPs reduced the plaque fiber cap by degrading ECM, so the P-family activity was associated with plaque rupture.
3、 再狭窄： 冠状动脉介入治疗（PCI) 的发展为治疗缺血性心脏病提供了一个强有力的 方法， 但许多病人在几个月内症状再次发作， 因为在原来的部位发生了再狭窄。 这是由于中 层血管平滑肌细胞的迁移和快速增长， 产生内膜纤维细胞增生的特征病变。 有报道在动物模 型中球囊损伤上调了受损动脉明胶酶的表达。 PCI扩张所致斑块中细胞外基质的降解便于循环 血液和外源性凝血途径的激活与血管壁中的组织因子相接触， 以及血管平滑肌细胞的迁移。 这说明在冠状动脉增加的應 PS水平与血管平滑肌细胞的迁移和血栓形成所致的血管重塑和再 狭窄有关。 3. Restenosis: The development of coronary intervention (PCI) provides a powerful method for treating ischemic heart disease, but many patients relapse within a few months because of restenosis in the original site. . This is due to the migration and rapid growth of the middle vascular smooth muscle cells, resulting in characteristic lesions of intimal fibroblast proliferation. Balloon injury has been reported to upregulate the expression of damaged arterial gelatinase in animal models. Degradation of extracellular matrix in plaques caused by PCI expansion facilitates circulation Activation of blood and exogenous coagulation pathways is in contact with tissue factor in the vessel wall, as well as migration of vascular smooth muscle cells. This suggests that increased PS levels in the coronary arteries are associated with vascular smooth muscle cell migration and vascular remodeling and restenosis due to thrombosis.
二、 动脉粥样硬化模型构建 ' Second, the construction of atherosclerosis model '
大鼠常用高脂饲养法， 高脂饲养至少三个月， 5 %鸡蛋黄 （富含胆固醇， 捏碎）， 10% 猪油， 0. 2 %甲硫氧嘧啶， 1 %胆固醇； 3个月后， 腹主动脉形成明显的斑块。 用球囊损伤方法 研究血管重构及再狭窄。  Rats are usually fed with high fat, high fat for at least three months, 5% egg yolk (rich in cholesterol, crushed), 10% lard, 0.2% methylthiouracil, 1% cholesterol; 3 months After the abdominal aorta forms obvious plaques. Vascular remodeling and restenosis were studied using balloon injury methods.
三、 检测指标 Third, the detection indicators
根据囊 Ps可能的作用机理， 应用醒 P抑制剂于大鼠的球囊损伤模型， 考察 MMP抑制剂对该 病的影响， 本实验是以大鼠的颈动脉为研究对象， 造模后立刻幵始腹腔注射刺槐亭 300mg/kg, 持续 70天。 相关检测指标如下：  According to the possible mechanism of action of capsular Ps, the effect of MMP inhibitor on the disease was investigated by using the smectin P inhibitor in the balloon injury model of rats. This experiment is based on the carotid artery of rats, and immediately after modeling. The sputum sputum was injected into the abdominal cavity 300 mg/kg for 70 days. The relevant testing indicators are as follows:
1、 内腔变窄和新内膜形成： 在造模后的不同天数时处死动物， 取动脉片断， 固定包埋 切片， 测量不同处理组的内腔变窄， 新内膜形成， 脉管损伤及重构的程度。 内腔变窄从三个 方面考虑： 绝对内腔横断面积 （LA) ； LA比率 （LA比率 =LA损失 /对照的内弹性膜面积， LA ratio=LA injury/IEL area of reference) ； 由新内膜形成的内腔横断面积变窄的比率 =(IEL area- LA) /IEL area。 新内膜形成也用内膜到中间层的比例评价， 即 I/M ratio=新内膜的绝 对面积 /中间层的绝对面积。  1. Narrowing of the lumen and neointimal formation: Animals were sacrificed at different days after modeling, arterial fragments were taken, and embedded sections were fixed. The lumens of different treatment groups were narrowed, neointimal formation, and vascular injury were measured. And the degree of refactoring. The narrowing of the lumen is considered in three aspects: absolute lumen cross-sectional area (LA); LA ratio (LA ratio = LA loss / control inner elastic membrane area, LA ratio = LA injury / IEL area of reference); The ratio of the lumen cross-sectional area of the film formation is narrowed = (IEL area- LA) / IEL area. The neointimal formation was also evaluated by the ratio of the inner membrane to the intermediate layer, i.e., I/M ratio = absolute area of the neointima or absolute area of the intermediate layer.
2、 血管壁重构： 重构的程度通过球囊损伤部分总的血管壁面积 （EEL AREA) 与接连处 没有损伤的标准部分的血管壁面积 （EEL AREA) 比率确定， 即重构比率 （remodeling ratio, RR)。血管壁面积（EEL AREA)是通过周长计算得到（A=C2/4 π )。缩窄性重构（阴性） RR<0. 95， 扩大性重构 (阳性) RR>1. 05o 2. Reconstruction of the vessel wall: The degree of remodeling is determined by the ratio of the total vessel wall area (EEL AREA) of the balloon lesion to the wall area of the standard portion of the joint without damage (EEL AREA), ie the remodeling ratio (remodeling) Ratio, RR). The vessel wall area (EEL AREA) is calculated from the circumference (A = C 2 / 4 π ). Constrictive remodeling (negative) RR<0.95, expanded remodeling (positive) RR>1. 05o
3、 酶谱分析： 大鼠处死后取颈动脉血管在缓冲液中冷冻保存， 匀浆离心后测蛋白含量， 并用等量的蛋白在明胶饱和的 SDS-PAGE中分离， 染色后用密度分析仪分析各条带的密度而表 示酶活性。  3. Analysis of zymogram: After the rats were sacrificed, the carotid blood vessels were cryopreserved in buffer, the protein content was measured after homogenization, and the same amount of protein was separated in gelatin-saturated SDS-PAGE. After staining, the density analyzer was used. The density of each band was analyzed to indicate the enzyme activity.
四、 结果分析 Fourth, the results analysis
1、 内腔变窄和新内膜形成  1, lumen narrowing and neointimal formation
在给药后 35天和 70天， 内腔面积和新膜面积以及内腔面积比率均有显著差异， 而在给药 后 3天差异不明显 （如表 3) 。 表 3 组织形态学数据 At 35 and 70 days after administration, there was a significant difference in lumen area and new membrane area as well as lumen area ratio, but the difference was not significant 3 days after administration (see Table 3). Table 3 Histomorphology data
Figure imgf000017_0001
Figure imgf000017_0001
a p=0. 0001 b p=0. 001 0 p=0. 01 Ap=0. 0001 b p=0. 001 0 p=0. 01
在动物球囊血管损伤后 35、 70天后， 联合用药组和单独给药组与未给药得阳性对照组相 比， 绝对新内膜面积、 I/M比率明显减小， 内腔面积比率明显增加。 在造模三个月后 Movat染 色可见与给药组相比有明显的内腔变窄和新膜形成。  After 35 and 70 days after the balloon injury of the animal's balloon, the absolute neointimal area and I/M ratio were significantly reduced and the ratio of lumen area was significantly higher in the combination group and the single administration group than in the non-administered control group. increase. After three months of modeling, Movat staining showed significant lumen narrowing and new membrane formation compared to the drug-administered group.
2、 血管壁重构  2, blood vessel wall reconstruction
由表 1中重构比率的数据可以看出， 在第 3天、 35天和 70天， 给药组与对照组的重构比率 均有显著差异。由表 2重构分类的数据可以看出，给药组与对照组相比，缩窄性重构明显减少, 说明药物对血管重构的缩窄有抑制作用。 表 4重构分类 As can be seen from the data of the reconstitution ratio in Table 1, the reconstitution ratios of the administration group and the control group were significantly different on the 3rd day, the 35th day, and the 70th day. It can be seen from the data of the reconstituted classification in Table 2 that the narrowing remodeling was significantly reduced in the drug-administered group compared with the control group, indicating that the drug inhibited the narrowing of vascular remodeling. Table 4 Reconstruction Classification
Figure imgf000018_0001
Figure imgf000018_0001
3、 酶谱分析  3, zymogram analysis
球囊损伤后 3天取大鼠的颈动脉进行酶谱分析， 通过密度分析仪分析 MMP-2带的相对密 度。 阳性对照组明显比 3个给药组和阴性对照组大 （如图 1 ) 。 说明用药可以减少 MMP的活性。  The carotid artery of the rat was taken for zymography analysis 3 days after the balloon injury, and the relative density of the MMP-2 band was analyzed by a density analyzer. The positive control group was significantly larger than the three drug-administered groups and the negative control group (Fig. 1). Explain that medication can reduce the activity of MMP.
五、结论： 应用匪 P抑制剂于大鼠的动脉粥样硬化球囊损伤模型， 以大鼠的颈动脉为研究 对象， 证明腹腔注射匪 P抑制剂剌槐亭对此疾病有治疗和缓解作用。  V. Conclusion: The atherosclerotic balloon injury model of 匪P inhibitor in rats was used to study the carotid artery of rats. It was proved that the intraperitoneal injection of 匪P inhibitor 剌槐亭 has a therapeutic and mitigating effect on this disease. .
实施例 4外源性基质金属蛋白酶抑制剂治疗子宫内膜异位症的实验研究  Example 4 Experimental study of exogenous matrix metalloproteinase inhibitors in the treatment of endometriosis
子宫内膜移位症是子宫内膜腺体浸润正常组织后发生的。子宫内膜移位症是一个浸润的 过程。 基质金属蛋白酶对于细胞外基质的重塑是必需的。 基质金属蛋白酶参与正常的组织代 谢周转， 包括***时的内膜破裂。 充分证据表明金属蛋白酶和它们的组织抑制剂参与子 宫内膜移位症的病理过程， 与子宫内膜移位症的发展有关。 目前发现的和子宫内膜移位症相 关的金属蛋白酶和它们的组织抑制剂包括 MMP-1， MMP-2, MMP-3, 薩 P- 9， TACE和 TIMP - 1， TIMP- 3。 本研究借助大鼠子宫内膜异位症 （endometriosis, EMs ) 模型， 进行外源性基质金 属蛋白酶抑制剂的治疗。 通过治疗前后异位内膜病灶大小的变化了解外源性基质金属蛋白酶 抑制剂对子宫内膜异位症的治疗效果。 并通过检测抑制剂治疗组干预结束后 3个月的血清性 激素水平、 肝肾功能及对重要脏器的影响， 来了解该治疗方法的副反应及疾病的复发情况。 为寻找治疗 EMs新的有效药物奠定理论基础， 为其临床治疗 EMs提供理论依据。  Endometrial translocation occurs after the endometrial glands infiltrate normal tissues. Endometriosis is a process of infiltration. Matrix metalloproteinases are essential for the remodeling of extracellular matrices. Matrix metalloproteinases are involved in normal tissue metabolic turnover, including intimal rupture during the menstrual cycle. There is sufficient evidence that metalloproteinases and their tissue inhibitors are involved in the pathological process of endometrial translocation and are associated with the development of endometrial translocation. The metalloproteinases and their tissue inhibitors currently associated with endometrial translocation include MMP-1, MMP-2, MMP-3, Sa P-9, TACE and TIMP-1, TIMP-3. In this study, a rat model of endometriosis (EMs) was used to treat exogenous matrix metalloproteinase inhibitors. The effects of exogenous matrix metalloproteinase inhibitors on endometriosis were investigated by changes in the size of ectopic endometrial lesions before and after treatment. The side effects of the treatment and the recurrence of the disease were investigated by measuring the serum hormone levels, liver and kidney function, and the effects on vital organs 3 months after the end of the intervention in the inhibitor treatment group. In order to find a theoretical basis for the treatment of new effective drugs for EMs, it provides a theoretical basis for clinical treatment of EMs.
外源性基质金属蛋白酶抑制剂为飞燕草甙元。  The exogenous matrix metalloproteinase inhibitor is delphinidin.
1. 大鼠子宫内膜异位症模型的建立  1. Establishment of a rat model of endometriosis
根据 Vernon采用自体子宫内膜盆腔移植法， 改进建立大鼠子宫内膜异位症动物模型。 建模后四周， 再次开腹， 观察移植物的生长情况。 EM模型建模成功肉眼观察标准： 移植灶体 积增大， 呈透明的结节状、 囊状、 有澄清液体积聚， 移植物被结締组织覆盖并有血管形成， 质地软， 作为大体判定标准。 并取其异位病灶进行组织学观察。 病理组织学观察： 可见移植 的内膜存活， 表面上皮增生呈柱状， 腺体稀疏， 腺腔形态欠规则， 但仍可见腺上皮及间质细 胞， 腺上皮呈柱状、 立方状或扁平状， 部分可见核下空泡。 间质致密， ***小而稀疏。 建模成功后的大鼠随机分成五组， 每组 10只。 实验组 A为外源性抑制剂飞燕草甙元治 疗组 : 50mg/kg， 腹腔注射，一日一次，共四周。 B为外源性抑制剂飞燕草甙元治疗组：100mg/kg, 腹腔注射， 一日一次， 共四周。 C为外源性抑制剂飞燕草甙元治疗组： 150mg/kg, 腹腔注射， 一日一次， 共四周。 An animal model of rat endometriosis was established according to Vernon's autologous endometrial pelvic transplantation. Four weeks after modeling, the laparotomy was performed again to observe the growth of the graft. The EM model was successfully modeled by the naked eye. The size of the graft was increased, and it was transparent, nodular, cystic, with clear liquid accumulation. The graft was covered by connective tissue and formed with blood vessels. The texture was soft and was used as a general criterion. The ectopic lesions were taken for histological observation. Histopathological observation: It can be seen that the intima of the transplant survived, the surface epithelial hyperplasia was columnar, the gland was sparse, the shape of the glandular cavity was not regular, but the glandular epithelium and interstitial cells were still visible. The glandular epithelium was columnar, cubic or flat. Visible vacuoles under the nucleus. The interstitial is dense and the interstitial cells are small and sparse. Rats after successful modeling were randomly divided into five groups of 10 each. Experimental group A was an exogenous inhibitor of Feiyancaoyuanyuan treatment group: 50mg/k g , intraperitoneal injection, once a day for a total of four weeks. B is the exogenous inhibitor Feiyancaoyuan treatment group: 100mg/kg, intraperitoneal injection, once a day, for a total of four weeks. C is the exogenous inhibitor Feiyancaoyuan treatment group: 150mg/kg, intraperitoneal injection, once a day, for a total of four weeks.
对照组 D为阳性对照组： ***释放激素激动剂 (LHRH-A)—那法瑞林 0. lmg/kg,皮 下注射， 一日一次， 共四周。 E为阴性对照组： 0. 9%生理盐水 0. 5ml/kg，腹腔注射， 一日一次， 共四周。  Control group D was a positive control group: gonadotropin-releasing hormone agonist (LHRH-A) - nafarelin 0. lmg/kg, subcutaneous injection, once a day for four weeks. E is a negative control group: 0. 9% normal saline 0. 5ml / kg, intraperitoneal injection, once a day, a total of four weeks.
2. 观测指标：  2. Observation indicators:
1) 外源性抑制剂、 那法瑞林对实验性子宫内膜异位症模型异位病灶体积的影响 通过对各组大鼠治疗前后异位病灶体积变化的测定， 结果显示： 治疗前各组异位病灶体 积无显著性差异 （P>0. 05); 而治疗后外源性抑制剂治疗组显示剂量为 50mg/kg的作用较弱， 剂量为 100mg/kg、 150mg/kg治疗组疗效较好。 B、 C治疗组和西药对照组异位病灶体积明显 缩小或萎缩， 抑制率明显升高， 与模型组相比差异非常显著（P<0. 05)， 外源性抑制剂治疗组 异位病灶与西药对照组相比则无明显统计学意义（P>0. 05)， 由此可见， 外源性基质金属抑制 剂、 那法瑞林治疗 EMs， 其异位内膜生长受到了明显的抑制， 而且外源性基质金属抑制剂与 那法瑞林的抑制作用相似。  1) The effect of exogenous inhibitors and nafarelin on the volume of ectopic lesions in experimental endometriosis model. The volume of ectopic lesions before and after treatment in each group of rats was measured. The results showed: There was no significant difference in the volume of ectopic lesions (P>0.05). However, the treatment group of exogenous inhibitors showed a weaker dose of 50 mg/kg after treatment. The doses of 100 mg/kg and 150 mg/kg were effective. better. The volume of ectopic lesions in B, C treatment group and western medicine control group was significantly reduced or atrophied, and the inhibition rate was significantly increased. The difference was significant compared with the model group (P<0.05). The exogenous inhibitor treatment group ectopic lesions Compared with the western medicine control group, there was no statistically significant difference (P>0.05). It can be seen that exogenous matrix metal inhibitor and narfarrein treated EMs, and the ectopic endometrial growth was significantly inhibited. And the exogenous matrix metal inhibitors are similar to the inhibitory effects of nafarelin.
2) 外源性抑制剂、 那法瑞林对实验性子宫内膜异位症模型在位内膜及异位内膜的组织 形态及超微结构的影响  2) Effects of exogenous inhibitors and nafarelin on the histomorphology and ultrastructure of eutopic endometrium and ectopic endometrium in experimental endometriosis model
大体观察  General observation
分别于各组大鼠给药前（术后 4周时）和药物治疗后（给药后 4周时）测量子宫分叉处、 卵巢及腹壁等处异位内膜的长、 宽、 高， 计算其总体积（Vl、 V2), 计算各组药物对异位内膜 的抑制率。  The length, width and height of the ectopic endometrium at the uterus bifurcation, ovary and abdominal wall were measured before administration (4 weeks after surgery) and after drug treatment (4 weeks after administration). The total volume (Vl, V2) was calculated, and the inhibition rate of the ectopic endometrium of each group of drugs was calculated.
各组动物处死后， 打开腹腔见模型组大鼠腹腔内粘连广泛， 10 只鼠均可见移植物， 约 1 - 5 mm3大小不等， 有的为含血性液的囊性小泡， 有的为含无色液呈透明状的囊性小泡， 有的 呈囊实性， 有 3只大鼠移植物与大网、 肠管、 子宫、 ***广泛粘连， 形成团块， 有 2只 大鼠移植物表面形成***包裹， 有 1只异位病灶缩小， 萎缩。 外源性抑制剂治疗组大鼠 腹腔内粘连很少， 腹壁及分叉处病灶均都萎缩（其内未见液体）， 80 %移植物已消失， 仅呈痕 迹， 双侧卵巢移植物明显缩小 50%— 70%， 有 3只出现了 1一 3mm3左右的白色质硬结节。 那 法瑞林组大鼠腹腔内移植物吸收好， 有的明显缩小， 有的已消失， 与外源性抑制剂治疗组类 似。正常对照组（假手术组）见盆腔结构完整， 仅有一例出现肠系膜与腹壁切口处轻微粘连， 但易于分离。 After the animals in each group were sacrificed, the abdominal cavity was opened. The rats in the model group had extensive adhesions in the peritoneal cavity. The grafts were visible in 10 rats, ranging from 1 to 5 mm 3 , and some were cystic vesicles containing bloody fluids. It is a cystic vesicle containing transparent colorless liquid, and some of them are cystic. There are 3 rat grafts which are widely adhered to the large net, intestinal tube, uterus and pelvic peritoneum, forming a mass, and there are 2 rats. The connective tissue was formed on the surface of the graft, and one ectopic lesion was shrunk and atrophied. In the treatment group of exogenous inhibitors, there were few intra-abdominal adhesions in the rats, and the lesions in the abdominal wall and bifurcation were atrophy (no liquid was seen in it), 80% of the grafts had disappeared, only traces were found, and bilateral ovarian grafts were significantly reduced. 50% - 70%, there are 3 white hard nodules around 1 to 3mm 3 . The intraperitoneal grafts of the farelin group were well absorbed, some were significantly reduced, some had disappeared, and the exogenous inhibitor treatment group Like. In the normal control group (sham operation group), the pelvic structure was intact, and only one case showed slight adhesion between the mesentery and the abdominal wall incision, but it was easy to separate.
光镜观察  Light microscope observation
石蜡切片经 HE染色可见- Paraffin sections were visible by HE staining -
①正常大鼠子宫内膜粘膜上皮完整， 上皮细胞为高柱状或柱状， 粘膜层内***分布 均匀， 腺体数量多， 腺腔完整， 腺上皮细胞多为柱状， 可见到核下空泡和顶将分泌。 1 Normal rat endometrial mucosa epithelium is intact, epithelial cells are highly columnar or columnar, the interstitial cells in the mucosal layer are evenly distributed, the number of glands is large, the glandular cavity is intact, and the glandular epithelial cells are mostly columnar, and the vacuoles under the nucleus are visible. And the top will be secreted.
②模型组可见移植的内膜存活， 上皮细胞明显增生， 呈柱状、 立方状或扁平状， 部分可 见到核下空泡和顶桨分泌，表现出与在位子宫内膜接近同步的周期改变， ***小而稀疏， 生长不活跃， 腺体数量减少， 有的腺体形态改变， 呈缝隙状， 有的腺腔极度扩大， 有***状 突起， 有的腺腔中可见到含铁血黄素细胞及泡沫细胞， 有的腺体消失， 血管丰富。  2 The model group showed that the intima of the transplant survived, and the epithelial cells proliferated in a columnar, cubic or flat shape, and some of the vacuoles and the top paddle were secreted, showing a cyclical change close to the eutopic endometrium. The interstitial cells are small and sparse, the growth is not active, the number of glands is reduced, some glands are changed in shape, and there are gaps. Some glandular cavities are extremely enlarged, there are papillary processes, and some hemosiderin can be seen in the glandular cavity. Cells and foam cells, some glands disappear, blood vessels are rich.
③外源性抑制剂治疗组在位内膜组织腺腔形态改变，但腺体结构完整，腺上皮排列整齐， 间质致密； 异位内膜组织呈缝隙状改变， 表面上皮短立方状， 上皮细胞排列紊乱， 内膜腺体 减少， 腺上皮萎缩， 表面血管减少， ***小而稀疏。  3 The exogenous inhibitor treatment group had morphological changes in the lining of the endometrial tissue, but the gland structure was intact, the glandular epithelium was neatly arranged, and the interstitial was dense; the ectopic endometrial tissue was slit-like, the surface epithelium was short cubic, epithelium The cells are arranged disorderly, the endometrial glands are reduced, the glandular epithelium is atrophied, the surface vessels are reduced, and the interstitial cells are small and sparse.
④那法瑞林治疗 在位内膜组织腺体形态改变， 有的扩张如球形， 有的萎缩无腺腔， 上 皮细胞不完整； 异位内膜组织上皮细胞变性、 坏死， 囊肿囊腔缩小， 囊壁变薄， 腺体数目减 少， 腺腔小， 腺上皮略有萎缩。  4 narfarlin treatment of eutopic endometrial gland morphological changes, some expansion such as spherical, some atrophy without glandular cavity, epithelial cells are incomplete; ectopic endometrial tissue epithelial cell degeneration, necrosis, cyst cystic reduction, The wall of the capsule is thinner, the number of glands is reduced, the glandular cavity is small, and the glandular epithelium is slightly atrophied.
3. 结论  3. Conclusion
应用醒 Ρ抑制剂在大鼠子宫内膜异位症模型中，证明腹腔注射 ΜΜΡ抑制剂飞燕草甙元对此 疾病有治疗和缓解作用。  In the rat model of endometriosis, a sputum inhibitor was used to demonstrate that the intraperitoneal injection of the bismuth inhibitor, delphinidin, has a therapeutic and palliative effect on this disease.
实施例 5基质金属蛋白酶（ΜΜΡ) 抑制剂在兔骨关节炎模型中的应用  Example 5 Application of Matrix Metalloproteinase (ΜΜΡ) Inhibitor in Rabbit Osteoarthritis Model
骨性关节炎 (Osteoarthritis, OA)是关节软骨的变性， 破坏及骨质增生为特征的慢性关 节病， 是我国最常见的关节性疾病， 也是引起老年人疼痛和残疾的主要原因之一。 其发病机 制尚不清楚， 但关于其软骨降解的机制已得到多方面阐述。 OA治疗的基本目的是缓解症状， 改善功能， 延缓进展及矫正畸形， 对 OA应着眼于早诊断、 早治疗、 长疗程， 即在患者出现轻 度症状时就应釆用综合治疗。多种丽 Ps对关节软骨细胞外基质有降解作用，使关节软骨基质 结构改变， 抗外力作用下降， 最终导致关节软骨进行性破坏。 因此应用 MMP抑制剂治疗骨关 节炎是实现软骨保护以延缓病情发展的重要手段。  Osteoarthritis (OA) is a chronic joint disease characterized by degeneration, destruction and bone hyperplasia of articular cartilage. It is the most common joint disease in China and one of the main causes of pain and disability in the elderly. Its pathogenesis is still unclear, but the mechanism of its cartilage degradation has been described in many ways. The basic purpose of OA treatment is to relieve symptoms, improve function, delay progression and correct deformity. OA should focus on early diagnosis, early treatment, long course of treatment, that is, comprehensive treatment should be used when patients have mild symptoms. A variety of Ps degrades the extracellular matrix of articular cartilage, changes the structure of the articular cartilage matrix, and reduces the resistance to external forces, eventually leading to the progressive destruction of articular cartilage. Therefore, the use of MMP inhibitors in the treatment of osteoarthritis is an important means to achieve cartilage protection to delay the development of the disease.
〈实验进度〉  <Experiment progress>
第一周： 24只大耳白兔分别行 ACL手术。 第三一五周： 动物随机分成三组， 分别为 A组（关节腔注射生理盐水）， B组（关节腔注 射 100mg/ml野黄芩素溶液）， C组 （关节腔注射 lOOmg/tnl五羟黄酮溶液）。 The first week: 24 large white rabbits underwent ACL surgery. The third week and the fifth week: Animals were randomly divided into three groups, group A (intra-articular injection of normal saline), group B (joint cavity injection of 100 mg/ml wild scutella solution), group C (joint injection of lOOmg/tnl pentoxide) Flavonoid solution).
六周后： 处死。 进行临床表现观察， 大体观察， 病理学观察， 统计学分析。  After six weeks: Execution. Clinical manifestations, gross observation, pathological observation, and statistical analysis were performed.
〈模型建立〉  <Model Establishment>
将模兔用 3%戊巴比妥钠（30mg/kg)耳缘静脉注射麻醉， 仰卧于手术台上固定， 右膝关 节局部脱毛， 备皮， 消毒， 铺巾， 严格无菌操作。 取膝关节内侧长约 4cm的纵切口， 探査关 节腔内无原发病变后切断内侧副韧带， 进一步切除内侧半月板和前后交叉韧带， 注意勿损伤 关节软骨面， 彻底止血， 冲洗关节腔， 行抽屉实验阳性后， 逐层缝合关节囊， 皮下， 皮肤， 无菌敷料包扎伤处， 不予固定。 术后每天肌注青霉素 20万 U， 伤口换药， 连续七天以预防感 染。 根据临床上超疲劳活动量提前出现 OA的发病特点， 手术后 1周， 每天强迫动物活动 30 分钟， 分两次驱赶， 4周后即获得稳定的 0A模型。  The rabbits were anesthetized with 3% sodium pentobarbital (30 mg/kg) in the ear vein, fixed on the operating table, localized in the right knee joint, prepared for skin, disinfected, draped, and strictly aseptic. Take a longitudinal incision about 4 cm long inside the knee joint, and explore the medial collateral ligament after no primary lesion in the joint cavity, further remove the medial meniscus and anterior and posterior cruciate ligaments, be careful not to damage the articular cartilage surface, completely stop bleeding, flush the joint cavity, After the drawer test is positive, the joint capsule is sutured layer by layer, subcutaneous, skin, and sterile dressing are used to dress the wound and will not be fixed. After surgery, 200,000 U of penicillin was intramuscularly injected every day, and the wound was changed for 7 days to prevent infection. According to the clinical characteristics of super-fatigue activity, the onset of OA occurred in advance. One week after the operation, the animals were forced to move for 30 minutes every day, and they were driven twice, and a stable 0A model was obtained after 4 weeks.
〈骨关节炎临床表现的观察〉  <Observation of clinical manifestations of osteoarthritis>
于第六周给药结束后， 用 Lequesne MG的膝关节临床评估级别进行评估。  After the end of the sixth week of dosing, the evaluation of the knee joint clinical evaluation level of Lequesne MG was performed.
①局部疼痛剌激反应： 用手指挤压膝关节，按刺激反应程度不同分为 4级。  1 Local pain stimuli response: Squeeze the knee joint with your fingers and divide it into 4 grades according to the degree of stimuli.
I级： 无异常疼痛反应； II级： 患肢收缩； III级： 患肢收缩、 痉挛， 伴轻度全身反应， 如周身颤抖， 回头舔吮等； W级：患肢剧烈收缩、 痉挛， 全身颤抖， 乱窜、 挣扎。  Grade I: No abnormal pain response; Grade II: Contraction of the affected limb; Grade III: Contraction of the affected limb, convulsion, mild systemic reaction, such as trembling around the body, turning back, etc.; W: severe contraction of the affected limb, paralysis, whole body Trembling, snarling, struggling.
②步态改变： 按患肢行走、 奔跑时的步态分为 4级。  2 Gait changes: According to the walking gait of the affected limb, the gait is divided into 4 levels.
I级： 患肢无跛行， 跑动正常， 蹬地有力； II级： 患肢奔跑时轻度跛行， 蹬地有力； III 级： 患肢参与行走， 但跛行明显； IV级： 患肢不能参与行走， 不能触地、 蹬地。  Grade I: The affected limbs are innocent, running normally, and powerful; Grade II: Mild limp while running on the affected limb, strong and powerful; Grade III: The affected limb is involved in walking, but the movement is obvious; Grade IV: The affected limb cannot participate Walking, can't touch the ground, squatting.
③关节活动范围:按患肢膝关节活动范围分为 4级 (伸直为 0° )。  3 Joint range of motion: According to the range of knee joint activity of the affected limb, it is divided into 4 grades (straightening to 0°).
I级： 90° 以上； II级： 45° 〜90° ； III级： 15° ~45° IV级： 15° 。  Class I: 90° or more; Class II: 45° to 90°; Class III: 15° to 45° Class IV: 15°.
④关节肿胀： 按膝关节肿胀程度分为 3级： I级:无肿胀， 骨性标记清楚可见； II级:轻 度肿胀， 骨性标记变浅； ΙΠ级：明显肿胀， 骨性标记消失。  4 joint swelling: according to the degree of swelling of the knee joint is divided into 3 levels: Grade I: no swelling, bone markers are clearly visible; Grade II: mild swelling, shallow bone markers; ΙΠ grade: significant swelling, bone markers disappear.
表 5 3组兔膝关节骨关节炎临床表现评分结果 (n=8)  Table 5 Results of clinical manifestations of rabbit knee osteoarthritis in 3 groups (n=8)
组 疼痛刺激反应 步态改变 关节涪动范围 关节肿胀 别 I II III IV I II III IV I II III IV I II II Group pain stimuli response gait changes joint mobility range joint swelling I II III IV I II III IV I II III IV I II II
A组 0 1 2 3 0 1 4 1 0 2 3 1 1 3 2Group A 0 1 2 3 0 1 4 1 0 2 3 1 1 3 2
B组 1 3 4 0 2 4 2 0 3 3 2 0 4 4 0Group B 1 3 4 0 2 4 2 0 3 3 2 0 4 4 0
C组 2 4 2 0 3 4 1 0 4 2 1 1 4 3 1 以上 4组指标， A组分别与 B组、 C组相比， p<0. 01。 Group C 2 4 2 0 3 4 1 0 4 2 1 1 4 3 1 The above four groups of indicators, group A compared with group B, group C, p <0.01.
〈大体观察〉  <General observation>
手术 6周后处死大白兔， 观察膝关节有无关节积液和滑膜肿胀充血， 并于解剖显微镜下 观察股骨髁关节面的病理改变， 按以下原则评分： 0分， 关节面光整， 色泽如常； 1分， 关节 面粗糙， 有小的裂隙且色泽灰暗； 2分， 关节面糜烂， 软骨缺损深达软骨表中层； 3分， 关节 面溃疡形成， 缺损深达软骨深层； 4分， 软骨剥脱， 软骨下骨质暴露。  After 6 weeks of operation, the white rabbits were sacrificed. The joints of the knee joints and synovial swelling were observed. The pathological changes of the femoral condyle surface were observed under a dissecting microscope. The scores were scored according to the following principles: 0 points, articular surface smoothness, color As usual; 1 point, the joint surface is rough, there are small cracks and the color is gray; 2 points, the articular surface is erosive, the cartilage defect is deep to the middle layer of the cartilage; 3 points, the articular surface ulcer is formed, the defect is deep to the deep cartilage; 4 points, cartilage Exfoliation, subchondral bone exposure.
Figure imgf000022_0001
Figure imgf000022_0001
上表数据， Α组与 Β组、 C组相比， 在 p〈0. 05水平上有显著差异。  In the above table data, there was a significant difference in the p<0.05 level between the Α group and the Β group and the C group.
〈组织学评价〉  <Histological Evaluation>
10%***固定的膝关节在脱钙剂中脱钙大约一周。 脱钙完全后膝关节在前沿平面中 横断， 因此中间的和侧面的循环得以保持， 且两个半边在石蜡中包埋。 初级的切片以 150 μ ηι 的间隔用两步切断。这些切片以甲苯胺蓝染色， 光镜下观察， 用于评价软骨破坏和骨赘形成。 软骨破坏用如下***评价： 1=只有最小表面区域， 2=轻微扩展到中上部区域， 3=适当地深 入中间的区域， 4=标志到深部区域但不是高潮， 5=严重的完全增厚退化到高潮。软骨破坏的 数量根据组织学部分表面的 1/3、 2/3或 3/3确定， 以上的分数分别乘以 1、 2或 3， 以分别 反应参与的胫骨板的范围。骨赘用一个目镜千分尺测量的大小评分： 1、 2或 3分别为轻度的、 中度的或严重的。  The 10% formalin-fixed knee joint was decalcified in the decalcifying agent for approximately one week. After the decalcification is complete, the knee joint traverses in the leading plane, so the intermediate and lateral circulation is maintained, and the two halves are embedded in the paraffin. The primary sections were cut in two steps at intervals of 150 μηη. These sections were stained with toluidine blue and observed under light microscopy for evaluation of cartilage destruction and callus formation. Cartilage destruction was evaluated by the following system: 1 = only the minimum surface area, 2 = slightly extended to the upper middle area, 3 = appropriately deep into the middle area, 4 = marked to the deep area but not the climax, 5 = severe full thickening degradation To the climax. The number of cartilage destruction is determined by 1/3, 2/3 or 3/3 of the histological surface, and the above scores are multiplied by 1, 2 or 3, respectively, to reflect the extent of the participating tibial plates. The size of the epiphysis measured with an eyepiece micrometer: 1, 2, or 3 are mild, moderate, or severe, respectively.
Figure imgf000022_0002
Figure imgf000022_0002
上表中数据 Α组与 Β组、 C组相比， 在 p<0. 05水平上有显著差异。  The data in the above table is significantly different from the Β group and the C group at the p<0.05 level.
〈结论〉  <in conclusion>
应用 MMP抑制剂于在兔骨关节炎模型中， 证明关节腔注射 MMP抑制剂野黄芩素 和五羟黄酮溶液对此疾病有治疗和缓解作用。  In the rabbit osteoarthritis model, MMP inhibitors were used to demonstrate the treatment and alleviation of the disease by intra-articular injection of MMP inhibitors of scutellarin and quercetin solution.

Claims

权利要求书 Claim
1、式 I化合物或其药学上可接受的盐在制备用于抑制锌离子金属蛋白酶的药物 中的应用， 所述式 I化合物是  A use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting a zinc ion metalloproteinase, said compound of formula I
Figure imgf000023_0001
Figure imgf000023_0001
其中 R1, R2和 R3独立的是氢原子， 取代或未取代的(：「 C18烷基， 被一个或几个不连续 0 原子间隔的取代或未取代的 C2- C18烷基，或被 -CO-、 - C00-、 - 0C0_、 - 0C00 -、 -C0-N (R4) -、 - N (R4) - CO-、 - N (R4) - C0_N (R4) -、 - [N (R4) ] 2- CO-、 -N (R4) - COO-间隔的取代或未取代的 C2- Cl8垸基， 取代基独立的选自 _0R5、 - 0C0- R5、 - COO- R5、 - N (R4) - R5、 _N (R4) - CO- R5和 -C0-N (R4) -R5 ; C2- Cl2链烯基或被一个或几个不连续 0原子间隔的 C2- C12链烯基； 取代或 未取代的 C6-C2。的芳基， 或是取代或未取代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立 的选自羟基、 卤素、 0、 S、 d- C8垸基、 d- 烷硫基、 Ct-Cs烷氧基、 Ce- C2。的芳基、 C4_C2。 的含 0、 S或 N的杂芳基、 C6-C2。的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基； - C0-0R4 或 -C0-N (R4) 2; 或者 R1和 R2， 或 R2和 R3共同形成取代或未取代的 Ce- C2。的芳基， 或是取代 或未取代的 CrC2。的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤素、 0、 S、 C -C8 垸基、 d- (：8垸硫基、 C「 (：8垸氧基、 C6_C2。的芳基、 C4- C2。的含 0、 S或 N的杂芳基、 C6-C2。 的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基； R4和 R5相互独立的是氢， C,- Cl8烷基， 被一个或几个不连续 0原子间隔的 C2- C18烷基， C3-C12环烷基， C2- Cl8链烯基， 取代或未 取代的 C6-C2。的芳基， 或是取代或未取代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立的 选自羟基、 卤素、 0、 S、 d-Cs烷基、 d- 烷硫基、 d- C8烷氧基、 C6- C2。的芳基、 C4-C2。 的含 0、 S或 N的杂芳基、 C6- C2。的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基。 Wherein R 1 , R 2 and R 3 are independently a hydrogen atom, substituted or unsubstituted (: "C 18 alkyl, substituted or unsubstituted C 2 - C 18 alkane interrupted by one or several discrete 0 atoms" Base, or by -CO-, - C00-, - 0C0_, - 0C00 -, -C0-N (R 4 ) -, - N (R 4 ) - CO-, - N (R 4 ) - C0_N (R 4 -, - [N (R 4 ) ] 2 - CO-, -N (R 4 ) - COO- spacer substituted or unsubstituted C 2 - C l8 fluorenyl, the substituents are independently selected from _0R 5 , - 0C0- R 5 , - COO- R 5 , - N (R 4 ) - R 5 , _N (R 4 ) - CO- R 5 and -C0-N (R 4 ) - R 5 ; C 2 - C l2 Alkenyl or C 2 -C 12 alkenyl separated by one or several discrete 0 atoms; substituted or unsubstituted C 6 -C 2 aryl, or substituted or unsubstituted C 4 - C 2 containing 0, S or N heteroaryl substituents independently selected from hydroxy, halo, 0, S, d- C 8 alkyl with, d- alkylthio, Ct-Cs alkoxy, C e - C 2 · aryl, C 4 _C 2 . Containing 0, S or N heteroaryl, C 6 -C 2 aryloxy and C 4 - C 2 . Containing 0, S or N Heteroaryloxy; -C0-0R 4 or -C0-N (R 4 ) 2; or R 1 and R 2 , or R 2 and R 3 together form a substituted or unsubstituted C e - C 2 . aryl group, or a substituted or unsubstituted CrC 2 group containing 0, S or N heteroaryl group , the substituent is independently selected from the group consisting of hydroxy, halogen, 0, S, C-C 8 fluorenyl, d- (: 8 thiol, C " ( 8 methoxy, C 6 _C 2 aryl, C ... 4 - C 2 containing 0, S or N heteroaryl, C 6 -C 2 aryl group and a C 4 - C 2 containing 0, S or N heteroaryl group; R & lt 4 and R 5 independently of one another are hydrogen, C, - C l8 alkyl, substituted with one or several discrete intervals 0 atoms C 2 - C 18 alkyl, C 3 -C 12 cycloalkyl, C 2 - C a l8 alkenyl group, a substituted or unsubstituted C 6 -C 2 aryl group, or a substituted or unsubstituted C 4 - C 2 heteroaryl group containing 0, S or N, independently selected as a substituent from hydroxyl, halo, 0, S, d-Cs-alkyl, d- alkylthio, d- C 8 alkoxy, C 6 - C 2. Aryl, C 4 -C 2 . a heteroaryl group containing 0, S or N, C 6 - C 2 . Aryloxy and C 4 - C 2 . A heteroaryloxy group containing 0, S or N.
2、如权利要求 1所述的应用， 其中抑制锌离子金属蛋白酶的药物用于调节基质 金属蛋白酶 （MMPs )、 ADAMs或 ADAM- TS参与的生理及病理过程。  The use according to claim 1, wherein the drug for inhibiting zinc ion metalloproteinase is for regulating physiological and pathological processes involved in matrix metalloproteinases (MMPs), ADAMs or ADAM-TS.
3、 如权利要求 2所述的应用， 其中基质金属蛋白酶（MMPs )、 ADAMs或 ADAM - TS 参与的生理及病理过程是血管新生，伤口愈合， 器官移植， 对受精过程和再生能力的 控制， 骨的重建或疼痛。 3. Use according to claim 2, wherein matrix metalloproteinases (MMPs), ADAMs or ADAM-TS The physiological and pathological processes involved are angiogenesis, wound healing, organ transplantation, control of fertilization and regeneration, bone remodeling or pain.
4、如权利要求 1所述的应用， 其中抑制锌离子金属蛋白酶的药物用于治疗和预 防癌症， 心血管疾病， 关节炎， 牙周疾病， 多发性硬化症， 炎症， 角膜溃疡， 细菌性 脑膜炎， 糖尿病综合症， 肾病， 神经退行性疾病， AIDS, 疱疹， 过敏症， 子宫内膜异 位症， 骨质疏松或哮喘。  The use according to claim 1, wherein the drug for inhibiting zinc ion metalloproteinase is used for treating and preventing cancer, cardiovascular disease, arthritis, periodontal disease, multiple sclerosis, inflammation, corneal ulcer, bacterial meninges Inflammation, Diabetes Syndrome, Kidney Disease, Neurodegenerative Disease, AIDS, Herpes, Allergies, Endometriosis, Osteoporosis or Asthma.
5、如权利要求 1所述的应用，其中抑制锌离子金属蛋白酶的药物用于治疗和预 防癌症、 心血管疾病或关节炎。  The use according to claim 1, wherein the drug for inhibiting zinc ion metalloproteinase is used for the treatment and prevention of cancer, cardiovascular disease or arthritis.
6、如权利要求 1所述的应用， 其中抑制锌离子金属蛋白酶的药物用于抗衰老或 抗菌。  The use according to claim 1, wherein the drug for inhibiting zinc ion metalloproteinase is used for anti-aging or antibacterial.
7、式 I化合物或其盐在保健食品、化妆品或日常用品中作为添加剂用于抑制锌 离子金属蛋白酶的应用， 所述式 I化合物为  7. The compound of the formula I or a salt thereof is used as an additive in a health food, a cosmetic or a daily product for inhibiting the application of a zinc ion metalloproteinase, wherein the compound of the formula I is
Figure imgf000024_0001
Figure imgf000024_0001
其中 R1, R2和 R3独立的是氢原子， 取代或未取代的 d- C18烷基， 被一个或几个不连续 0 原子间隔的取代或未取代的 C2-C18垸基，或被- CO-、 -C00-、 - 0C0-、 - 0C00-、 - C0-N (R4) -、 - N (R4) - CO-、 -N (R4) -C0-N (R4) -, - [N (R4) ]2- CO-、 -N (R4) - COO -间隔的取代或未取代的 C2-C18烷基， 取代基独立的选自- 0R5、 - 0C0- Rs、 - COO- R5、 -N (R4) - R5、 - N (R4) - CO- R5和 -C0-N( 4) -R5; C2- Cl2链烯基或被一个或几个不连续 0原子间隔的 C2- C12链烯基； 取代或 未取代的 C3- C2。的芳基， 或是取代或未取代的 (VC2Q的含 0、 S或 N的杂芳基， 取代基独立 的选自羟基、 卤素、 0、 S、 d- C8烷基、 d- ( 8烷硫基、 d- C8烷氧基、 C6-Ca)的芳基、 C-Cao 的含 0、 S或 N的杂芳基、 GrC2。的芳基氧基和 C C2。的含 0、 S或 N的杂芳基氧基； - C0_0 或 -C0-N (R4) 2; 或者 Rl和 R2， 或 R2和 R3共同形成取代或未取代的 C6-C2。的芳基， 或是取代 或未取代的 C4-C2。的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤素、 0、 S、 C -Ca 烷基、 d- C8烷硫基、 C「C8烷氧基、 C6- C2。的芳基、 C4- C2。的含 0、 S或 N的杂芳基、 C6- C2„ 的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基； R4和 R5相互独立的是氢， d- 烷基， 被一个或几个不连续 0原子间隔的 C2- C18烷基， （3-(]12环烷基， C2- C18链稀基， 取代或未 取代的 Ce-C2。的芳基， 或是取代或未取代的 C4-C2。的含 0、 S或 N的杂芳基， 取代基独立的 选自羟基、 卤素、 0、 S、 C「C8烷基、 d- C8烷硫基、 - 垸氧基、 Ce-C2。的芳基、 CrC2。 的含 0、 S或 N的杂芳基、 Ce_C2。的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基。 Wherein R 1 , R 2 and R 3 are independently a hydrogen atom, a substituted or unsubstituted d-C 18 alkyl group, a substituted or unsubstituted C 2 -C 18 fluorenyl group interrupted by one or several discrete 0 atoms. , or by -CO-, -C00-, - 0C0-, - 0C00-, - C0-N (R 4 ) -, - N (R 4 ) - CO-, -N (R 4 ) -C0-N ( R 4) -, - [N (R 4)] 2 - CO-, -N (R 4) - COO - a substituted or unsubstituted spaced C 2 -C 18 alkyl substituents independently selected from - 0R 5 , - 0C0- R s , - COO- R 5 , -N (R 4 ) - R 5 , - N (R 4 ) - CO- R 5 and -C0-N( 4 ) -R 5 ; C 2 - C l 2 alkenyl or C 2 -C 12 alkenyl separated by one or several discrete 0 atoms; substituted or unsubstituted C 3 - C 2 . An aryl group, either substituted or unsubstituted (VC 2Q containing a 0, S or N heteroaryl group, the substituents are independently selected from the group consisting of hydroxyl, halogen, 0, S, d-C 8 alkyl, d- ( An aryl group of 8 alkylthio, d-C 8 alkoxy, C 6 -C a ), a heteroaryl group containing 0, S or N of C-Cao, an aryloxy group of GrC 2 and CC 2 . a heteroaryloxy group containing 0, S or N; - C0_0 Or -C0-N (R 4 ) 2; or R l and R 2 , or R 2 and R 3 together form a substituted or unsubstituted C 6 -C 2 . An aryl group, either substituted or unsubstituted C 4 -C 2 . Containing 0, S or N heteroaryl substituents independently selected from hydroxy, halo, 0, S, C -Ca alkyl, d- C 8 alkylthio, C "C 8 alkoxy, C 6 - C 2 aryl, C 4 - C 2 . Containing a heteroaryl group of 0, S or N, an aryloxy group of C 6 - C 2 „ and C 4 - C 2 . a heteroaryloxy group containing 0, S or N; independently of R 4 and R 5 is hydrogen, d-alkyl, C 2 -C 18 alkyl, interrupted by one or more discrete 0 atoms, ( 3 -(] 12 -cycloalkyl, C 2 -C 18 chain-dense, substituted or unsubstituted C e -C 2 aryl, or substituted or unsubstituted C 4 -C 2 . a heteroaryl group of S or N, the substituent being independently selected from the group consisting of hydroxy, halogen, 0, S, C "C 8 alkyl, d-C 8 alkylthio, -decyloxy, C e -C 2 . group, CrC 2 containing 0, S or N heteroaryl, C e _C 2 aryl groups and C 4 -... C 2 containing 0, S or N heteroaryl group.
8、 如权利要求 1至 7中任一项所述的应用， 其中式 I化合物是焦性末食子酸、 末食子酸丙酯、 单宁酸、 杨梅黄酮、 黄芩苷、 末食子酸、 五羟黄酮、 野黄芩素、 小木 麻黄素、 刺槐亭、 栎草亭、 杨梅苷、 没食子酸月桂酯、 没食子儿茶精、 飞燕草甙元或 苄丝肼。  The use according to any one of claims 1 to 7, wherein the compound of the formula I is pyrophoric acid, propyl galactate, tannic acid, myricetin, baicalin, and terminal acid , quercetin, wild baicalein, small casuarina, locust pavilion, valerian, myricetin, lauryl gallate, gallic tea, delphinidin or benserazide.
9、 如权利要求 1至 7中任一项所述的应用， 其中式 I化合物是焦性末食子酸。 9. Use according to any one of claims 1 to 7, wherein the compound of formula I is pyrophoric acid.
10、 用作锌离子金属蛋白酶抑制剂的药物组合物， 包含式 I化合物或其药学上 可接受的盐以及药学上可接受的载体、 稀释剂或赋形剂， A pharmaceutical composition for use as a zinc ion metalloproteinase inhibitor, comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient,
Figure imgf000025_0001
Figure imgf000025_0001
其中 R1, R2和 R3独立的是氢原子， 取代或未取代的(^- C18烷基， 被一个或几个不连续 0 原子间隔的取代或未取代的 C2- C18烷基，或被 -C0-、 - C00-、 -0C0-、 - OCOC -C0-N (R4) -、 - N (R4) - CO-、 - N (R4) - CO- N (R4) -、 - [N (R4) ] 2- C0_、 -N (R4) - C00-间隔的取代或未取代的 C2-Cl8烧基， 取代基独立的选自- 0R5、 _0C0- R5、 _C00- R5、 - N (R4) - R5、 - N (R4) -CO- R5和 - CO- N (R4) - R5; C2-C12链烯基或被一个或几个不连续 0原子间隔的 C2- C12链烯基； 取代或 未取代的( 6-(：2。的芳基， 或是取代或未取代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立 的选自羟基、 卤素、 0、 S、 d- C8烷基、 d- (：8垸硫基、 CrC^氧基、 C6-C2。的芳基、 CrC2。 的含 0、 S或 N的杂芳基、 Ce-C2。的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基； - C0 - 0R4 或- C0-N (R4) 2; 或者 R1和 R2, 或 R2和 R3共同形成取代或未取代的 C6-C2。的芳基， 或是取代 或未取代的 C4_C2。的含 0、 S或 N的杂芳基， 取代基独立的选自羟基、 卤素、 0、 S、 C「C8 烷基、 Cr Cs烷硫基、 d- C8垸氧基、 C6- C2。的芳基、 C4-C2Q的含 0、 S或 N的杂芳基、 C6-C2。 的芳基氧基和 C4- C2。的含 0、 S或 N的杂芳基氧基； R4和 R5相互独立的是氢， （ ds烷基， 被一个或几个不连续 0原子间隔的 C2-C18垸基， C3- ( 12环垸基， C2-Cls链烯基， 取代或未 取代的 C6-C2。的芳基， 或是取代或未取代的 C4- C2。的含 0、 S或 N的杂芳基， 取代基独立的 选自羟基、 卤素、 0、 S、 d- C8烷基、 C「。8垸硫基、 Cr C8烧氧基、 C6_C2。的芳基、 CrC2。 的含 0、 S或 N的杂芳基、 Ce- C2。的芳基氧基和 C4-C2。的含 0、 S或 N的杂芳基氧基。 Wherein R 1 , R 2 and R 3 are independently a hydrogen atom, a substituted or unsubstituted (^-C 18 alkyl group, a substituted or unsubstituted C 2 -C 18 alkane interrupted by one or several discrete 0 atoms) Base, or by -C0-, -C00-, -0C0-, - OCOC -C0-N (R 4 ) -, - N (R 4 ) - CO-, - N (R 4 ) - CO- N (R 4 ) -, - [N (R 4 ) ] 2 - C0_, -N (R 4 ) - C00- spacer substituted or unsubstituted C 2 -C l8 alkyl group, the substituents are independently selected from - 0R 5 , _0C0- R 5 , _C00- R 5 , - N (R 4 ) - R 5 , - N (R 4 ) -CO- R 5 and - CO- N (R 4 ) - R 5 ; C 2 -C 12 chain An alkenyl group or a C 2 -C 12 alkenyl group interrupted by one or more discrete 0 atoms; a substituted or unsubstituted ( 6- (: 2 aryl group) or a substituted or unsubstituted C 4 - C 2 containing 0, S or N heteroaryl substituents independently selected from hydroxy, halo, 0, S, d- C 8 alkyl, d- (: 8 embankment alkylthio, CrC ^ alkoxy, C 6- C 2 . of aryl, CrC 2 , heteroaryl containing 0, S or N, aryloxy of C e -C 2 and C 4 - C 2 containing 0, S or N Heteroaryloxy; - C0 - 0R 4 Or - C0-N (R 4 ) 2; or R 1 and R 2 , or R 2 and R 3 together form a substituted or unsubstituted C 6 -C 2 . An aryl group, either substituted or unsubstituted C 4 _C 2 . a heteroaryl group containing 0, S or N, the substituent being independently selected from the group consisting of hydroxyl, halogen, 0, S, C "C 8 alkyl, C r C s alkylthio, d-C 8 decyloxy, C 6 - C 2 · aryl, C 4 -C 2Q containing 0, S or N heteroaryl, C 6 -C 2 aryloxy and C 4 - C 2 . Containing 0, S or a heteroaryloxy group of N; R 4 and R 5 are each independently hydrogen, ( ds alkyl, C 2 -C 18 fluorenyl, interrupted by one or several discrete 0 atoms, C 3 - ( 12 ring 垸a C 2 -C ls alkenyl group, a substituted or unsubstituted C 6 -C 2 aryl group, or a substituted or unsubstituted C 4 - C 2 heteroaryl group containing 0, S or N The substituent is independently selected from the group consisting of hydroxy, halogen, 0, S, d-C 8 alkyl, C". 8 thiol, C r C 8 alkoxy, C 6 _C 2 aryl, CrC 2 . containing 0, S or N heteroaryl, C e -.. C 2 aryl groups and C 4 -C 2 containing 0, S or N heteroaryl group.
PCT/CN2007/000742 2006-03-14 2007-03-08 Compounds capable of inhibiting zinc ion metalloproteinases WO2007104242A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNA2006100657305A CN1837169A (en) 2006-03-14 2006-03-14 Compound capable of inhibiting zinc ion metalloproteinases
CN200610065730.5 2006-03-14

Publications (1)

Publication Number Publication Date
WO2007104242A1 true WO2007104242A1 (en) 2007-09-20

Family

ID=37014701

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2007/000742 WO2007104242A1 (en) 2006-03-14 2007-03-08 Compounds capable of inhibiting zinc ion metalloproteinases

Country Status (2)

Country Link
CN (1) CN1837169A (en)
WO (1) WO2007104242A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2030615A3 (en) * 2007-08-13 2009-12-02 ELFORD, Howard L. Ribonucleotide reductase inhibitors for use in the treatment or prevention of neuroinflammatory or autoimmune diseases
US8029815B2 (en) 2004-04-28 2011-10-04 Elford Howard L Methods for treating or preventing restenosis and other vascular proliferative disorders

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100902768B1 (en) * 2007-05-22 2009-06-15 바이오스펙트럼 주식회사 Agents for Preventing Hair Loss Comprising Delphinidin as an Active Ingredient
CN101591321B (en) * 2009-06-25 2011-08-17 昆明制药集团股份有限公司 Method for preparing 5,6,7,4'-tetrahydroxyflavone
CN103989694A (en) * 2014-05-29 2014-08-20 中国人民解放军第四军医大学 Application of myricetrin for preventing and treating osteoporosis
CN106727470B (en) * 2016-12-09 2019-10-01 中国药科大学 Application of the benserazide hydrochloride in the drug of preparation treatment acute inflammation
CN107550901A (en) * 2017-10-31 2018-01-09 上海华堇生物技术有限责任公司 The medicinal usage of robinetin
CN116077359A (en) * 2021-01-26 2023-05-09 上海澄穆生物科技有限公司 Use of tretinoin receptor agonist compounds in cosmetic compositions
CN115957225A (en) * 2022-12-23 2023-04-14 广东植肤生物科技有限公司 MMP-1 expression inhibitor, skin external composition and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072319A1 (en) * 2000-03-29 2001-10-04 Purdue Research Foundation Tea catechin formulations and processes for making same
US20040186167A1 (en) * 2003-01-24 2004-09-23 Dou Q. Ping Polyphenol proteasome inhibitors, synthesis, and methods of use
WO2004105740A1 (en) * 2003-05-30 2004-12-09 Astellas Pharma Inc. Polyhydroxy phenols and their use in bindng p-selectin
CN1559392A (en) * 2004-03-10 2005-01-05 牛凤兰 Applicaton of gallic acid in prepn. of medicine for antitumor
WO2005000330A1 (en) * 2003-05-28 2005-01-06 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Angiogenic agents from plant extracts, gallic acid, and derivatives
US6936726B2 (en) * 2000-12-22 2005-08-30 Consejo Superior De Investigaciones Cientificas Flavanol and cysteamine conjugates
JP2005281164A (en) * 2004-03-29 2005-10-13 Api Co Ltd Gallic ester compound, method for producing the same, anticancer agent and preparation

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072319A1 (en) * 2000-03-29 2001-10-04 Purdue Research Foundation Tea catechin formulations and processes for making same
US6936726B2 (en) * 2000-12-22 2005-08-30 Consejo Superior De Investigaciones Cientificas Flavanol and cysteamine conjugates
US20040186167A1 (en) * 2003-01-24 2004-09-23 Dou Q. Ping Polyphenol proteasome inhibitors, synthesis, and methods of use
WO2005000330A1 (en) * 2003-05-28 2005-01-06 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Angiogenic agents from plant extracts, gallic acid, and derivatives
WO2004105740A1 (en) * 2003-05-30 2004-12-09 Astellas Pharma Inc. Polyhydroxy phenols and their use in bindng p-selectin
CN1559392A (en) * 2004-03-10 2005-01-05 牛凤兰 Applicaton of gallic acid in prepn. of medicine for antitumor
JP2005281164A (en) * 2004-03-29 2005-10-13 Api Co Ltd Gallic ester compound, method for producing the same, anticancer agent and preparation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8029815B2 (en) 2004-04-28 2011-10-04 Elford Howard L Methods for treating or preventing restenosis and other vascular proliferative disorders
EP2030615A3 (en) * 2007-08-13 2009-12-02 ELFORD, Howard L. Ribonucleotide reductase inhibitors for use in the treatment or prevention of neuroinflammatory or autoimmune diseases
US9526707B2 (en) 2007-08-13 2016-12-27 Howard L. Elford Methods for treating or preventing neuroinflammation or autoimmune diseases

Also Published As

Publication number Publication date
CN1837169A (en) 2006-09-27

Similar Documents

Publication Publication Date Title
WO2007104242A1 (en) Compounds capable of inhibiting zinc ion metalloproteinases
EP0750512B1 (en) A medical use of matrix metalloproteinase inhibitors for inhibiting tissue contraction
KR100518106B1 (en) Quinazolinone-containing pharmaceutical compositions for prevention of neovascularization and for treating malignancies
JP5504119B2 (en) Methods for treating and preventing diseases of biological conduits
EA016250B1 (en) Use of plasminogen for treating infection diseases
Nasi et al. Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis
Zhou et al. Chondroprotective effects of palmatine on osteoarthritis in vivo and in vitro: A possible mechanism of inhibiting the Wnt/β-catenin and Hedgehog signaling pathways
JP2006507297A (en) Treatment and prevention of abnormal scar formation in keloids and other skin or internal wounds or lesions
JP2006506321A (en) Methods for inhibiting increased vascular permeability
Kim et al. The role of MMP-9 in integrin-mediated hippocampal cell death after pilocarpine-induced status epilepticus
Wang et al. Scutellarin suppresses cartilage destruction in osteoarthritis mouse model by inhibiting the NF-κB and PI3K/AKT signaling pathways
Ng et al. Plasminogen deficiency results in poor clearance of non-fibrin matrix and persistent activation of hepatic stellate cells after an acute injury
Im et al. The role of cathepsins in ocular physiology and pathology
WO2009068926A1 (en) Use of urokinase type plasminogen activator inhibitors for the treatment of corneal disorders
KR101739757B1 (en) Sulodexide for use in the treatment of pathologies wherein metalloproteinases are involved
Fernandes et al. Effects of tenidap on the progression of osteoarthritic lesions in a canine experimental model. Suppression of metalloprotease and interleukin‐1 activity
Mainardi The role of connective tissue degrading enzymes in human pathology
JP4673309B2 (en) Malignant tumor local invasion inhibitor containing batroxobin
JP6336067B2 (en) Intraarticular indication of pepstatin in the case of arthropathy
CN112569338B (en) Application of TDFA in preparation of medicine for preventing and/or treating ocular surface inflammatory diseases
JP2009538611A (en) Chymotrypsin obtained from Lucilia sericata larvae and its use for the treatment of wounds
EP1033130A1 (en) Urokinase production inhibitors, angiogenesis inhibitors and methods of prevention or treatment with both
RU2277411C1 (en) Pharmaceutical composition for treating eye diseases as a result of microcirculation disorders and/or inflammatory processes
Georgoulas Novel methods for the modulation of wound healing after glaucoma filtration surgery
Tong Effect of Wnt16 Manipulation on the Severity of Osteoarthritis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07720350

Country of ref document: EP

Kind code of ref document: A1