WO2010031248A1 - 噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途 - Google Patents

噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途 Download PDF

Info

Publication number
WO2010031248A1
WO2010031248A1 PCT/CN2009/001036 CN2009001036W WO2010031248A1 WO 2010031248 A1 WO2010031248 A1 WO 2010031248A1 CN 2009001036 W CN2009001036 W CN 2009001036W WO 2010031248 A1 WO2010031248 A1 WO 2010031248A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
treat
relieve
protein
group
Prior art date
Application number
PCT/CN2009/001036
Other languages
English (en)
French (fr)
Other versions
WO2010031248A8 (zh
Inventor
李松
钟武
王莉莉
郑志兵
崔浩
肖军海
程罡
谢云德
张冰
Original Assignee
北京摩力克科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京摩力克科技有限公司 filed Critical 北京摩力克科技有限公司
Priority to JP2011527183A priority Critical patent/JP6050586B2/ja
Priority to EP09813969A priority patent/EP2341048B1/en
Priority to US13/120,009 priority patent/US8338616B2/en
Publication of WO2010031248A1 publication Critical patent/WO2010031248A1/zh
Publication of WO2010031248A8 publication Critical patent/WO2010031248A8/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • 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
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • Thiazolium salt compound and use thereof for treating aging-related diseases
  • the present invention relates to a thiazolyl salt compound, a process for the preparation thereof, a composition containing the same as an active ingredient and a carrier, excipient or diluent acceptable for the pharmaceutical industry and/or the cosmetic industry, and the compound for prevention or treatment Use of diseases or symptoms associated with AGEs (Advanced Glycosylation Endproducts, AGEs), (i) increase skin elasticity or reduce skin wrinkles, (ii) treat diabetes, (Hi) treat or relieve the sequelae of diabetes, (iv) treatment or relief Kidney damage, (V) treatment or relief of vascular injury, (vi) treatment or relief of hypertension, (vii) treatment or relief of retinopathy, (viii) treatment or relief of lens protein damage, (ix) treatment or relief of cataract, (X Treating or relieving peripheral neuropathy, (xi) treating or relieving osteoarthritis; or improving the use of cardiovascular system sclerosis; or enhancing the sensitivity of cardiovascular and drug therapy in elderly and diabetic; or treating chronic heart failure; or for preparation Use
  • a Chinese thiazolium salt compound such as 3-benzyloxycarbonylmethyl-4-methyl-thiazole-3-bromide, of formula III, is disclosed in Chinese patent application CN200610002391.6.
  • the object of the present invention is to find and develop a small molecule cleavage agent for AGEs, which is used to cleave already formed AGEs to prevent protein cross-linking, to cleave the already cross-linked protein, thereby promoting protein metabolism, and further improving the Various pathological changes caused by increased body weight, including increased skin elasticity or skin wrinkles, treatment of diabetes or treatment or relief of diabetes sequelae, kidney damage, vascular injury, hypertension, retinopathy, lens protein damage, cataract, peripheral neuropathy Or osteoarthritis; or improve the hardening of the cardiovascular system; or treat chronic heart failure; or enhance the sensitivity of cardiovascular therapy in the elderly and diabetes.
  • the glycosylated protein which is acted upon by the protein cross-linking structure cleavage agent is not limited to human proteins, but also includes plant proteins or animal proteins in crops, thereby expanding the use of plant proteins and animal proteins in crops.
  • the present inventors have found that compounds of formula I are useful in the treatment and/or prevention of a variety of diseases caused by glycosylation of proteins; for improving cardiovascular system sclerosis; for enhancing the sensitivity of cardiovascular therapy in elderly and diabetic; For the treatment of chronic heart failure.
  • a compound of the formula I or a solvated composition thereof has the preferred compound 3-benzyloxycarbonylacyl-4-methyl-thiazole-3-bromide disclosed in CN200610002391.6 (see formula III) Equivalent AGEs cleavage activity and more stable pharmacokinetic properties.
  • a first aspect of the invention relates to a compound of formula I, or a solvate thereof,
  • M is Na or K
  • X is Br, CI or I
  • Another aspect of the invention relates to a process for the preparation of a compound of formula I, which comprises:
  • X CI, Br, I Formula II wherein: X is Br or Cl, I;
  • M is Na or K
  • X is Br or ⁇ , I.
  • Bases referred to in the above methods including but not limited to sodium hydroxide; carbonic acid Sodium hydrogenate; sodium carbonate; potassium hydroxide; potassium carbonate.
  • a further aspect of the invention relates to a composition comprising at least one compound of formula I or a solvate thereof, in which carrier excipients are conventionally employed.
  • the carrier or excipient includes, but is not limited to, a carrier or excipient commonly used in medicines, cosmetics or foods.
  • One aspect of the invention relates to the use of at least one compound of formula I or a solvate thereof for the manufacture of a medicament for the prevention and/or treatment of various diseases caused by glycosylation of a protein.
  • the present invention also relates to a method of preventing and/or treating various diseases caused by glycation aging of a protein comprising administering a prophylactically and/or therapeutically effective amount of at least one compound of the formula I or a solvate thereof, in need of the above prevention and/or Or treated patients.
  • glycosylated protein to which the compound of the present invention acts is not limited to human proteins, but also includes plant proteins or animal organ proteins in crops, and thus the compounds or compositions disclosed in the present invention can be used for preservation purposes.
  • the compound of the formula I of the present invention or a solvate thereof is preferably the following compound:
  • compositions of the present invention may be exemplified by pharmaceutical compositions or cosmetic forms or other forms.
  • the pharmaceutical composition of the invention comprises an effective amount of a compound of the formula I according to the invention or dissolved A pharmaceutically acceptable carrier and one or more suitable pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphate, glycerin, sorbic acid, potassium sorbate, saturated plants.
  • the compound of the present invention is a potent cross-linking protein cleavage agent which has a good ability to cleave glycosylated aging protein, and thus can be used for, but not limited to, (i) increasing skin elasticity or reducing skin wrinkles, ( ⁇ ) treatment Diabetes, (m) treatment or relief of sequelae of diabetes, (iv) treatment or relief of kidney damage, (V) treatment or relief of vascular injury, (vi) treatment or relief of hypertension, (vii) treatment or relief of retinopathy, (viii Treatment or relief of lens protein damage, (ix) treatment or relief of cataracts, (X) treatment or relief of peripheral neuropathy, (xi) treatment or relief of osteoarthritis.
  • the compounds of the invention are excellent for improving the hardening of the cardiovascular system.
  • the compounds of the present invention are capable of enhancing the sensitivity of cardiovascular drug therapy in elderly and diabetic patients.
  • the compounds of the invention have the effect of treating chronic heart failure.
  • the invention may also be extended to prevent or reverse tooth coloration due to non-enzymatic glycosylation reactions in the oral cavity.
  • the regimen containing the compound of the present invention can be varied depending on the use involved.
  • Non-enzymatic reactions that occur in the mouth can cause tooth coloration.
  • the anti-corrosion agents currently used can accelerate this carbonization reaction and further cause coloration of the teeth.
  • the compounds of the present invention and pharmaceutical compositions thereof can be used in the oral cavity. Especially used as an additive in oral cleaning solutions and toothpastes.
  • the compound of the present invention can be applied to a mouthwash and a toothpaste in a suitable form of a non-toxic and pharmaceutically acceptable carrier.
  • composition of the compound of the present invention can be administered in any of the following ways: oral, spray inhalation, rectal administration, nasal administration, buccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, sheath Inside, intraventricular, intrasternal and intracranial injection or input, or by means of an explant reservoir.
  • oral, intraperitoneal or intravenous administration is preferred.
  • the compounds of the invention may be formulated in any orally acceptable form including, but not limited to, tablets, capsules, aqueous solutions or aqueous suspensions.
  • the carrier used for the tablet generally comprises lactose and corn starch, and a lubricant such as magnesium stearate may also be added.
  • the diluent used in the capsule preparation generally comprises lactose and dried corn starch.
  • Aqueous suspension formulations are usually prepared by admixing the active ingredient with a suitable emulsifier or suspension. If desired, some sweetening, aroma or coloring agents may be added to the above oral formulation.
  • the compounds of the present invention can be formulated into different topical preparations according to different affected faces or organs.
  • the form is specifically described as follows:
  • the compound of the present invention can be formulated into a preparation form of a micronized suspension or solution, and the carrier used is an isotonic pH of sterile saline, which may or may not be added.
  • Preservatives such as benzyl chloride alkoxide.
  • the compound can also be formulated in the form of a cream such as a Vaseline cream.
  • the compounds of the invention When applied topically to the skin, the compounds of the invention may be formulated in the form of a suitable ointment, lotion or cream preparation in which the active ingredient is suspended or dissolved in one or more carriers.
  • Carriers which may be used in ointment preparations include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; and detergents or creams which may be used include, but are not limited to, minerals Oil, sorbitan monostearate, Tween 60, cetyl esters wax, hexadecene aryl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the compounds of the present invention can also be administered in the form of a sterile injectable preparation, including sterile aqueous or oily suspension or sterile injection solutions.
  • a sterile injectable preparation including sterile aqueous or oily suspension or sterile injection solutions.
  • carriers and solvents which can be used include water, Ringer's solution and isotonic sodium chloride solution.
  • sterilized, fixed oils may be employed as a solvent or suspension medium such as a monoglyceride or a diglyceride.
  • the dosage and method of use of the compounds of the invention depends on a number of factors, including the age, weight, sex, natural health, nutritional status of the compound, the strength of the compound, the time of administration, the rate of metabolism, the severity of the condition, and Subjective judgment of the doctor.
  • the preferred dosage is between 0.01 and 100 mg/k body weight per day, with the optimal dose being between 20 mg/kg and 30 mg/kg body weight per day.
  • Figure 1 is an X-ray diffraction structure of 3-carboxymethyl-4-methylthiazole b-sodium salt.
  • the AGEs cross-linked structure was prepared by cross-linking AGE-BSA with rat tail rubber protein coated on a 96-well microtiter plate.
  • the cleavage effect of the compound on AGEs cross-linking was evaluated by ELISA.
  • the tail collagen is prepared by 96-well microtiter plate:
  • Normal Wister rats (body weight 200 ⁇ 20g), acutely sacrificed, tailed, and the following tail collagen preparation process was carried out at 4 °C.
  • the cercaria collagen filaments were extracted, washed with physiological saline and the non-collagen silk tissue was removed, washed with steamed water three times, and chopped and soaked in 4.
  • C is 0.1% glacial acetic acid for 1 week, and shaking is often performed during the period.
  • the mixture was centrifuged at 8000 g for 30 min, and the collagen solution of the supernatant was collected, and the protein content was determined after dilution.
  • a 96-well microtiter plate (Costar) was coated with 70 g of collagen per well at 4 ° C for 24 h, and the coating solution was discarded. The sample was air-dried and aseptic wrap under sterile conditions. Coated, stored at 4 ° C for later use.
  • Bovine serum albumin BSA (V) ( Roch ) 50mg/ml and 0.5M glucose were incubated in 0.2M PBS (pH 7.4) at 37 ° C under sterile conditions for 3-4 months in the dark to form a glycosyl group.
  • BSA is BSA-AGEs.
  • aglycosylated BSA was prepared from glucose-free BSA. Then dialyzed in 0.01M PBS (pH 7.4) dialysate to remove unreacted glucose, fluorescent scanning (Exi/Em (395/460nm)) and SDS-PAGE to identify BSA-AGEs, and Proteiny method for protein quantification .
  • the tail collagen was coated with a 96-well plate, and the acidic collagen was neutralized with a pH of 7.4 PBS;
  • test compound was diluted with pH 7.4 PBS, and ⁇ /well was added to each of the AGE-BSA cross-linking and BSA wells, and PBS100/well was added as the same method. Lysis control, incubation at 37 ° C
  • the reaction was terminated by 2 mol/L H 2 S0 4 ; in OMmin, the OD value was read by zero-setting the blank hole of the plate at 450 nm of the BOBRAD Model 550 plate reader.
  • the average OD value was a 4-well average.
  • Correction OD OD of AGE-BSA well - OD average of BSA well
  • the cleavage rate is expressed as a percentage decrease in OD value:
  • Table 1 ELISA assay for compound AGE-BSA - Collagen cross-linking rate of compound cracking rate (OD reduction %)
  • Blood cell treatment method After 16 weeks of diabetic rats, the common carotid artery was taken for anesthesia, and heparin was anticoagulated. After centrifugation at 1000g for 3min at 4°C, the lower layer of red blood cells (RBC) was removed; and 0.1mol/L PBS (pH7.4) was washed three times. Centrifuge at 1000g for 3min at 4°C; remove the lower layer of RBC for the experiment.
  • RBC red blood cells
  • In vitro administration method 0.1 mol/L isotonic PBS (pH 7.4) was used as a negative control, and each test compound was used as a solvent to prepare various concentrations of the drug solution. Add 100 ⁇ l of RBC per 900 ⁇ l of the drug solution or solvent control, shake gently for 16-18 hours at 37 ° C; centrifuge at 1000 °C for 3 min at 4 ° C, discard the supernatant, and wash the plate 4 times with O.lmol/L PBS (pH 7.4). Residual compound; 1000 g, centrifuged at 4 ° C for 3 min, the lower layer of RBC was taken, and 1:100 dilution was used for ELISA assay.
  • RBC surface cross-linked IgG content immunosorbent assay procedure Multiscreen- HA 0.45 ⁇ 96-well plate (Millipore), closed with Superblock (300 ⁇ 1 I Hole), 37 ° C for 1 hour; then drained under 5 mmHg negative pressure, PBST full-plate wash 3 times, 0.1 M PBS (pH 7.4) wash plate 2 times, each plate for 1 min; RBC (50 ⁇ 1/well), another PBS background control well (OD0); negative pressure drain; 0.1mol/L PBS (pH7.4) 150 ⁇ 1 wash four times, each plate for 1min. After vacuuming, add 1:500 diluted sheep anti-mouse IgG-HRP
  • reaction solution 150 ⁇ l/well was quickly aspirated and transferred to a common 96-well microtiter plate, and the OD value was measured at 490 nm.
  • Corrected OD OD average of the RBC sample to be tested - OD average of the PBS background hole without RBC, the drug lysis rate is expressed as a percentage decrease in OD 49 () nm value: (PBS hole OD 49 onm - test compound OD4 9. nm ) I PBS pore OD 49 . nm xl00%. Cracking rate (% reduction in OD)
  • Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (0.8%, 50 mg/kg), endotracheal intubation, heparin anticoagulation, intubation of the right common carotid artery, and connected to a Biopac physiological recorder via a pressure transducer to record blood pressure; The sternal midline thoracotomy, separation of the ascending aorta, with a suitable pulsed Doppler probe, the pulse Doppler flowmeter connected to the Biopac physiological recorder and real-time via Biopac's own software (Acknowledge, Version 3) Record and calculate hemodynamic parameters: systolic blood pressure (SBP), diastolic Pressure (DBP), heart rate (HR), cardiac output (CO), heart index (CI), total peripheral resistance (TPR), total peripheral resistance index (TPR Index), stroke volume (SV), and systemic arterial compliance Sex (SAC), etc. After the operation was stabilized for lOmin, the parameters were continuously recorded, and the average value of
  • TPR mean arterial pressure (MAP) / CO
  • Cardiac index CO corrected for body surface area
  • TPR index TPR corrected for body surface area
  • LVSP left ventricular systolic pressure
  • LVEDP left ventricular end diastolic pressure
  • Example 8 Experiment to improve left ventricular function in rats
  • Rats were anesthetized with sodium pentobarbital (0.8%, 50 mg/kg), tracheal intubation, heparin anticoagulation, and the right common carotid artery was cannulated to the left ventricle and connected to the Biopac physiological recorder via a pressure transducer.
  • the left ventricular pressure curve was recorded and recorded in real time by Biopac's own software (Acknowledge, Version 3): heart rate, left ventricular systolic pressure peak (LVSP), left ventricular maximum pressure change rate ( ⁇ dp/dtmax), left ventricular end diastolic pressure ( LVEDP).
  • LVSP left ventricular systolic pressure peak
  • ⁇ dp/dtmax left ventricular maximum pressure change rate
  • LVEDP left ventricular end diastolic pressure
  • the heart rate, LVSP, +dp/dt, -dp/dt of the diabetic model group were significantly decreased (P ⁇ 0.01), and LVEDP was significantly increased (P ⁇ 0.01), indicating that the diabetic rats Left ventricular dysfunction.
  • the cercaria collagen filaments were extracted under a water bath, washed with physiological saline and the non-collagen tissue was removed, and after lyophilization, stored at -70 ° C until use.
  • the lyophilized tail collagen was cut, and 2 mg of tail collagen was accurately weighed, and ⁇ pepsin (solvent: 0.5 mol/L acetic acid) was added to give a final concentration of 5 ⁇ ⁇ pepsin/mg tail collagen, 4 ° C vibration. Shake for 2 hours, centrifuge at 40,000g for 60min, accurately determine After the supernatant volume, 500 ⁇ l of the supernatant and all the precipitates were separately transferred to 5 ml of ampoule, 6 mol/L of HC1 was added, the tube was sealed, placed in a constant temperature drying oven, and hydrolyzed at 110 ° C for 24 hours.
  • tail collagen% total hydroxyproline content in the supernatant + the total hydroxyproline content of the precipitate X 0 compared with the normal control group, the solubility of tail collagen in the diabetic model group was significantly decreased.
  • the rat heart was quickly taken, the heart and the right ventricle were removed, only the left ventricle was left, the trimmed tissue piece was placed in a mortar, and a small amount of liquid nitrogen was added to rapidly grind, and the tissue was softened and added. A small amount of liquid nitrogen continues to grind until it is ground Fine powder, stored at -70 ° C for later use. About 100 mg of myocardial powder was weighed, and 1 ml of 200 g/ml pepsin (solvent: 0.5 mol/L acetic acid) was added, and shaken at 37 ° C for 2 and 24 hours, respectively.
  • solvent 0.5 mol/L acetic acid
  • the myocardial collagen solubility is obtained by the following formula:
  • pepsin digestive liquid supernatant hydroxyproline content ⁇ ⁇ / '" muscle collagen %% a 24- hour gastric protein Sahua liquid supernatant galeolic acid content
  • the left ventricular myocardial collagen solubility was significantly lower in the diabetic model group (42.8 ⁇ 4.3% vs. 68.9 ⁇ 14.1%, P ⁇ 0.01).
  • the A 18m g /kg dose group significantly increased myocardial collagen solubility in diabetic rats (54.7 ⁇ 11.0, 53.7 ⁇ 11.9, 57.7 ⁇ 7.3 vs. 42.8 ⁇ 4.3%, P ⁇ 0.01).
  • Example 5-10 indicate that A has the function of cleavage of AGEs cross-linking structure in vitro; it can significantly reduce the fluorescence content of AGEs in aorta, left ventricular myocardium and kidney of long-term diabetic rats, and improve myocardial collagen and tail collagen dissolution. Sexuality, and can improve aortic compliance in diabetic rats, reduce total peripheral resistance, increase cardiac output, and significantly improve left ventricular function. Therefore, the compound A can cleave the formed AGEs, reconstitute the vascular structure, reverse the diabetes-induced cardiovascular cirrhosis and dysfunction, and is a novel AGEs lysing agent.
  • Example 11 Experiments to enhance the sensitivity of cardiovascular drugs in elderly and diabetic patients
  • DM-HTN diabetic-hypertensive control
  • A (18 mg/kg) groups.
  • the rats were intragastrically administered once a day, and the diabetic-hypertensive control group was given an equal amount of distilled water for 4 weeks.
  • the blood was taken, the thoracic aorta, liver and kidney were stored at -70 °C for use in the detection of tissue biochemical indicators and gene expression. Another part of the kidney was fixed with 4% paraformaldehyde for pathological staining.
  • Nifedipine powder was formulated into 5 mg/ml solution with DMSO, then diluted with 15% ethanol-10% DMSO-25% PEG400 into a 500 pg/ml solution, and then diluted to 125 pg/ml, 62.5 pg/ml, 31.25 pg. /ml, 15.62 g/ml, 7.8pg/ml solution.
  • the antihypertensive drug was administered at a low to high gradient concentration, and the degree of blood pressure in the diabetic-hypertensive rat was observed. From the second dose of nifedipine (15.62 g/ml), the antihypertensive effect of the compound A group began to increase compared with the diabetes-hypertensive model group. By the third dose (31.25 g/ml), the nitrate The depressor amplitude of fendipine in the compound group A was significantly higher than that in the model group (19.49 ⁇ 13.29 vs. 9.35 ⁇ 6.46 mmHg, P ⁇ 0.05).
  • the compound A had a stronger enhancement effect (29.99 ⁇ 9 ⁇ 06 vs. 18.92 ⁇ 10.54 mmHg), ⁇ ⁇ 0.05 compared with the model group. This indicates that AGEs lytic compound A can increase the sensitivity of diabetic-hypertensive rats to nifedipine after treatment.
  • the first batch diabetic rats were treated with heart failure for 20 weeks NaCl, divided into 4 groups, model control group, valsartan group (VAL, ig, 10 mg/kg), compound A group (ig, 18 mg). /kg ). Once daily, for 16 weeks.
  • VAL valsartan group
  • compound A group ig, 18 mg. /kg ).
  • the second batch treatment of diabetic rats with heart failure and 20-minute NaCl treatment
  • Synchronous normal drinking water control rats were divided into 5 groups, normal control drinking water normal control rats (Normal Control), model control group (Model Control), compound A group (ig, 9 mg/kg), compound A group (ig, 36 mg). /k ). Once daily for 10 weeks.
  • the ventricular function was evaluated by ventricular intubation technique.
  • Evaluation index (1) Morphological indicators: left ventricular posterior wall thickness (LVF d), left ventricular end diastolic diameter (LVDd), end systolic diameter (LVDs); (2) functional indicators: ejection fraction (EF), Short axis shortening rate (FS), ventricular early blood flow filling speed (E), atrial filling speed (A) and E / A ratio; Doppler tissue imaging; apical mitral anterior valve on apical four-chamber view The ring is the sampling point, the early diastolic peak (Ea peak), the late diastolic peak (Aa peak), and the Ea/Aa ratio reflect the left ventricular systolic and diastolic motion.
  • the processed data was analyzed using SPSS statistical software, and all data were expressed as mean ⁇ SD.
  • the results of the experiment showed that the significance test of the difference between the groups was statistically processed by one-way ANOVA analysis, and P ⁇ 0.05 was considered as significant difference.
  • Left ventricular morphological parameters Echocardiographic measurements at 30 weeks of modeling (10 weeks of dosing) showed that the valsartan group (10 mg/kg) was administered 10 weeks after gavage compared with the diabetic model control rats treated with NaCl drinking water. In the compound A group (18 mg/kg), the PWd of the rats was slightly thickened, and the LVDd and LVDs were slightly decreased.
  • Echocardiographic measurements at 36 weeks (16 weeks of administration) showed that the morphological changes of the left ventricle in each drug-administered group were compared with those in the diabetic model-controlled rats treated with NaCl drinking water. The results were similar at 30 weeks (10 weeks of administration).
  • echocardiography showed that the valsartan group was administered 16 weeks after gavage compared with the saline-controlled diabetic control rats.
  • A can improve the diastolic function of the ventricles of diabetic rats treated with NaCl.
  • LVSP left ventricular systolic pressure
  • LVEDP left ventricular end diastolic pressure
  • BNP B-type natriuretic peptide
  • NC normal control
  • MC model control

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Ophthalmology & Optometry (AREA)
  • Diabetes (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Birds (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Vascular Medicine (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Urology & Nephrology (AREA)
  • Emergency Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Immunology (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Thiazole And Isothizaole Compounds (AREA)

Description

噻唑镟盐类化合物及其治疗蛋白老化相关疾病的用途 技术领域
本发明涉及噻唑表翁盐类化合物， 其制备方法， 含有它们作为 有效成分及制药业和 /或化妆品行业可接受的载体、 赋形剂或稀 释剂的组合物以及所述化合物在预防或治疗与 AGEs ( Advanced Glycosylation Endproducts， AGEs )有关的疾病或症状中的用 途， （i )增加皮肤弹性或者减少皮肤皱纹， （ii ) 治疗糖尿病， ( Hi ) 治疗或緩解糖尿病的后遗症， （iv ) 治疗或緩解肾脏损 伤， （V) 治疗或緩解血管损伤， （vi ) 治疗或緩解高血压， （vii) 治疗或緩解视网膜病变， （viii) 治疗或緩解晶状体蛋白损伤， ( ix ) 治疗或緩解白内障， （X) 治疗或緩解周围神经病， （xi ) 治疗或緩解骨关节炎； 或改善心血管系统硬化的用途； 或增强老 年及糖尿病心血管药物治疗敏感性用途； 或治疗慢性心衰用途； 或者用于制备防止或逆转牙齿着色的口腔用制剂的用途； 或者用 于制备各种农作物植物蛋白、 动物蛋白保鲜剂的用途。 背景技术
中国发明专利 CN200610002391.6 中披露了一类噻唑盐化合 物， 如式 III所示的 3-苄氧羰酰基甲基 -4-甲基 -噻唑 -3-溴酸盐。
Figure imgf000003_0001
(式 III )
3-苄氧羰酰基甲基 -4-甲基 -噻唑 -3-溴酸盐通过大鼠口服该化 合物药物代谢动力学研究、 比格犬口服该化合物药物代谢动力学 研究和小鼠体内组织分布等综合药物代谢动力学呈现出很好的
AGEs裂解作用。 但开发并寻找更筒单的对 AGEs有很好裂解作 用的化合物仍是十分需要的。 发明内容
本发明的目的是寻找并且开发作用于 AGEs 的小分子裂解 剂， 用来裂解已经形成的 AGEs从而阻止蛋白交联， 对已经交联 的蛋白进行裂解， 从而促进蛋白的代谢， 进一步改善由于 AGEs 在体内的增高从而导致的各种病理改变， 包括增加皮肤弹性或者 减少皮肤皱紋， 治疗糖尿病或者治疗或緩解糖尿病的后遗症、 肾 脏损伤、 血管损伤、 高血压、 视网膜病变、 晶状体蛋白损伤、 白 内障、 周围神经病或者骨关节炎； 或者改善心血管系统的硬化； 或者治疗慢性心衰； 或者增强老年及糖尿病心血管药物治疗的敏 感性。 同时， 这种蛋白交联结构裂解剂所作用的糖基化蛋白不局 限于人体蛋白， 还包括农作物中的植物蛋白或者动物蛋白， 因而 可以扩展用于农作物中植物蛋白和动物蛋白的保鲜用途。
本发明已经发现通式 I的化合物可以用于治疗和 /或预防由蛋 白糖基化造成的多种疾病； 用于改善心血管系统硬化； 用于增强 老年及糖尿病心血管药物治疗的敏感性； 用于治疗慢性心衰。
本发明发现， 通式 I 化合物或其溶剂化组合物具有跟 CN200610002391.6 中披露的优选化合物 3-苄氧羰酰基 ψ基 -4-甲 基 -噻唑 -3-溴酸盐 （见式 III )相当的 AGEs裂解活性和更稳定的 药物代谢动力学的性质。
因此， 本发明第一方面涉及通式 I 的化合物， 或其溶剂化 物，
Figure imgf000005_0001
其中：
M是 Na或 K
X是 Br， CI或 I;
本发明另一方面涉及制备通式 I化合物方法 , 其包括:
a)将 4-曱基噻唑与氯乙酸或溴乙酸反应
Figure imgf000005_0002
X=CI, Br, I 式 II 其中： X是 Br或 Cl、 I;
b) 将式 II化合物与碱反应得到式 I化合物
Figure imgf000005_0003
式 II 式 I 其中：
M是 Na或 K
X是 Br或 α、 I。
上述方法中所涉及到的碱: 包括但不局限于氢氧化钠； 碳酸 氢钠； 碳酸钠； 氢氧化钾； 碳酸钾。
本发明再一方面涉及一种组合物， 其包括至少一种式 I化合物 或其溶剂化物， 组合物中常用载体赋形剂。 所述载体或赋形剂包括 但不限于： 药物， 化妆品或食品中常用的栽体或赋形剂。
本发明一方面涉及至少一种式 I化合物或其溶剂化物用于制备 预防和 /或治疗蛋白糖基化所导致的各种疾病的药物的用途。
本发明还涉及预防和 /或治疗蛋白糖基化老化所导致的各种 疾病的方法， 其包括将预防和 /或治疗有效量的至少一种式 I 化 合物或其溶剂化物给予需要上述预防和 /或治疗的患者。
本发明化合物可作用的糖基化蛋白不局限于人体蛋白， 还包 括农作物中的植物蛋白或者动物器官蛋白， 因而本发明公开的化 合物或组合物可以用于保鲜用途。
根据本发明， 本发明式 I 化合物或其溶剂化物优选为下面的 化合物：
Figure imgf000006_0001
本发明的组合物举例讲可为药物组合物或化妆品形式或其它 形式。
本发明的药物组合物包括有效剂量的本发明式 I化合物或溶 剂化物和一种或多种适宜的可药用载体。 这里的药用载体包括但 不限于： 离子交换剂， 氧化铝， 硬脂酸铝， 卵磷脂， 血清蛋白如 人血白蛋白， 缓冲物质如磷酸盐， 甘油， 山梨酸， 山梨酸钾， 饱 和植物脂肪酸的部分甘油酯混合物， 水， 盐或电解质， 如硫酸鱼 精蛋白， 磷酸氢二钠， 磷酸氢钾， 氯化钠， 锌盐， 胶态氧化硅， 三硅酸镁， 聚乙烯吡咯烷酮， 纤维素物质， 聚乙二醇， 羧甲基纤 维素钠， 聚丙烯酸酯， 蜂蜡， 羊毛脂。
本发明化合物是一类强效交联蛋白裂解剂， 具有很好的裂解 糖基化老化蛋白的能力， 因此可以用于但不局限于 （i ) 增加皮 肤弹性或者减少皮肤皱紋， （ϋ ) 治疗糖尿病， （m ) 治疗或緩 解糖尿病的后遗症， （iv ) 治疗或緩解肾脏损伤， （V) 治疗或緩 解血管损伤， ( vi ) 治疗或緩解高血压， （vii) 治疗或緩解视网膜 病变， （viii) 治疗或緩解晶状体蛋白损伤， （ix ) 治疗或緩解白 内障， （X) 治疗或緩解周围神经病， （xi ) 治疗或緩解骨关节 炎。
本发明的化合物能够很好的改善心血管系统的硬化。
本发明的化合物能够 ^好增强老年及糖尿病心血管药物治疗 敏感性。
本发明的化合物具有治疗慢性心衰的作用。
本发明也可以扩展应用于阻止或逆转由于口腔中的非酶促糖 基化反应导致的牙齿着色。 含有本发明的化合物的用药方案可以 根据所涉及的用途进行变化。
发生在口腔中的非酶促反应可以导致牙齿着色。 目前所使用 的抗蛀蚀剂可以加速这种碳基化反应进一步导致了牙齿的着色。 最近有一类具有抗蛀蚀功能的阳离子杀菌剂用于常规口腔清洗。 这些阳离子抗菌剂有阿莱西丁， 十六烷基吡啶氯酸盐等等。 而这 些制剂可以加速糖基化反应中关键的一步 Maillard 反应， 进而 加速牙齿的着色 （Nordbo, J. Dent. Res., 58: 1429 (1979) ) 。 并 且有报道在体外观察到了洗必泰和洁而灭能够催化糖基化反应 (褐化反应） 。 由于 Maillard 反应， 洗必泰加入糖和氨基酸的 混合物中加速了色素的形成。
基于上述原因， 本发明所涉及的化合物及其药物組合物可以 用于口腔。 特别是用作口腔清洗液和牙膏中的添加剂。
在有关本发明化合物的上述用途中， 可以采用无毒且药学上 可接受的载体的适当形式应用于洁口液和牙膏中。
本发明化合物的药物组合物可以以下面的任意方式施用： 口 服， 喷雾吸入， 直肠用药， 鼻腔用药， 颊部用药， 局部用药， 非 肠道用药， 如皮下，静脉，肌内，腹膜内，鞘内，心室内，胸骨内和颅内 注射或输入,或借助一种外植储器用药。 其中优选口服、 腹膜内 或静脉内给药方式。
当口服用药时，本发明化合物可制成任意口服可接受的制剂 形式，包括但不限于片剂、 胶嚢、 水溶液或水悬浮液。 其中，片剂 使用的载体一般包括乳糖和玉米淀粉，另外也可加入润滑剂如硬 脂酸镁。 胶嚢制剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。 水悬浮液制剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合 使用。 如果需要，以上口服制剂形式中还可加入一些甜味剂、 芳 香剂或着色剂。
当局部用药时，特别是治疗局部外敷容易达到的患面或器官， 如眼睛、 皮肤或下肠道神经性疾病时，可根据不同的患面或器官 将本发明化合物制成不同的局部用药制剂形式，具体说明如下： 当眼部局部施用时,本发明化合物可配制成一种微粉化悬浮 液或溶液的制剂形式，所使用载体为等渗的一定 pH 的无菌盐水， 其中可加入也可不加防腐剂如氯化苄基烷醇盐。 对于眼用，也可 将化合物制成膏剂形式如凡士林膏。 当皮肤局部施用时,本发明化合物可制成适当的软膏、 洗剂 或霜剂制剂形式,其中将活性成分悬浮或溶解于一种或多种载体 中。 软膏制剂可使用的载体包括但不限于:矿物油，液体凡士林，白 凡士林，丙二醇，聚氧化乙烯，聚氧化丙烯，乳化蜡和水;洗剂或霜剂 可使用的载体包括但不限于：矿物油,脱水山梨糖醇单硬脂酸酯，吐 温 60，十六烷酯蜡，十六碳烯芳醇，2-辛基十二烷醇，苄醇和水。
本发明化合物还可以无菌注射制剂形式用药，包括无菌注射 水或油悬浮液或无菌注射溶液。 其中,可使用的载体和溶剂包括 水、 林格氏溶液和等渗氯化钠溶液。 另外，灭菌的非挥发油也可 用作溶剂或悬浮介质,如单甘油酯或二甘油酯。
另外需要指出，本发明化合物的使用剂量和使用方法取决于 诸多因素,包括患者的年龄、 体重、 性别、 自然健康状况、 营养 状况、 化合物的活性强度、 服用时间、 代谢速率、 病症的严重程 度以及诊治医师的主观判断。 优选的使用剂量介于 0.01 ~ 100mg/k 体重 /天， 其中最优剂量在 20mg/kg-30mg/kg 体重 / 天。 附图说明
图 1为 3-羧曱基 -4-甲基噻唑溴化 t钠盐的 X -衍射结构图 实施例
下面的实施例是本发明说明性优选实施方案，对本发明不构 成任何限制。
化合物熔点由 SRY-1 型熔点仪测定， 温度未经校正。 1H- NMR光谱由 BrukerARX400或 USVarianUnityInova600型核磁 仪测定， FAB质借由 Zabspect高分辨质讲仪测定。 实施例 1: 3-羧甲基 -4-甲基-噻唑溴化镟盐的制备
15.6g 4-甲基噻唑溶于 50ml无水丙酮中， 加入 21g溴乙酸， 搅拌 3d， 过滤， 得固体， 乙醇重结晶得到白色固体， 干燥， 共 得到 26g， 产率 72%， mP=240.6 ~ 241.6°C„
MS[M]+=158.2m/e;lH-NMR (400MHz,DMSO) 2.48(d,3H);
5.55(s,2H); 8.09(d,lH); 10.25 (d,lH), 14.05 ( brs,lH ) 。 实施例 2: 3-羧甲基 -4-甲基-噻唑氯化锵盐的制备
15.6g 4-甲基噻唑溶于 50ml无水丙酮中， 加入 15g氯乙酸， 搅拌 5d， 过滤， 得固体， 乙醇重结晶得到白色固体， 干燥， 共 得到 20.4g， 产率 66.9%， mP=262.3 ~ 263.6°C。
MS[M]+=158.2m/e;lH-NMR (400MHz,DMSO) 2.42(d, 3H);
4.84(s,2H); 7.93(d,lH); 10.01 (d,lH), 13.98 ( brs，lH ) 。 实施例 3: 3-羧曱基 -4-甲基-噻唑溴化 、钠盐 （A ) 的制备
20g 3-羧甲基 -4-甲基 -噻唑溴化镟盐白色固体， 悬浮于 90- 100ml 无水甲醇中， 随后加入等摩尔数的碳酸氢钠， 室温搅拌 8 小时， 过滤， 母液再加入无水乙醇 150ml， 静置析晶， 得到 15g 产物， 白色晶体， 产率 75%。
MS[M]+=158.2m/e;lH-NMR (400MHz,DMSO) 2.50(d, 3H); 4.82(s?2H); 7.93(d,lH); 10.00 (d,lH).
元素分析 C6H7NO2SBr(260.08)理论值： C， 27.71; H, 2.71;
N,5.39; Br, 30.72%
实测值： C， 27.50; H, 2.836; N, 5.304; Br, 30.891
X-单晶衍射测定晶体结构
取 3-羧甲基 -4-甲基 -噻唑溴化镶钠盐白色结晶 5mg, 加 1ml 无水乙醇加热溶解， 得无色溶液， 过滤， 滤液在 30°C恒温箱放 置 5d, 慢慢析出白色柱状结晶， 待晶粒慢慢长大生长为单晶， 进行 X-单晶衍射测定晶体结构。 结构见图 1
结晶学数据： C12H14Br2N2Na204S2， Mr = 520.20, 三斜晶 系， 空间群 P-l， 晶体学参数： a = 8.9000(18)A ， α = 92.82(3) deg., b = 9.4095(19) A, β = 109.00(3) deg.,
c = 12.028(2) A, δ = 104.11(3) deg.。 实施例 4: 3-羧甲基 -4-甲基-噻唑氯化 、钠盐的制备
20g 3-羧甲基 -4-甲基 -噻唑氯化镟盐白色固体， 悬浮于 90- 120ml 无水甲醇中， 随后加入等摩尔数的碳酸氢钠， 室温搅拌 8 小时， 过滤， 母液再加入无水乙醇 150ml, 静置析晶， 得到 19g 产物， 白色晶体， 产率 79%。 实施例 5: 裂解 AGE-BSA-胶原交联结构的 ELISA 筛选试 验
以 AGE-BSA与包被在 96孔酶标板上大鼠尾胶蛋白交联、 体外制备 AGEs 交联结构， 采用 ELISA 方法评价化合物对 AGEs交联的裂解作用。
尾胶原包被 96孔酶标板制备：
正常 Wister大鼠 （体重 200±20g ) ， 急性处死， 取尾， 4°C 下进行以下尾胶原蛋白制备过程。 首先， 抽取尾腱胶原丝， 用生 理盐水洗涤并去除非胶原丝组织， 再经 蒸水洗 3次， 剪碎、 浸 泡于 4。C的 0.1%冰醋酸中 1 周， 期间时常进行震摇。 最后以 8000g 离心 30min， 收集离心上清胶原蛋白溶液， 稀释后测定蛋 白含量。 以每孔 70 g 胶原蛋白满孔包被 96 孔酶标板 ( Costar ) ， 4°C、 24h, 弃去包被液， 无菌条件下风干、 保鲜膜 包被， 4°C贮存备用。
AGE-BSA制备：
牛血清白蛋白 BSA ( V ) ( Roch ) 50mg/ml及 0.5M葡萄糖 在 0.2MPBS ( PH7.4 ) 中， 37°C无菌条件下， 避光孵育 3-4个月， 使其形成糖基化 BSA即 BSA-AGEs。 同时， 以无葡萄糖的 BSA 制备无糖基化 BSA。 然后在 0.01M PBS(pH7.4)透析液中透析， 除去未反应的葡萄糖， 荧光扫描 （ Exi/Em(395/460nm) )及 SDS- PAGE鉴定 BSA-AGEs形成， 同时采用 Lowery方法进行蛋白定 量。
分析测定方法流程：
尾胶原包被 96孔板， 用 pH7.4PBS满孔中和酸性胶原 lh;
SuperBlock ( PIERCE ) 37°C , 封闭 lh; PBST ( PBS-Tween ) 洗板 3次， 每次振荡 1分钟； 用 PBS稀释 AGE-BSA, 以获得最 大交联度浓度的 AGE-BSAlOO l加入 96孔板的 A、 B、 C, D行 的孔中， 相同浓度的 BSA加入 E、 F、 G、 H行的孔中， 1列前
3 孔中 PBS 作为系统和试剂空白， 37°C下， 使之与胶原交联
4h ; PBST 洗板 4 次， 间隔振荡 1 min ; 受试化合物采用 pH7.4PBS稀释， 取 ΙΟΟμΙ/孔分别加于 AGE-BSA交联和 BSA孔 各 4孔， 同样方式加入 PBSlOO l/孔作为非裂解对照， 37°C孵育
16h; PBST洗板 4次， 间隔振荡 1 min; 加 80μ1/孔兔抗 BSA抗 体 （ 1:500 ) 37°C , 50min; PBST 洗板 4 次， 间隔振荡 1 min;
加入 80μ1/孔辣根过氧化物酶标记羊抗兔 IgG ( 1：1000 ) 37°C , 50min; PBST洗板 3次， 间隔振荡 1 min; 加底物液 TMB ( 3, 3，， 5， 5，-四甲基联苯胺） ΙΟΟμΙ/孔室温， 闭光 20min ; 用
2mol/L H2S04终止反应； lOmin 内， 在 BOBRAD Model550读 板机 450nm下， 板空白孔调零读取 OD值。
数据分析：
-10-
4- 平均 OD值采用 4孔平均值。
校正 OD=AGE-BSA孔的 OD平均值 -BSA孔的 OD平均值 裂解率以 OD值降低的百分率表示：
【（ PBS孔的 OD平均值 -受试药物孔 OD平均值） /PBS孔的 OD平均值】x%
根据上述步骤， 受试化合物在 0.1、 0.3、 lmmol/L或较低浓 度下裂解率结果见表 1 (结果均为 3 次以上筛选结果的平均 值） ： 表 1: ELISA测定化合物对 AGE-BSA-胶原交联的裂解率 化合物 裂解率 （ OD降低值％ )
0.01 ( umol/L ) 0.1 ( umol/L )
A 8.1±4.76, n = 5 11.3±7.70, n = 5 实施例 6: 体外裂解红细胞表面交联 IgG的实验
血细胞处理方法： 16 周糖尿病大鼠麻醉后颈总动脉取血， 加肝素抗凝， 4°C 1000g 离心 3min， 取下层红细胞 ( RBC ) ； 0.1mol/L PBS ( pH7.4 ) 洗三次， 每次 4°C 1000g离心 3min; 取 下层 RBC用于实验。
体外给药方法： 以 0.1mol/L 等渗 PBS ( pH7.4 ) 为阴性对 照， 各受试化合物以其为溶剂配制成不同浓度药液。 每 900μ1 药 液或溶剂对照中加入 lOOplRBC , 37°C轻微振摇 16-18 小时； 1000g, 4°C离心 3min, 弃上清， O.lmol/L PBS ( pH7.4 ) 洗板 4 次除去残留化合物； 1000g , 4°C离心 3min， 取下层 RBC , 1： 100稀释用于 ELISA测定。
RBC表面交联 IgG含量免疫吸附法测定流程： Multiscreen- HA 0.45μιη 96孔板 ( Millipore ) ， 以 Superblock封闭 ( 300μ1 I 孔） ， 37°C 1 小时； 然后在 5mmHg 负压的奈件下抽干， PBST 满孔洗板 3次， 0.1MPBS ( pH7.4 ) 洗板 2次， 每次振板 lmin; 加入待测 RBC ( 50μ1/孔） ， 另设 PBS本底对照孔 （ OD0 ) ； 负 压抽干； 0.1mol/L PBS ( pH7.4 ) 150μ1 洗四次， 每次振板 lmin。 负压抽干后加入 1 : 500 稀幹的羊抗小鼠 IgG-HRP
( 50μ1/孔） ， 室温静置 2h , 抽干； 0.1mol/L PBS ( pH7.4 ) 150μ1/孔洗 3 次， 每次振板 lmin; 抽干； 力 P邻苯二胺 ( OPD ) 底物显色液（ΙΟΟμΙ/孔） ， 室温避光放置 30min， 2mol/L H2S04
( ΙΟΟμΙ/孔） 终止反应； 快速吸出反应液 （ 150μ1/孔） 转入普通 96孔酶标板， 于 490nm下测定 OD值。
受试化合物裂解率的计算：
校正 OD = 待测 RBC样品的 OD平均值 - 无 RBC的 PBS 本底孔的 OD平均值， 药物裂解率以 OD49()nm值降低的百分率表 示： （ PBS 孔 OD49onm-受试化合物 OD49。nm ) I PBS 孔 OD49。nmxl00%。 裂解率（ OD降低值％ )
化合物
ΙμΜ 10μΜ 30μΜ ΙΟΟμΜ
A 25.6±2.1 26.9±3.0 21.6±5.0 26.0±2.7 实施例 7: 改善大鼠血管顺应性的实验
大鼠腹腔注射戊巴比妥钠 （0.8%， 50 mg/kg )麻醉， 气管插 管， 肝素抗凝， 右颈总动脉插管， 通过压力换能器连于 Biopac 生理记录仪， 记录血压； 胸骨正中行开胸术， 分离升主动脉， 套 以合适的脉冲多普勒探头， 将脉冲多普勒血流计连于 Biopac 生 理记录仪上并通过 Biopac 自带软件 ( Acknowledge , Version 3 ) 实时记录并计算血流动力学各参数： 收缩压 （SBP ) 、 舒张 压 （DBP) 、 心率 （HR) 、 心输出量 （CO) 、 心指数（CI) 、 总外周阻力 （TPR) 、 总外周阻力指数（TPR Index ) 、 每搏输 出量 （ SV ) 及系统动脉顺应性 （ SAC ) 等。 待术后稳定 lOmin, 连续记录各参数， 并求得各参数连续 30s 的均值作为各 参数的测量值。
部分血流动力学参数计算公式：
TPR =平均动脉压 ( MAP ) / CO
SAC = SV/ ( SBP-DBP )
与正常对照組比较， 糖尿病模型组大鼠体重和心率均显箸下 降 （Ρ<0.01 ) , 但收缩压和舒张压均无明显改变 （ Table 1- 1) ， 各脏器脏体系数均显著增加 （P<0.01) ( Table 1-2 ) 。 与 模型大鼠比较， 给药組大鼠体重、 心率、 收缩压、 舒张压及各脏 体系数均无显著变化。 表 1-1 在用 A治疗 4星期的糖尿病大鼠上测得的血压和心率 剂量 收缩压 舒张压 心率 组 体重 （g)
(mg/kg) (mmHg) (mmHg) (beats/min) 正常 447±29 105.7±10.1 72.5±11.4 340.0±20.2 糖尿病 346±20* 108.8±11.3 70.4±14.4 297.6±37.6*
9(i.g.) 341±27 105.3±12.0 68.8±14.0 265.2±25.8
A 18(i.g.) 344士 24 108.7±14.7 72.8±17.2 282.2±31.9
342±28 99.2±13.2 60.7±16.1 261.0±32.3
^<0.01 vs.正常组 器官参数 (11=9-12， Mean±SD)
器官^ Ife/g xlO"3)
组
心脏 心室 左心室 左肾 右肾 正常 2.49±0.08 2.23±0.07 1.76±0.08 2.69±0.16 2.77±0.17 糖尿病 3.47±0.34« 3.04±0.29** 2.45±0.28* 5.14±0.34* 5.28±0.36*
3.42±0.21 2.99±0.14 2.29±0.10 5.20±0.42 5.33±0.39
A 3.44±0.19 3.01±0.17 2.34±0.12 5.16±0.42 5.22±0.39
3.44±0.18 3.03±0.16 2.32±0.15 5.07±0.37 5.19±0.33
# P < 0.01 vs.正常组 采用多普勒血流测定仪对大鼠 SBP、 DBP、 HR进行检测， 并计算大鼠 CO、 CI及 SAC。 从表 1-3 可以看出， 与正常大鼠 比较， 糖尿病模型大鼠 CO、 CI及 SAC显著降低（P < 0.01 ) , TPR和 TPRI显箸增高 （P < 0.01 ) ； 说明糖尿病大鼠的总外周 阻力显著增加， 心输出量和系统顺应性显著降低， 表明长期糖尿 病导致了大鼠心血管系统的硬化或其它结构和功能的紊乱。 与糖 尿病模型组比较， 给药 4周后， 所有给药组的 CO， CI和 SAC 有显著提高； TPR和 TPRI均有显著降低。 表明 A有改善长期 糖尿病大鼠血管硬化的作用。
-14- 、 在糖尿病大鼠及用 A处理 4星期的糖尿病大鼠上测得的血流动力学 （n=9-12)
CO CI TPR TPR index SAC 组
(ml/min) (ml/min per cm2) (103.dyne.sec/cms) (dyne.sec/cm3) (103ml/mmHg) 正常 124.6±20.3 0.214±0.040 83.8±21.1 142.8±33.8 13.8=13.6
^^J^病 60.3±7.9" 0.134±0.016* 109.87±19.0SS 241.3±19.0 ** 5.55±0.94 B
A 9(i.g.) 84.9±13.6** 0.194db0.031** 78.6±19.7** 181.6±51.3** 9.05±1.44**
18(i.g.) 79.2±10.6** 0.179±0.026** 87.4±22.3* 198.2±56.2* 8.02±1.27**
77.7±10.( 0.179±0.024** 77.0±19.6** 176.4±44.6** 7.92±l.lf
Cardiac index: CO corrected for body surface area; TPR index: TPR corrected for body surface area.
<0.05, mP<0.01 vs. Normal; *P<0.05, **P<0.01 vs. Diabetic 表 1"4 Α对糖尿病大鼠 LV功能的影响 （n=8-10)
剂量 HR LVSP Pos dp/dt Neg dp/dt LVEDP 组
(mg/kg) (beat/min) (mmHg) (mmHg/s) (mmHg/s) (mmHg) 正常 430.46±23.82 191.68±22.03 15861.8^093.2 12724.3±2299.0 1.50±0.87 糖尿病 359.56^32.91* 153.49i20.05* 9410.2士 1294.2s 6684.2±1400.7S 8.16±3.05s
9 (i-g.) 361.80±39.74 161.54±13.98 12201.2士 1872.2** 7762.8±1320.2* 5.73±1.44**
A 18 (i.g.) 377.35±17.54 170.65±8.94** 11712.8±1370.9** 7660.8±1154.8 6.87±1.26*
36 (i.g.) 368.99=131.66 168.25±15.48* 11425.3±1660.0* 7675.4±2216.6 6.79±2.47*
LVSP:左心室收缩压； LVEDP:左心室末端舒张压.
UP < 0.05, mP < 0.01 vs. Normal; *P < 0.05, **P < 0.01 vs. Diabetic
实施例 8: 改善大鼠左心室功能的实验
大鼠腹腔注射戊巴比妥钠 （0.8%， 50 mg/kg)麻醉， 气管插 管， 肝素抗凝， 右颈总动脉插管至左心室， 通过压力换能器连于 Biopac生理记录仪， 记录左心室压力曲线， 通过 Biopac 自带软 件 （Acknowledge, Version 3) 实时记录： 心率、 左室收缩压峰 值 （LVSP) 、 左室内压最大变化速率 （±dp/dtmax) 、 左室舒 张末期压 （LVEDP) 。 待术后稳定 lOmin, 连续记录上述参 数， 并求得各参数连续 30s的均值作为各参数的测量值。 结果见 表 1-4·
与正常对照组相比， 糖尿病模型组大鼠心率、 LVSP、 +dp/dt、 -dp/dt均显著下降（ P< 0.01 ) ， 同时 LVEDP显著升高 (P<0.01) , 表明糖尿病大鼠的左室功能异常。 与糖尿病模型 组比， 所有给药组在给药四周后 +dp/dt 均能够显著升高； LVEDP显著降低 （Ρ<0·05或 P<0.01) ； 除 A 9mg/kg组外均 有显箸提高 LVSP (P<0.05或 P<0.01) 的作用； A 9mg/kg 组 的 -dp/dt有显著提高 ( P < 0.05 ) ， A18mg/kg、 36mg/kg 两个剂 量组 P值分别为 0.055和 0.057， 也存在升高趋势。 表明， A能 够改善糖尿病引起的心脏收缩功能障碍， 能够改善糖尿病引起的 心脏舒张功能障碍， 有显著性改善左室功能的作用。 （Table 1- 4) 实施例 9: 大鼠尾胶原溶解性实验
水浴下抽取尾腱胶原丝， 生理盐水清洗并去除非胶原组织， 冷冻干燥后， -70°C贮存备用。
将冻干的尾胶原剪碎， 精确称取 2mg尾胶原， 加入 ΙΟμ^ιηΙ 胃蛋白酶（溶剂： 0.5mol/L 乙酸） ， 使其终浓度为 5μδ 胃蛋白 酶 /mg尾胶原， 4°C振摇 2小时， 40000g 离心 60min, 精确测定 上清体积后， 将 500μ1上清及全部沉淀分别移至 5ml安瓿中， 加 入 6mol/L HC1, 封管后放入恒温干燥箱中， 110°C水解 24 小 时。
测定水解液中羟脯氨酸浓度： ①取各管水解液 100μ1， 加入 约 50μ1 10mol/L NaOH调 pH值至 6.0, 用柠檬酸緩冲液（ 50g 柠檬酸 ·Η20, 72.36g 无水乙酸钠， 34gNaOH, 11.52ml 水醋酸 混合加水至 1200ml， 加入 300ml 正丙醇） 850μ1。 ②加入氯胺 Τ ( 1.41g氯胺 T溶于 10ml蒸馏水后， 分别加入 10ml正丙醇及 80ml 柠檬酸緩冲液） 500μ1， 混匀后， 室温反应 10min。 ③加 入 3.15mol/L高氯酸 500μ1, 迅速混匀， 室温 5min。 ④加入 10 % P-DMAB (对二甲氨基苯甲醛 lg， 加 3.15mol/L高氯酸 2.6ml 溶解后用正丙醇稀释至 10ml, 即用即配） 500μ1， 迅速混匀， 75Ό水浴 10min。 ⑤冰水中迅速冷却反应液， 醉标仪 570nm测 定吸光度值。 ⑥对应同步测定的羟脯氨酸标准曲线 （0、 0.5、 1、 2、 3、 4、 5、 6 g/ml )换算出水解液中羟脯氨酸含量。 尾胶 原溶解性按如下公式求得：
上清总羟脯氨酸含量
尾胶原溶解性％ =上清总羟脯氨酸含量 +沉淀总羟脯氨酸含量 X 0 与正常对照组比较， 糖尿病模型组大鼠尾胶原溶解性显箸降
4氐（19.7 ± 7,2 vs. 79.8 ± 12.0 %, Ρ < 0·01 ) 。 与糖尿病模型组比 较， A 18mg/kg, 36mg/kg 剂量组能够显著提高糖尿病大鼠尾胶 原溶解性 （33.7 ± 17.8， 37.5 ± 11.1 vs. 19.7 ± 7.2% , P < 0.01 ) 。
实施例 10: 增加大鼠心肌胶原溶解性实验
血管顺应性实验结束后， 迅速取大鼠心脏， 修剪剔除心耳及 右心室， 只保留左心室， 将修剪好的組织块放入研钵， 加入少量 液氮迅速研磨， 待组织将变软再加少量液氮继续研磨， 直至研成 细粉， -70°C贮存备用。 称量约 lOOmg 心肌粉末， 加入 1ml 200 g/ml 胃蛋白酶（溶剂： 0.5mol/L 乙酸） ， 37°C分别振摇 2 和 24小时。 40000g离心 60min, 分别取 2和 24小时胃蛋白酶 消化液上清， 水解并测定羟脯氨酸含量 （水解及测定方法同 上） 。 心肌胶原溶解性按如下公式求得：
、、Bn te t。/ ― 2小时胃蛋白酶消化液上清羟脯氨酸含量 ..ι ηηη/ '"肌胶原 生％一 24小时胃蛋白薩化液上清轻薩酸含量
与正常对照組比较， 糖尿病模型组大鼠左心室心肌胶原溶解 性显著降低 （42.8 ± 4.3% vs. 68.9 ± 14.1% ， P < 0.01 ) 。 与糖 尿病模型组比较， A 18mg/kg剂量组均能够显著提高糖尿病大鼠 心肌胶原溶解性 （ 54.7 ± 11.0， 53.7 ± 11.9, 57.7 ± 7.3 vs. 42.8 ± 4.3 %, P < 0.01 ) 。
实施例 5 ~ 10的结果表明 A在体外具有裂解 AGEs交联结 构的作用； 在体内能够显著减少长期糖尿病大鼠主动脉、 左心室 心肌、 腎脏 AGEs荧光含量， 同时提高心肌胶原、 尾胶原溶解 性， 且能够提高糖尿病大鼠主动脉顺应性， 降低总外周阻力， 增 加心输出量， 显著改善左心室功能。 因此该化合物 A可以裂解 已经形成的 AGEs交联， 重构血管结构， 逆转糖尿病诱发的心血 管系统硬化及功能紊乱， 是一个新型 AGEs裂解剂。 实施例 11: 增强老年及糖尿病心血管药物治疗敏感性的实 验
在糖尿病-高血压大鼠模型上进行 AGEs 裂解剂对硝苯地平 降压作用的影响实验。 实验动物及糖尿病、 高血压模型， 选取与 对照组比较确认模型成立的大鼠进行实验。 实验期间自由进食， 不使用降糖、 降压药物进行干预。
动物分组及给药方式 A 用蒸馏水溶解， 现用现配。 大鼠随机分为糖尿病-高血压 对照 （DM-HTN ) 、 A ( 18mg/kg ) 组。 每日灌胃给药一次， 糖 尿病 -高血压对照組给予等量蒸馏水， 连续 4 周。 实验完成后取 血， 胸主动脉、 肝脏、 肾脏, -70°C保存备用， 用于组织生化指 标及基因表达的检测， 另取一部分肾脏 4%多聚甲醛固定， 用于 病理染色。
硝苯地平溶液的配制
硝苯地平粉末用 DMSO 配成 5mg/ml的溶液， 然后用 15% 乙醇 -10%DMSO-25%PEG400 稀释成 500pg/ml的溶液， 再依次 稀释成 125pg/ml 、 62.5pg/ml 、 31.25pg/ml 、 15.62 g/ml 、 7.8pg/ml的溶液。
AGEs裂解剂对硝苯地平降压作用的影响研究实验方法 大鼠用乌氯合剂 i.p.麻醉， 肝素抗凝， 右侧颈总动脉插管通 过压力换能器连于 Biopac 生理记录仪。 右侧股静脉插留置针， 浓度由低至高分 5次緩慢注射硝苯地平溶液， 连续记录动脉收缩 压、 舒张压、 脉压差以及心率变化情况。
AGEs裂解剂对糖尿病-高血压大鼠血压的影响
在体测定大鼠血压结果显示， 给予化合物 A 4 周对糖尿病- 高血压大鼠心率、 血压无明显变化。 表明 AGEs 裂解剂对糖尿 病-高血压大鼠血压无直接影响。 （ Table 2-2 ) 表 2-2 大鼠上测得的血压和心率
( n=12 Mean士 SD )
HR SBP DBP MBP PP
组
( beat/min ) ( mmHg ) ( mmH ) ( mmHg ) ( mmHg )
DM-HTN 423.8±38.5 165.8±14.1 145.9±13.4 152.3±16.9 19.8±4.6
A 408.7±35.7 166.6±12.4 144.2±11.1 153.5±13.7 22.9±3.8
-19- AGEs裂解剂对硝苯地平降压作用的影响
采用从低到高梯度浓度给予降压药， 观察其降低糖尿病-高 血压大鼠血压的程度。 从给予硝苯地平的第二个剂量 ( 15.62 g/ml )起， 化合物 A 组降压作用较糖尿病 -高血压模型 组开始有增加趋势， 到第三个剂量 （31.25 g/ml ) 时， 硝苯地平 对化合物 A 组大鼠的降压幅度都较模型组有显著提高 ( 19.49±13.29 vs. 9.35±6.46mmHg， P < 0.05 ) , 在第四个剂量 ( 62.5 g/ml ) 时， 化合物 A 组有较强的增强效果（ 29·99±9·06 vs. 18.92±10.54mmHg ) , 与模型组相比 Ρ < 0.05。 表明 AGEs裂 解剂化合物 A 治疗后能够增加糖尿病 -高血压大鼠对硝苯地平的 敏感程度。
在本实验中， 糖尿病高血压大鼠化合物 A预给药 4周， 之后 采用经典的降压药物评价方法， 从低到高浓度依次给予作用于血 管平滑肌细胞上的降压药物硝苯地平， 随着给药浓度的增大， 硝 苯地平对 A 化合物 A 组大鼠的降压效果逐渐好于高血压模型 组， 待硝苯地平浓度增至 31.25 g/ml 时， AGEs 裂解剂化合物 A组的降压幅度均比高血压模型组有显著的统计学差别， 表明， AGEs裂解剂化合物 A确具增强硝苯地平地降压作用， 能增强老 年及糖尿病心血管药物治疗的敏感性。 实施例 12: 治疗慢性心衰的药效学实验
动物分组及给药方式
第一批： 采用心力衰竭成模 20 周 NaCl处理糖尿病大鼠， 分 4 组， 模型对照组 （Model Control ) 、 纈沙坦组 （ VAL， ig, 10mg/kg ) 、 化合物 A组 （ig， 18mg/kg ) 。 每日给药一次， 连续 16周。 采用无创超声心动图检测方法进行药效评价。
第二批： 釆用心力衰竭成模 20 周 NaCl 处理糖尿病大鼠及 同步常规饮水正常对照大鼠， 分 5组， 同步常规饮水正常对照大 鼠 （Normal Control ) 、 模型对照组 （Model Control ) 、 化合 物 A组 （ ig， 9mg/kg ) 、 化合物 A組 （ ig, 36mg/k ) 。 每日给药 一次， 连续 10 周。 采用心室插管技术进行左室功能进行药效评 价。
评价指标： （1)形态学指标： 左室后壁厚度 (LVF d)、 左室舒 张末期内径 (LVDd)、 收缩末期内径 (LVDs); (2〉功能学指标:射 血分数（ EF)、 短轴缩短率（ FS)、 心室早期血流充盈速度 (E)、 心 房充盈速度 (A)和 E /A比值； 多普勒组织成像； 在心尖四腔切面 上， 以二尖瓣前瓣瓣环为取样点， 舒张早期运动峰值 （ Ea 峰） 、 舒张晚期运动峰值（Aa峰） ， 和 Ea /Aa比值反映左室整 体的收缩和舒张运动。
应用 SPSS 统计软件分析处理数据， 所有数据均采用均数士 标准差 （Mean±SD ) 表示。 实验结果组间差异的显著性检验均 采用单因素方差分析 ( one-way ANOVA analysis ) 进行统计学 处理， 以 P<0.05为有显著性差异。
第一批模型药效评价结果
超声心动图评价测定结果：
左室形态学指标： 在造模 30周 （给药 10周） 时超声心动图 测定显示， 与 NaCl饮水处理的糖尿病模型对照大鼠比较， 灌胃 10 周后缬沙坦组 （10mg/kg ) 、 化合物 A 组 （18mg/kg ) 大鼠 PWd略有增厚、 LVDd和 LVDs略有降低。
在造模 36 周 （给药 16 周） 时超声心动图测定显示， 与 NaCl饮水处理的糖尿病模型对照大鼠比较， 灌胃 16周后， 各给 药组上述左室形态学指标变化趋势与造模 30周 （给药 10周） 时 测定结果相似。
左室舒张功能指标 在造模 30 周 （给药 10 周） 时， 超声心动图测定显示， 与
NaCl饮水处理的糖尿病模型对照大鼠比较， 灌胃 10周后， 缬沙 坦組 （10mg/kg ) 、 化合物 A ( 18mg/k )组大鼠 E/A 比值均显 著降低 （模型 2.5±0.31；缬沙坦组 1.24±0.32；化合物 A 组
1.32±0.31 , P<0.05 或 P<0.01 ) ， 同时 Ea/Aa 比值均显著提高
( P<0.05 或 Ρ<0·01，） ； 表明在造模 30 周时， 缬沙坦、 化合物
Α给药 10周均能够显著改善 NaCI饮水处理的糖尿病大鼠左室 舒张功能。
在造模 36周 （给药 16周） 时， 超声心动图测定显示， 与 NaCl 饮水处理的糖尿病模型对照大鼠比较， 灌胃 16 周后， 纈沙坦组
( 10mg/kg )化合物 A组 ( 18mg/kg ) 組大鼠 E/A比值均显著降 低（模型 2.5±0.52；缬沙坦组 1.32±0.25；化合物 A组 1.45±0.18,
P<0.05 或 P<0.01 ) ； 且缬沙坦 （10mg/kg ) 能够显著提高大鼠
Ea/Aa 比值 （ Ρ<0·01 ) ； 表明在造模 36 周时， 缬沙坦、 化合物
Α给药 16周均能够显著改善 NaCl饮水处理的糖尿病大鼠左室 舒张功能。
第二批模型评价结果
化合物 A模型大鼠左室功能及血浆 BNP水平的影响
如表 3-1所示， 与正常对照组相比， NaCl饮水处理的糖尿 病大鼠 +dp/dt及 -dp/dt均显著降低， 同时 LVEDP以及血浆 BNP 水平显著升高， 表现出心室的收舒张功能均显著降低。 化合物 A 组 （9， 36mg/k ) 均能够显箸升高大鼠 +dp/dt 及 -dp/dt ( P<0.05 或 P<0.01 ) ， 同时显箸降低 LVEDP ( P<0.01 ) 。 表明， 化合物
A能够改善 NaCl饮水处理的糖尿病大鼠心室的舒张功能。
-22- * A对 NaCl处理的糖尿病大鼠 LV功能的作用（n-8-10)
Figure imgf000025_0001
LVSP: 左心室收缩压； LVEDP: 左心室末端舒张压； BNP: B-type natriuretic peptide; NC: 正常 对照； MC:模型对照
<0.05,bp<0.01 vs. NC; <0.05, d <0.01 vs. MC

Claims

Figure imgf000026_0001
一种具有通式 I的化合物， 或其溶剂化物，
Figure imgf000026_0002
其中：
M是 Na或 K,
X是 Br或 Cl、 I。
2、 权利要求 1的化合物， 其为：
3-羧甲基 -4-甲基-溴化噻唑镚、钠盐， 或
3-羧甲基 -4-甲基-氯化噻唑責钠盐。
3、 一种组合物， 其包含权利要求 1或 2的化合物及药学上可 接受的载体或赋形剂。
4、 权利要求 1或 2化合物在制备用于治疗或緩解蛋白老化相 关疾病的产品中用途， 所述蛋白老化相关疾病， 包括但不局限 于：
( i ) 增加皮肤弹性或者减少皮肤皱纹， （ii ) 治疗糖尿病， ( iii) 治疗或缓解糖尿病的后遗症， （iv) 治疗或緩解肾脏损 伤， （V) 治疗或緩解血管损伤， （vi) 治疗或緩解高血压， （vii) 治疗或緩解视网膜病变， （viii) 治疗或緩解晶状体蛋白损伤， ( ix) 治疗或緩解白内障， （X) 治疗或緩解周围神经病， （xi) 治疗或緩解骨关节炎。
5、 权利要求 1或 2所述的化合物用于制备在动物体内牙齿着 色的逆转剂或者其它用于防止和逆转牙齿着色的口腔用制剂的用 途。
6、 权利要求 1 或 2 所述的化合物用于制备农作物中植物蛋 白、 动物蛋白保鲜剂的用途。
PCT/CN2009/001036 2008-09-22 2009-09-15 噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途 WO2010031248A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2011527183A JP6050586B2 (ja) 2008-09-22 2009-09-15 チアゾリウム塩化合物及びタンパク質老化疾患を治療する用途
EP09813969A EP2341048B1 (en) 2008-09-22 2009-09-15 Thiazolium salt compound and its use for treating protein aging associated diseases
US13/120,009 US8338616B2 (en) 2008-09-22 2009-09-15 Thiazoliums and their use for treating protein aging associated diseases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2008102116577A CN101684106B (zh) 2008-09-22 2008-09-22 噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途
CN200810211657.7 2008-09-22

Publications (2)

Publication Number Publication Date
WO2010031248A1 true WO2010031248A1 (zh) 2010-03-25
WO2010031248A8 WO2010031248A8 (zh) 2011-05-05

Family

ID=42039079

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2009/001036 WO2010031248A1 (zh) 2008-09-22 2009-09-15 噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途

Country Status (6)

Country Link
US (1) US8338616B2 (zh)
EP (1) EP2341048B1 (zh)
JP (1) JP6050586B2 (zh)
CN (1) CN101684106B (zh)
HK (1) HK1142607A1 (zh)
WO (1) WO2010031248A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11180463B2 (en) * 2013-07-09 2021-11-23 Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China Thiazole inner salt compounds, and preparation methods and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112015014993A2 (pt) * 2012-12-20 2017-07-11 Colgate Palmolive Co composição para higiene oral
MX352361B (es) 2012-12-20 2017-11-22 Colgate Palmolive Co Composicion para el cuidado oral conteniendo liquidos ionicos.

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1185736A (zh) * 1995-01-18 1998-06-24 奥尔顿有限公司 噻唑鎓化合物用于预防和逆转高级糖基化终产物形成的用途
WO2002062301A2 (en) * 2001-02-07 2002-08-15 Farrington Pharmaceuticals, Llc Method and composition for rejuvinating cells, tissues, organs, hair and nails
CN1534027A (zh) * 2003-04-02 2004-10-06 中国人民解放军军事医学科学院毒物药 新的取代五元氮杂环盐类化合物及其治疗蛋白老化相关疾病的用途
CN101007789A (zh) * 2006-01-27 2007-08-01 北京摩力克科技有限公司 新的取代五元氮杂环盐类化合物及其治疗蛋白老化相关疾病的用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5185334A (en) * 1989-07-31 1993-02-09 Schering Corporation 2,2-disubstituted glycerol and glycerol-like compounds, compositions and methods of use
PE20030968A1 (es) * 2002-02-28 2004-01-12 Novartis Ag Derivados de 5-feniltiazol como inhibidores de cinasas
GB0320197D0 (en) * 2003-08-28 2003-10-01 Novartis Ag Organic compounds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1185736A (zh) * 1995-01-18 1998-06-24 奥尔顿有限公司 噻唑鎓化合物用于预防和逆转高级糖基化终产物形成的用途
WO2002062301A2 (en) * 2001-02-07 2002-08-15 Farrington Pharmaceuticals, Llc Method and composition for rejuvinating cells, tissues, organs, hair and nails
CN1534027A (zh) * 2003-04-02 2004-10-06 中国人民解放军军事医学科学院毒物药 新的取代五元氮杂环盐类化合物及其治疗蛋白老化相关疾病的用途
CN101007789A (zh) * 2006-01-27 2007-08-01 北京摩力克科技有限公司 新的取代五元氮杂环盐类化合物及其治疗蛋白老化相关疾病的用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NORDBO, J. DENT. RES., vol. 58, 1979, pages 1429

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11180463B2 (en) * 2013-07-09 2021-11-23 Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China Thiazole inner salt compounds, and preparation methods and uses thereof

Also Published As

Publication number Publication date
US20110178141A1 (en) 2011-07-21
EP2341048A4 (en) 2012-02-29
JP2012502929A (ja) 2012-02-02
CN101684106B (zh) 2013-06-12
WO2010031248A8 (zh) 2011-05-05
EP2341048B1 (en) 2013-03-06
EP2341048A1 (en) 2011-07-06
JP6050586B2 (ja) 2016-12-21
CN101684106A (zh) 2010-03-31
US8338616B2 (en) 2012-12-25
HK1142607A1 (en) 2010-12-10

Similar Documents

Publication Publication Date Title
US20110009460A1 (en) Compositions and methods for treating amyotrophic lateral sclerosis
US20210260090A1 (en) Use of sglt-2 inhibitors for the prevention and/or treat-ment of cardiac diseases in felines
CA2826884A1 (en) Method for improving endothelial function and decreasing cardiovascular morbidity using shilajit
CN115702000A (zh) 用于预防或治疗代谢性疾病或肌肉疾病的包含乳杆菌衍生性囊泡的组合物
WO2010031248A1 (zh) 噻唑鎓盐类化合物及其治疗蛋白老化相关疾病的用途
JP4635339B2 (ja) 心拡張障害治療剤
US11180463B2 (en) Thiazole inner salt compounds, and preparation methods and uses thereof
NO316494B1 (no) Anvendelse av forbindelse for fremstilling av medikament for profylakse og behandling av diabetiske komplikasjoner
EP1243261A1 (en) Use of a hydantoin derivative in a pharmaceutical composition against hypoalbuminaemia
WO2007085203A1 (en) New substituted pentabasic azaheterocyclic compounds and its use of treating protein aging disease
AU749673B2 (en) Use of glycosaminoglycans for producing pharmaceutical preparations for treating diabetes-associated diseases of the eye
JP2020188689A (ja) Ｔｏｘｉｃ ＡＧＥｓ生成抑制剤
CN117599151B (zh) 益心舒片联合活性肽预防和/或治疗慢性心力衰竭的用途
CN109640957A (zh) 一氧化氮合酶途径对口腔健康的调节
WO2024135348A1 (ja) 動脈スティフネス増大抑制剤
KR102489247B1 (ko) 리나릴 아세테이트를 유효성분으로 포함하는 근감소증의 예방 또는 치료용 조성물
KR20230170579A (ko) 식물성 오메가3 개지치씨유를 유효성분으로 포함하는 혈전 질환 예방 또는 치료용 약학 조성물
JP2024089152A (ja) 動脈スティフネス増大抑制剤
TW202116297A (zh) 組合療法之方法、組成物與套組
CN116650476A (zh) 一种化合物、组合物及其在制备治疗心脏病的药物中的应用
KR20130091604A (ko) 시난디온 ａ를 포함하는 항염증용 조성물
TW201902490A (zh) 一種多醣混合物用於製備高血糖症藥物的用途
EP1990053A1 (en) Pharmaceutical composition for enhancing adiponectin production
WO2012060860A1 (en) Treatment of ocular and cerebral ischemia

Legal Events

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

Ref document number: 09813969

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2011527183

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 13120009

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2009813969

Country of ref document: EP