WO2018030762A1 - Pharmaceutical composition for stroke treatment based on ampk inhibition - Google Patents

Pharmaceutical composition for stroke treatment based on ampk inhibition Download PDF

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WO2018030762A1
WO2018030762A1 PCT/KR2017/008569 KR2017008569W WO2018030762A1 WO 2018030762 A1 WO2018030762 A1 WO 2018030762A1 KR 2017008569 W KR2017008569 W KR 2017008569W WO 2018030762 A1 WO2018030762 A1 WO 2018030762A1
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zinc
thiazol
indol
amino
ylmethylene
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PCT/KR2017/008569
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French (fr)
Korean (ko)
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김양희
박황서
고재영
엄재원
김태윤
서보라
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세종대학교산학협력단
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Priority claimed from KR1020170098417A external-priority patent/KR101911785B1/en
Application filed by 세종대학교산학협력단 filed Critical 세종대학교산학협력단
Priority to EP17839780.8A priority Critical patent/EP3498277B1/en
Priority to ES17839780T priority patent/ES2901059T3/en
Priority to US16/323,963 priority patent/US20190167647A1/en
Publication of WO2018030762A1 publication Critical patent/WO2018030762A1/en

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    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings

Definitions

  • the present invention relates to a pharmaceutical composition for treating stroke, and more particularly, to a pharmaceutical composition for treating stroke based on AMPK inhibitory function.
  • Stroke is a collective term for local neurological symptoms that are suddenly caused by abnormal brain blood flow.
  • the brain takes up only 2% of its body weight by weight, but the blood flow to the brain is 15% of cardiac output and 20% of the body's oxygen consumption.
  • the brain uses only glucose as an energy source, so necrosis easily occurs even if the energy supply is interrupted for a while. Therefore, abnormalities in cerebral blood flow are closely related to brain damage.
  • Brain damage caused by stroke includes various toxic mechanisms such as excitotoxicity, oxidative stress, apoptosis and zinc neurotoxicity. Excavation is necessary.
  • Korean Patent Laid-Open Publication No. 1283416 discloses a neuroprotective method in which an AMPK inhibitor Compound C or FAS inhibitor C75 is administered to an ischemic mouse to significantly reduce the size of the infarct so that function is preserved after stroke or ischemic injury. .
  • the present invention is to solve various problems, including the above problems, it is an object of the present invention to provide a new stroke treatment based on the AMPK inhibitory function effective for various mechanisms of stroke.
  • these problems are exemplary, and the scope of the present invention is not limited thereby.
  • the present invention provides a pharmaceutical composition for treating stroke, which contains a compound having a structural formula of Formula 1 as an active ingredient:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 7 carbon atoms, an amine group, a carboxyl group or R 2 And R 3 together form a —O— (CH 2 ) n —O— ring or a substituted or unsubstituted benzene ring (n is an integer from 1 to 3), R 6 is hydrogen or a methyl group, and R 7 is hydrogen Or halogen)
  • FIG. 1 is a photograph showing that the neurotoxicity was increased after zinc treatment in cultured cerebral cortical neurons (A) and the degree of cytotoxicity after treatment with Compound C (+ Cpd C), an AMPK inhibitor, through TUNEL staining and LDH assay. Quantified graphs (B and C).
  • FIG. 2 is a Western blot photograph (A) and an enzyme activity assay (B) graph confirming that AMPK activity is increased after zinc treatment in cultured cerebral cortical neurons.
  • FIG. 3 shows Western blot photographs (A) and AMPK inhibitor Compound C (+ Cpd C), which showed increased expression of apoptosis promoting gene and caspase-3 activity after zinc treatment in cultured cerebral cortical neurons.
  • Western blot photograph (B) shows that both increase and caspase-3 activity was decreased.
  • Figure 4 is the purchase of the AMPK activity assay kit (CycLex, Japan) and recombinant AMPK ( ⁇ 2 / ⁇ 1 / ⁇ 1; CycLex, Japan) to measure the enzyme activity and compared to the compound C effect 40 showing a similar or stronger inhibitory effect It is a graph which selected two candidate compounds.
  • FIG. 5 is a graph showing whether various neurotoxic inhibition is observed by inducing various neurotoxicity in cultured cerebral cortical neurons, treating 7 selected compounds and quantifying the degree of cytotoxicity through LDH assay.
  • Figure 6 is a graph comparing the brain damage of the experimental animals and the control animals compared with the control group to observe the brain damage inhibitory effect by administering drug # 28 to the animal model of the stroke.
  • FIG. 7 is a graph showing the results of an acute toxicity test by administering the final selected drug # 28 to rats and extracting spleen (A), liver (B) and kidney (C).
  • FIG. 8 is a graph illustrating a neuroprotective effect by searching for compounds having a structure similar to that of the previously selected drug # 28 and treating 25 similar compounds selected to zinc-induced cerebral cortical neurons.
  • FIG. 9 is a graph illustrating the neuroprotective effect of 12 drugs that showed a significant drug effect on zinc toxicity, and were treated with NMDA-induced neurotoxicity in cerebral cortical neurons.
  • FIG. 10 is a graph illustrating the neuroprotective effect of 12 drugs selected in cerebral cortical neurons of mice induced with neurotoxicity by H 2 O 2 .
  • FIG. 11 is a graph illustrating treatment of # 28 drug and clioquinol of the present invention to neurons by concentration, and analysis of free zinc concentration with a pZn meter.
  • 15 is a photograph of a neuron treated with 4CO1, 4CO7, and # 28 drugs and treated with FluoZin-3 staining material, followed by confocal laser microscopy (A).
  • Figure 16 is a graph of neuronal cells treated with hydrogen peroxide (H 2 O 2 ), zinc chelator TPEN or CaEDTA, calcium chelator ZnEDTA and whether neurotoxin reduction.
  • 17 is a photomicrograph of the toxic inhibitory effect of the drug after treatment with hydrogen peroxide (H 2 O 2 ), TPEN, # 28, 4C01, 4C07 drug to FluoZin-3.
  • FIG. 18 is a graph illustrating whether the iontocarrier is inhibited in toxicity by treating ionomycin and # 28 drugs in concentrations in neurons.
  • 19 is a graph analyzing the chelation effect on calcium treated with Ca 2 + , # 28 drug and EDTA on the test tube and treated with the fluorescent Fura-2 dye.
  • 20 is a graph analyzing the activity of caspase-3 by treating TPEN, zinc, # 28 drug and clioquinol in neurons (A) and neurotoxicity (LDH secretion) (B).
  • FIG. 21 is a graph illustrating the change in the concentration of zinc in neurons by treating neuronal cultures with normal cultures, zinc-added cultures, and zinc-free cultures, treatment with clioquinol and # 28 drugs, and treatment with the fluorescent substance ZinPyr-1. .
  • AMP-activated protein kinase is a heterologous trimer protein consisting of a catalyst ⁇ subunit ( ⁇ 1 or ⁇ 2), and two regulatory subunits ( ⁇ and ⁇ ). AMPK is phosphorylated and activated at low cellular energy levels, and then regulates cell metabolism to restore gene levels over time by regulating gene expression over time. Increasing the AMP / ATP ratio, changing the cell pH and redox state, and increasing the creatine / phosphocreatin ratio are known to activate AMPK.
  • the present invention provides a pharmaceutical composition for treating stroke, which contains a compound having a structural formula of Formula 1 as an active ingredient:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 7 carbon atoms, an amine group or a carboxyl group, or R 2 and R 3 together form a —O— (CH 2 ) n —O— ring or a substituted or unsubstituted benzene ring (n is an integer from 1 to 3), R 6 is hydrogen or a methyl group, and R 7 is Hydrogen or halogen)
  • the substituted alkyl group may be a trifluoromethyl group and the halogen may be iodine, bromine or chlorine.
  • the compound is (5Z) -5- (1H-Indol-3-ylmethylene) -2- ⁇ [2- (trifluoromethyl) phenyl] amino ⁇ -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2- ⁇ [3- (trifluoromethyl) phenyl] amino ⁇ -1,3-thiazol-4 (5H) -one, ( 5Z) -2-[(3-Bromophenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol- 3-ylmethylene) -2-[(4-methylphenyl) amino] -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2-[(3 -
  • the stroke may be a hemorrhagic stroke, an ischemic stroke or a metal toxicity stroke, and the metal may be lead, mercury, or manganese. (manganese), arsenic, thallium, iron, iron, zinc, cadmium, bismuth or tin, the ischemic stroke may be excitatory neuronal death or It may be caused by oxidative neuronal death.
  • the effective amount of the compound in the pharmaceutical composition of the present invention may vary depending on the type of affected part of the patient, the site of application, the number of treatments, the treatment time, the dosage form, the condition of the patient, the type of supplement, and the like.
  • the amount used is not particularly limited, but may be 0.01 ⁇ g / kg / day to 10 mg / kg / day.
  • the daily dose may be administered once or in two or three times a day at appropriate intervals or may be administered intermittently at intervals of several days.
  • the compound may be contained at 0.1-100% by weight based on the total weight of the composition.
  • the pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the preparation of the pharmaceutical compositions may be used as additives for the preparation of solids or liquids.
  • the additive for preparation may be either organic or inorganic.
  • excipients include lactose, sucrose, white sugar, glucose, cornstarch, starch, talc, sorbet, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide.
  • binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin, and the like.
  • the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hardened vegetable oil, and the like.
  • coloring agent if it is normally permitted to add to a pharmaceutical, all can be used. These tablets and granules can be appropriately coated according to sugar, gelatin coating and other needs. Moreover, preservatives, antioxidants, etc. can be added as needed.
  • compositions of the present invention may be prepared in any formulation commonly prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. . These formulations are described in Remington's Pharmaceutical Science, 15 th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a prescription generally known for all pharmaceutical chemistries.
  • the compound may be administered orally or parenterally, and preferably, by parenteral administration, intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection Intramuscular injection, intraperitoneal injection, and the like.
  • the mouse cerebral cortical neurons used in the present invention were extracted and cultured from the brains of mouse embryos and added Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) to which 5% fetal bovine serum (FBS) and 5% horse serum (HS) were added. , US) was incubated at 95% humidity, 5% CO 2 and 37 °C temperature conditions. The cells were grown to a density of 2 ⁇ 10 4 cells in a 24-well tissue culture plate for activation and differentiation of cells and cultured in MEM medium without FBS and HS before treatment with zinc and compounds.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HS horse serum
  • zinc toxicity is one of the representative causes of stroke, and it was confirmed whether zinc toxicity induced in the cultured cerebral cortical neurons was reduced by AMPK inhibitor treatment.
  • the cultured cerebral cortical neurons according to one embodiment of the present invention treated with 300 ⁇ M of zinc (ZnCl 2 ) for 10 minutes and then removed and cytotoxicity (cytotoxicity) after 10 hours TUNEL staining or LDH (Lactate Dehydrogenase)
  • the cells were treated with AMPK inhibitor Compound C (+ Cpd C, Tocris) at 20 ⁇ M and then neurotoxic reduction was observed.
  • TUNEL staining confirmed that neurotoxicity was increased after zinc treatment in cerebral cortical neurons (Fig. 1A).
  • the cytotoxicity was quantified by TUNEL staining and LDH analysis and treated with Compound C (+ Cpd C), an AMPK inhibitor. It was confirmed that zinc neurotoxicity was significantly reduced (FIGS. 1B and 1C).
  • the AMPK activity was observed by Western blot and enzyme activity analysis after zinc treatment in cortical neurons.
  • the cultured cerebral cortical neurons were treated with 300 ⁇ M of zinc (ZnCl 2 ) for 10 minutes and then removed. After 0.5, 1, 2, 4, and 6 hours, the cell samples were loaded on polyacrylamide gels with protein leather and protein Isolate according to size. The antibody was then processed, washed and read.
  • Example 3 AMPK Inhibition of zinc toxicity through inhibition of activity
  • zinc toxicity was induced by treating cerebral cortical neurons with 300 ⁇ M of zinc (ZnCl 2 ) for 2, 3, 4, 5 and 6 hours.
  • the protein of each sample was isolated and subjected to Western blot, and the relationship between zinc toxicity and apoptosis was confirmed by treatment with Compound C (+ Cpd C), an AMPK inhibitor, at 20 ⁇ M.
  • the primary screening was performed on compounds that may act as AMPK inhibitors.
  • AMPK activity is related to the zinc toxicity mechanism, and other studies have reported an increase in AMPK activity and Bim expression in the excitatory toxicity mechanism.
  • the AMPK enzyme is an enzyme in which three subunits of ⁇ , ⁇ and ⁇ form a complex structure, and the alpha subunit has kinase enzymatic activity.
  • Previous studies have shown that AMPK alpha2 significantly inhibits neurotoxicity caused by ischemia in knock-out mice, unlike alpha1. Therefore, in the present invention, candidate chemicals capable of inhibiting AMPK enzyme activity by targeting alpha2 were selected through structure-based virtual screening, and finally 208 candidate compounds were selected.
  • Example 4-1 The 208 compounds selected in Example 4-1 were obtained from a compound library manufacturer (Interbioscreen, Russia) and subjected to secondary screening through observation of their inhibitory effect on AMPK enzyme activity.
  • AMPK enzyme activity was measured using AMPK activity assay kit (CycLex, Japan) and recombinant AMPK ( ⁇ 2 / ⁇ 1 / ⁇ 1; CycLex, Japan).
  • AMPK activity assay kit CycLex, Japan
  • recombinant AMPK ⁇ 2 / ⁇ 1 / ⁇ 1; CycLex, Japan
  • 40 drugs showed a similar or better inhibitory effect than compound C at 10 ⁇ M.
  • the 40 drugs are shown in Table 1 below.
  • the neuroprotective effect was observed by treating each of the 40 selected drugs with zinc at 12 hr after treating and removing 300 ⁇ M of zinc for 10 minutes to cultured cerebral cortical neurons.
  • the degree of death of the cells was quantified by LDH analysis, and each effect was expressed as an average value, and four separate experiments were performed for each of the selected drugs (FIG. 4).
  • the neurotoxicity model used includes excitatory toxicity, oxidative damage, and apoptosis, which are thought to be the cause of stroke.
  • excitatory toxicity models NMDA was treated, and the oxidative damage model was iron.
  • Toxicity and H 2 O 2 toxicity model was used, and apoptosis model was used as TPEN, staurosporine and etoposide toxicity model.
  • the TPEN is a zinc chelator and is known to induce typical apoptosis in neurons.
  • Storosporin is one of the representative apoptosis inducing enzymes as a kinase inhibitor and etoposide is DNA. It is a drug known to cause cell death due to damage.
  • Compound # 35 and Compound # 28 had an inhibitory effect on all toxicity (FIG. 5).
  • Compound # 28 from the library vendor was (Z) -5-((1H-indol-3-yl) methylene) -2-((3-hydroxyphenyl) amino) thiazol-4 (5H)-having the structure Verified that it is one:
  • Example 5 Inhibitory effect on brain injury in stroke animal model
  • Example 4-4 In order to confirm whether the compound selected as # 28, which was finally selected in Example 4-4, was effective in an animal model, the compound was treated with a stroke model animal that induced brain injury, and then whether the brain damage inhibitory effect was observed was examined. .
  • a permanent model of cerebral artery was constructed using male Sprague-Dawley (SD) rats aged 8-9 weeks. CBF was measured using laser Doppler flowmetry 30 minutes before and after 10 minutes after permanent middle cerebral artery occlusion (MCAO) and is shown in Table 3 below. Subsequently, the final selected AMPK inhibitor candidate compound # 28 (75 ng in 3 ⁇ l) or vehicle (10% DMSO) was 0.8 mm rear and 1.2 mm flank of the brain at 3.8 mm depth 15 minutes after MCAO. Intracerebroventricular injection was performed. After that, the degree of motor deficit of rats was evaluated.
  • the criteria for evaluation indicate that the deficit does not indicate a deficiency . Mild, opposite rotations are classified as moderate and loss of circling or loss of reflexes are classified as severe deficit.
  • the rat model was obtained 24 hours after induction of ischemia in the rat model and stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) to measure the degree of cerebral infarction.
  • TTC 2,3,5-triphenyl tetrazolium chloride
  • the final selected compound # 28 was injected into rats and acute toxicity results were observed.
  • SD rats 8-9 week old male Sprague-Dawley (SD) rats were injected intravenously with Compound # 28, the final selected AMPK inhibitor candidate compound, at 75 ⁇ g / kg or vehicle (10% DMSO) per rat. intravenous injection) and repeated four times.
  • Example 7 Detection of similar compounds and observation of zinc toxicity inhibitory effect
  • neurotoxicity models for observing neurotoxicity inhibitory effects include excitotoxicity and oxidative damage, which are classified as a cause of stroke.
  • the excitatory toxicity model is NMDA (N-methyl-D-aspartate).
  • H 2 O 2 hydrogen peroxide
  • NMDA 50 ⁇ M
  • H 2 O 2 100 ⁇ M
  • (10 ⁇ ) and # 28, a previously selected drug, were treated (20 ⁇ ). Since the neurons were stained with propidium iodide (PI), the number of stained cells was quantified to measure the degree of cell death. Cells dead after the staining are stained with PI because of lack of selective permeability of the cell membrane, while healthy cells are not stained.
  • PI propidium iodide
  • the # 28 drug of the present invention was treated with the zinc fluorescent material ZnAF (2.5 ⁇ M) on a test tube by concentration (2.5-20 ⁇ M) and the control group was a very powerful zinc chelator.
  • ZnAF zinc fluorescent material
  • concentration 2.5-20 ⁇ M
  • the control group was a very powerful zinc chelator.
  • clioquinol 1 ⁇ 5 ⁇ M was used and free zinc concentration was measured using a pZn meter (NeuroBioTex Inc).
  • the cortical neurons of the cultured mice were pre-treated with FluoZin-3 staining material and exposed to high concentrations of zinc, followed by 4CO1 (20 ⁇ M) and 4CO7 (20 ⁇ M). And # 28 (20, 50 ⁇ M) drug was used to observe the image using a confocal laser microscope and the fluorescence size was quantitatively measured using a fluorometer.
  • TPEN a zinc chelator that easily enters cells
  • EDTA a zinc chelator that easily enters cells
  • free zinc ions are maintained in the cells, leading to neuronal death.
  • caspase-3 protease activity is decreased during apoptosis.
  • the # 28 drug and clioquinol of the present invention not only acts as a chelator, but also acts as an ion permeable carrier, which is introduced into the cell in combination with zinc and then the zinc is released from the drug because the concentration of free zinc in the cytoplasm is too low. It was expected that the concentration of free zinc was maintained to decrease the neurotoxicity by TPEN, and the activity and neurotoxicity (LDH secretion) of caspase-3 were observed by treatment with zinc, # 28 drug and clioquinol.
  • ZinPyr-1 is a fluorescent dye that has a lower Kd value than FluoZin-3 and is used to measure zinc concentrations at lower concentrations. ZinPyr-1 is used to determine whether the # 28 drug of the present invention can act as an ion permeable carrier. It was. Specifically, after treating ZinPyr-1 to the cultured cerebral cortical neurons, # 28 drug (0.05 ⁇ M) and clioquinol (0.5 ⁇ M) were treated to a commonly used neuronal cell culture and the untreated group was used as a control. The change of zinc ion concentration in neurons was measured.
  • the increase of zinc by clioquinol was observed in normal culture conditions, but the increase by # 28 drug was not clearly observed. Therefore, after adding zinc at a concentration of 0.5 ⁇ M to the cell culture solution, the zinc concentration was observed in the cells. As a result, a significant increase in zinc was observed by treatment with clioquinol and # 28 drug (FIG. 21). Therefore, the # 28 drug of the present invention may also act as an ion permeable carrier and increase zinc to a low level when compared to clioquinol.
  • clioquinol has a high zinc affinity, which can chelate high concentrations of zinc, but it is well known that toxicity can be caused by easily increasing cytoplasmic zinc concentration due to the ion permeability of the drug itself. Therefore, Clioquinol is not suitable as a stroke treatment, but the # 28 drug of the present invention functions as an ion permeable carrier at a level that does not significantly increase the level of cytoplasmic zinc, and if the free zinc in the cytoplasm is increased to a level at which toxicity is induced, clay It is believed that zinc homeostasis is regulated to an appropriate level through nitridation.
  • AMPK enzyme plays an important role in zinc toxicity, which is considered to be one of the causes of stroke, and has been screened several times in a new compound candidate group that inhibits AMPK enzyme activity.

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Abstract

The present invention provides a novel composition for stroke treatment, which treats a stroke by treating with a novel compound inhibiting the AMPK activity of zinc neurotoxicity, which is a main cause of strokes.

Description

AMPK 억제기능에 기반한 뇌졸중 치료용 약학적 조성물Pharmaceutical composition for stroke treatment based on AMPK inhibitory function
본 발명은 뇌졸중 치료용 약학적 조성물에 관한 것으로서, 더 상세하게는 AMPK 억제기능에 기반한 새로운 뇌졸중 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for treating stroke, and more particularly, to a pharmaceutical composition for treating stroke based on AMPK inhibitory function.
뇌졸중(腦卒中, stroke)은 뇌혈류 이상으로 인해 갑작스레 유발된 국소적인 신경학적 증상을 통칭하는 말이다. 뇌는 몸 전체에서 무게로는 체중의 2%만 차지하지만, 뇌로 가는 혈류량은 심박출량의 15%, 산소 소모량은 몸 전체 산소 소모량의 20%나 된다. 게다가 뇌는 에너지원으로 포도당만을 사용하므로 에너지 공급이 잠시만 중단되어도 쉽게 괴사가 일어난다. 따라서 뇌혈류의 이상은 뇌손상과 밀접한 관련이 있다. 뇌졸중에 의한 뇌손상은 흥분독성(excitotoxicity), 산화성손상(oxidative stress), 아폽토시스(apoptosis) 및 아연독성(zinc neurotoxicity) 등의 다양한 독성기전을 포함하고 있어 뇌손상을 막기 위해서는 다양한 신경독성에 모두 효과적인 약물의 발굴이 필요하다.Stroke is a collective term for local neurological symptoms that are suddenly caused by abnormal brain blood flow. The brain takes up only 2% of its body weight by weight, but the blood flow to the brain is 15% of cardiac output and 20% of the body's oxygen consumption. In addition, the brain uses only glucose as an energy source, so necrosis easily occurs even if the energy supply is interrupted for a while. Therefore, abnormalities in cerebral blood flow are closely related to brain damage. Brain damage caused by stroke includes various toxic mechanisms such as excitotoxicity, oxidative stress, apoptosis and zinc neurotoxicity. Excavation is necessary.
현재, 뇌졸중 치료를 위한 많은 연구가 진행되었으나 아직까지 뚜렷한 치료제 개발이 되고 있지 않다. 뇌허혈-재관류 손상은 여러 가지 복잡한 신경세포사 기전에 입각한 약물 개발 및 효과 평가를 위해 시험관내 모델이나 동물 모델과 같은 객관적인 평가 시스템이 필요하나 약물치료에 다른 생체 징후의 변화나 부작용을 평가할 수 있는 방법이 전 임상 연구에서는 매우 제한적이어서 임상 시험 시 부작용으로 인한 실패를 초래할 가능성이 높다. 특히 NMDA 길항제(antagonist)를 중심으로 많은 임상연구들이 진행되었으나 모두 실패하였다. 이와 관련하여 대한민국 공개특허 제1283416호는 AMPK 저해제 화합물 C 또는 FAS 저해제 C75를 허혈성 마우스에 투여하여 경색부의 크기를 유의하게 감소시켜 뇌졸중 또는 허혈성 상해 후 기능이 보존 되게 하는 신경보호 방법에 대해 개시하고 있다. At present, a lot of research has been conducted for the treatment of stroke, but there is no clear therapeutic development. Cerebral ischemia-reperfusion injury requires an objective evaluation system, such as an in vitro model or an animal model, for drug development and effectiveness evaluation based on a variety of complex neuronal cell death mechanisms. In previous preclinical studies, they are very limited and are likely to cause side effects in clinical trials. In particular, many clinical studies have been conducted, especially NMDA antagonists, but all have failed. In this regard, Korean Patent Laid-Open Publication No. 1283416 discloses a neuroprotective method in which an AMPK inhibitor Compound C or FAS inhibitor C75 is administered to an ischemic mouse to significantly reduce the size of the infarct so that function is preserved after stroke or ischemic injury. .
그러나, 상기 선행기술의 경우 허혈 모델 동물에 대하여만 뇌신경보호 효과를 입증한 것으로서 허혈이 아닌 다른 기전에 의한 뇌졸중에 대한 치료효과까지 보증하는 것은 아니다. 본 발명은 상기와 같은 문제점을 포함하여 여러 문제점들을 해결하기 위한 것으로서, 다양한 기전에 의한 뇌졸중에 효과적인 AMPK 억제기능에 기반한 새로운 뇌졸중 치료제를 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.However, in the case of the prior art, the neuroprotective effect is demonstrated only for ischemic model animals, and it does not guarantee the therapeutic effect against stroke due to mechanism other than ischemia. The present invention is to solve various problems, including the above problems, it is an object of the present invention to provide a new stroke treatment based on the AMPK inhibitory function effective for various mechanisms of stroke. However, these problems are exemplary, and the scope of the present invention is not limited thereby.
본 발명은 일 관점에 따르면, 하기 화학식 1의 구조식을 갖는 화합물을 유효성분으로 함유하는, 뇌졸중 치료용 약학적 조성물이 제공된다:According to one aspect, the present invention provides a pharmaceutical composition for treating stroke, which contains a compound having a structural formula of Formula 1 as an active ingredient:
[화학식 1][Formula 1]
Figure PCTKR2017008569-appb-I000001
Figure PCTKR2017008569-appb-I000001
(상기 식에서 R1 내지 R5는 각각 독립적으로 수소, 히드록시기, 할로겐, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알킬기, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알콕시기, 아민기, 카르복실기이거나 R2 및 R3는 함께 -O-(CH2)n-O- 고리 또는 치환 또는 비치환된 벤젠고리를 형성하며(n은 1 내지 3의 정수), R6는 수소 또는 메틸기이고, R7은 수소 또는 할로겐이다)Wherein R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 7 carbon atoms, an amine group, a carboxyl group or R 2 And R 3 together form a —O— (CH 2 ) n —O— ring or a substituted or unsubstituted benzene ring (n is an integer from 1 to 3), R 6 is hydrogen or a methyl group, and R 7 is hydrogen Or halogen)
상기한 바와 같이 이루어진 본 발명의 일 실시예에 따르면, 뇌졸중의 주요 원인인 아연신경독성의 AMPK 활성을 억제하는 새로운 화합물을 처리하여 뇌졸중 치료효과를 구현할 수 있다. 물론 이러한 효과에 의해 본 발명의 범위가 한정되는 것은 아니다.According to one embodiment of the present invention made as described above, by treating a new compound that inhibits AMPK activity of zinc neurotoxicity, which is the main cause of stroke, it is possible to implement a stroke treatment effect. Of course, the scope of the present invention is not limited by these effects.
도 1은 배양된 대뇌피질 신경세포에 아연처리 후 신경독성이 증가되었음을 TUNEL 염색으로 확인한 사진(A)과 AMPK 억제제인 Compound C(+Cpd C) 처리 후 세포독성 정도를 TUNEL 염색과 LDH assay를 통해 정량화한 그래프이다(B 및 C).1 is a photograph showing that the neurotoxicity was increased after zinc treatment in cultured cerebral cortical neurons (A) and the degree of cytotoxicity after treatment with Compound C (+ Cpd C), an AMPK inhibitor, through TUNEL staining and LDH assay. Quantified graphs (B and C).
도 2는 배양된 대뇌피질 신경세포에 아연처리 후 AMPK 활성이 증가됨을 확인한 Western blot 사진(A)과 enzyme activity assay(B) 그래프이다. FIG. 2 is a Western blot photograph (A) and an enzyme activity assay (B) graph confirming that AMPK activity is increased after zinc treatment in cultured cerebral cortical neurons.
도 3은 배양된 대뇌피질 신경세포에 아연처리 후 아폽토시스 촉진 유전자인 Bim의 발현증가와 caspase-3 활성을 관찰한 Western blot 사진(A)과 AMPK 억제제인 Compound C(+Cpd C) 처리 후 Bim 발현증가와 caspase-3 활성이 모두 감소된 것을 확인한 Western blot 사진(B)이다. FIG. 3 shows Western blot photographs (A) and AMPK inhibitor Compound C (+ Cpd C), which showed increased expression of apoptosis promoting gene and caspase-3 activity after zinc treatment in cultured cerebral cortical neurons. Western blot photograph (B) shows that both increase and caspase-3 activity was decreased.
도 4는 AMPK activity assay kit(CycLex, Japan)와 recombinant AMPK(α2/β1/γ1; CycLex, Japan)를 구입하여 효소활성을 측정하고 compound C 효과와 비교하여 비슷하거나 좀 더 강한 억제효과를 나타내는 40개의 후보 화합물을 선별한 그래프이다.Figure 4 is the purchase of the AMPK activity assay kit (CycLex, Japan) and recombinant AMPK (α2 / β1 / γ1; CycLex, Japan) to measure the enzyme activity and compared to the compound C effect 40 showing a similar or stronger inhibitory effect It is a graph which selected two candidate compounds.
도 5는 배양된 대뇌피질 신경세포에 다양한 신경독성을 유발한 뒤, 선별된 7개의 화합물을 처리하고 세포 독성 정도를 LDH assay를 통해 정량화하여 다양한 신경 독성 억제 여부를 관찰한 그래프이다. FIG. 5 is a graph showing whether various neurotoxic inhibition is observed by inducing various neurotoxicity in cultured cerebral cortical neurons, treating 7 selected compounds and quantifying the degree of cytotoxicity through LDH assay.
도 6은 뇌졸중 동물모델에 최종 선별된 약물 #28을 투여하여 뇌손상 억제효과를 관찰한 그래프와 실험동물의 뇌손상 정도를 대조군과 비교한 그림이다. Figure 6 is a graph comparing the brain damage of the experimental animals and the control animals compared with the control group to observe the brain damage inhibitory effect by administering drug # 28 to the animal model of the stroke.
도 7은 최종 선별된 약물 #28를 랫트에 투여하고 비장(A), 간(B) 및 신장(C)을 적출하여 급성 독성 테스트 결과를 나타낸 그래프이다.FIG. 7 is a graph showing the results of an acute toxicity test by administering the final selected drug # 28 to rats and extracting spleen (A), liver (B) and kidney (C).
도 8은 기존에 선별된 약물 #28과 유사한 구조를 갖는 화합물을 검색하여 선별된 25개의 유사화합물을 아연독성이 유발된 생쥐의 대뇌피질 신경세포에 처리하여 신경보호 효과를 관찰한 그래프이다. FIG. 8 is a graph illustrating a neuroprotective effect by searching for compounds having a structure similar to that of the previously selected drug # 28 and treating 25 similar compounds selected to zinc-induced cerebral cortical neurons.
도 9는 아연독성에 대해 유의하게 약물효과를 나타낸 12개의 약물을 선정하여 NMDA에 의해 신경독성이 유발된 생쥐의 대뇌피질 신경세포에 처리하여 신경보호 효과를 관찰한 그래프이다. FIG. 9 is a graph illustrating the neuroprotective effect of 12 drugs that showed a significant drug effect on zinc toxicity, and were treated with NMDA-induced neurotoxicity in cerebral cortical neurons.
도 10은 H2O2에 의해 신경독성이 유발된 생쥐의 대뇌피질 신경세포에 선별된 12개의 약물을 처리하여 신경보호 효과를 관찰한 그래프이다.FIG. 10 is a graph illustrating the neuroprotective effect of 12 drugs selected in cerebral cortical neurons of mice induced with neurotoxicity by H 2 O 2 .
도 11은 신경세포에 본 발명의 #28 약물 및 클리오퀴놀을 농도별로 처리하고 자유 아연 농도를 pZn meter로 분석한 그래프이다. FIG. 11 is a graph illustrating treatment of # 28 drug and clioquinol of the present invention to neurons by concentration, and analysis of free zinc concentration with a pZn meter.
도 12는 test tube 상에서 아연, 클리오퀴놀 및 #28 약물을 처리하고 형광 염색물질인 Newport green DCF(Kd(Zn)=1 uM)을 사용하여 자유 아연 농도 변화를 분석한 그래프이다. FIG. 12 is a graph illustrating the change of free zinc concentration using zinc, clioquinol, and # 28 drug on a test tube and using Newport green DCF (Kd (Zn) = 1 uM) as a fluorescent dye.
도 13은 test tube 상에서 아연, 클리오퀴놀 및 #28 약물을 처리하고 형광 염색물질인 FluoZin-3(Kd(Zn)=15 nM)을 사용하여 자유 아연 농도 변화를 분석한 그래프이다. FIG. 13 is a graph illustrating the change in free zinc concentration using zinc, clioquinol, and # 28 drug on a test tube, and using a fluorescent dye, FluoZin-3 (Kd (Zn) = 15 nM).
도 14는 신경세포에 아연, 클리오퀴놀, 피리치온 및 #28 약물을 처리하고 아연신경독성 억제효과를 분석한 그래프이고(A) DTDP 및 #28 약물을 처리하고 아연신경독성 억제효과를 분석한 그래프이다(B).14 is a graph of treatment of zinc, clioquinol, pyrithione, and # 28 drugs in neuronal cells and the effects of zinc neurotoxicity inhibition (A) treatment of the DTDP and # 28 drugs and analysis of zinc neurotoxicity inhibitory effects. It is a graph (B).
도 15는 신경세포에 4CO1, 4CO7 및 #28 약물을 처리 후 FluoZin-3 염색물질을 처리하여 공초점 레이저 현미경으로 관찰한 사진이고(A) 형광크기를 정량적으로 분석한 그래프이다(B).15 is a photograph of a neuron treated with 4CO1, 4CO7, and # 28 drugs and treated with FluoZin-3 staining material, followed by confocal laser microscopy (A).
도 16은 신경세포에 과산화수소(H2O2), 아연 킬레이터인 TPEN 또는 CaEDTA, 칼슘 킬레이터 ZnEDTA를 처리하고 신경독소 감소여부를 분석한 그래프이다. Figure 16 is a graph of neuronal cells treated with hydrogen peroxide (H 2 O 2 ), zinc chelator TPEN or CaEDTA, calcium chelator ZnEDTA and whether neurotoxin reduction.
도 17은 신경세포에 과산화수소(H2O2), TPEN, #28, 4C01, 4C07 약물을 처리하고 FluoZin-3 염색한 후 상기 약물의 독성 억제 효과를 현미경으로 관찰한 사진이다. 17 is a photomicrograph of the toxic inhibitory effect of the drug after treatment with hydrogen peroxide (H 2 O 2 ), TPEN, # 28, 4C01, 4C07 drug to FluoZin-3.
도 18은 신경세포에 이오노마이신 및 #28 약물을 농도별로 처리하여 이온투과담체의 독성 억제여부를 분석한 그래프이다. FIG. 18 is a graph illustrating whether the iontocarrier is inhibited in toxicity by treating ionomycin and # 28 drugs in concentrations in neurons.
도 19는 test tube 상에서 Ca2 +, #28 약물 및 EDTA를 처리하고 형광물질인 Fura-2 dye 처리하여 칼슘에 대한 킬레이트화 효과를 분석한 그래프이다. 19 is a graph analyzing the chelation effect on calcium treated with Ca 2 + , # 28 drug and EDTA on the test tube and treated with the fluorescent Fura-2 dye.
도 20은 신경세포에 TPEN, 아연, #28 약물 및 클리오퀴놀을 처리하여 caspase-3의 활성을 분석한 그래프이고(A) 신경독성(LDH 분비)을 분석한 그래프이다(B).20 is a graph analyzing the activity of caspase-3 by treating TPEN, zinc, # 28 drug and clioquinol in neurons (A) and neurotoxicity (LDH secretion) (B).
도 21은 신경세포 배양액을 일반 배양액, 아연 첨가 배양액, 아연 제거 배양액으로 분류하고 클리오퀴놀 및 #28 약물의 처리하고 형광물질인 ZinPyr-1을 처리하여 신경세포 내 아연 농도의 변화를 분석한 그래프이다.FIG. 21 is a graph illustrating the change in the concentration of zinc in neurons by treating neuronal cultures with normal cultures, zinc-added cultures, and zinc-free cultures, treatment with clioquinol and # 28 drugs, and treatment with the fluorescent substance ZinPyr-1. .
용어의 정의:Definition of Terms:
본 문서에서 사용되는 "AMPK(AMP-activated protein kinase)"는 촉매 α 서브유닛(α1 또는 α2), 및 2개의 조절 서브유닛(β 및 γ)으로 구성된 이형삼량체 단백질(heterologous trimer protein)이다. AMPK는 세포 에너지 수준이 낮을 때 인산화되고 활성화되며 다시 세포 대사 작용을 조절하여 유전자 발현을 장기간에 걸쳐 조절하여 ATP의 수준을 회복한다. AMP/ATP 비의 증가, 세포 pH 및 산화 환원 상태의 변화 및 크레아틴/포스포크레아틴 비의 증가가 AMPK를 활성화시키는 것으로 공지되어 있다.As used herein, "AMP-activated protein kinase" (AMPK) is a heterologous trimer protein consisting of a catalyst α subunit (α1 or α2), and two regulatory subunits (β and γ). AMPK is phosphorylated and activated at low cellular energy levels, and then regulates cell metabolism to restore gene levels over time by regulating gene expression over time. Increasing the AMP / ATP ratio, changing the cell pH and redox state, and increasing the creatine / phosphocreatin ratio are known to activate AMPK.
발명의 상세한 설명:Detailed description of the invention:
본 발명은 일 관점에 따르면, 하기 화학식 1의 구조식을 갖는 화합물을 유효성분으로 함유하는, 뇌졸중 치료용 약학적 조성물이 제공된다:According to one aspect, the present invention provides a pharmaceutical composition for treating stroke, which contains a compound having a structural formula of Formula 1 as an active ingredient:
[화학식 1][Formula 1]
Figure PCTKR2017008569-appb-I000002
Figure PCTKR2017008569-appb-I000002
(상기 식에서 R1 내지 R5는 각각 독립적으로 수소, 히드록시기, 할로겐, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알킬기, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알콕시기, 아민기, 카르복실기이거나, R2 및 R3는 함께 -O-(CH2)n-O- 고리 또는 치환 또는 비치환된 벤젠고리를 형성하며(n은 1 내지 3의 정수), R6는 수소 또는 메틸기이고, R7은 수소 또는 할로겐이다)Wherein R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 7 carbon atoms, an amine group or a carboxyl group, or R 2 and R 3 together form a —O— (CH 2 ) n —O— ring or a substituted or unsubstituted benzene ring (n is an integer from 1 to 3), R 6 is hydrogen or a methyl group, and R 7 is Hydrogen or halogen)
상기 뇌졸중 치료용 약학적 조성물에 있어서, 상기 치환된 알킬기는 트리플루오로메틸기일 수 있고 상기 할로겐은 요오드, 브롬 또는 염소일 수 있다. In the pharmaceutical composition for treating stroke, the substituted alkyl group may be a trifluoromethyl group and the halogen may be iodine, bromine or chlorine.
상기 뇌졸중 치료용 약학적 조성물에 있어서, 상기 화합물은 (5Z)-5-(1H-Indol-3-ylmethylene)-2-{[2-(trifluoromethyl) phenyl]amino}-1,3-thiazol-4(5H)-one,(5Z)-5-(1H-Indol-3-ylmethylene)-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-4(5H)-one,(5Z)-2-[(3-Bromophenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one,(5Z)-5-(1H-Indol-3-ylmethylene)-2-[(4-methylphenyl)amino]-1,3-thiazol-4(5H)-one, (5Z)-5-(1H-Indol-3-ylmethylene)-2-[(3-methylphenyl)amino]-1,3-thiazol-4(5H)-one,(5Z)-2-Anilino-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one, no]-1,3-thiazol-4(5H)-one,(5E)-5-[(2-Methyl-1H-indol-3-yl)methylene]-2-[(4-methylphenyl)amino]-1,3-thiazol-4(5H)-one,3-{[(5Z)-5-(1H-Indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid,2-{[(5E)-5-(1H-Indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid, (5Z)-2-[(2-Chlorophenyl)amino]-5-[(2-methyl-1H-indol-3-yl)methylene]-1,3-thiazol-4(5H)-one, 또는 2-Hydroxy-5-{[(5Z)-5-(1H-indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid일 수 있다. In the pharmaceutical composition for treating stroke, the compound is (5Z) -5- (1H-Indol-3-ylmethylene) -2-{[2- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2-{[3- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one, ( 5Z) -2-[(3-Bromophenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol- 3-ylmethylene) -2-[(4-methylphenyl) amino] -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2-[(3 -methylphenyl) amino] -1,3-thiazol-4 (5H) -one, (5Z) -2-Anilino-5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H)- one, no] -1,3-thiazol-4 (5H) -one, (5E) -5-[(2-Methyl-1H-indol-3-yl) methylene] -2-[(4-methylphenyl) amino ] -1,3-thiazol-4 (5H) -one, 3-{[(5Z) -5- (1H-Indol-3-ylmethylene) -4-oxo-4,5-dihydro-1,3-thiazol -2-yl] amino} benzoic acid, 2-{[(5E) -5- (1H-Indol-3-ylmethylene) -4-oxo-4,5-dihydro-1,3-thiazol-2-yl] amino} benzoic acid, (5Z) -2-[(2-Chlorophenyl) amino] -5-[(2-methyl-1H-indol-3-yl) methylene] -1,3-thiazol-4 (5H)- one, or 2-Hydroxy-5-{[(5Z) -5- (1H-indol-3-y lmethylene) -4-oxo-4,5-dihydro-1,3-thiazol-2-yl] amino} benzoic acid.
상기 뇌졸중 치료용 약학적 조성물에 있어서, 상기 뇌졸중은 출혈성(hemorrhagic) 뇌졸중, 허혈성(ischemic) 뇌졸중 또는 금속 독성(metal toxicity) 뇌졸중일 수 있고, 상기 금속은 납(lead), 수은(mercury), 망간(manganese), 비소(arsenic), 탈륨(thallium), 철(iron), 아연(zinc), 카드뮴(cadmium), 비스무스(bismuth) 또는 주석(tin)일 수 있으며, 상기 허혈성 뇌졸중은 흥분성 신경세포사 또는 산화성 신경세포사에 의해 유발되는 것일 수 있다. In the pharmaceutical composition for treating stroke, the stroke may be a hemorrhagic stroke, an ischemic stroke or a metal toxicity stroke, and the metal may be lead, mercury, or manganese. (manganese), arsenic, thallium, iron, iron, zinc, cadmium, bismuth or tin, the ischemic stroke may be excitatory neuronal death or It may be caused by oxidative neuronal death.
본 발명의 약학적 조성물에서 상기 화합물의 유효량은 환자의 환부의 종류, 적용부위, 처리회수, 처리시간, 제형, 환자의 상태, 보조제의 종류 등에 따라 변할수 있다. 사용량은 특별히 한정되지 않지만, 0.01μg/kg/day 내지 10 mg/kg/day일일 수 있다. 상기 1일량은 1일에 1회, 또는 적당한 간격을 두고 하루에 2~3회에 나눠 투여해도 되고, 수일(數日) 간격으로 간헐(間歇)투여해도 된다.The effective amount of the compound in the pharmaceutical composition of the present invention may vary depending on the type of affected part of the patient, the site of application, the number of treatments, the treatment time, the dosage form, the condition of the patient, the type of supplement, and the like. The amount used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The daily dose may be administered once or in two or three times a day at appropriate intervals or may be administered intermittently at intervals of several days.
본 발명의 약학적 조성물에서 상기 화합물은, 조성물 총 중량에 대하여 0.1-100 중량%로 함유될 수 있다. 본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 또한, 약학적 조성물의 제조에는 고체 또는 액체의 제제용 첨가물을 사용할 수 있다. 제제용 첨가물은 유기 또는 무기 중 어느 것이어도 된다.In the pharmaceutical composition of the present invention, the compound may be contained at 0.1-100% by weight based on the total weight of the composition. The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, the preparation of the pharmaceutical compositions may be used as additives for the preparation of solids or liquids. The additive for preparation may be either organic or inorganic.
부형제로서는 예를 들면 유당, 자당, 백당, 포도당, 옥수수 전분(cornstarch), 전분, 탈크, 소르비트, 결정 셀룰로오스, 덱스트린, 카올린, 탄산칼슘 및 이산화규소 등을 들 수 있다. 결합제로서는 예를 들면 폴리비닐알코올, 폴리비닐에테르, 에틸셀룰로오스, 메틸셀룰로오스, 아라비아고무, 트래거캔스(tragacanth), 젤라틴, 셀락(shellac), 히드록시프로필셀룰로오스, 히드록시프로필메틸셀룰로오스, 구연산칼슘, 덱스트린 및 펙틴(pectin) 등을 들 수 있다. 활택제로서는 예를 들면 스테아린산마그네슘, 탈크, 폴리에틸렌글리콜, 실리카, 경화식물유 등을 들 수있다. 착색제로서는 통상 의약품에 첨가하는 것이 허가되어 있는 것이라면 모두 사용할 수 있다. 이들의 정제, 과립제에는 당의(糖衣), 젤라틴코팅, 기타 필요에 따라 적절히 코팅할 수 있다. 또한, 필요에 따라 방부제, 항산화제 등을 첨가할 수있다.Examples of excipients include lactose, sucrose, white sugar, glucose, cornstarch, starch, talc, sorbet, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hardened vegetable oil, and the like. As a coloring agent, if it is normally permitted to add to a pharmaceutical, all can be used. These tablets and granules can be appropriately coated according to sugar, gelatin coating and other needs. Moreover, preservatives, antioxidants, etc. can be added as needed.
본 발명의 약학적 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며(예: 문헌 [Remington's Pharmaceutical Science, 최신판; Mack Publishing Company, Easton PA), 제제의 형태는 특별히 한정되는 것은 아니다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌[Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042(Chapter 87: Blaug, Seymour)에 기술되어 있다. The pharmaceutical compositions of the present invention may be prepared in any formulation commonly prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. . These formulations are described in Remington's Pharmaceutical Science, 15 th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a prescription generally known for all pharmaceutical chemistries.
본 발명의 약학적 조성물에서 상기 화합물은 경구 또는 비경구로 투여되는 것이 가능하며, 바람직하게는 비경구 투여로 정맥내 주입, 피하 주입, 뇌실내 주입(intracerebroventricular injection), 뇌척수액내 주입(intracerebrospinal fluid injection), 근육내 주입 및 복강 주입 등으로 투여할 수 있다.In the pharmaceutical composition of the present invention, the compound may be administered orally or parenterally, and preferably, by parenteral administration, intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection Intramuscular injection, intraperitoneal injection, and the like.
이하, 실시예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms, and the following embodiments are intended to complete the disclosure of the present invention, the scope of the invention to those skilled in the art It is provided to inform you completely.
일반적 방법General method
대뇌피질 신경세포(Cerebral Cortex Neurons) 배양Cerebral Cortex Neurons Culture
본 발명에서 사용한 마우스 대뇌피질 신경세포는 마우스 배아의 뇌로부터 추출 배양하여 5% 우태아혈청(FBS)과 5% 마혈청(HS)을 첨가한 Dulbecco's modified Eagle's medium(DMEM, Gibco, Grand Island, NY, US)을 이용하여 95% 습도, 5% CO2 및 37℃ 온도 조건에서 배양하였다. 상기 세포의 활성화와 분화를 위해 24-well 조직 배양 플레이트에서 2 x104 세포의 밀도로 증식하였고 아연 및 화합물을 처리하기 전에는 FBS와 HS가 없는 MEM 배지에서 배양하였다.The mouse cerebral cortical neurons used in the present invention were extracted and cultured from the brains of mouse embryos and added Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) to which 5% fetal bovine serum (FBS) and 5% horse serum (HS) were added. , US) was incubated at 95% humidity, 5% CO 2 and 37 ℃ temperature conditions. The cells were grown to a density of 2 × 10 4 cells in a 24-well tissue culture plate for activation and differentiation of cells and cultured in MEM medium without FBS and HS before treatment with zinc and compounds.
실시예Example 1:  One: AMPKAMPK 억제제에 의한 세포독성 감소확인 Reduction of Cytotoxicity by Inhibitors
본 발명의 일 실시예에 따라 아연독성(Zinc toxicity)은 뇌졸중의 대표적인 원인기전 중 하나로서, 상기 배양된 대뇌피질 신경세포에서 유발된 아연독성이 AMPK 억제제 처리에 따른 독성 감소여부를 확인하였다. According to an embodiment of the present invention, zinc toxicity is one of the representative causes of stroke, and it was confirmed whether zinc toxicity induced in the cultured cerebral cortical neurons was reduced by AMPK inhibitor treatment.
구체적으로, 본 발명의 일 실시예에 따른 배양된 대뇌피질 신경세포에 300 μM의 아연(ZnCl2)을 10분간 처리한 후 제거하고 10시간 후에 세포독성(cytotoxicity)을 TUNEL 염색 또는 LDH(Lactate Dehydrogenase) 분석을 통해 관찰하였고 상기 세포에 AMPK 억제제인 Compound C(+Cpd C, Tocris)를 20 μM로 처리한 후 신경독성 감소를 관찰하였다. Specifically, the cultured cerebral cortical neurons according to one embodiment of the present invention treated with 300 μM of zinc (ZnCl 2 ) for 10 minutes and then removed and cytotoxicity (cytotoxicity) after 10 hours TUNEL staining or LDH (Lactate Dehydrogenase) The cells were treated with AMPK inhibitor Compound C (+ Cpd C, Tocris) at 20 μM and then neurotoxic reduction was observed.
그 결과, 대뇌피질 신경세포에 아연 처리 후 신경독성이 증가되었음을 TUNEL 염색으로 확인하였고(도 1A) 세포독성 정도를 TUNEL 염색과 LDH 분석을 통해 정량화하고 AMPK 억제제인 Compound C(+Cpd C) 처리에 의해 아연신경독성이 현저히 감소했음을 확인하였다(도 1B 및 1C). As a result, TUNEL staining confirmed that neurotoxicity was increased after zinc treatment in cerebral cortical neurons (Fig. 1A). The cytotoxicity was quantified by TUNEL staining and LDH analysis and treated with Compound C (+ Cpd C), an AMPK inhibitor. It was confirmed that zinc neurotoxicity was significantly reduced (FIGS. 1B and 1C).
실시예Example 2: 아연처리 후  2: after zinc treatment AMPKAMPK 활성 관찰 Active observation
본 발명의 일 실시예에 따라 아연독성과 AMPK 효소 활성간의 상관관계를 파악하고자 대뇌피질 신경세포에 아연 처리 후 AMPK 활성을 웨스턴 블랏과 효소활성 분석을 통해 관찰하였다.In order to determine the correlation between zinc toxicity and AMPK enzyme activity according to an embodiment of the present invention, the AMPK activity was observed by Western blot and enzyme activity analysis after zinc treatment in cortical neurons.
2-1: 2-1: 웨스턴Weston 블랏Blot (Western blot)(Western blot)
배양된 대뇌피질 신경세포에 300 μM의 아연(ZnCl2)을 10분간 처리한 후 제거하고 0.5, 1, 2, 4, 6시간 후에 상기 세포 샘플을 단백질 레더와 함께 폴리아크릴아마이드 겔에 로딩하고 단백질 크기에 따라 분리하였다. 그 후, 항체를 처리하고 세척한 뒤 판독하였다. The cultured cerebral cortical neurons were treated with 300 μM of zinc (ZnCl 2 ) for 10 minutes and then removed. After 0.5, 1, 2, 4, and 6 hours, the cell samples were loaded on polyacrylamide gels with protein leather and protein Isolate according to size. The antibody was then processed, washed and read.
그 결과, 아연처리 후 30분 후부터 AMPK alpha-1과 alpha-2의 트레오닌 잔기(threonine residue)에 인산화됨이 관찰되었는데 이는 AMPK 활성(phosphorylated AMPK)을 의미한다. 그러나 다른 세린 잔기(serine residue)의 인산화는 관찰되지 않았다(도 2A).As a result, 30 minutes after zinc treatment, phosphorylation was observed at threonine residues of AMPK alpha-1 and alpha-2, which means AMPK activity (phosphorylated AMPK). However, no phosphorylation of other serine residues was observed (FIG. 2A).
2-2: 효소활성 분석(enzyme activity assay)2-2: enzyme activity assay
대뇌피질 신경세포에 상기와 같은 조건으로 아연을 처리한 후 0.5, 1, 2, 4 및 6시간 경과에 따라 각각 단백질 추출물을 얻어 AMPK activity assay kit(CycLex, Japan)를 이용하여 효소활성을 측정하였다.After treatment with zinc in cerebral cortical neurons under the above conditions, protein extracts were obtained at 0.5, 1, 2, 4 and 6 hours, respectively, and enzyme activity was measured using an AMPK activity assay kit (CycLex, Japan). .
그 결과, 아연 처리에 따른 AMPK 효소의 활성은 시간 의존적(Time-dependent) 으로 증가하는 것을 관찰하였다(도 2B). As a result, it was observed that the activity of the AMPK enzyme with zinc treatment increased time-dependently (FIG. 2B).
실시예Example 3:  3: AMPKAMPK 활성억제를 통한 아연독성 억제 Inhibition of zinc toxicity through inhibition of activity
본 발명의 일 실시예에 따라 대뇌피질 신경세포에 아연을 처리하여 AMPK 효소 활성이 증가하는 것이 세포사멸(apoptosis)과 관련이 있는지 관찰하였다. In accordance with an embodiment of the present invention, it was observed whether the increase in AMPK enzyme activity was correlated with apoptosis by treating zinc cortical neurons.
구체적으로, 대뇌피질 신경세포에 300 μM의 아연(ZnCl2)을 2, 3, 4, 5 및 6시간 동안 처리하여 아연독성을 유발하였다. 상기 각 샘플의 단백질을 분리하여 웨스턴 블랏을 실시하였고 AMPK 억제제인 Compound C(+Cpd C)를 20 μM로 처리하여 아연독성과 아폽토시스와의 관계를 확인하였다. Specifically, zinc toxicity was induced by treating cerebral cortical neurons with 300 μM of zinc (ZnCl 2 ) for 2, 3, 4, 5 and 6 hours. The protein of each sample was isolated and subjected to Western blot, and the relationship between zinc toxicity and apoptosis was confirmed by treatment with Compound C (+ Cpd C), an AMPK inhibitor, at 20 μM.
그 결과, 아연 처리 후 3시간 이후부터 BH3-only Bcl family 중에 하나인 아폽토시스 촉진 단백질(pro-apoptotic protein)인 Bim의 발현 증가가 관찰되었고caspase-3 활성도 관찰할 수 있었다(도 3A). 그러나 아연독성에 의한 Bim의 증가와 caspase-3 활성 증가가 AMPK 억제제인 compound C 처리에 의해 모두 감소된 것을 확인하였다(도 3B). 그러므로 AMPK 효소는 아연독성 기전에서 아폽토시스(apoptosis)와 관련되어 있으며 AMPK 효소의 활성억제는 아연독성에 의한 아폽토시스를 억제하는 것을 의미한다. As a result, the expression of Bim, a pro-apoptotic protein, which is one of the BH3-only Bcl family, was observed from 3 hours after zinc treatment and caspase-3 activity was also observed (FIG. 3A). However, it was confirmed that both the increase of Bim and the increase of caspase-3 activity due to zinc toxicity were reduced by compound C treatment, which is an AMPK inhibitor (FIG. 3B). Therefore, AMPK enzymes are associated with apoptosis in the zinc toxicity mechanism, and the inhibition of AMPK enzyme activity means inhibition of apoptosis by zinc toxicity.
실시예Example 4:  4: AMPKAMPK 활성억제 화합물 스크리닝 및 억제효과 확인 Screening and inhibitory effect of activity inhibitory compounds
4-1: 구조기반 가상 스크리닝(Structure-based virtual screening)4-1: Structure-based virtual screening
본 발명의 일 실시예에 따라 AMPK 활성억제제로 작용할 가능성이 있는 화합물을 대상으로 1차 스크리닝을 실시하였다. According to one embodiment of the present invention, the primary screening was performed on compounds that may act as AMPK inhibitors.
구체적으로, 상기 선행연구를 통해 AMPK 활성이 아연독성 기전에 관련되어 있음을 확인하였고, 다른 연구논문에서도 흥분독성 기전에서 AMPK 활성 및 Bim 발현 증가가 보고되었다. AMPK 효소는 α, β 및 γ 3개의 서브유닛이 복합구조를 이루어 작용하는 효소로, 이중 alpha 서브유닛이 키나아제 효소활성을 가지고 있다. 기존의 연구결과에서 AMPK alpha2가 alpha1과 달리 넉-아웃 마우스(knock-out mice)에서 허혈(ischemia)에 의한 신경독성을 현저히 억제한다고 보고하였다. 따라서 본 발명은 alpha2를 타겟으로 하여 AMPK 효소활성을 억제할 가능성이 있는 후보 케미컬(chemicals)을 구조기반 가상 스크리닝(Structure-based virtual screening)을 통해 선별하였고 결론적으로 208개의 후보 화합물이 선별되었다. Specifically, it was confirmed through the preceding studies that AMPK activity is related to the zinc toxicity mechanism, and other studies have reported an increase in AMPK activity and Bim expression in the excitatory toxicity mechanism. The AMPK enzyme is an enzyme in which three subunits of α, β and γ form a complex structure, and the alpha subunit has kinase enzymatic activity. Previous studies have shown that AMPK alpha2 significantly inhibits neurotoxicity caused by ischemia in knock-out mice, unlike alpha1. Therefore, in the present invention, candidate chemicals capable of inhibiting AMPK enzyme activity by targeting alpha2 were selected through structure-based virtual screening, and finally 208 candidate compounds were selected.
4-2: 4-2: AMPKAMPK 효소활성 분석( Enzyme activity analysis AMPKAMPK enzyme activity assay) enzyme activity assay)
상기 실시예 4-1에서 선별된 화합물 208개를 화합물 라이브러리 제조회사(Interbioscreen, Russia)로부터 입수하여 이들의 AMPK 효소활성 억제효과 관찰을 통해 2차 스크리닝을 실시하였다.The 208 compounds selected in Example 4-1 were obtained from a compound library manufacturer (Interbioscreen, Russia) and subjected to secondary screening through observation of their inhibitory effect on AMPK enzyme activity.
구체적으로, AMPK 효소활성은 AMPK activity assay kit(CycLex, Japan)와 recombinant AMPK(α2/β1/γ1; CycLex, Japan)를 이용하여 측정하였다. 상기 선별된 208개의 화합물과 기존에 잘 알려진 AMPK 억제제 compound C의 AMPK 효소활성 억제 효과를 측정한 결과, 40개의 약물이 10 μM에서 compound C와 비슷하거나 더 좋은 억제효과를 나타내는 것을 확인하였다. 상기 40개의 약물을 하기 표 1에 표시하였다. Specifically, AMPK enzyme activity was measured using AMPK activity assay kit (CycLex, Japan) and recombinant AMPK (α2 / β1 / γ1; CycLex, Japan). As a result of measuring the inhibitory effect of AMPK enzyme activity of the selected 208 compounds and the well-known AMPK inhibitor compound C, it was confirmed that 40 drugs showed a similar or better inhibitory effect than compound C at 10 μM. The 40 drugs are shown in Table 1 below.
Figure PCTKR2017008569-appb-T000001
Figure PCTKR2017008569-appb-T000001
4-3: 아연독성 억제효과 관찰4-3: Observation of zinc toxicity inhibitory effect
상기 실시예 4-2에서 선별된 화합물 40 개의 아연 신경독성 억제효과 관찰을 통해 3차 스크리닝을 실시하였다. Tertiary screening was performed by observing the zinc neurotoxic inhibitory effect of 40 compounds selected in Example 4-2.
구체적으로, 배양된 마우스의 대뇌피질 신경세포에 300 μM의 아연을 10분간 처리하고 제거한 후 12시간이 경과한 시점에서 상기 선별한 40개의 약물들을 각각 아연과 함께 처리하여 신경보호효과를 관찰하였다. 상기 세포의 사멸 정도는 LDH 분석을 통해 정량화하였는데 각각의 효과를 평균하여 수치로 나타내었고 선별된 각각의 약물들에 대해 별도의 실험을 4회 수행하였다(도 4).Specifically, the neuroprotective effect was observed by treating each of the 40 selected drugs with zinc at 12 hr after treating and removing 300 μM of zinc for 10 minutes to cultured cerebral cortical neurons. The degree of death of the cells was quantified by LDH analysis, and each effect was expressed as an average value, and four separate experiments were performed for each of the selected drugs (FIG. 4).
그 결과, 도 4에 나타난 바와 같이, 일부 화합물은 아연독성에 의한 세포사멸을 억제하였는데, 하기 표 2에 나타난 바와 같이 이 중 7개의 화합물(#6, #11, #14, #17, #28, #35 및 #37)이 아연독성을 유의하게 억제하는 것으로 확인되었다. As a result, as shown in Figure 4, some compounds inhibited apoptosis due to zinc toxicity, as shown in Table 2 of the seven compounds (# 6, # 11, # 14, # 17, # 28) , # 35 and # 37) were found to significantly inhibit zinc toxicity.
Figure PCTKR2017008569-appb-T000002
Figure PCTKR2017008569-appb-T000002
4-4: 다양한 신경독성에 대한 억제효과 관찰4-4: Observation of inhibitory effects on various neurotoxicities
상기 실시예 4-3에서 선별한 7개 화합물의 신경독성 억제효과 관찰을 통해 4차 스크리닝을 실시하였다.  Fourth screening was performed by observing the neurotoxic inhibitory effect of the seven compounds selected in Example 4-3.
구체적으로, 배양된 마우스의 대뇌피질 신경세포에 50 μM NMDA, 50 μM FeCl2, 100 μM H2O2, 2 μM TPEN(N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, Sigma), 500 nM 스토로스포린(staurosporine, Abchem) 및 10 μM 에토포사이드(etoposide, Sigma)를 각각 처리 하여 세포독성을 유발한 후 상기 선별된 7개 화합물을 20 μM의 농도로 처리하여 세포사멸 억제 효과를 관찰하였고 세포사멸 정도는 LDH assay를 통해 정량화하였다. 상기 사용한 신경독성 모델은 뇌졸중의 원인기전으로 생각되는 흥분독성, 산화성 손상 및 세포사멸(apoptosis) 등이 포함되어 있는데 그 중 흥분독성 모델로는 NMDA를 처리하였고, 산화성손상 모델로는 철(iron) 독성과 H2O2 독성 모델을 사용하였으며, 세포사멸 모델로는 TPEN, 스토로스포린(staurosporine) 및 에토포사이드(etoposide) 독성모델을 이용하였다. 상기 TPEN은 아연 킬레이터(zinc chelator)로 신경세포에서 전형적인 세포사멸을 유발하는 것으로 잘 알려져 있고, 스토로스포린은 효소활성 억제제(kinase inhibitor)로 역시 대표적인 세포사멸 유발물질 중 하나이며 에토포사이드는 DNA 손상으로 인한 세포사멸을 유발한다고 알려진 약물이다. Specifically, in the cultured cerebral cortical neurons 50 μM NMDA, 50 μM FeCl 2 , 100 μM H 2 O 2 , 2 μM TPEN (N, N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine, Sigma), 500 nM storosporine (Abchem) and 10 μM etoposide (Sigma) were treated respectively to induce cytotoxicity, and then the selected seven compounds were treated at a concentration of 20 μM to inhibit apoptosis The effect was observed and the degree of cell death was quantified by LDH assay. The neurotoxicity model used includes excitatory toxicity, oxidative damage, and apoptosis, which are thought to be the cause of stroke. Among the excitatory toxicity models, NMDA was treated, and the oxidative damage model was iron. Toxicity and H 2 O 2 toxicity model was used, and apoptosis model was used as TPEN, staurosporine and etoposide toxicity model. The TPEN is a zinc chelator and is known to induce typical apoptosis in neurons. Storosporin is one of the representative apoptosis inducing enzymes as a kinase inhibitor and etoposide is DNA. It is a drug known to cause cell death due to damage.
그 결과, 상기 선별된 7개의 약물을 함께 처리하여 신경독성 감소에 따른 신경보호 효과를 관찰하였는데 특히, 화합물 #35 및 화합물 #28이 모든 독성에 억제효과를 나타냄을 알 수 있었다(도 5). 라이브러리 구입처로부터 상기 화합물 #28은 하기와 같은 구조를 갖는 (Z)-5-((1H-indol-3-yl)methylene)-2-((3-hydroxyphenyl)amino)thiazol-4(5H)-one임을 확인하였다:As a result, the selected seven drugs were treated together to observe the neuroprotective effect of reducing neurotoxicity. In particular, it was found that Compound # 35 and Compound # 28 had an inhibitory effect on all toxicity (FIG. 5). Compound # 28 from the library vendor was (Z) -5-((1H-indol-3-yl) methylene) -2-((3-hydroxyphenyl) amino) thiazol-4 (5H)-having the structure Verified that it is one:
Figure PCTKR2017008569-appb-I000003
Figure PCTKR2017008569-appb-I000003
실시예Example 5: 뇌졸중 동물모델에서 뇌손상 억제효과 관찰 5: Inhibitory effect on brain injury in stroke animal model
상기 실시예 4-4에서 최종 선별된 화합물인 #28이 실제 동물 모델에서도 효과적인지 확인하기 위해 상기 화합물을 뇌손상을 유발한 뇌졸중 모델 동물에 처리한 후, 뇌손상 억제효과가 나타나는지 여부를 조사하였다.In order to confirm whether the compound selected as # 28, which was finally selected in Example 4-4, was effective in an animal model, the compound was treated with a stroke model animal that induced brain injury, and then whether the brain damage inhibitory effect was observed was examined. .
구체적으로, 8-9 주령이 된 수컷 Sprague-Dawley(SD) 랫트(rat)를 이용하여 영구 중뇌동맥(MCA) 폐쇄모델을 제작하였다. 영구 중뇌동맥 폐쇄(MCAO) 전 30분 및 폐쇄 후 10분경과 시점에서 레이저 도플러 혈류측정기(Laser-Doppler flowmetry)를 이용하여 대뇌 혈류(CBF)를 측정하였고 하기 표 3에 표시하였다. 그 후, 상기 최종 선별된 AMPK 억제제 후보 화합물인 #28(3 ㎕ 내 75 ng) 또는 비히클(10% DMSO)을 MCAO 후 15분에 3.8 mm 깊이로 0.8 mm 후면 및 브레그마의 측면 1.2 mm의 뇌실내 주입(intracerebroventricular injection)하였다. 이 후, 상기 랫트의 운동 결핍(motor deficit) 정도를 평가 하였는데 평가의 기준(Longa et al., 1989)은 결핍을 나타내지 않는 것은 정상(Normal), 수직으로 정지할 때 앞발 연장의 실패는 경증(mild), 반대쪽으로 회전하는 것은 보통(moderate) 및 회전 손실(loss of circling) 또는 반사작용의 손실은 심각한 결핍(severe deficit)으로 분류하였다. 또한, 상기 랫트 모델에 허혈(ischemia)을 유도 후 24시간 경과한 시점에서 뇌를 수득하였고 2% 2,3,5-triphenyl tetrazolium chloride(TTC)로 염색하여 뇌경색(cerebral infarction) 정도를 측정하였다. Specifically, a permanent model of cerebral artery (MCA) was constructed using male Sprague-Dawley (SD) rats aged 8-9 weeks. CBF was measured using laser Doppler flowmetry 30 minutes before and after 10 minutes after permanent middle cerebral artery occlusion (MCAO) and is shown in Table 3 below. Subsequently, the final selected AMPK inhibitor candidate compound # 28 (75 ng in 3 μl) or vehicle (10% DMSO) was 0.8 mm rear and 1.2 mm flank of the brain at 3.8 mm depth 15 minutes after MCAO. Intracerebroventricular injection was performed. After that, the degree of motor deficit of rats was evaluated. The criteria for evaluation (Longa et al., 1989) indicate that the deficit does not indicate a deficiency . Mild, opposite rotations are classified as moderate and loss of circling or loss of reflexes are classified as severe deficit. In addition, the rat model was obtained 24 hours after induction of ischemia in the rat model and stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) to measure the degree of cerebral infarction.
그 결과, 본 발명을 통해 발굴된 최종 후보 화합물인 화합물 #28이 뇌졸중 동물모델 중 하나인 영구 중뇌동맥 폐쇄(permanent middle cerebral artery occulusion)에 따른 뇌손상을 현저하게 억제시킴을 최종 확인하였다(도 6).As a result, it was finally confirmed that Compound # 28, the final candidate compound discovered through the present invention, significantly suppresses brain damage caused by permanent middle cerebral artery occulusion, which is one of the stroke animal models (FIG. 6). ).
Figure PCTKR2017008569-appb-T000003
Figure PCTKR2017008569-appb-T000003
실시예Example 6: 급성 독성 테스트(Acute toxicity test) 6: Acute toxicity test
상기 최종 선별된 화합물인 #28을 랫트에 주입하고 급성 독성 결과를 관찰하였다. The final selected compound # 28 was injected into rats and acute toxicity results were observed.
구체적으로, 8-9 주령된 수컷 Sprague-Dawley(SD) 랫트에 상기 최종 선별된 AMPK 억제제 후보 화합물인 화합물 #28을 랫트 1 마리 당 75 μg/kg 또는 비히클(10% DMSO)을 정맥내 주입(intravenous injection)하였고 이를 4회 반복하였다. Specifically, 8-9 week old male Sprague-Dawley (SD) rats were injected intravenously with Compound # 28, the final selected AMPK inhibitor candidate compound, at 75 μg / kg or vehicle (10% DMSO) per rat. intravenous injection) and repeated four times.
그 결과, 24시간 경과 후에도 죽는 랫트는 발견되지 않았고 랫트를 희생시켜 간, 비장 및 신장 장기들을 적출하여 무게를 측정하였고, 상기 랫트의 혈액을 채취하여 WBC, RBC, BUN, AST, ALT, CREA 및 GLU 등의 지표 결과를 확인하였으나 대조군과 비교하여 대부분의 지표가 정상으로 나타났으며 눈에 띄는 독성을 관찰되지 않았다(도 7). 상기 지표에 대한 결과를 하기 표 4에 표시하였다. As a result, the rats that died after 24 hours were not found and the liver, spleen and kidney organs were sacrificed and weighed by sacrifice. The blood of the rats was collected and WBC, RBC, BUN, AST, ALT, CREA and Although the results of indicators such as GLU were confirmed, most of the indicators were normal compared to the control group, and no noticeable toxicity was observed (FIG. 7). The results for the indicators are shown in Table 4 below.
Figure PCTKR2017008569-appb-T000004
Figure PCTKR2017008569-appb-T000004
실시예Example 7: 유사화합물 검색 및 아연독성 억제효과 관찰  7: Detection of similar compounds and observation of zinc toxicity inhibitory effect
본 발명의 일 실시예에 따라 상기 최종 선별된 #28의 구조 유사성을 기반으로 하여 이와 유사한 구조를 가지는 25개 유사 화합물을 화합물 라이브러리 제조회사(InterBioScreen, Russia; Akos, Germany)로부터 구입하였다. 이 후 상기 배양된 생쥐의 대뇌피질 신경세포에 아연독성을 유발하기 위하여 ZnCl2(400 μM)을 10분간 처리하였고 12.5시간 경과 한 뒤, 상기 선별된 25개의 화합물 및 기존에 선별된 약물인 #28을 처리(20 μM)하여 세포 생존능 분석(cell viability assay, Cell Counting Kit-8, Dojindo)을 통해 세포사(cell death) 억제 여부를 관찰하였다. 그 결과, 상기 처리한 25개의 새로운 화합물 중에서 3개의 약물(4A02, 4B02 및 4D01)을 제외한 나머지 22개 약물이 모두 신경보호 효과를 나타내었다(도 8). 상기 선별된 25개 화합물의 명칭과 구조를 하기 표 5에 표시하였다. Based on the structural similarity of the final selected # 28 according to an embodiment of the present invention 25 similar compounds having a similar structure was purchased from the compound library manufacturer (InterBioScreen, Russia; Akos, Germany). Subsequently, ZnCl 2 (400 μM) was treated for 10 minutes to induce zinc toxicity to the cultured cerebral cortical neurons, and after 12.5 hours, the 25 compounds selected and the previously selected drug # 28 Was treated (20 μM) and cell inhibition was observed through cell viability assay (Cell Counting Kit-8, Dojindo). As a result, all 22 drugs except the 3 drugs (4A02, 4B02 and 4D01) among the 25 new compounds treated showed neuroprotective effect (FIG. 8). The names and structures of the 25 compounds selected are shown in Table 5 below.
코드명Codename 구조식constitutional formula IUPAC 명칭IUPAC Name
4-A014-A01
Figure PCTKR2017008569-appb-I000004
Figure PCTKR2017008569-appb-I000004
 		(5Z)-5-(1H-Indol-3-ylmethylene)-2-{[2-(trifluoromethyl)phenyl]amino}-1,3-thiazol-4(5H)-one(5Z) -5- (1H-Indol-3-ylmethylene) -2-{[2- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one
4-A024-A02
Figure PCTKR2017008569-appb-I000005
Figure PCTKR2017008569-appb-I000005
 		(5Z)-5-(1H-Indol-3-ylmethylene)-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-4(5H)-one(5Z) -5- (1H-Indol-3-ylmethylene) -2-{[3- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one
4-A034-A03
Figure PCTKR2017008569-appb-I000006
Figure PCTKR2017008569-appb-I000006
 		(5Z)-2-[(3-Bromophenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(3-Bromophenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-A044-A04
Figure PCTKR2017008569-appb-I000007
Figure PCTKR2017008569-appb-I000007
 		(5Z)-5-(1H-Indol-3-ylmethylene)-2-[(4-methylphenyl)amino]-1,3-thiazol-4(5H)-one(5Z) -5- (1H-Indol-3-ylmethylene) -2-[(4-methylphenyl) amino] -1,3-thiazol-4 (5H) -one
4-A054-A05
Figure PCTKR2017008569-appb-I000008
Figure PCTKR2017008569-appb-I000008
 		(5Z)-5-(1H-Indol-3-ylmethylene)-2-[(3-methylphenyl)amino]-1,3-thiazol-4(5H)-one(5Z) -5- (1H-Indol-3-ylmethylene) -2-[(3-methylphenyl) amino] -1,3-thiazol-4 (5H) -one
4-A064-A06
Figure PCTKR2017008569-appb-I000009
Figure PCTKR2017008569-appb-I000009
 		(5Z)-2-Anilino-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-Anilino-5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-A074-A07
Figure PCTKR2017008569-appb-I000010
Figure PCTKR2017008569-appb-I000010
 		(5Z)-2-[(2,4-Dimethylphenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(2,4-Dimethylphenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-A084-A08
Figure PCTKR2017008569-appb-I000011
Figure PCTKR2017008569-appb-I000011
 		(5Z)-2-[(2-Chlorophenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(2-Chlorophenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-B014-B01
Figure PCTKR2017008569-appb-I000012
Figure PCTKR2017008569-appb-I000012
 		(5Z)-2-[(3,4-Dimethylphenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(3,4-Dimethylphenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-B024-B02
Figure PCTKR2017008569-appb-I000013
Figure PCTKR2017008569-appb-I000013
 		(5Z)-2-[(4-Hydroxyphenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(4-Hydroxyphenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-B034-B03
Figure PCTKR2017008569-appb-I000014
Figure PCTKR2017008569-appb-I000014
 		(5Z)-5-(1H-Indol-3-ylmethylene)-2-[(2-methylphenyl)amino]-1,3-thiazol-4(5H)-one(5Z) -5- (1H-Indol-3-ylmethylene) -2-[(2-methylphenyl) amino] -1,3-thiazol-4 (5H) -one
4-B044-B04
Figure PCTKR2017008569-appb-I000015
Figure PCTKR2017008569-appb-I000015
 		(5Z)-2-[(2,3-Dimethylphenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(2,3-Dimethylphenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
4-B054-B05
Figure PCTKR2017008569-appb-I000016
Figure PCTKR2017008569-appb-I000016
 		(5E)-5-(1H-Indol-3-ylmethylene)-2-(1-naphthylamino)-1,3-thiazol-4(5H)-one(5E) -5- (1H-Indol-3-ylmethylene) -2- (1-naphthylamino) -1,3-thiazol-4 (5H) -one
4-B064-B06
Figure PCTKR2017008569-appb-I000017
Figure PCTKR2017008569-appb-I000017
 		(5Z)-2-[(3-Chlorophenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one(5Z) -2-[(3-Chlorophenyl) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one
실시예Example 8: 신경독성 억제효과 관찰  8: Observation of neurotoxic inhibitory effect
본 발명의 일 실시예에 따라 아연독성에 대한 상기 25개 유사화합물의 신경보호 효과를 반복적으로 관찰한 결과, 12개의 약물이 지속적으로 유의성 있는 약물효과를 나타내어 다양한 신경독성 억제효과 관찰을 위해 상기 12개의 약물을 선별하여 처리하였다. As a result of repeatedly observing the neuroprotective effects of the 25 analogous compounds on zinc toxicity according to an embodiment of the present invention, 12 drugs continuously showed a significant drug effect to observe various neurotoxic inhibitory effects 12 Dog drugs were selected and treated.
구체적으로, 신경독성 억제효과를 관찰하기 위한 신경독성 모델은 뇌졸중의 원인기전으로 분류되는 흥분독성(excitotoxicity), 산화성손상 등이 포함되어 있는데 상기 흥분독성 모델로는 NMDA(N-methyl-D-aspartate)를 사용하였고, 산화성손상 모델로는 과산화수소(H2O2) 독성 모델을 사용하였다. 먼저, 상기 배양된 생쥐의 대뇌피질 신경세포에 NMDA(50 μM) 또는 H2O2(100 μM)를 각각 1.5시간, 4.5시간 처리하여 신경독성을 유발하였고 상기 신경세포에 상기 선별된 12개의 약물(10 μM) 및 기존에 선별된 약물인 #28을 처리(20 μM)하였다. 이후 상기 신경세포를 프로피디움 요오드화물(propidium iodide, PI)에 염색 후 염색된 세포수를 정량화하여 세포사 정도를 측정하였다. 상기 염색 후 죽은 세포는 세포막의 선별적 투과성이 없기 때문에 PI에 염색되는 반면, 건강한 세포는 염색되지 않는다.Specifically, neurotoxicity models for observing neurotoxicity inhibitory effects include excitotoxicity and oxidative damage, which are classified as a cause of stroke. The excitatory toxicity model is NMDA (N-methyl-D-aspartate). ), And the hydrogen peroxide (H 2 O 2 ) toxicity model was used as the oxidative damage model. First, NMDA (50 μM) or H 2 O 2 (100 μM) was treated for 1.5 hours and 4.5 hours for the cultured cerebral cortical neurons, respectively, to induce neurotoxicity. (10 μΜ) and # 28, a previously selected drug, were treated (20 μΜ). Since the neurons were stained with propidium iodide (PI), the number of stained cells was quantified to measure the degree of cell death. Cells dead after the staining are stained with PI because of lack of selective permeability of the cell membrane, while healthy cells are not stained.
그 결과, 1개의 약물(4B04, 4B07, 4B08)을 제외한 9개의 약물이 NMDA에 의한 신경독성을 유의성 있게 억제함으로써 신경보호 효과를 나타내는 것을 확인하였고(도 9) 2개의 약물(4B03, 4B04)을 제외한 10개의 약물이 H2O2에 의한 신경독성을 유의성 있게 억제하는 것을 관찰하였다(도 10). 이러한 결과는 먼저 선정된 약물인 #28과 비교하여 동일하거나 더 높은 신경보호 효과를 확인한 것이다. As a result, it was confirmed that nine drugs except one drug (4B04, 4B07, 4B08) exhibited a neuroprotective effect by significantly inhibiting neurotoxicity by NMDA (FIG. 9). Two drugs (4B03, 4B04) were identified. Ten drugs except were observed to significantly inhibit neurotoxicity by H 2 O 2 (FIG. 10). These results confirm the same or higher neuroprotective effect compared to # 28, the drug of choice.
실시예Example 9: 자유아연(free zinc) 농도 측정  9: Free zinc concentration measurement
9-1: 9-1: pZnpZn meter  meter
자유 아연 농도를 측정하기 위하여 Test tube 상에서 아연 형광 물질인 ZnAF(2.5 μM) 와 함께 본 발명의 #28 약물을 농도별(2.5 ~ 20 μM)로 처리하였고 대조군은 매우 강력한 아연 킬레이터(zinc chelator)로 알려진 클리오퀴놀(clioquinol, 1 ~ 5μM)을 사용하였으며 pZn meter(NeuroBioTex Inc)를 이용하여 자유아연 농도를 측정하였다.In order to measure the free zinc concentration, the # 28 drug of the present invention was treated with the zinc fluorescent material ZnAF (2.5 μM) on a test tube by concentration (2.5-20 μM) and the control group was a very powerful zinc chelator. Known as clioquinol (1 ~ 5μM) was used and free zinc concentration was measured using a pZn meter (NeuroBioTex Inc).
그 결과, 본 발명의 #28 약물에 의해 자유아연 농도가 농도의존적으로 감소하는 것을 관찰하였다(도 11).As a result, it was observed that the concentration of free zinc decreased by concentration # 28 of the present invention (Fig. 11).
9-2: 형광 염색물질 9-2: fluorescent dye
또한, Test tube 상에서 클리오퀴놀(20 μM) 또는 #28(20 μM) 약물과 함께 아연 형광 염색물질인 Newport green DCF(0.5 μM, Kd(Zn)=1 uM) 또는 FluoZin-3(0.5 μM, Kd(Zn)=15 nM)을 사용하여 자유 아연 농도 변화를 측정하였다.In addition, Newport green DCF (0.5 μM, Kd (Zn) = 1 uM) or FluoZin-3 (0.5 μM, Kd), a zinc fluorescent dye, with clioquinol (20 μM) or # 28 (20 μM) drug on the test tube. (Zn) = 15 nM) was used to determine the change in free zinc concentration.
그 결과, 대조군인 클리오퀴놀 및 #28 약물 모두 아연이온에 결합하여 자유 아연이온이 감소하는 것을 관찰하였다(도 12 및 13). 특히 #28 약물의 경우 높은 농도의 아연에 대해서 자유 아연 농도를 낮춰주는 효과가 현저하게 관찰되었으나 (도 12), 낮은 농도의 아연에 대해서는 뚜렷한 효과가 보이지 않았다(도 13). 이러한 결과는 #28 약물의 아연 친화도가 클리오퀴놀에 비해 현저히 낮음을 의미한다.As a result, it was observed that both the control group clioquinol and # 28 drug binds to the zinc ion and the free zinc ion is reduced (FIGS. 12 and 13). Especially in the case of # 28 drug, the effect of lowering the free zinc concentration was observed significantly for the high concentration of zinc (Fig. 12), but did not show a clear effect for the low concentration of zinc (Fig. 13). These results indicate that the zinc affinity of drug # 28 is significantly lower than that of clioquinol.
9-3: 아연 신경독성 억제 효과 9-3: zinc neurotoxic inhibitory effect
본 발명의 #28 약물 아연 신경독성 억제 효과가 아연 통로인 채널에 작용하는 것이 아니라 직접적인 아연 킬레이트화(zinc chelation)에 의한 결과인지 확인하기 위해 상기 배양된 생쥐의 대뇌피질 신경세포에 세포 내 아연을 유입시키는 아연 이온투과담체(zinc ionophore)로 잘 알려진 클리오퀴놀, 피리치온(pyrithione) 및 세포내 아연 분비를 유발하는 약물인 DTDP(2,2'-Dithiodipyridine)을 처리한 후 세포 밖에서 유입된 아연 증가 외에 세포 내에서 유발된 아연 신경독성 감소를 관찰하였다.To determine whether the # 28 drug zinc neurotoxic inhibitory effect of the present invention is a result of direct zinc chelation rather than acting on a channel that is a zinc pathway, intracellular zinc was added to the cortical neurons of the cultured mice. Zinc introduced outside the cell after treatment with clioquinol, pyrithione, and intracellular zinc secretion DTDP (2,2'-Dithiodipyridine), a well-known zinc ionophore In addition to the increase, a decrease in intracellular zinc neurotoxicity was observed.
그 결과, 이온투과담체에 의한 아연 신경독성이 #28 약물에 의해 현저히 감소하는 것으로 나타났다 (도 14). 즉, #28 약물 효과는 아연 통로에 작용하여 나타나는 것이 아니라 직접적인 아연 킬레이트화에 의한 결과라 예상할 수 있다.As a result, zinc neurotoxicity by the ion permeate carrier was found to be significantly reduced by the # 28 drug (Fig. 14). In other words, the # 28 drug effect may be expected to be a result of direct zinc chelation rather than action on the zinc pathway.
9-4: 현미경 관찰 9-4: Microscopy
신경세포 내 실제 자유 아연 농도가 증가하는지 관찰하기 위하여 상기 배양된 생쥐의 대뇌피질 신경세포에 FluoZin-3 염색물질을 미리 처리하고 고농도의 아연에 노출시킨 후 4CO1(20 μM), 4CO7(20 μM) 및 #28(20, 50 μM) 약물을 처리해서 공초점 레이저 현미경을 사용하여 이미지를 관찰하였고 형광광도계(fluorometer)를 사용하여 형광크기를 정량적으로 측정하였다. In order to observe the increase in the actual free zinc concentration in neurons, the cortical neurons of the cultured mice were pre-treated with FluoZin-3 staining material and exposed to high concentrations of zinc, followed by 4CO1 (20 μM) and 4CO7 (20 μM). And # 28 (20, 50 μM) drug was used to observe the image using a confocal laser microscope and the fluorescence size was quantitatively measured using a fluorometer.
그 결과, 아연 노출 16분 후부터 신경세포 내 자유 아연이 뚜렷이 증가하며 #28 약물에 의해 신경세포 내 자유 아연 증가가 현저히 감소됨을 관찰하였고(도 15A) 형광크기를 정량적으로 분석한 결과 #28 약물 이외에 유사화합물 4C01과 4C07도 유사한 수준으로 세포 내 자유 아연 증가를 억제할 수 있음을 확인하였다(도 16B).As a result, it was observed that free zinc in neurons increased markedly after 16 minutes of zinc exposure, and that the increase in free zinc in neurons was significantly decreased by the # 28 drug (FIG. 15A). Similar compounds 4C01 and 4C07 were also found to be able to inhibit the increase of free zinc in the cells to a similar level (Fig. 16B).
9-5: 9-5: H2O2H2O2 독성 및 아연  Toxic and Zinc 킬레이트화Chelation
과산화수소에 의한 신경독성 기전에서 세포 내 자유 아연이 증가하고 이때 아연 킬레이터를 처리하면 과산화수소 독성이 감소한다고 보고된 바 있고 종래 실험에서 #28 약물이 과산화수소에 의한 신경독성을 억제했다. 따라서 이때 #28 약물 효과가 아연 감소와 관련 있는지 관찰하기 위해 신경세포에 과산화수소(H2O2), 아연 킬레이터인 TPEN 또는 CaEDTA 및 칼슘 킬레이터인 ZnEDTA를 처리한 후 신경 독성(LDH 분비)을 확인하였다. 또한, FluoZin-3 염색을 통해 과산화수소 처리 후에 신경세포 내 자유 아연의 증가여부를 관찰하였고 TPEN 처리군은 대조군으로 사용하였다. #28 외에도 유사 화합물 4C01, 4C07의 아연 킬레이션 여부를 공초점 현미경으로 관찰하였다.In the neurotoxic mechanism induced by hydrogen peroxide, intracellular free zinc was increased and zinc chelator treatment was reported to reduce hydrogen peroxide toxicity. In a previous experiment, drug # 28 inhibited neurotoxicity caused by hydrogen peroxide. Therefore, neurotoxicity (LDH secretion) was treated after treatment with hydrogen peroxide (H 2 O 2 ), zinc chelator TPEN or CaEDTA, and calcium chelator ZnEDTA to observe whether the # 28 drug effect was associated with zinc reduction. Confirmed. FluoZin-3 staining also observed the increase of free zinc in neurons after hydrogen peroxide treatment, and the TPEN treatment group was used as a control. In addition to # 28, zinc chelation of similar compounds 4C01 and 4C07 was observed under confocal microscopy.
그 결과, 대표적인 아연 킬레이터 중 하나인 TPEN 처리에 의해 과산화수소에 의한 신경 독성이 감소하였고 다른 아연 킬레이터인 CaEDTA의 처리에 의해서도 독성이 유의성 있게 감소하여 H2O2 독성이 아연과 관련되어 있음을 나타내었다. 그러나 이미 아연이 결합된 EDTA(ZnEDTA)에 의해서는 독성이 억제되지 않았는데 이는 EDTA가 추가적으로 아연을 킬레이트화(chelation) 하지 못했기 때문으로 H2O2 신경독성은 아연 킬레이트화에 의해 감소되는 것으로 나타났다(도 16). As a result, neurotoxicity by hydrogen peroxide was reduced by TPEN treatment, one of the representative zinc chelators, and the toxicity was significantly reduced by treatment with other zinc chelators CaEDTA, indicating that H 2 O 2 toxicity was related to zinc. Indicated. But did not already inhibit the toxic by EDTA (ZnEDTA) a zinc-binding which H 2 O 2 due to failure to EDTA is additionally chelated (chelation) with zinc Neurotoxicity has been shown to be reduced by zinc chelation (FIG. 16).
또한, #28, 4C01, 4C07 약물 처리에 의해 H2O2 독성 과정에서 나타나는 신경세포 내 아연 증가가 감소됨을 관찰하였으므로 상기 약물의 독성 억제 효과가 아연 감소에 의한 결과로 사료된다(도 17). In addition, it was observed that the increase of zinc in neurons during H 2 O 2 toxicity process was reduced by drug treatments # 28, 4C01, and 4C07, so the toxic inhibitory effect of the drug is considered to be the result of zinc reduction (FIG. 17).
9-6: 9-6: NMDANMDA 독성 및  Toxicity and Ca2Ca2 + + 킬레이트화Chelation
종래 실험에서 신경세포의 NMDA 채널을 개방하여 세포 내로 칼슘이온이 유입되도록 하는 약물인 NMDA에 의한 흥분독성도 #28 약물에 의해 억제되었는데 상기 결과가 #28 약물 외에 Ca2 + 이온의 킬레이트화에 의한 결과인지 관찰하기 위해 칼슘 이온투과담체(calcium ionophore)인 직접적으로 세포 내 칼슘농도를 증가시키는 이오노마이신(ionomycin)을 처리 후 #28 약물(10 ~ 60μM)의 보호효과를 관찰하였다. 또한, 칼슘에 결합해서 형광을 나타내는 Fura-2 dye를 이용해서 Test tube 상에서 #28 약물이 칼슘에 결합해서 킬레이트화 효과를 나타내는지 관찰하였다. Prior the experiment to open the NMDA channel of nerve cells excitotoxic by drug NMDA such that calcium ions are introduced into the cells also was inhibited by the # 28 drugs is the result # 28 drugs in addition to by the chelation of Ca 2 + ions In order to observe whether the result was a protective effect of # 28 drug (10 ~ 60μM) after treatment with ionomycin (ionomycin) that directly increases the intracellular calcium concentration, calcium ionophore (calcium ionophore). In addition, the Fura-2 dye, which binds to the fluorescence of calcium, was used to observe whether the drug # 28 on the test tube showed the chelation effect by binding to calcium.
그 결과, #28 약물의 처리는 이온투과담체의 독성을 억제할 수 있음을 관찰하였으나 40 μM이상의 농도를 처리해야 보호효과가 나타남을 확인하였다(도 18). 또한 EDTA는 낮은 칼슘농도에서 킬레이트화 효과가 잘 나타나는 반면, #28 약물은 높은 농도의 칼슘에 대해 킬레이트화 효과가 관찰되었다(도 19). 따라서 #28 약물에 의한 NMDA 신경독성 억제 효과는 칼슘이온을 직접적으로 킬레이트화한 결과로 나타나는 현상이며 아연 이온보다 칼슘 이온에 대한 친화도가 더 낮음을 알 수 있었다. As a result, it was observed that the treatment of # 28 drug can suppress the toxicity of the ion permeable carrier, but it was confirmed that the treatment effect appeared only when the concentration of 40 μM or more was treated (FIG. 18). EDTA also showed good chelating effect at low calcium concentrations, while # 28 drug had a chelating effect on high calcium concentrations (FIG. 19). Therefore, NMDA neurotoxicity inhibitory effect by the # 28 drug is a result of the direct chelation of calcium ions and showed a lower affinity for calcium ions than zinc ions.
9-9- 6: TPEN6: TPEN 독성 및 아연  Toxic and Zinc 이온투과담체Ion permeation carrier
일반적으로 세포 내로 쉽게 유입되는 아연 킬레이터인 TPEN을 처리하면 전형적인 아폽토시스(apoptosis) 형태의 세포사가 나타나는데(EDTA는 세포 내로 유입되지 않음) 여기에 아연을 첨가해주면 세포 내 자유 아연이온이 유지되어 신경세포사가 감소되고 아폽토시스 과정에서 caspase-3 단백질 분해효소 활성도 감소된다. 본 발명의 #28 약물과 클리오퀴놀이 킬레이터의 역할 뿐만 아니라 이온투과담체 역할을 하여, 세포 내로 아연과 결합된 상태로 유입되고 이후 세포질 내 자유 아연 농도가 너무 낮기 때문에 약물로부터 아연이 떨어져 나와 세포질 내 자유아연 농도가 증가 유지되어 TPEN에 의한 신경독성이 감소될 것을 예상하고 아연, #28 약물 및 클리오퀴놀을 처리하여 caspase-3의 활성 및 신경독성(LDH 분비)를 관찰하였다. In general, treatment of TPEN, a zinc chelator that easily enters cells, results in typical apoptosis-like cell death (EDTA does not enter the cell). When zinc is added, free zinc ions are maintained in the cells, leading to neuronal death. And caspase-3 protease activity is decreased during apoptosis. The # 28 drug and clioquinol of the present invention not only acts as a chelator, but also acts as an ion permeable carrier, which is introduced into the cell in combination with zinc and then the zinc is released from the drug because the concentration of free zinc in the cytoplasm is too low. It was expected that the concentration of free zinc was maintained to decrease the neurotoxicity by TPEN, and the activity and neurotoxicity (LDH secretion) of caspase-3 were observed by treatment with zinc, # 28 drug and clioquinol.
그 결과, 아연, #28 약물 및 클리오퀴놀의 처리에 의해 TPEN에 의해 유도된 caspase-3 활성이 유의성 있게 감소하였고 TPEN에 의한 신경독성도 감소하는 것으로 나타났다(도 20). As a result, the treatment of zinc, # 28 drug and clioquinol significantly reduced caspase-3 activity induced by TPEN and also reduced neurotoxicity by TPEN (FIG. 20).
9-7: 아연 9-7: Zinc 이온투과담체Ion permeation carrier
ZinPyr-1은 형광염색물질로 FluoZin-3보다 Kd 값이 작아서 더 낮은 농도의 아연 변화를 측정하는데 사용되는데 본 발명의 #28 약물이 이온투과담체의 역할을 할 수 있는지 ZinPyr-1을 사용하여 관찰하였다. 구체적으로 상기 배양된 생쥐의 대뇌피질 신경세포에 ZinPyr-1을 처리한 후 일반적으로 사용하는 신경세포 배양액에 #28 약물(0.05 μM)과 클리오퀴놀(0.5 μM)을 처리하고 무처리군을 대조군으로 하여 신경세포 내 아연이온 농도의 변화를 측정하였다. ZinPyr-1 is a fluorescent dye that has a lower Kd value than FluoZin-3 and is used to measure zinc concentrations at lower concentrations. ZinPyr-1 is used to determine whether the # 28 drug of the present invention can act as an ion permeable carrier. It was. Specifically, after treating ZinPyr-1 to the cultured cerebral cortical neurons, # 28 drug (0.05 μM) and clioquinol (0.5 μM) were treated to a commonly used neuronal cell culture and the untreated group was used as a control. The change of zinc ion concentration in neurons was measured.
그 결과, 일반적인 배양액 조건에서는 클리오퀴놀에 의한 아연 증가가 관찰되었으나 #28 약물에 의한 증가는 뚜렷하게 관찰되지 않았다. 그래서 세포배양액에 추가적으로 0.5 μM 농도의 아연을 추가한 후 세포 내 아연농도 변화를 관찰한 결과 클리오퀴놀 및 #28 약물의 처리에 의해 유의한 아연 증가가 관찰되었다(도 21). 따라서 본 발명의 #28 약물도 이온투과담체 역할을 할 수 있다고 사료되며 클리오퀴놀과 비교할 때 낮은 수준으로 아연을 증가시키는 것으로 사료된다. As a result, the increase of zinc by clioquinol was observed in normal culture conditions, but the increase by # 28 drug was not clearly observed. Therefore, after adding zinc at a concentration of 0.5 μM to the cell culture solution, the zinc concentration was observed in the cells. As a result, a significant increase in zinc was observed by treatment with clioquinol and # 28 drug (FIG. 21). Therefore, the # 28 drug of the present invention may also act as an ion permeable carrier and increase zinc to a low level when compared to clioquinol.
실제 클리오퀴놀은 아연 친화도(affinity)가 높아서 높은 농도의 아연은 킬레이트화 할 수 있으나 약물 자체의 이온투과담체 기능 때문에 세포질 아연농도를 쉽게 증가시켜서 독성이 유발되는 것으로 잘 알려져 있다. 그래서 클리오퀴놀은 뇌졸중 치료제로 적합하지 않으나 본 발명의 #28 약물은 세포질 아연농도를 크게 증가시키지 않는 수준에서 이온투과담체 역할을 수행하며, 독성이 유발될 수준으로 세포질 내 자유 아연이 증가하게 되면 클레이트화를 통해 적절한 수준으로 아연 항상성을 조절하는 것으로 사료된다.In fact, clioquinol has a high zinc affinity, which can chelate high concentrations of zinc, but it is well known that toxicity can be caused by easily increasing cytoplasmic zinc concentration due to the ion permeability of the drug itself. Therefore, Clioquinol is not suitable as a stroke treatment, but the # 28 drug of the present invention functions as an ion permeable carrier at a level that does not significantly increase the level of cytoplasmic zinc, and if the free zinc in the cytoplasm is increased to a level at which toxicity is induced, clay It is believed that zinc homeostasis is regulated to an appropriate level through nitridation.
상기 결과들을 종합해보면, AMPK 효소가 뇌졸중의 원인기전 중 하나로 생각되는 아연독성에 중요한 역할을 하고 있음을 발견하여 AMPK 효소 활성을 억제기능을 가지고 있는 새로운 화합물 후보군에서 여러 차례 스크리닝을 실시하여 탁월한 아연독성에 효과를 보인 화합물 #28을 최종 선별하고 뇌졸중 동물모델에 처리한 결과 현저하게 뇌손상을 억제됨을 확인하였고 상기 #28 약물의 구조를 기반으로 유사화합물을 선별하여 다양한 신경독성 유발 후 처리한 결과 뛰어난 신경보호 효과를 나타내었으므로 이들 화합물들은 새로운 뇌졸중 치료제로 활용 가능하다. Taken together, we found that AMPK enzyme plays an important role in zinc toxicity, which is considered to be one of the causes of stroke, and has been screened several times in a new compound candidate group that inhibits AMPK enzyme activity. The final screening of Compound # 28, which was shown to be effective in the treatment of animal models of stroke, significantly inhibited brain damage, and the selection of similar compounds based on the structure of the # 28 drug resulted in various neurotoxic effects. Because of its neuroprotective effect, these compounds could be used as new stroke treatments.
본 발명은 상술한 실시예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.Although the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Claims (7)

  1. 하기 화학식 1의 구조식을 갖는 화합물을 유효성분으로 함유하는, 뇌졸중 치료용 약학적 조성물:A pharmaceutical composition for treating stroke, containing a compound having the structural formula of Formula 1 as an active ingredient:
    [화학식 1][Formula 1]
    Figure PCTKR2017008569-appb-I000018
    Figure PCTKR2017008569-appb-I000018
    (상기 식에서 R1 내지 R5는 각각 독립적으로 수소, 히드록시기, 할로겐, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알킬기, 탄소수 1 내지 7의 치환되거나 치환되지 않은 알콕시기, 아민기, 카르복실기이거나, R2 및 R3는 함께 -O-(CH2)n-O- 고리 또는 치환 또는 비치환된 벤젠고리를 형성하며(n은 1 내지 3의 정수), R6는 수소 또는 메틸기이고, R7은 수소 또는 할로겐이다).Wherein R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 7 carbon atoms, an amine group or a carboxyl group, or R 2 and R 3 together form a —O— (CH 2 ) n —O— ring or a substituted or unsubstituted benzene ring (n is an integer from 1 to 3), R 6 is hydrogen or a methyl group, and R 7 is Hydrogen or halogen).
  2. 제1항에 있어서, The method of claim 1,
    상기 치환된 알킬기는 트리플루오로메틸기인, 약학적 조성물. Wherein the substituted alkyl group is a trifluoromethyl group.
  3. 제1항에 있어서, The method of claim 1,
    상기 할로겐은 요오드, 브롬 또는 염소인, 약학적 조성물. Wherein said halogen is iodine, bromine or chlorine.
  4. 제1항에 있어서, The method of claim 1,
    상기 화합물은 (5Z)-5-(1H-Indol-3-ylmethylene)-2-{[2-(trifluoromethyl) phenyl]amino}-1,3-thiazol-4(5H)-one,(5Z)-5-(1H-Indol-3-ylmethylene)-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-4(5H)-one,(5Z)-2-[(3-Bromophenyl)amino]-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one,(5Z)-5-(1H-Indol-3-ylmethylene)-2-[(4-methylphenyl)amino]-1,3-thiazol-4(5H)-one, (5Z)-5-(1H-Indol-3-ylmethylene)-2-[(3-methylphenyl)amino]-1,3-thiazol-4(5H)-one,(5Z)-2-Anilino-5-(1H-indol-3-ylmethylene)-1,3-thiazol-4(5H)-one, no]-1,3-thiazol-4(5H)-one,(5E)-5-[(2-Methyl-1H-indol-3-yl)methylene]-2-[(4-methylphenyl)amino]-1,3-thiazol-4(5H)-one,3-{[(5Z)-5-(1H-Indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid,2-{[(5E)-5-(1H-Indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid, (5Z)-2-[(2-Chlorophenyl)amino]-5-[(2-methyl-1H-indol-3-yl)methylene]-1,3-thiazol-4(5H)-one, 또는 2-Hydroxy-5-{[(5Z)-5-(1H-indol-3-ylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}benzoic acid인, 약학적 조성물.The compound is (5Z) -5- (1H-Indol-3-ylmethylene) -2-{[2- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one, (5Z)- 5- (1H-Indol-3-ylmethylene) -2-{[3- (trifluoromethyl) phenyl] amino} -1,3-thiazol-4 (5H) -one, (5Z) -2-[(3-Bromophenyl ) amino] -5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2-[(4 -methylphenyl) amino] -1,3-thiazol-4 (5H) -one, (5Z) -5- (1H-Indol-3-ylmethylene) -2-[(3-methylphenyl) amino] -1,3- thiazol-4 (5H) -one, (5Z) -2-Anilino-5- (1H-indol-3-ylmethylene) -1,3-thiazol-4 (5H) -one, no] -1,3-thiazol -4 (5H) -one, (5E) -5-[(2-Methyl-1H-indol-3-yl) methylene] -2-[(4-methylphenyl) amino] -1,3-thiazol-4 ( 5H) -one, 3-{[(5Z) -5- (1H-Indol-3-ylmethylene) -4-oxo-4,5-dihydro-1,3-thiazol-2-yl] amino} benzoic acid, 2-{[(5E) -5- (1H-Indol-3-ylmethylene) -4-oxo-4,5-dihydro-1,3-thiazol-2-yl] amino} benzoic acid, (5Z) -2 -[(2-Chlorophenyl) amino] -5-[(2-methyl-1H-indol-3-yl) methylene] -1,3-thiazol-4 (5H) -one, or 2-Hydroxy-5- { [(5Z) -5- (1H-indol-3-ylmethylene) -4-oxo-4,5-dihydro-1,3-thiazol-2-yl] amino} benzoic The pharmaceutical composition is acid.
  5. 제1항에 있어서, The method of claim 1,
    상기 뇌졸중은 출혈성(hemorrhagic) 뇌졸중, 허혈성(ischemic) 뇌졸중 또는 금속 독성(metal toxicity) 뇌졸중인, 약학적 조성물.Wherein the stroke is a hemorrhagic stroke, an ischemic stroke or a metal toxicity stroke.
  6. 제5항에 있어서,The method of claim 5,
    상기 금속은 납(lead), 수은(mercury), 망간(manganese), 비소(arsenic), 탈륨(thallium), 철(iron), 아연(zinc), 카드뮴(cadmium), 비스무스(bismuth) 또는 주석(tin)인, 약학적 조성물.The metal may be lead, mercury, manganese, arsenic, thallium, iron, zinc, cadmium, bismuth, or tin ( tin).
  7. 제5항에 있어서, The method of claim 5,
    상기 허혈성 뇌졸중은 흥분성 신경세포사 또는 산화성 신경세포사에 의해 유발되는 것인, 약학적 조성물.The ischemic stroke is caused by excitatory neuronal death or oxidative neuronal death, pharmaceutical composition.
PCT/KR2017/008569 2016-08-09 2017-08-08 Pharmaceutical composition for stroke treatment based on ampk inhibition WO2018030762A1 (en)

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KR20080056730A (en) * 2005-09-16 2008-06-23 토렌트 파마슈티칼스 리미티드 Thiazolinones and oxazolinones and their use as ptp1b inhibitors
US20090163545A1 (en) * 2007-12-21 2009-06-25 University Of Rochester Method For Altering The Lifespan Of Eukaryotic Organisms
US20140031547A1 (en) * 2010-12-14 2014-01-30 Electrophoretics Limited CASEIN KINASE 1delta (CK 1delta) INHIBITORS AND THEIR USE IN THE TREATMENT OF NEURODE-GENERATIVE DISEASES SUCH AS TAUOPATHIES
CN104059060A (en) * 2014-05-30 2014-09-24 西安交通大学 5-(1H-indolyl-3-methylene)-1,3-thiazolidinyl-4-one derivatives, and synthesis method and application thereof
WO2015153959A2 (en) * 2014-04-04 2015-10-08 The Regents Of The University Of Michigan Small molecule inhibitors of mcl-1 and uses thereof

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US20090163545A1 (en) * 2007-12-21 2009-06-25 University Of Rochester Method For Altering The Lifespan Of Eukaryotic Organisms
US20140031547A1 (en) * 2010-12-14 2014-01-30 Electrophoretics Limited CASEIN KINASE 1delta (CK 1delta) INHIBITORS AND THEIR USE IN THE TREATMENT OF NEURODE-GENERATIVE DISEASES SUCH AS TAUOPATHIES
WO2015153959A2 (en) * 2014-04-04 2015-10-08 The Regents Of The University Of Michigan Small molecule inhibitors of mcl-1 and uses thereof
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