WO2016206551A1 - Src蛋白抑制剂在制备预防和/治疗阿尔兹海默病药物中的应用 - Google Patents

Src蛋白抑制剂在制备预防和/治疗阿尔兹海默病药物中的应用 Download PDF

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WO2016206551A1
WO2016206551A1 PCT/CN2016/085701 CN2016085701W WO2016206551A1 WO 2016206551 A1 WO2016206551 A1 WO 2016206551A1 CN 2016085701 W CN2016085701 W CN 2016085701W WO 2016206551 A1 WO2016206551 A1 WO 2016206551A1
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compound
formula
pharmaceutically acceptable
solvate
salt
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PCT/CN2016/085701
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English (en)
French (fr)
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冯泽旺
汪进良
刘岩
赵宣
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北京键凯科技有限公司
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Publication of WO2016206551A1 publication Critical patent/WO2016206551A1/zh
Priority to US15/848,918 priority Critical patent/US20180117043A1/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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

  • the present invention relates to the use of a Src protein inhibitor for the preparation of a medicament for the prevention and/or treatment of Alzheimer's disease, in particular the compound N-(2-chloro-6-methylphenyl)-2- ⁇ 6-[4-( Use of 3-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinylamino ⁇ -5-thiazolecarboxamide for the preparation of a medicament for the prevention and/or treatment of Alzheimer's disease.
  • AD Alzheimer's disease
  • APP amyloid precursor protein
  • PS1 presenilin-1
  • PS2 presenilin-2
  • ApoE4 mitochondria-encoded cytochrome oxidases I and II.
  • AD Alzheimer's disease
  • a ⁇ 42 is derived from amyloid precursor protein (APP), intact APP is type I transmembrane protein, APP is proteolytically firstly under the action of ⁇ -secretase or ⁇ -secretase, extracellular The region is cleaved to produce sAPP ⁇ (soluble APP)/sAPP ⁇ and CTF ⁇ (C-terminal fragments)/CTF ⁇ , and ⁇ -secretase cleaves CTF ⁇ /CTF ⁇ to generate a series of A ⁇ and CTF ⁇ with different molecular weights. Most of the PS mutations are passed. Affecting ⁇ -secretase (PS may be a component of ⁇ -secretase) leads to an increase in the etiology of A ⁇ 42 production.
  • PS amyloid precursor protein
  • AD Alzheimer's disease
  • a ⁇ 42 plays a key role in the development of AD.
  • Current in-depth studies on the metabolism and toxicity of A ⁇ 42 provide a wide range of potential drug targets for AD therapy.
  • ZHONG Yi reported in WO2012/103282 that a transgenic Drosophila and double transgenic mice were used to screen for a preferred target for the treatment of Alzheimer's disease, the epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • Inhibition of EGFR can ameliorate A ⁇ 42-induced early memory loss in transgenic Drosophila and mice, and pharmacological activity screening data show that EGFR inhibitors are being studied (eg EKB-569, CL-387785, HKI-272, BIBW 2992, HKI-357) , ZD-6474, AEE 788, XL647, BMS-599626, IPI-504, 17-AAG, JKF-006, JKF-011, JKF-027, GJ-06, GJ-06-1, GJ-12, GJ- 12-1) Effective in A ⁇ -induced memory loss in transgenic Drosophila and double transgenic mice treated with A ⁇ 42 expression.
  • Three patented compounds JKF-006, JKF-011 and JKF-027 were screened out in the patent.
  • N-(2-chloro-6-methylphenyl)-2- ⁇ 6-[4-(3-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinylamino ⁇ - 5-thiazolecarboxamide, dasatinib, is a second-generation tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia with Abl, Src, c-Kit, ephrin receptors and other tyrosine kinases. Targeted, but not targeted at EGFR or Her2.
  • Src protein inhibitors particularly dasatinib
  • in vitro test results show that it is preventing and/or treating Alzheimer's disease and Inhibition of A treatment has a significant increase in cytotoxic activity compared to currently reported EGFR inhibitors.
  • the present invention provides the use of a Src protein inhibitor for the preparation of a medicament for the treatment and/or prevention of Alzheimer's disease (AD) diseases.
  • AD Alzheimer's disease
  • the invention also provides the use of a Src protein inhibitor for the preparation of a medicament for the treatment and/or prevention of a neurodegenerative disease.
  • the invention also provides the use of a Src protein inhibitor for the preparation and/or improvement of a cognitive dysfunction or a learning and memory dysfunction drug.
  • the invention also provides the use of a Src protein inhibitor for the preparation of a medicament for inhibiting the preparation of A to produce cytotoxicity.
  • the invention also provides the use of a Src protein inhibitor for the manufacture and/or use of a medicament for vascular dementia or vascular cognitive impairment.
  • the invention also provides the use of a Src protein inhibitor for the preparation of a drug for degeneration of cholinergic neurons.
  • the Src protein inhibitor of the present invention is a Src/Abl double-effect inhibitor. More preferably, the Src protein inhibitor of the present invention is selected from the group consisting of dasatinib or a pharmaceutically acceptable salt thereof, bosutinib or a pharmaceutically acceptable salt thereof.
  • the invention provides N-(2-chloro-6-methylphenyl)-2- ⁇ 6-[4-(3-hydroxyethyl)-1-piperazinyl]-2 a solvate, ester or ether, precursor or metabolite of -methyl-4-pyrimidinylamino ⁇ -5-thiazolecarboxamide (as shown in Formula I) or a pharmaceutically acceptable salt, solvate or salt thereof Use in the preparation of a medicament for the treatment and/or prevention of Alzheimer's disease (AD) diseases.
  • AD Alzheimer's disease
  • the present invention provides a solvate, ester or ether, precursor or metabolite of a compound of Formula I or a pharmaceutically acceptable salt, solvate or salt thereof for the preparation of a therapeutic and/or neurodegenerative disease Application in medicine.
  • the present invention provides a solvate, an ester or an ether, a precursor or a metabolite of a compound of the formula I or a pharmaceutically acceptable salt, solvate or salt thereof for the preparation of a treatment and/or prevention for improving cognitive dysfunction Or the application of drugs that learn to lose memory.
  • the present invention provides a solvate, ester or ether, precursor or metabolite of the compound of Formula I or a pharmaceutically acceptable salt, solvate or salt thereof for the preparation of a medicament for inhibiting the toxicity of A or ether cytotoxicity.
  • the present invention provides a solvate, ester or ether, precursor or metabolite of the compound of Formula I or a pharmaceutically acceptable salt, solvate or salt thereof, in the preparation and/or vascular dementia or vascular recognition.
  • the present invention provides a solvate, ester or ether, precursor or metabolite of a compound of formula I or a pharmaceutically acceptable salt, solvate or salt thereof for the preparation of a therapeutic and/or prophylactic cholinergic neuron Application in drugs for degenerative diseases.
  • the surprising discovery of the present invention is that the compound of the formula I is compared with the JKF-027 reported in WO2012/103282 for the antiviral effect of the anti-A ⁇ 42 protein mouse cerebral cortex neuron, using the primary isolation of the 17-day pregnant mouse rat brain. Cortical neurons, A ⁇ 42 oligomers (10 ⁇ mol/L) and different concentrations of test drugs were used for 24 h to detect the in vitro anti-A ⁇ 42 oligomer toxicity effects of different concentrations of the compound of formula I and JKF-027.
  • the compounds of the formula I according to the invention are to be understood as comprising the amorphous forms of the compounds of the formula I and any crystalline forms, such as the amorphous forms of dasatinib and any crystalline forms disclosed in US Pat. No. 6,596,746 B, US Pat. No. 2,050,215, 795 A, WO 2009 053 854 A1, CN 102086 195 A, CN 104341410 A.
  • the "pharmaceutically acceptable salt” as used in the present invention means a derivative in which the parent compound is modified by forming an acid salt thereof.
  • acceptable salts include, but are not limited to, inorganic or organic acid salts.
  • Pharmaceutically acceptable salts include the conventional non-toxic salts formed by the parent compound, for example, salts formed from non-toxic inorganic or organic acids.
  • the conventional non-toxic salts include salts derived from inorganic acids such as hydrochlorides, hydrobromides, sulfates, phosphates, nitrates, and the like; and salts prepared from organic acids, such as acetates.
  • a pharmaceutically acceptable salt of a compound of formula I as described herein refers to a hydrochloride, hydrobromide, sulfate, phosphate, nitrate, acetate, propionate salt of a compound of formula I.
  • succinate glycolate, stearate, lactate, malate, tartrate, citrate, ascorbate, pamoate, maleate, hydroxymaleate, benzene Kimalylate, glutamate, benzoate, salicylate, fumarate, tosylate, methanesulfonate, oxalate and isethionate.
  • the salt can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic or organic acids.
  • the acid includes acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, horse Acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like.
  • the solvates of the present invention may be solvates well known in the art, and the solvates of the compounds of formula I may be hydrates such as monohydrates, ethanol solvates, isopropanol solvates and the like.
  • esters or ethers of the present invention may be esters or ethers well known in the art, and the esters or ethers of the compounds of formula I may form ethers or esters of the compounds by conventional chemical reactions in the art.
  • the precursor or metabolite of the present invention may be a precursor or metabolite known in the art as long as the precursor or metabolite is metabolized in vivo to form a compound.
  • the compound of formula I is a compound of formula I in free base form, or a pharmaceutically acceptable salt thereof.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a solvate, ester or ether, precursor or metabolite of a compound of formula I or a pharmaceutically acceptable salt, solvate or salt thereof, and a One or more pharmaceutically acceptable additives.
  • the pharmaceutical composition of the present invention includes any composition prepared by mixing the compound of the present invention and a pharmaceutically acceptable additive.
  • “Pharmaceutically acceptable” means that an additive such as a carrier, diluent or excipient must be compatible with the other formulation ingredients and not deleterious to the recipient thereof.
  • the compounds of the invention may be administered orally, parenterally (for example, intramuscularly, intraperitoneally, intravenously, ICV, intracisternal injection or infusion, subcutaneous injection or implantation), by inhalation spray, nasally, vaginally, rectally, Administration by sublingual or topical routes of administration, and they may be formulated alone or in combination with conventional non-toxic pharmaceutically acceptable carriers, adjuvants and excipients, etc., in suitable dosages suitable for the various routes of administration. Unit preparation.
  • compositions of the compounds of the invention for administration may be presented in the form of suitable dosage units and may be prepared by any methods known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with one or more pharmaceutically acceptable additives.
  • the pharmaceutical compositions are prepared by uniformly and intimately mixing the active ingredient with a liquid additive or a finely divided solid additive or both, and then shaping the product into the desired formulation.
  • the active ingredient is included in the pharmaceutical composition in an amount that produces the desired therapeutic effect on the disease process or condition.
  • compositions containing the active ingredient can be formulated into a suitable oral form such as a tablet, an aqueous or oily suspension, a dispersible powder or granule, an emulsion, a hard or soft capsule, a syrup, and the like.
  • suitable oral form such as a tablet, an aqueous or oily suspension, a dispersible powder or granule, an emulsion, a hard or soft capsule, a syrup, and the like.
  • Compositions for oral administration can be prepared by any method well known in the art of pharmaceutical formulation, which compositions may contain one or more additives selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives.
  • Tablets contain the active ingredient with non-toxic pharmaceutically acceptable additives suitable for the manufacture of tablets.
  • additives may be inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulation and disintegrants (such as corn starch or alginic acid), binders (such as starch, gel or arabic). Gum) and lubricants (such as magnesium stearate, stearic acid or talc).
  • the tablets may be uncoated or coated by known techniques to prolong disintegration and absorption in the gastrointestinal tract, thereby providing a long lasting therapeutic effect.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be used, and a sustained release tablet may be prepared by coating, and may be formulated into an immediate release tablet or the like.
  • the preparation for oral use may also be a hard capsule, the active ingredient of which is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or a soft capsule, the active ingredient of which is combined with an aqueous or oily medium (such as peanut oil, liquid paraffin) Or olive oil) mixed.
  • an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin
  • a soft capsule such as peanut oil, liquid paraffin) Or olive oil
  • Aqueous suspensions contain the active materials and non-toxic pharmaceutically acceptable additives suitable for use in the manufacture of aqueous suspensions.
  • the additive is a suspending agent (such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum arabic), a dispersing or wetting agent (such as lecithin) , polyoxyethylene stearate, heptadecene vinyl hexadecanol, polyoxyethylene sorbitan monooleate).
  • the aqueous suspension may also contain one or more preservatives (such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate), one or more colorants, one or more flavoring agents. And one One or more sweeteners (such as sucrose or saccharin).
  • preservatives such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate
  • colorants such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate
  • flavoring agents such as ethyl p-hydroxybenzoate
  • One or more sweeteners such as sucrose or saccharin.
  • An oily suspension can be prepared by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin).
  • the oily suspensions may contain a thickening agent (such as beeswax, hard paraffin or cetyl alcohol).
  • a thickening agent such as beeswax, hard paraffin or cetyl alcohol.
  • the above sweeteners and flavoring agents may be added, and an antioxidant such as ascorbic acid may be added.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsifier.
  • the oil phase may be a vegetable oil (such as olive oil or peanut oil) or a mineral oil (such as liquid paraffin) or a mixture thereof.
  • Suitable emulsifiers may be natural gums (such as acacia or tragacanth), natural phospholipids (such as soy lecithin) and esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate). And a condensation product of the partial ester with ethylene oxide (such as polyoxyethylene sorbitan monooleate).
  • the emulsifier may also contain a sweetener or a flavoring agent.
  • a syrup can be formulated with a sweetener such as glycerin, propylene glycol, sorbitol or sucrose.
  • the syrup may also contain a wetting agent, a preservative and a flavoring agent, and a coloring agent.
  • compositions may be in the form of a sterile injectable aqueous or oily suspension.
  • the suspension may be formulated according to procedures well known in the art using such suitable dispersing or wetting agents and suspending agents.
  • the pharmaceutical composition may also be in the form of a suppository for rectal administration.
  • the suppository can be prepared by mixing the drug with a suitable non-irritating additive which is solid at ordinary temperatures but liquid at the rectal temperature.
  • the additives may be cocoa butter and polyethylene glycol.
  • creams, ointments, gums, liquid preparations or suspensions, and the like, containing the compounds of the invention may be employed.
  • transdermal patches can also be used for topical administration.
  • the present invention also provides N-(2-chloro-6-methylphenyl)-2- ⁇ 6-[4-(3-hydroxyethyl)-1-piperazinyl]-2-methyl- a solvate, ester or ether, precursor or metabolite of 4-pyrimidinylamino ⁇ -5-thiazolecarboxamide (as shown in Formula I) or a pharmaceutically acceptable salt, solvate or salt thereof, in the preparation of a therapeutic and/or Or use in a medicament for preventing a disease of Alzheimer's disease (AD), which comprises a solvate, ester or ether, precursor or metabolism of a compound of formula I or a pharmaceutically acceptable salt, solvate or salt thereof And one or more pharmaceutically acceptable additives.
  • AD Alzheimer's disease
  • the medicament of the present invention may be an oral preparation, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion preparation, subcutaneous injection or implant preparation, by inhalation spray, nasal, vaginal, rectal, tongue
  • the preparation is administered subcutaneously or topically.
  • the solvate, ester or ether, precursor or metabolite of the compound of formula I or a pharmaceutically acceptable salt, solvate or salt thereof in the medicament is from 0.1 to 99.9% by mass, more preferably It is 1-90%, more preferably 5-80%, and most preferably 10-70%.
  • Figure 1 is a histogram of the first in vitro pharmacodynamic test of Compound I against A?42 protein.
  • Figure 2 is a bar graph of the second in vitro pharmacodynamic test of Compound I against A?42 protein.
  • Figure 3 is a bar graph of the third in vitro pharmacodynamic test of Compound I against A?42 protein.
  • Fig. 4 is a histogram of the average in vitro efficacy test of compound I anti-A ⁇ 42 protein.
  • the 2-chloro-6-methylaniline used in the examples was purchased from Hangzhou Dakang Chemical Co., Ltd., and (E)-3-ethoxyacryloyl chloride was purchased from Hunan Huateng Pharmaceutical Co., Ltd., 4,6-dichloro- 2-Methylpyrimidine was purchased from Changzhou Ruisheng Chemical Co., Ltd., 1-(2-hydroxyethyl)piperazine was purchased from Shanghai Haiqu Chemical Co., Ltd., and the remaining reagents were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.
  • N-(2-Chloro-6-methylphenyl)-2-[(6-chloro-2-methyl-4-pyrimidinyl)amino]-5-thiazolecarboxamide (V) (52.5 g, 133 mmol Dissolved in dimethyl sulfoxide (500 mL), 1-(2-hydroxyethyl)piperazine (60.9 g, 265 mmol) and N,N-diisopropylethylamine (34.7 g, 267 mmol). The reaction solution was heated to 80 ° C, and then stirred and reacted for 1 h.
  • Example 2 Compound I anti-Amyloid- ⁇ protein mouse cerebral cortical neuron toxicity test
  • the compound JKF-027 used in the test of the present invention was prepared according to the method reported in the literature: WO2012/103282 (publication date: August 2, 2012)
  • the cerebral cortical neurons were isolated from fetal rats by Amyloid- ⁇ 42 oligomers, and the anti-A ⁇ 42 protein toxicity of Compound I was detected.
  • Test preparation sterile surgical instruments, pipetting guns, ice packs, petri dishes, 70% alcohol and cotton balls, centrifuge tubes, 12-well plates (coated), trypsin, HBSS solution, DNase, growth medium, maintenance Medium.
  • Test operation The pregnant mouse uterus was taken out for 17 days of pregnancy, and the embryos were separated one by one.
  • the cortex of the fetal rat was removed, placed in HBSS and washed several times. After adding the enzyme digestion treatment, the supernatant was discarded. Add growth medium.
  • the culture dish was tilted, and slowly pipetted with a 1 mL pipette. After standing, the upper suspension cells were transferred to a centrifuge tube, and the above operation was repeated three times.
  • a ⁇ 42 monomer oligomer mixture H4 was added to each group. For 24 hours, and set a duplicate hole. Twelve cell culture wells were used in one test and the test was repeated once.
  • the test grouping is shown in Table 1.
  • the cell viability was measured by WST-8, and the A ⁇ 42 concentration of the neuron survival rate of 30%-60% was selected as the formal modeling concentration to determine the neuroprotective activity of JKF-027.
  • the plates were incubated for 2-4 hours in an incubator.
  • the absorbance at 450 nm was measured with a microplate reader.
  • the results of the first independent test are shown in Table 3 and Figure 1; the results of the second independent test are shown in Table 4 and Figure 2; the results of the third independent test are shown in Table 5 and Figure 3; the average values of the three independent tests are shown in Table 6 and Figure 4. From the test results, the three tests have quite good repeatability and the results are more convincing.
  • the abscissa represents different administration concentrations, 1 represents 0.001 ⁇ M; 2 represents 0.01 ⁇ mol/L; 3 represents 0.1 ⁇ mol/L; 4 represents 1.0 ⁇ mol/L; 5 represents 10 ⁇ mol/L; 6 represents 100 ⁇ mol/L; /L.
  • the anti-A ⁇ 42 protein toxicity curves of different tested drugs showed a trend of increasing first and then decreasing, and there was an effective dose interval.
  • the optimal anti-toxic concentration range of JKF-027 is 0.1-10 ⁇ mol/L.
  • Compound I has a wide range of anti-toxic effective concentrations.
  • the anti-toxic effects between multiple groups of effective concentrations are significantly different from those of the model. There is no significant difference between the effective concentrations.
  • -1000 ⁇ mol / L has a significant anti-toxic effect.
  • the optimal anti-toxic concentration range of JKF-027 is 0.1-10 ⁇ mol/L. Compared with JKF-027, the concentration of anti-toxicity of compound I was wide, and there was no significant difference in anti-toxicity between multiple groups of effective concentrations. The anti-toxic effect in the range of 0.01-1000 ⁇ mol/L compared with the model group. Both have significant differences, so the effect of anti-Alzheimer's disease is much better than JKF-027.

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Abstract

本发明提供了Src蛋白抑制剂N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺(式I)在制备预防和/或治疗阿尔兹海默病药物中的应用，利用原代分离的胎鼠大脑皮层神经元，Aβ42寡聚体和受试药品作用24h，检测不同浓度式Ⅰ化合物体外抗Aβ42寡聚体毒性药效，结果表明式Ⅰ化合物的有效浓度范围宽，且在有效浓度范围内的细胞存活率高。

Description

Src蛋白抑制剂在制备预防和/治疗阿尔兹海默病药物中的应用 技术领域
本发明涉及Src蛋白抑制剂在制备预防和/治疗阿尔兹海默病药物中的应用，特别是化合物N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺在制备预防和/或治疗阿尔兹海默病药物中的应用。
背景技术
老年痴呆(又称为阿尔茨海默症，AD)是中枢神经系统一种常见的退行性疾病，病人临床上首先表现为近期记忆力障碍，继而持续性智能衰退，判断推理能力丧失，运动障碍等。早在1976年，Davies等就发现，AD病人中枢神经元和神经突触特别是参与胆碱能神经通路的神经元和神经突触存在功能异常和凋亡现象。随着分子生物学的发展，先后发现与AD相关的基因有APP(amyloid precursor protein)、PS1(presenilin-1)、PS2(presenilin-2)、ApoE4和线粒体编码的细胞色素氧化酶Ⅰ和Ⅱ等。AD的主要病理改变是区域性神经元丧失、脑组织萎缩，出现大量的淀粉样斑块(amyloid plaques)，亦称老年斑(Senile Plaque,SP)和神经纤维缠结(neurofibritary tangles)沉积，在老年斑周围围绕着营养障碍性轴突、激活的胶质细胞和星形细胞。AD病人痴呆的程度与淀粉样斑块的发展呈正相关，老年斑的主要成分是β淀粉样肽(β-amyloid peptide,β-amyloid protein)-Aβ42，其分子量约4.2KD，由39～43个氨基酸残基组成，Aβ42来自淀粉样前体蛋白(amyloid precursor protein,APP)，完整的APP是Ⅰ型跨膜蛋白，APP进行蛋白水解首先是在α-secretase或β-secretase的作用下，其胞外区发生裂解，产生sAPPα(soluble APP)/sAPPβ和CTFα(C-terminal fragments)/CTFβ，γ-secretase剪切CTFα/CTFβ，生成分子量大小不等的一系列Aβ和CTFγ，大部分PS突变是通过影响γ-secretase(PS可能是γ-secretase的组成部分)而导致病因性的Aβ42生成增加。
在进行威胁健康的流行病学调查中，AD在老年性疾病中的关注程度已跃居前位，成为仅次于心血管病、癌症、脑卒中之后威胁老年人生命健康的高发病。尽管科学家们对AD进行了多年不懈的研究，但如今这一最常见的痴呆疾病仍旧不能被治愈。目前在市场上销售的AD治疗药仅能改善AD患者的症状，延缓疾病的发展，但是不能阻止或者逆转疾病进程。因此，针对AD疾病的发病原因，而不是症状的新作用机制的新药开发仍是国内外制药公司努力的重点方向。
Aβ42在AD的发生发展中起着关键作用，当前针对Aβ42代谢和毒性的深入研究为AD治疗提供了广泛的药物潜在靶点。ZHONG Yi在WO2012/103282中报道了利用转基因果蝇和双转基因小鼠筛选出了一个治疗阿尔茨海默病的优选靶点——表皮生长因子受体(EGFR)。抑制EGFR可改善在转基因果蝇和小鼠中Aβ42诱导的早期记忆丧失，药理活性筛选数据显示，在研EGFR抑制剂(如EKB-569、CL-387785、HKI-272、BIBW 2992、HKI-357、ZD-6474、AEE 788、XL647、BMS-599626、IPI-504、17-AAG、JKF-006、JKF-011、JKF-027、GJ-06、GJ-06-1、GJ-12、GJ-12-1)在治疗Aβ42表达的转基因果蝇和双转基因小鼠的Aβ诱导的记忆丧失中有效。专利中筛选出三个疗效较优的化合物JKF-006、JKF-011、JKF-027。
Figure PCTCN2016085701-appb-000001
N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺，即达沙替尼，用于治疗慢性髓性白血病，是第二代酪氨酸激酶抑制剂，其以Abl，Src，c-Kit，ephrin受体以及其它络氨酸激酶为靶点，但是并不针对EGFR或Her2为靶点。
本发明发现，Src蛋白抑制剂，特别是达沙替尼，可用于预防和/或治疗阿尔兹海默症的药物，并且体外试验结果显示，其在预防和/或治疗阿尔茨海默病和抑制A治疗阿细胞毒性活性比现有报道的EGFR抑制剂有显著提高。
发明内容
本发明提供了Src蛋白抑制剂在制备治疗和/或预防老年痴呆症(AD)疾病的药物中的应用。
本发明还提供了Src蛋白抑制剂在制备治疗治疗和/或预防神经系统退行性疾病的药物中的应用。
本发明还提供了Src蛋白抑制剂在制备和/或改善认知功能障碍或学习记忆能力减退的药物中的应用。
本发明还提供了Src蛋白抑制剂在制备抑制A制备制细胞毒性的药物中的应用。
本发明还提供了Src蛋白抑制剂在制备和/或血管性痴呆或血管性认知障碍的药物中的应用。
本发明还提供了Src蛋白抑制剂在制备和/或胆碱能神经元退行性病变的药物中的应用。
优选的，本发明所述的Src蛋白抑制剂为Src/Abl双效抑制剂。更优选的，本发明所述的Src蛋白抑制剂选自达沙替尼(dasatinib)或其药学上可接受的盐、伯舒替尼(bosutinib)或其药学上可接受的盐。
在一个具体实施方式中，本发明提供了N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺(如式Ⅰ所示)或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备治疗和/或预防老年痴呆症(AD)疾病的药物中的应用。
Figure PCTCN2016085701-appb-000002
进一步的，本发明还提供式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备治疗和/或神经系统退行性疾病的药物中的应用。
进一步的，本发明还提供式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备治疗和/或预防改善认知功能障碍或学习记忆能力减退的药物中的应用。
进一步的，本发明还提供式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备抑制A制或醚细胞毒性的药物中的应用。
进一步的，本发明还提供式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备和/或血管性痴呆或血管性认知障碍的药物中的应用。
进一步的，本发明还提供式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备治疗和/或预防胆碱能神经元退行性病变的药物中的应用。
本发明惊奇的发现，式Ⅰ所示化合物与WO2012/103282中报道的JKF-027进行抗Aβ42蛋白小鼠大脑皮层神经元毒性药效比较试验，利用原代分离的怀孕17天昆明鼠胎鼠大脑皮层神经元，Aβ42寡聚体(10μmol/L)和不同浓度的受试药品作用24h，检测不同浓度式Ⅰ化合物和JKF-027的体外抗Aβ42寡聚体毒性药效。结果显示，JKF-027的有效浓度范围仅为0.1-10μmol/L；而式Ⅰ化合物的有效浓度为0.01-1000μmol/L，剂量跨度比较大，是JKF-027的104倍，并且这些浓度的细胞存活率均接近和超过JKF-027的最有效浓度，总体效果更好。
本发明所述的式Ⅰ化合物应理解为包括式Ⅰ化合物的无定型物和任意晶型，例如US6596746B、US20050215795A、WO2009053854A1、CN102086195A、CN104341410A中公开的达沙替尼的无定型物和任意晶型。
本发明所述的“药学上可接受的盐”是指其母体化合物通过形成其酸盐而得到修饰的衍生物。可接受的盐的实例包括但不限于无机或者有机酸盐。药学上可接受的盐包括母体化合物形成的常规无毒盐，例如，由无毒无机酸或者有机酸形成的盐。例如，所述常规的无毒盐包括由无机酸得到的盐，例如盐酸盐、氢溴酸盐、硫酸盐、磷酸盐和硝酸盐等等；以及由有机酸制备的盐，例如醋酸盐、丙酸盐、琥珀酸盐、乙醇酸盐、硬脂酸盐、乳酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、双羟萘酸盐、马来酸盐、羟基马来酸盐、苯基马来酸盐、谷氨酸盐、安息香酸盐、水杨酸盐、富马酸盐、甲苯磺酸盐、甲磺酸盐、草酸盐和羟乙磺酸盐等。例如本发明所述的式Ⅰ所示化合物药学上可接受的盐是指式Ⅰ所示化合物的盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、硝酸盐、醋酸盐、丙酸盐、琥珀酸盐、乙醇酸盐、硬脂酸盐、乳酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、双羟萘酸盐、马来酸盐、羟基马来酸盐、苯基马来酸盐、谷氨酸盐、安息香酸盐、水杨酸盐、富马酸盐、甲苯磺酸盐、甲磺酸盐、草酸盐和羟乙磺酸盐。
当本发明化合物为碱性时，所述盐可以由药学上可接受的无毒酸进行制备，包括无机或者有机酸。所述酸包括乙酸、苯磺酸、苯甲酸、樟脑磺酸、柠檬酸、乙磺酸、富马酸、葡糖酸、谷氨酸、氢溴酸、盐酸、羟乙磺酸、乳酸、马来酸、苹果酸、扁桃酸、甲磺酸、硝酸、双羟萘酸、泛酸、磷酸、琥珀酸、硫酸、酒石酸和对甲苯磺酸等等。
本发明所述的溶剂化物可以为本领域公知的溶剂化物，所述的式Ⅰ化合物的溶剂化物可以为水合物，例如一水合物、乙醇溶剂化物、异丙醇溶剂化物等。
本发明所述的酯或醚可以为本领域公知的酯或醚，所述的式Ⅰ化合物的酯或醚可以采用本领域常规化学反应可以形成该化合物的醚或酯。
本发明所述的前体或代谢物可以本领域公知的前体或代谢物，只要所述的前体或代谢物通过体内代谢转化形成化合物即可。
在本发明一个优选的实施方式中，式Ⅰ所示化合物为游离碱形式的式I化合物，或其可药用盐。
本发明还涉及一种药物组合物，所述药物组合物包含如式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物以及一种或多种药学上可接受的添加剂。
本发明的药物组合物包括任何通过混合本发明化合物和药学上可接受的添加剂而制备的组合物。“药学上可接受的”意指载体、稀释剂或者赋形剂等添加剂必须与其它制剂成分相容并且对其受者无害。
本发明化合物可以通过口服、胃肠外(例如，肌内、腹膜内、静脉内、ICV、脑池内注射或者输注、皮下注射或者植入)、通过吸入喷雾剂、经鼻、阴道、直肠、舌下或者局部给药途径给药，并且可以将它们单独或者与常规无毒药学上可接受的载体、助剂和赋形剂等添加剂一起配制成含有适用于各种给药途径的适宜剂量的单元制剂。
用于给药的本发明化合物的药物组合物可以适宜地的剂量单元形式存在，并且可以通过药学领域任何熟知的方法进行制备。所有方法都包括使活性成分与包括一种或者多种药学上可接受的添加剂混合的步骤。通常，药物组合物通过以下方式进行制备：使活性成分与液体添加剂或者细碎的固体添加剂或者二者均匀和密切混合，然后将产品成形为期望的制剂。在药物组合物中包含对疾病过程或者症状产生预期疗效量的所述活性成分。
含有活性成分的药物组合物可做成适用的口服形式，例如片剂、含水或者含油混悬剂、可分散粉剂或颗粒剂、乳剂、硬胶囊或软胶囊、糖浆剂等。可使用药物制剂领域熟知任何方法制备用于口服的组合物，所述组合物可以含有一种或者多种选自甜味剂、增香剂、着色剂和防腐剂的添加剂。
片剂含有活性成分与适宜于制造片剂的无毒药学上可接受的添加剂。这些添加剂可以为惰性稀释剂(如碳酸钙、碳酸钠、乳糖、磷酸钙或磷酸钠)、粒化和崩解剂(如玉米淀粉或海藻酸)、粘合剂(如淀粉、凝胶或阿拉伯胶)以及润滑剂(如硬脂酸镁、硬脂酸或滑石)。所述片剂可以未包衣，或通过已知工艺进行包衣以延长在胃肠道内的崩解和吸收，从而提供长期持续的治疗作用。例如，可使用延时材料(如单硬脂酸甘油酯或者二硬脂酸甘油酯)，可通过包衣制成缓控释片剂，可配制成速释片等。
用于口服应用的制剂还可以为硬胶囊，其活性成分与惰性固体稀释剂(如碳酸钙、磷酸钙或高岭土)混合，或软胶囊，其活性成分与含水或含油介质(如花生油、液体石蜡或橄榄油)混合。
含水混悬剂含有活性物质和适用于制造含水混悬剂的无毒药学上可接受的添加剂。所述添加剂为助悬剂(如羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、海藻酸钠、聚乙烯吡咯烷酮、阿拉伯胶)、分散或润湿剂(如卵磷脂、聚氧乙烯硬脂酸酯、十七烷乙烯氧基十六醇、聚氧乙烯山梨醇单油酸酯)。所述含水混悬剂还可以含有一种或多种防腐剂(如对羟基苯甲酸乙酯或对羟基苯甲酸正丙酯)、一种或多种着色剂、一种或多种增香剂和一 种或多种甜味剂(如蔗糖或糖精)。
含油混悬剂可以通过将活性成分悬浮在植物油(如花生油、橄榄油、芝麻油或椰子油)或矿物油(如液体石蜡)中进行制备。所述含油混悬剂可以含有增稠剂(如蜂蜡、硬石蜡或鲸蜡醇)。可以加入上述的甜味剂和增香剂，可以加入抗氧化剂(如抗坏血酸)。
本发明药物组合物还可以为水包油乳化剂形式。所述油相可以为植物油(如橄榄油或花生油)或矿物油(如液体石蜡)或它们的混合物。适宜的乳化剂可以为天然树胶(如阿拉伯胶或黄蓍胶)、天然磷脂(如大豆卵磷脂)和源于脂肪酸与己糖醇酸酐的酯或偏酯(如单油酸山梨醇酐酯)以及所述偏酯与乙烯氧的缩合产物(如聚氧乙烯单油酸山梨醇酐酯)。所述乳化剂还可以含有甜味剂或增香剂。
糖浆剂可以利用甜味剂(如甘油、丙二醇、山梨醇或蔗糖)进行配制。所述糖浆剂还可以含有润湿剂、防腐剂和增香剂以及着色剂。
药物组合物可以为无菌可注射含水或含油混悬剂形式。所述混悬剂可以根据本领域熟知的工艺，利用上述适宜的分散剂或润湿剂和助悬剂进行配制。
药物组合物还可以为用于直肠给药的栓剂形式。所述栓剂可以通过将药物与在常温下为固体但在直肠温度下为液体的适宜的无刺激性添加剂混合制备。所述的添加剂可以为可可脂和聚乙二醇。
对于局部应用，可以使用含本发明化合物的乳膏剂、膏剂、胶状物、液体制剂或者混悬剂等等。类似地，透皮贴片也可以用于局部给药。
因此，本发明还提供了N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺(如式Ⅰ所示)或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备治疗和/或预防老年痴呆症(AD)疾病的药物中的应用，所述的药物包含式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物以及一种或多种药学上可接受的添加剂。
本发明所述的药物可以为口服制剂、肌内、腹膜内、静脉内、ICV、脑池内注射或者输注制剂、皮下注射或者植入制剂、通过吸入喷雾剂、经鼻、阴道、直肠、舌下或者局部给药制剂。
优选的，所述的药物中式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物按质量百分比含量为0.1-99.9％，更优选的为1-90％，更优选的为5-80％，最优选的为10-70％。
附图说明
图1化合物Ⅰ抗Aβ42蛋白第1次体外药效试验柱状图。
图2化合物Ⅰ抗Aβ42蛋白第2次体外药效试验柱状图。
图3化合物Ⅰ抗Aβ42蛋白第3次体外药效试验柱状图。
图4化合物Ⅰ抗Aβ42蛋白3次独立重复体外药效试验均值柱状图。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，必须说明，下述实施例是用于说明本发明而不是对本发明的限制，根据本发明的实质对本发明进行的改进都属于本发明要求保护的范围。本发明所描述术语都是专业科学领域所普通应用的科学术语，并不以任何方式局限本发明范围。
实施例中所用的2-氯-6-甲基苯胺购自杭州达康化工有限公司，(E)-3-乙氧基丙烯酰氯购自湖南华腾制药有限公司，4,6-二氯-2-甲基嘧啶购自常州瑞盛化工有限公司，1-(2-羟乙基)哌嗪购自上海海曲化工有限公司，其余试剂均购自国药集团化学试剂北京有限公司。
实施例1：N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺(化合物I)的制备
Figure PCTCN2016085701-appb-000003
步骤1：(E)-N-(2-氯-6-甲基苯基)-3-乙氧基丙烯酰胺(化合物Ⅱ)的制备
将2-氯-6-甲基苯胺(34.8g,246mmol)溶于CH2Cl2(300mL)中，室温搅拌溶解，然后加入吡啶(18.6g,235mmol)。将反应液用冰水浴冷却至0℃，缓慢滴加(E)-3-乙氧基丙烯酰氯(39.6g,294mmol)，然后反应液在冰水浴中继续搅拌反应3h。减压蒸除溶剂，将残余物用冰水浴冷却至0℃，搅拌加入蒸馏水(180mL)，继续搅拌30min。过滤，用蒸馏水(180mL×2)洗涤滤饼，干燥得白色固体(E)-N-(2-氯-6-甲基苯基)-3-乙氧基丙烯酰胺(Ⅱ)55.8g，收率94.6％。
熔点：170～172℃
核磁共振检测：
1H NMR(400MHz,DMSO-d6):1.27(t,3H),2.16(s,3H),3.94(q,2H),5.58(d,1H),7.18～7.23(m,2H),7.32(d,1H),7.46(d,1H),9.29(s,1H)。
步骤2：2-氨基-N-(2-氯-6-甲基苯基)-5-噻唑甲酰胺(化合物Ⅳ)的制备
将(E)-N-(2-氯-6-甲基苯基)-3-乙氧基丙烯酰胺(Ⅱ)(51.6g,215mmol)溶于蒸馏水(240mL)和1，4-二氧六环(120mL)的混合溶液中，放入低温浴槽中冷却至-10℃，加入N-溴代丁二酰亚胺(42.2g,237mmol)，升温至室温后继续搅拌3h。加入硫脲(16.5g,217mmol)，将反应液加热至80℃后保温搅拌2h。将反应液冷却至室温后，缓慢滴加氨水，调溶液pH值至8～9，减压浓缩至有大量固体析出，向反应液中加入蒸馏水(200mL)，将反应液降温至0℃，抽滤，用蒸馏水(200mL×2)洗涤滤饼，干燥得黄褐色固体2-氨基-N-(2-氯-6-甲基苯基)-5-噻唑甲酰胺(Ⅳ)55.1g，收率95.7％。
熔点：207～209℃
核磁共振检测：
1H NMR(400MHz,DMSO-d6):2.21(s,3H),7.24(m,2H),7.36(d,1H),7.69(s,2H),7.87(s,1H),9.66(s,1H)。
步骤3：N-(2-氯-6-甲基苯基)-2-[(6-氯-2-甲基-4-嘧啶基)氨基]-5-噻唑甲酰胺(化合物Ⅴ)的制 备
将2-氨基-N-(2-氯-6-甲基苯基)-5-噻唑甲酰胺(Ⅳ)(45.0g,168mmol)溶于无水四氢呋喃(450mL)中，加入4,6-二氯-2-甲基嘧啶(32.9g,202mmol)，用冰水浴冷却至10℃以下。向反应液中缓慢加入叔丁醇钠(56.3g,586mmol)，继续搅拌反应3h。用盐酸将溶液pH值调至6，有结晶析出。继续搅拌1.5h，过滤，滤饼用蒸馏水(100mL)洗涤，干燥得淡黄色固体N-(2-氯-6-甲基苯基)-2-[(6-氯-2-甲基-4-嘧啶基)氨基]-5-噻唑甲酰胺(Ⅴ)59.3g，收率89.5％。
熔点：>300℃
核磁共振检测：
1H NMR(400MHz,DMSO-d6):2.23(s,3H),2.57(s,3H),6.93(s,1H),7.27(m,2H),7.39(d,1H),8.28(s,1H),10.00(s,1H),12.20(s,1H)。
步骤4：N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲 酰胺(化合物Ⅰ)的制备
将N-(2-氯-6-甲基苯基)-2-[(6-氯-2-甲基-4-嘧啶基)氨基]-5-噻唑甲酰胺(Ⅴ)(52.5g,133mmol)溶于二甲基亚砜(500mL)中，加入1-(2-羟乙基)哌嗪(60.9g,265mmol)和N,N-二异丙基乙胺(34.7g,267mmol)。将反应液加热至80℃后保温搅拌反应1h。向反应液中加入蒸馏水(1000mL)，冰水浴冷却至15℃，缓慢搅拌析晶1h。过滤，干燥得淡黄色固体61.2g。将该固体加入到80％乙醇水溶液(650mL)中，加热至80℃，保温搅拌直至固体完全溶 解，自然冷却至15℃，并保温搅拌1h，过滤，干燥得白色固体N-(2-氯-6-甲基苯基)-2-{6-[4-(3-羟乙基)-1-哌嗪基]-2-甲基-4-嘧啶基氨基}-5-噻唑甲酰胺(Ⅰ)50.6g，收率75.2％。
HPLC纯度：99.4％
熔点：282～283℃
核磁共振检测：
1H NMR(400MHz,DMSO-d6):2.24(s,3H),2.42(s,3H),2.46(t,2H),2.50(t,4H),3.50(m,4H),3.54(q,2H),4.41(t,1H),6.05(s,1H),7.23(t,1H),7.27(d,1H),7.39(d,1H),8.21(s,1H),9.85(s,1H),11.43(s,1H)。
实施例2：化合物Ⅰ抗Amyloid-β蛋白小鼠大脑皮层神经元毒性药效试验
本发明测试所用的化合物JKF-027依据文献报道的方法制备得到：WO2012/103282(公开日：2012年08月02日)
1)试验目的
利用Amyloid-β42寡聚体致胎鼠原代分离脑皮层神经元损伤，检测化合物Ⅰ抗Aβ42蛋白毒性作用。
2)试验材料
(1)供试品基本信息
Figure PCTCN2016085701-appb-000004
(2)供试品配制
配制方法：稀释化合物Ⅰ和JKF-027成7个浓度。
配制后暂存条件及有效期：现用现配。
3)试验方法
(1)胎鼠神经元原代分离
试验准备：消毒的手术器械、移液枪、冰袋、培养皿、70％酒精及棉球、离心管、12孔板(已包被)、胰酶、HBSS液、DNA酶、生长培养基、维持培养基。
试验操作：取出怀孕17天的孕鼠子宫，逐个分离胚胎。取出胎鼠的脑皮层，将其放入HBSS中并清洗几次。加入酶消化处理后弃去上清。加入生长培养基。倾斜培养皿，用1mL移液枪缓慢吹打，静止后取上层单个悬浮细胞转移至离心管，重复上述操作三次。
(2)预试验：Aβ42致神经元损伤的毒性浓度试验
在神经元培养第6天，将细胞分成6组，Aβ42浓度分别为0、10、20、40、80、160μmol/L，每组均加入Aβ42单体寡聚体混合物(H4)，作用时间均为24个小时，并设置一个复孔。一次试验使用12个细胞培养孔，试验重复1次，试验分组如表1所示。
表1 造模的浓度分组表(μmol/L)
Figure PCTCN2016085701-appb-000005
根据试验结果，利用WST-8检测细胞活性，筛选神经元成活率30％-60％的Aβ42浓度作为正式造模浓度，测定JKF-027的神经保护活性。
(3)化合物Ⅰ的神经保护活性测定
加样：由细胞存活率能够看到，当高于80μmol/L浓度时，由于造模药浓度太高相互聚集形成胶状物质，影响蛋白之间交互作用，导致寡聚体聚合效果下降，高浓度的Aβ42细胞毒性效果下降。而低于10μmol/L后造模药浓度较低，在相同聚合时间内寡聚体聚合效率较低，细胞毒性下降。10μmol/L-80μmol/L的浓度范围的细胞存活率都能达到预先设定的要求，由于在人体内Aβ42含量非常低，因此我们选择10μmol/L造模药浓度来构建细胞损伤模型。在神经元培养第6天加入相同浓度的Aβ42以及不同浓度的不同受试药物培养24h，用WST-8检测细胞活性。并设置1个复孔及2个阴性对照孔和2个正常对照孔。试验重复2次。试验分组如表2所示。
表2 药物的浓度分组表(μmol/L)
Figure PCTCN2016085701-appb-000006
(4)用WST-8检测细胞活性
向每孔中加入相应量的WST-8溶液。
将培养板在培养箱内孵育2-4小时。
用酶标仪测定在450nm处的吸光度。
4)细胞活性检测
细胞活力*(％)＝[A(加药)-A(空白)]/[A(未加药)–A(空白)]×100％
A(加药)：具有细胞、WST溶液和药物溶液的孔的吸光度
A(空白)：具有培养基和WST溶液而没有细胞的孔的吸光度
A(未加药)：具有细胞、WST溶液而没有药物溶液的孔的吸光度
*细胞活力：细胞增殖活力或细胞毒性活力
5)试验结果
第1次独立试验结果见表3和图1；第2次独立试验结果见表4和图2；第3次独立试验结果见表5和图3；三次独立试验结果平均值见表6和图4。从试验结果来看，三次试验具有相当好的重复性，结果更加具有说服力。横坐标代表不同的给药浓度，1代表0.001μM；2代表0.01μmol/L；3代表0.1μmol/L；4代表1.0μmol/L；5代表10μmol/L；6代表100μmol/L；7代表1000μmol/L。不同浓度的不同受试药物抗Aβ42蛋白毒性曲线呈现先上升后下降的趋势，且都有一个有效剂量区间。相比模型组，JKF-027最佳抗毒浓度范围为0.1-10μmol/L。相比于JKF-027，化合物Ⅰ抗毒有效浓度范围广，多组有效浓度之间抗毒作用与造模组相比都具有差异显著性，有效浓度之间不存在明显的差异，在浓度0.01-1000μmol/L之间都具有明显抗毒作用。
从3次重复试验结果来看，3次检测数值规律基本一致，药物细胞试验的趋势都呈现很正常的峰值曲线，两种受试药品均有效。JKF-027的最佳抗毒浓度范围为0.1-10μmol/L。相比于JKF-027，化合物Ⅰ的抗毒作用浓度范围广，多组有效浓度之间抗毒作用都不存在明显的差异，在浓度0.01-1000μmol/L范围内抗毒作用相比于模型组都具有明显差异，因此抗阿尔兹海默症作用效果较JKF-027要好很多。
表3 第1次独立试验存活率数据
Figure PCTCN2016085701-appb-000007
表4 第2次独立试验存活率数据
Figure PCTCN2016085701-appb-000008
表5 第3次独立试验存活率数据
Figure PCTCN2016085701-appb-000009
表6 根据3次独立重复试验得到的细胞存活率平均值
Figure PCTCN2016085701-appb-000010

Claims (10)

  1. Src蛋白抑制剂在制备下述药物中的应用：
    (1)治疗和/或预防阿尔茨海默病或老年痴呆症；
    (2)治疗和/或预防神经系统退行性疾病；
    (3)改善认知功能障碍或学习记忆能力减退；
    (4)抑制A抑制忆细胞毒性；
    (5)治疗和/或预防血管性痴呆或血管性认知障碍；
    (6)治疗和/或预防胆碱能神经元退行性病变。
  2. 权利要求1所述的应用，所述的Src蛋白抑制剂为Src/Abl双效抑制剂。
  3. 权利要求1所述的应用，所述的Src蛋白抑制剂选自达沙替尼(dasatinib)或其药学上可接受的盐、伯舒替尼(bosutinib)或其药学上可接受的盐。
  4. 式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物在制备下述药物中的应用：
    Figure PCTCN2016085701-appb-100001
    (1)治疗和/或预防阿尔茨海默病或老年痴呆症；
    (2)治疗和/或预防神经系统退行性疾病；
    (3)改善认知功能障碍或学习记忆能力减退；
    (4)抑制A抑制忆细胞毒性；
    (5)治疗和/或预防防血管性痴呆或血管性认知障碍；
    (6)治疗和/或预防胆碱能神经元退行性病变。
  5. 权利要求4所述的应用，所述的式Ⅰ所示化合物为游离碱形式的式I化合物，或其可药用盐。
  6. 权利要求4所述的应用，所述的药学上可接受的盐为盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、硝酸盐、醋酸盐、丙酸盐、琥珀酸盐、乙醇酸盐、硬脂酸盐、乳酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、双羟萘酸盐、马来酸盐、羟基马来酸盐、苯基马来酸盐、谷氨酸盐、安息香酸盐、水杨酸盐、富马酸盐、甲苯磺酸盐、甲磺酸盐、草酸盐和羟乙磺酸盐。
  7. 权利要求4所述的应用，所述的溶剂化物或盐的溶剂化物是指式Ⅰ所示化合物的水合物或式Ⅰ所示化合物的盐的水合物。
  8. 权利要求4所述的应用，所述的药物包含如式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物以及一种或多种药学上可接受的添加剂。
  9. 权利要求4所述的应用，所述的药物为口服制剂、肌内、腹膜内、静脉内、ICV、脑池内注射或者输注制剂、皮下注射或者植入制剂、通过吸入喷雾剂、经鼻、阴道、直肠、舌下或者局部给药制剂。
  10. 权利要求4所述的应用，所述的药物中式Ⅰ所示化合物或其药学上可接受的盐、溶剂化物或盐的溶剂化物、酯或醚、前体或代谢物按质量百分比含量为0.1-99.9％，优选的为1-90％，更优选的为5-80％，最优选的为10-70％。
PCT/CN2016/085701 2015-06-24 2016-06-14 Src蛋白抑制剂在制备预防和/治疗阿尔兹海默病药物中的应用 WO2016206551A1 (zh)

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