WO2015154721A1 - 异类叶升麻苷衍生物及其制造方法与用途 - Google Patents

异类叶升麻苷衍生物及其制造方法与用途 Download PDF

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WO2015154721A1
WO2015154721A1 PCT/CN2015/076381 CN2015076381W WO2015154721A1 WO 2015154721 A1 WO2015154721 A1 WO 2015154721A1 CN 2015076381 W CN2015076381 W CN 2015076381W WO 2015154721 A1 WO2015154721 A1 WO 2015154721A1
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formula
group
compound
hydrogen
independently selected
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苏慕寰
林汉钦
胡明宽
李易达
王昭日
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Sinphar Tian Li Pharmaceutical Co Ltd
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Sinphar Tian Li Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the present invention relates to a heterologous leaf asparagine derivative, a method for producing the same, and a method for producing the same, and particularly to a heterologous leaf asparagine derivative, a method for producing the same, and a medicament comprising a heterologous leaf cystein derivative use.
  • Isoacteoside is a kind of phenylpropanoid glycoside compound which is found in many plants, such as Cistanche deserticola.
  • the structure of heterologous leaf aspirin comprises dihydrooxyphenethyl-D-glucoside, phenyl acrylate and monosaccharide.
  • heterophylloside has neuroprotection, liver protection, anti-oxidation and biological activity to reduce aggregation of amyloid peptides.
  • the Caffeoyl group is located in the sixth position (for example, heterophylloside), ⁇ -amyloid peptides (A ⁇ ) are inhibited.
  • the activity of aggregation is better, and if it is in the fourth position (e.g., acteoside), the activity is lowered. This result shows that the position of the caffeoyl group has a great influence on the activity of the heterophylloside.
  • one of the possible bisphenol groups of catechol has metal chelation for transition metals (such as copper, iron and zinc).
  • the Phenylethanoid group in the first position also has a catechol group, which should also have similar metal chelation, and may be an essential group for activity.
  • the present invention provides for the treatment or prevention of amyloid peptide-related diseases (eg, neuroprotection, reduction of amyloid peptide aggregation, neurodegenerative diseases, and ocular lesions) by synthesizing a series of derivatives of heterophylloside Etc.)
  • amyloid peptide-related diseases eg, neuroprotection, reduction of amyloid peptide aggregation, neurodegenerative diseases, and ocular lesions
  • heterophyllophyllin derivative having the structure of formula (I):
  • R 1 and R 2 are independently selected from hydrogen, halogen, hydroxy, or alkoxy
  • R 3 and R 4 are independently selected from hydroxy, alkoxy or acyloxy
  • R 5 is independently It is selected from a hydroxy or acyloxy group.
  • R 1 and/or R 2 when R 1 and/or R 2 is a hydrocarbyloxy group, it is independently selected from an alkoxy group, an alkenyloxy group or an aryloxy group.
  • R 1 and/or R 2 when R 1 and/or R 2 is an alkoxy group, it is a methoxy group.
  • R 1 and/or R 2 when R 1 and/or R 2 is an alkenyloxy group, it is an allyloxy group.
  • R 1 and/or R 2 when R 1 and/or R 2 is an aryloxy group, it is a benzyloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is a hydrocarbyloxy group, it is independently selected from an alkenyloxy group or an aryloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an alkenyloxy group, it is an allyloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an aryloxy group, it is a benzyloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an acyloxy group, it is an acetoxy group.
  • R 3 and R 4 are the same substituent.
  • R 5 when R 5 is an acyloxy group, it is an acetoxy group.
  • R 5 is the same substituent.
  • the heterophylloin is a structure selected from the group consisting of:
  • Another aspect of the present invention is to provide a medicament for use in the treatment or prevention of a disease associated with an amyloid peptide, wherein the medicament comprises the aforementioned heterophylloside derivative.
  • the disease associated with the amyloid peptide is a neurodegenerative disease.
  • the disease associated with the amyloid peptide is Alzheimer's disease, Mild Cognitive Impairment, Lewy body dementia, Down syndrome (Down's syndrome), hereditary cerebral hemorrhage complicated with amyloidosis (Dutch type); Guam Parkinson-Dementia complex, progressive supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson's disease, frontotemporal dementia, Pick's disease, amyotropic lateral sclerosis, inclusion body myositis Inclusion-body myositis), adult-onset diabetes, senile myocardial amyloidosis or endocrine tumors.
  • the amyloid peptide is a beta-type starch peptide.
  • Yet another aspect of the present invention provides a use of a medicament for the prevention of ocular lesions, wherein the medicament comprises the aforementioned heterophylloside derivative.
  • the ocular lesion is neuronal degeneration, visual cortical defect, glaucoma, cataract, ocular amyloidosis, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis or grid dystrophy.
  • a process for producing a heterophyllophylline derivative comprising reacting a compound having the structure of the formula (II) with ⁇ -D-glucose pentaacetate to form a formula (III) a compound of the structure wherein formula (II) is:
  • R 1 and R 2 are independently selected from hydrogen, chlorine, methoxy or benzyloxy.
  • the reaction is carried out in a potassium oxide-methanol solution to form a compound having the structure of the formula (IV-1), wherein the formula (IV-1) is:
  • R 3 and R 4 are independently selected from hydrogen or allyloxy
  • a compound having the structure of the formula (III) is dissolved in methanol and mixed with sodium methoxide to Forming a compound having the structure of the formula (IV-1) wherein R 3 and R 4 are independently selected from hydrogen, chlorine, methoxy or benzyloxy, or (3) a compound having the structure of the formula (III)
  • the acetyl chloride and methanol-dichloromethane are reacted to form a compound having the structure of the formula (IV-2), wherein the formula (IV-2) is:
  • R 5 and R 6 are independently selected from hydrogen or chlorine. Then, a compound having the structure of the formula (IV-1) or a compound having the structure of the formula (IV-2) and di-o-acetyl-ferulic acid chloride, di-o-allyl-ferulic acid chloride The compound or di-o-benzyl ferulic acid chloride is reacted in a solution containing dichloromethane and pyridine to form a compound having any structure of the formula (V-1) to (V-4), wherein the formula (V) -1) is:
  • R 7 and R 8 are independently selected from hydrogen or allyloxy, and the formula (V-2) is:
  • R 9 and R 10 are independently selected from hydrogen, methoxy or benzyloxy, and the formula (V-3) is:
  • R 11 and R 12 are independently selected from hydrogen, methoxy or benzyloxy, and the formula (V-4) is:
  • R 13 and R 14 are independently selected from hydrogen or chlorine.
  • the manufacturing method further comprises reacting a compound having the structure of the formula (V-1) with a cuprous chloride and palladium chloride in a mixture of methanol and water to form a formula (VI-1).
  • a compound of the structure wherein the formula (VI-1) is:
  • R 15 and R 16 are independently selected from hydrogen or a hydroxyl group.
  • the manufacturing method further comprises reacting a compound having the structure of the formula (V-2) with methylamine-containing methanol to form a compound having the structure of the formula (VI-1), wherein the formula (VI) -1) is:
  • R 15 and R 16 are independently selected from hydrogen, chlorine, methoxy or benzyloxy.
  • the manufacturing method further comprises reacting a compound having a structure of the formula (V-4) with methylamine-containing methanol to form a compound having the structure of the formula (VI-2), wherein the formula (VI) -2) is:
  • R 17 and R 18 are independently selected from hydrogen or chlorine.
  • the heterologous leaf asparagine derivative of the present invention is a method for treating or preventing an amyloid peptide-related disease and preventing eye by modifying the chemical structure of the heterophylloside For lesions and other uses.
  • FIGS. 1A to 1D are diagrams showing test results of inhibition of accumulation of amyloid peptides by a heterologous leaf asparagine derivative according to an embodiment of the present invention, and a graph showing results of cell survival analysis;
  • 2A to 2B are graphs showing the results of an experiment for inhibiting the accumulation of an amyloid peptide in a heterologous leaf asparagine derivative according to an embodiment of the present invention, and a graph showing the results of cell survival analysis;
  • 3A to 3B are diagrams showing the results of an experiment for inhibiting aggregation of an amylin-like peptide by a heterologous leaf asparagine derivative according to an embodiment of the present invention
  • 4A to 4B are graphs showing the results of an experiment for inhibiting aggregation of an amylin-like peptide by a heterologous leaf asparagine derivative according to an embodiment of the present invention
  • Figure 5 is a graph showing the results of the test for degradation of the heterologous leaf asparagine derivative A ⁇ according to the embodiment of the present invention.
  • 6A to 6B are graphs showing the results of an experiment for preventing eye lesions by a heterologous leaf asparagine derivative according to an embodiment of the present invention.
  • heterophyllophyllin derivative having the structure of formula (I):
  • R 1 and R 2 are independently selected from hydrogen, halogen, hydroxy, or alkoxy
  • R 3 and R 4 are independently selected from hydroxy, alkoxy or acyloxy
  • R 5 is independently It is selected from a hydroxy or acyloxy group.
  • hydrocarboxyl group refers to a group formed by linking a hydrocarbon group to an oxygen atom, wherein the hydrocarbon group is an organic compound composed of carbon and hydrogen, and contains an alkane, an alkene or an alkyne. , cyclic hydrocarbons and aromatic hydrocarbons.
  • Acyloxy group refers to a group formed by linking an acyl group to an oxygen atom, wherein the acyl group means a functional group derived by removing one or more hydroxyl groups from an oxyacid.
  • amyloid peptide-related diseases refers to neurodegenerative diseases, Alzheimer's disease, Mild Cognitive Impairment, and Lewy Body Dementia. (Lewy body dementia), Down's syndrome, hereditary cerebral hemorrhage complicated with amyloidosis (Dutch type); Guam Parkinson-Dementia complex Nuclear palsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson's disease, frontotemporal dementia, Pick disease, amyotrophic lateral sclerosis (amyotropic) Lateral sclerosis), inclusion-body myositis, adult-onset diabetes, senile myocardial amyloidosis or endocrine tumors.
  • ocular lesion refers to neuronal degeneration, visual cortical defect, glaucoma, cataract, ocular amyloidosis, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis or mesh. Malnutrition.
  • R 1 and/or R 2 are halogen, it is chlorine.
  • R 1 and/or R 2 are a hydrocarbyloxy group, they are independently selected from alkoxy, alkenyloxy or aryloxy.
  • Alkoxy group refers to a group formed by linking an alkyl group to an oxygen atom.
  • Alkenoxy group refers to a group formed by linking an alkenyl group to an oxygen atom.
  • Aryloxy group means a group formed by linking an aryl group to an oxygen atom, wherein the aryl group means any functional group derived from an aromatic ring.
  • R 1 and/or R 2 when R 1 and/or R 2 is an alkoxy group, it is a methoxy group.
  • the methoxy group has a structure of -O-CH 3 and is represented by "OMe" in the following chemical formula.
  • R 1 and/or R 2 when R 1 and/or R 2 is an alkenyloxy group, it is an alloxy group. Allyloxy has The structure is represented by "OAll" in the following chemical formula.
  • R 1 and/or R 2 when R 1 and/or R 2 is an aryloxy group, it is a benzyloxy group.
  • Benzyloxy has The structure is represented by "OBn" in the following chemical formula.
  • R 3 and/or R 4 when R 3 and/or R 4 is a hydrocarbyloxy group, it is independently selected from an alkenyloxy group or an aryloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an alkenyloxy group, it is an allyloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an aryloxy group, it is a benzyloxy group.
  • R 3 and/or R 4 when R 3 and/or R 4 is an acyloxy group, it is an acetoxy group.
  • Acetoxy has The structure is represented by "OAc" in the following chemical formula.
  • R 3 and R 4 are the same substituent.
  • R 5 when R 5 is an acyloxy group, it is an acetoxy group.
  • R 5 is the same substituent.
  • the heterophylloside is a structure selected from the group consisting of:
  • Another aspect of the present invention provides a use of a medicament for treating or preventing an amyloid peptide-related disease, wherein the medicament comprises the aforementioned heterophylloside derivative.
  • the amyloid peptide is a beta-amyloid peptide (A ⁇ ).
  • the ⁇ -amyloid peptide is an amyloid precursor protein (APP) via different secretases, from the original 770 amino acid protein to the ⁇ -amyloid peptide containing 37 to 49 amino acids. .
  • the common ⁇ -type starch peptides A ⁇ 40 and A ⁇ 42 in which A ⁇ 42 has higher hydrophobicity, are more likely to form ⁇ -amyloid plaque than A ⁇ 40 .
  • the deposited A ⁇ fibers are neurotoxic and can cause a series of complex reactions such as changes in synapses, phosphorylation of Tau protein, reduction of neurotransmitters, proliferation of glial cells, and inflammatory responses.
  • beta-amyloid plaque accumulation is thought to be caused
  • One of the causes of Alzheimer's disease so many of the current drugs for the treatment or prevention of Alzheimer's disease and the deterioration of the symptoms have been developed to interfere with the production of ⁇ -amyloid peptides. It prevents the formation of ⁇ -amyloid plaques by reducing the production of ⁇ -amyloid peptides, inhibiting the accumulation of ⁇ -amyloid peptides in the extracellular, and inhibiting the aggregation of ⁇ -amyloid peptides.
  • the heterologous leaf asparagine derivative of the present invention can inhibit the aggregation of the ⁇ -amyloid peptide, and further has the effects of neuroprotection and treatment of neurodegenerative diseases.
  • Yet another aspect of the present invention provides a use of a medicament for the prevention of ocular lesions, wherein the medicament comprises the aforementioned heterophylloside derivative.
  • Retinal Pigment Epidermis is located between the retinal neuroepithelial layer and the choroid, and has many physiological functions. For example: retinal barrier, phagocytic function, involvement in visual cycle metabolism, antioxidant function and secretion of growth factors. Retinal pigment epithelial cells are the same as other tissues, and are easily damaged by oxidative stress, causing cell death and causing retinopathy, visual dysfunction, and severe visual function loss.
  • retinal pigment epithelial cells are often used to study cell patterns of retinopathy such as diabetic retinopathy or age-related macular degeneration.
  • the heterologous leaf asparagine derivative of the invention can reduce free radicals, avoid oxidative stress damage, and further has the functions of anti-oxidation, protection of retinal cells and prevention of ocular lesions.
  • the heterologous leaf asparagine derivative of the present invention is capable of treating or preventing a disease associated with an amylin-like peptide, neuroprotection, treating a neurodegenerative disease, and preventing an ocular lesion by changing the chemical structure of the isoflavone And other effects.
  • the heterophylloside derivatives of the examples were synthesized by the following steps A to G.
  • Step A contains the following steps:
  • R 1 of compound 1a is hydrogen and R 2 is hydrogen;
  • R 1 of compound 1b is hydrogen and R 2 is chlorine;
  • R 1 of compound 1c is methoxy (OMe), R 2 is methoxy;
  • compound 1d R 1 is hydrogen, R 2 is benzyloxy (OBn);
  • R 1 of compound 1e is benzyloxy, R 2 is benzyloxy;
  • R 1 of compound 1f is benzyloxy and R 2 is hydrogen.
  • Step 2 The reaction mixture of Step 1 was stirred for 6 hours and then stirred vigorously with saturated aqueous sodium bicarbonate for 30 min.
  • the structure of formula (III) is:
  • R 1 of compound 2a is hydrogen and R 2 is hydrogen
  • R 1 of compound 2b is hydrogen and R 2 is chlorine
  • R 1 of compound 2c is methoxy and R 2 is methoxy
  • R 1 of compound 2d is Hydrogen
  • R 2 is a benzyloxy group
  • R 1 of the compound 2e is a benzyloxy group
  • R 2 is a benzyloxy group
  • R 1 of the compound 2f is a benzyloxy group, and R 2 is hydrogen.
  • step A The reaction process of step A is as follows:
  • Step B contains the following steps:
  • Step 2 Mix the intermediate product of Step 1 with Potassium carbonate (K 2 CO 3 ), allyl bromide, and acetone. The mixture was refluxed with a calcium chloride drying tube (CaCl 2 ) in a silicone oil bath for 10 hours and then cooled to room temperature.
  • K 2 CO 3 Potassium carbonate
  • CaCl 2 calcium chloride drying tube
  • R 1 of compound 3d is hydrogen
  • R 2 is allyloxy (OAll)
  • R 1 of compound 3e is allyloxy
  • R 2 is allyloxy
  • R 1 of compound 3f is allyloxy
  • R 2 is hydrogen
  • step B The reaction process of step B is as follows:
  • Step C contains the following steps:
  • R 1 of compound 3a is hydrogen and R 2 is hydrogen;
  • R 1 of compound 3b is hydrogen and R 2 is chlorine;
  • R 1 of compound 3c is methoxy, R 2 is methoxy; and R 1 of compound 3g is hydrogen.
  • R 2 is a benzyloxy group
  • R 1 of the compound 3h is a benzyloxy group
  • R 2 is a benzyloxy group
  • R 1 of the compound 3i is a benzyloxy group
  • R 2 is hydrogen.
  • step C is as follows:
  • Step D contains the following steps:
  • step 2 The mixture of step 1 was neutralized with Triethanolamine (TEA). Next, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography using ethyl acetate (EA) as a solvent to give compound 3j and 3k having the structure of formula (IV-2).
  • the structure of formula (IV-2) is:
  • R 1 of compound 3j is hydrogen and R 2 is hydrogen; and R 1 of compound 3k is hydrogen and R 2 is chlorine.
  • step D is as follows:
  • Step E contains the following steps:
  • step 1 of step E can be divided into the following three conditions:
  • R 1 of compound 4d is hydrogen and R 2 is allyloxy
  • R 1 of compound 4e is allyloxy
  • R 2 is allyloxy
  • R 1 of compound 4f is allyloxy, R 2 It is hydrogen.
  • R 1 of compound 4a is hydrogen and R 2 is hydrogen;
  • R 1 of compound 4b is hydrogen and R 2 is chlorine;
  • R 1 of compound 4c is methoxy and R 2 is methoxy;
  • R 1 of compound 4g is Hydrogen,
  • R 2 is a benzyloxy group;
  • R 1 of the compound 4h is a benzyloxy group, and R 2 is a benzyloxy group.
  • R 1 of compound 4i is benzyloxy and R 2 is hydrogen
  • R 1 of compound 4m is hydrogen
  • R 2 is benzyloxy
  • R 1 of compound 4n is hydrogen
  • R 2 is hydrogen
  • R 1 of compound 4o Is a methoxy group and R 2 is a methoxy group.
  • R 1 of compound 4j is hydrogen and R 2 is hydrogen; and R 1 of compound 4k is hydrogen and R 2 is chlorine.
  • reaction process for forming compounds 4d to 4f in step E is as follows:
  • Step F contains the following steps:
  • the compounds 4d to 4e are separately stirred with a mixture of cuprous chloride (CuCl) and palladium chloride (PdCl 2 ) in methanol and water at room temperature to obtain a compound 5d having the structure of the formula (VI-1). ⁇ 5f.
  • the structure of formula (VI-1) is:
  • R 1 of the compound 5d is hydrogen and R 2 is a hydroxyl group
  • R 1 of the compound 5e is a hydroxyl group
  • R 2 is a hydroxyl group
  • R 1 of the compound 5f is a hydroxyl group, and R 2 is hydrogen.
  • reaction process for forming compounds 5d to 5f of operation F is as follows:
  • Step G includes the following steps:
  • Step 2 The reaction mixture of Step 1 was stirred for 20 minutes and then concentrated under vacuum. The residue was purified by silica gel column chromatography using methanol/dichloromethane mixed solvent (1:20, v/v) to give compounds 5a to 5c, 5g and 5h having the structure of formula (VI-1) and Compounds 5j and 5k having the structure of the formula (VI-2) wherein the yield of the compound 5a is 90%.
  • R 1 of compound 5a is hydrogen and R 2 is hydrogen; R 1 of compound 5b is hydrogen and R 2 is chlorine; R 1 of compound 5c is methoxy, R 2 is methoxy; and R 1 of compound 5g is hydrogen R 2 is a benzyloxy group; and R 1 of the compound 5h is a benzyloxy group, and R 2 is a benzyloxy group.
  • the structure of formula (VI-2) is:
  • R 1 of compound 5j is hydrogen and R 2 is hydrogen; and R 1 of compound 5k is hydrogen and R 2 is chlorine.
  • reaction process for the formation of compounds 5a to 5c, 5g and 5h of step G is as follows:
  • a stock solution having a concentration of 10 mM was formulated into a solution of different sample concentrations.
  • the stock solution is prepared to a concentration of 5 ⁇ M, and 0.5 ⁇ L of the stock solution is quantified to 1 mL; the concentration is 10 ⁇ M.
  • 1 ⁇ L of the stock solution was quantified to 1 mL; the concentration was 20 ⁇ M, and 2 ⁇ L of the stock solution was quantified to 1 mL; when the concentration was 50 ⁇ M, 5 ⁇ L of the stock solution was quantified to 1 mL; when the concentration was 100 ⁇ M, 1 ⁇ L of the stock solution was quantified to 0.1 mL.
  • the concentration of 200 ⁇ M was determined to be 2 ⁇ L of the stock solution to 0.1 mL.
  • Experimental Example 1 to Experimental Example 4 were to evaluate the pharmacological effects of the heterologous leaf apocynin derivatives of the present invention by the following three directions, including: 1) whether the cell-producing ⁇ -amyloid peptide (A ⁇ ) can be reduced; 2) It can promote the cell to clear the activity of the ⁇ -amyloid peptide enzyme, thereby increasing the efficiency of scavenging the ⁇ -amyloid peptide; and 3) Whether it can inhibit the aggregation of A ⁇ 40 and A ⁇ 42 .
  • a ⁇ cell-producing ⁇ -amyloid peptide
  • the experimental example is divided into two stages: the first stage uses a lower sample concentration for preliminary screening; the second stage is based on the results of the first stage, further selecting an effective sample to increase the test concentration of the sample without affecting cytotoxicity. In the case, the optimal concentration and the best effect of inhibiting the accumulation of A ⁇ 40 in the test sample were obtained.
  • Human neuroblastoma cells (SH-SY5Y-APP695) expressing Swedish mutant human APP695 were cultured in 3.5 cm culture dishes to a cell concentration of 90% full, and in a 24-well plate at the time of passage 4 x 10 5 cells per well.
  • the culture medium was replaced with 300 ⁇ L of chemical-defined medium every other day, which was DMEM/F12 medium containing 5 mM Hepes buffer, 0.6% glucose, 3 mM NaHCO 3 , 2.5 ⁇ M Glutamine, 100 ⁇ g/mL of transferrin, 20 nM of progesterone, 60 ⁇ M of putrescine, 30 nM of sodium selenite, and 2 ⁇ g/mL of heparin.
  • test sample 3 ⁇ L was added to each well, and each concentration sample was 4 sets each. After the cells were placed in an incubator (37 ° C, 5% CO 2 ) for 24 hours, the culture solution was collected and analyzed by immunoassay reagent (Human A ⁇ 1-40 Immunoassay kits, Cat. KHB3482, Life Technologies). The A ⁇ 40 content in the post-culture medium was evaluated by cell viability assay (MTT assay) for toxicity to the cells after sample treatment.
  • MTT assay cell viability assay
  • the test concentrations of Sample 1 were 5 ⁇ M and 10 ⁇ M, and the test concentrations of Samples 2 to 8 were 10 ⁇ M and 20 ⁇ M.
  • the A ⁇ 40 content in the SH-SY5Y-APP695 cell culture medium to which no test sample was added was determined as a control group and set to 100%, and the A ⁇ 40 content in the culture medium treated with each test sample was compared with the control group. And expressed as a percentage. Further, a ⁇ -secretase inhibitor ( ⁇ -SI) was used as a positive control group.
  • Figs. 1A to 1D in which Figs. 1A and 1C are graphs showing the results of the test for inhibiting the accumulation of A? 40 , and Figs.
  • FIGS. 1B and 1D are graphs showing the results of cell viability analysis after the sample treatment.
  • samples 3, 4 and 6 have the effect of inhibiting the accumulation of A ⁇ 40 , wherein the inhibition of the accumulation of A ⁇ 40 in the cells by sample 3 was observed. The effect is the best, and the effect of inhibiting the accumulation of A ⁇ 40 is increased as the concentration of the sample increases.
  • sample concentrations of samples 3, 4, and 6 were increased, and the optimal concentration and the best effect of inhibiting the accumulation of A ⁇ 40 in the test samples were obtained without affecting the cytotoxicity.
  • sample 3 inhibited accumulation of about 20% at 20 ⁇ M without causing cytotoxicity
  • sample 6 inhibited accumulation of about 40% at 50 ⁇ M, while increasing sample concentration was not.
  • the effect of the sample 4 on inhibiting the accumulation of A ⁇ can be further promoted.
  • heterologous leaf asparagine derivatives of the present invention have an effect of inhibiting the accumulation of A ⁇ 40 .
  • Experimental Example 3 is to verify the efficacy of the sample in inhibiting A ⁇ 40 aggregation and inhibiting A ⁇ 42 oligomerization.
  • a ⁇ 40 aggregation The A ⁇ 40 solution was reconstituted in DMSO to 10 mg/mL. Each group of reactions contained 0.5 ⁇ l of 10 mg/mL A ⁇ 40 and 4.5 ⁇ L of the test sample diluted with Dulbecco's Phosphate-Buffered Saline (D-PBS). The concentration of sample 1 was 10 ⁇ M and 100 ⁇ M, and the concentrations of samples 2 to 8 were 20 ⁇ M and 200 ⁇ M. The total volume of the reaction was 5 ⁇ L, and each concentration was 6 groups. After reacting for 4 hours in an incubator at 37 ° C, 200 ⁇ L of Thioflavin T working solution (ThT working solution) was added and thoroughly mixed.
  • Thioflavin T working solution Thioflavin T working solution
  • the flavin T of 10 ⁇ M of the flavin T was dissolved in a potassium phosphate buffer (Potassium phosphate buffer, pH 6.0). 200 ⁇ L of the mixture was placed in a 96-well disk with a black transparent bottom, and the fluorescence intensity of thioflavin T (Ex: 440 nm, Em: 482 nm) was measured from the bottom of the plate to determine the degree of aggregation of A ⁇ 40 .
  • This experimental example measures the extent of A ⁇ aggregation by the thioflavin T assay (ThT assay). Since ThT and Congo red derivatives can form bonds with the polymeric form of A) ⁇ protein, the higher the degree of A ⁇ aggregation, the greater the number of bonded ThTs.
  • the degree of change in A ⁇ aggregation can be estimated.
  • the readings without any test sample reaction ie, containing only 0.5 ⁇ L of A ⁇ 40 and 4.5 ⁇ L of D-PBS, wherein the final concentration of A ⁇ 40 was 1 mg/mL
  • the reading was set to 100. %, and a heterologous leaf aspirin (IsoA) was in the control group.
  • the readings of the samples are compared to the control and expressed as a percentage.
  • Fig. 3A is a graph showing the results of experiments in which low concentration samples 1 to 9 inhibit A ⁇ 40 aggregation.
  • the concentration of sample 1 was 10 ⁇ M
  • the concentration of samples 2 to 9 was 20 ⁇ M
  • the concentration of IsoA was 10 ⁇ g/mL.
  • the readings measured in the control group to which no sample was added were set to 100%, and the remaining values were adjusted accordingly.
  • Fig. 3B is a graph showing the results of experiments in which high concentration samples 1 to 9 inhibit A ⁇ 40 aggregation.
  • the concentration of sample 1 was 100 ⁇ M
  • the concentration of samples 2 to 9 was 200 ⁇ M
  • the concentration of IsoA was 100 ⁇ g/mL.
  • the fourth experimental example is to increase the sample concentration and verify the efficacy of the sample to inhibit the aggregation of A ⁇ 42 .
  • a ⁇ 42 was reconstituted in DMSO to 2.5 mg/mL, and samples 1 to 9 were diluted to an appropriate concentration in D-PBS.
  • the concentration of sample 1 was 10 ⁇ M and 100 ⁇ M
  • the concentrations of samples 1 to 9 were 20 ⁇ M and 200 ⁇ M
  • the concentrations of IsoA were 10 ⁇ g/mL and 100 ⁇ g/mL.
  • Each group of reactions contained 1 ⁇ L of A ⁇ 42 (final concentration: 0.25 mg/mL) and 9 ⁇ L of the test sample, 8 samples of each concentration of each sample, and the mixture was uniformly mixed and then reacted at 37 ° C for 30 minutes.
  • Fig. 4A is a graph showing the results of experiments in which low concentration samples 1 to 9 inhibit A ⁇ 42 aggregation.
  • the concentration of sample 1 was 10 ⁇ M
  • the concentration of samples 2 to 9 was 20 ⁇ M
  • the concentration of IsoA was 10 ⁇ g/mL.
  • the amount of aggregation of A? 42 was measured by the thioflavin T test, and the reading measured by the control group to which no sample was added was set to 100%, and the remaining values were adjusted correspondingly.
  • Fig. 4B is a graph showing the results of experiments in which high concentration samples 1 to 9 inhibit A ⁇ 42 aggregation.
  • the concentration of sample 1 was 100 ⁇ M
  • the concentration of samples 2 to 9 was 200 ⁇ M
  • the concentration of IsoA was 100 ⁇ g/mL.
  • the amount of aggregation of A? 42 was measured by the thioflavin T test, and the reading measured by the control group to which no sample was added was set to 100%, and the remaining values were adjusted correspondingly.
  • sample 2 can inhibit about 20% of A ⁇ 42 aggregation at a concentration of 20 ⁇ M, while sample 4 and sample 8 can inhibit about 50% and 60%, respectively, at a concentration of 20 ⁇ M.
  • a ⁇ 42 aggregates.
  • sample 2 inhibited about 40% of A ⁇ 42 aggregation at a concentration of 200 ⁇ M, and sample 4 inhibited about 70% of A ⁇ 42 aggregation at a concentration of 200 ⁇ M, while sample 8 was A concentration of 200 ⁇ M completely inhibits any aggregation of A ⁇ 42 .
  • heterologous leaf asparagine derivative of the present invention has an effect of inhibiting aggregation of A ⁇ 40 and inhibiting aggregation of A ⁇ 42 .
  • the present invention also utilizes the effects of different oxidative stress on retinal epithelial cells, and observes the protective effect of the heterologous leaf asparagine derivatives of the present invention on retinal pigment epithelial cells.
  • the fifth experimental example is to verify that the sample activates extracellular decomposition of A ⁇ 40 enzyme activity, and improves the ability of the enzyme to decompose A ⁇ 40 and reduce the extracellular A ⁇ 40 content.
  • mice neuroblasts (Neuro-2a) were attached to a T175 culture dish, and then exchanged for 30 hours in 30 mL of chemically defined medium overnight, and the cultured cells were removed 24 hours later.
  • the component-defined medium called conditioned medium, was centrifuged at 13,000 rpm for five minutes and the supernatant was taken.
  • 10 ng of A ⁇ 40 and the test drug were added to 300 ⁇ l of the conditioned medium, and after reacting at 37 ° C for 24 hours, the residue in each reaction was measured with an immunoassay reagent (Human A ⁇ 1-40 Immunoassay kits, Cat. KHB3482, Life Technologies).
  • the amount of A ⁇ is used to examine whether the test drug can promote the activity of enzyme-degrading A ⁇ in the medium.
  • the test A ⁇ content was set to 100% without any test drug (only 10 ng A ⁇ ), and the A ⁇ content obtained after drug treatment was compared with the control group, and the percentage was Said.
  • the A ⁇ 40 content value obtained without adding any drug (C) was set to 100%, and the remaining values were adjusted correspondingly, and the results were expressed as percentages.
  • Sample 9 was effective in promoting the decomposition of extracellular A ⁇ 40 at concentrations of 50 ⁇ M and 100 ⁇ M.
  • DMEM/F12 cell culture medium Life Techonlogies
  • FBS fetal bovine serum
  • Samples 2 and 8 were diluted with Dimethyl sulfoxide (DMSO) to the test concentration 200 times the next day, and then an appropriate amount of DMEM/F 12 medium containing 5% fetal bovine serum was added and diluted to 2 of the test concentration.
  • DMSO Dimethyl sulfoxide
  • Tert-butyl hydroperoxide is an organic hydrogen peroxide that is metabolized by free radicals, causing damage to cells caused by lipid oxidation and covalent bonding of cellular molecules. Therefore, it is widely used in oxidative stress-induced cells. In the study of injury.
  • human retinal pigment epithelial cells cause 30% to 40% cell death in the presence of tBHP, but samples 2 (concentration 6.25M, 12.5M, and 25M, respectively) and samples were added. 8 (concentration 8.75M, 17.5M, and 35M, respectively) can significantly inhibit the death of human retinal pigment epithelial cells, and even the survival rate is better than the control group.
  • heterologous leaf asparagine derivative of the present invention has a protective effect against oxidative stress damage of human retinal pigment epithelial cells caused by t-butyl hydroperoxide.
  • the present invention provides a heterophyllophyllin derivative, which comprises a drug for inhibiting aggregation of an amylin-like peptide and preventing ocular lesions.

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Abstract

一种异类叶升麻苷衍生物及其制备方法与用途。所述异类叶升麻苷衍生物具有式（Ⅰ）的结构：其中，R 1及R 2是独立选自氢、卤素、羟基或烃氧基，R 3和R 4是独立选自羟基、烃氧基或酰氧基，R 5是独立选自羟基或酰氧基。包含所述异类叶升麻苷衍生物的药物具有治疗或预防类淀粉胜肽相关疾病及预防眼部病变等的用途。

Description

异类叶升麻苷衍生物及其制造方法与用途 技术领域
本发明是有关于一种异类叶升麻苷衍生物及其制造方法与用途，特别是有关于一种异类叶升麻苷衍生物及其制造方法与包含异类叶升麻苷衍生物的药物的用途。
背景技术
异类叶升麻苷(Isoacteoside)属于苯丙素苷类化合物的一种，存在于许多植物中，例如肉苁蓉中亦含有此成分。异类叶升麻苷的结构包含双氢氧苯乙基-D-葡萄糖甙、苯丙烯酸基酯以及单醣。
依据目前许多文献研究显示，异类叶升麻苷具有神经保护、肝脏保护、抗氧化以及降低类淀粉胜肽聚集的生物活性。根据WO 2011/157059 A1的研究活性测试结果，得知咖啡酰基团(Caffeoyl group)位于第六位(如：异类叶升麻苷)时，抑制β-类淀粉胜肽(β-amyloid peptides，Aβ)聚集的活性较佳，若在第四位(如：类叶升麻苷，acteoside)，则活性降低。此结果显示咖啡酰基团的位置对异类叶升麻苷的活性影响甚巨。
另，在异类叶升麻苷降低Aβ凝集的药理作用机制中，可能之一为邻苯二酚(Catechol)的双酚基团对过渡金属(例如铜、铁及锌等)具有金属螯合作用；而位于第一位置的苯乙醇(Phenylethanoid)基团也具有邻苯二酚基团，应亦具有类似的金属螯合作用，可能是活性必要基团。
因此，本发明藉由合成一系列异类叶升麻苷的衍生物，其具有治疗或预防类淀粉胜肽相关疾病(例如神经保护、降低类淀粉胜肽聚集、神经退化性疾病、以及眼部病变等)的功效。
发明内容
本发明的一方面是提供一种异类叶升麻苷衍生物，其具有式(I)的结构：
Figure PCTCN2015076381-appb-000001
在式(I)中，R1及R2是独立选自氢、卤素、羟基、或烃氧基，R3及R4是独立选自羟基、烃氧基或酰氧基，R5是独立选自羟基或酰氧基。
根据本发明的一实施方式，当R1及/或R2为烃氧基时，其是独立选自烷氧基、烯氧基或芳氧基。
根据本发明的一实施方式，当R1及/或R2为烷氧基时，其是甲氧基。
根据本发明的一实施方式，当R1及/或R2为烯氧基时，其是烯丙氧基。
根据本发明的一实施方式，当R1及/或R2为芳氧基时，其是苄氧基。
根据本发明的一实施方式，当R3及/或R4为烃氧基时，其是独立选自烯氧基或芳氧基。
根据本发明的一实施方式，当R3及/或R4为烯氧基时，其是烯丙氧基。
根据本发明的一实施方式，当R3及/或R4为芳氧基时，其是苄氧基。
根据本发明的一实施方式，当R3及/或R4为酰氧基时，其是乙酰氧基。
根据本发明的一实施方式，R3及R4是相同的取代基。
根据本发明的一实施方式，当R5为酰氧基时，其是乙酰氧基。
根据本发明的一实施方式，R5是相同的取代基。
根据本发明的一实施方式，异类叶升麻苷衍生物是选自以下的结构：
Figure PCTCN2015076381-appb-000002
Figure PCTCN2015076381-appb-000003
本发明的另一方面是提供一种药物用于治疗或预防与类淀粉胜肽相关疾病的用途，其中药物包含前述的异类叶升麻苷衍生物。
较佳的，该与类淀粉胜肽相关疾病为神经退化性疾病。
较佳的，该与类淀粉胜肽相关疾病为阿兹海默氏病(Alzheimer′s disease)、轻度认知障碍(Mild Cognitive Impairment)、路易体性痴呆(Lewy body dementia)、唐氏症候群(Down′s syndrome)、遗传性脑出血并发淀粉样变性病(荷兰型(Dutch type))；关岛帕金森痴呆复合症(the Guam Parkinson-Dementia complex)、进行性核上麻痹、多发性硬化、库贾氏病(Creutzfeld Jacob disease)、帕金森氏病(Parkinson′s disease)、额颞叶型失智症、皮克病、肌萎缩侧索硬化症(amyotropic lateral sclerosis)、包涵体肌炎(inclusion-body myositis)、成人发病的糖尿病、老年性心肌淀粉样变性病或内分泌肿瘤。
根据本发明的一实施方式，类淀粉胜肽是β-类淀粉胜肽。
本发明的又一方面是提供一种药物用于预防眼部病变的用途，其中药物包含前述的异类叶升麻苷衍生物。
较佳的，该眼部病变为神经元退化、视觉皮质缺损、青光眼、白内障、眼部淀粉样变性病、黄斑部病变性、视神经玻璃疣、视神经病变、视神经炎或网格状营养不良。
本发明的再一方面是提供一种异类叶升麻苷衍生物的制造方法，包含将具有式(II)的结构的化合物与β-D-葡萄糖五乙酸酯反应，以形成具有式(III)的结构的化合物，其中式(II)为：
Figure PCTCN2015076381-appb-000004
式(III)为：
Figure PCTCN2015076381-appb-000005
在式(II)以及式(III)中，R1及R2是独立选自氢、氯、甲氧基或苄氧基。接着，(1)将具有式(III)的结构的化合物与钯碳以及甲醇的混合物反应，移除钯碳并纯化后，与碳酸钾、烯丙基溴以及丙酮混合，经回流后，于氢氧化钾-甲醇溶液中反应，以形成具有式(IV-1)的结构的化合物，其中式(IV-1)为：
Figure PCTCN2015076381-appb-000006
在式(IV-1)中，R3及R4是独立选自氢或烯丙氧基，(2)将具有式(III)的结构的化合物溶于甲醇中，并与甲醇钠混合，以形成具有式(IV-1)的结构的化合物，其中R3及R4是独立选自氢、氯、甲氧基或苄氧基，或(3)将具有式(III)的结构的化合物与乙酰氯以及甲醇-二氯甲烷反应，以形成具有式(IV-2)的结构的化合物，其中式(IV-2)为：
Figure PCTCN2015076381-appb-000007
在式(IV-2)中，R5及R6是独立选自氢或氯。然后，将具有式(IV-1)的结构的化合物或具有式(IV-2)的结构的化合物与二-邻-乙酰基阿魏酸氯化物、二-邻-烯丙基阿魏酸氯化物或二-邻-苄基阿魏酸氯化物于含有二氯甲烷及吡啶的溶液中反应，以形成具有式(V-1)～(V-4)任一结构的化合物，其中式(V-1)为：
Figure PCTCN2015076381-appb-000008
在式(V-1)中，R7及R8是独立选自氢或烯丙氧基，式(V-2)为：
Figure PCTCN2015076381-appb-000009
在式(V-2)中，R9及R10是独立选自氢、甲氧基或苄氧基，式(V-3)为：
Figure PCTCN2015076381-appb-000010
在式(V-3)中，R11及R12是独立选自氢、甲氧基或苄氧基，式(V-4)为：
Figure PCTCN2015076381-appb-000011
在式(V-4)中，R13及R14是独立选自氢或氯。
根据本发明的一实施方式，制造方法更包含将具有式(V-1)的结构的化合物与氯化亚铜及氯化钯于甲醇及水的混合物中反应，以形成具有式(VI-1)的结构的化合物，其中式(VI-1)为：
Figure PCTCN2015076381-appb-000012
在式(VI-1)中，R15及R16是独立选自氢或羟基。
根据本发明的一实施方式，制造方法更包含将具有式(V-2)的结构的化合物与含甲胺的甲醇反应，以形成具有式(VI-1)的结构的化合物，其中式(VI-1)为：
Figure PCTCN2015076381-appb-000013
在式(VI-1)中，R15及R16是独立选自氢、氯、甲氧基或苄氧基。
根据本发明的一实施方式，制造方法更包含将具有式(V-4)的结构的化合物与含甲胺的甲醇反应，以形成具有式(VI-2)的结构的化合物，其中式(VI-2)为：
Figure PCTCN2015076381-appb-000014
在式(VI-2)中，R17及R18是独立选自氢或氯。
本发明的异类叶升麻苷衍生物是藉由改变异类叶升麻苷的化学结构，使包含本发明的异类叶升麻苷衍生物的药物具有治疗或预防类淀粉胜肽相关疾病及预防眼部病变等用途。
附图说明
为使本发明的特征、优点与实施例能更明显易懂，所附附图的说明如下：
图1A～图1D是本发明实施例的异类叶升麻苷衍生物抑制类淀粉胜肽累积的试验结果图以及细胞存活率分析结果图；
图2A～图2B分别是本发明实施例的异类叶升麻苷衍生物抑制类淀粉胜肽累积的试验结果图以及细胞存活率分析结果图；
图3A～图3B是本发明实施例的异类叶升麻苷衍生物抑制类淀粉胜肽聚集的试验结果图；
图4A～图4B是本发明实施例的异类叶升麻苷衍生物抑制类淀粉胜肽聚集的试验结果图；
图5是本发明实施例的异类叶升麻苷衍生物Aβ降解的试验结果图；
图6A～图6B是本发明实施例的异类叶升麻苷衍生物预防眼睛病变的试验结果图。
具体实施方式
为了使本揭示内容的叙述更加详尽与完备，下文针对了本发明的实施态样与具体实施例提出了说明性的描述；但这并非实施或运用本发明具体实施例的唯一形式。以下所揭露的各实施例，在有益的情形下可相互组合或取代，也可在一实施例中附加其他的实施例，而无须进一步的记载或说明。在以下描述中，将详细叙述许多特定细节以使读者能够充分理解以下的实施例。然而，可在无此等特定细节的情况下实践本发明的实施例。
本发明的一方面是提供一种异类叶升麻苷衍生物，其具有式(I)的结构：
Figure PCTCN2015076381-appb-000015
在式(I)中，R1及R2是独立选自氢、卤素、羟基、或烃氧基，R3及R4是独立选自羟基、烃氧基或酰氧基，R5是独立选自羟基或酰氧基。
值得注意的是，本文所述的「烃氧基(Hydrocarboxyl group)」是指烃基与氧原子连结后的生成基团，其中烃基是由碳和氢组成的有机化合物，包含烷烃、烯烃、炔烃、环烃及芳烃。「酰氧基(Acyloxy group)」是指酰基与氧原子连结后的生成基团，其中酰基是指从含氧酸中除去一个或多个羟基基团的衍生的官能团。
又，本文所述的「与类淀粉胜肽相关疾病」是指神经退化性疾病、阿兹海默氏病(Alzheimer′s disease)、轻度认知障碍(Mild Cognitive Impairment)、路易体性痴呆(Lewy body dementia)、唐氏症候群(Down′s syndrome)、遗传性脑出血并发淀粉样变性病(荷兰型(Dutch type))；关岛帕金森痴呆复合症(the Guam Parkinson-Dementia complex)、进行性核上麻痹、多发性硬化、库贾氏病(Creutzfeld Jacob disease)、帕金森氏病(Parkinson′s disease)、额颞叶型失智症、皮克病、肌萎缩侧索硬化症(amyotropic lateral sclerosis)、包涵体肌炎(inclusion-body myositis)、成人发病的糖尿病、老年性心肌淀粉样变性病或内分泌肿瘤。
再，本文所述的「眼部病变」是指神经元退化、视觉皮质缺损、青光眼、白内障、眼部淀粉样变性病、黄斑部病变性、视神经玻璃疣、视神经病变、视神经炎或网格状营养不良。
在本发明的一实施方式中，其中当R1及/或R2为卤素时，其是氯。
在本发明的一实施方式中，其中当R1及/或R2为烃氧基时，其是独立选自烷氧基、烯氧基或芳氧基。
值得注意的是，本文所述的「烷氧基(Alkoxy group)」是指烷基与氧原子连结后的生成基团。「烯氧基(Alkenoxy group)」是指烯基与氧原子连结后的生成基团。「芳氧基(Aryloxy group)」是指芳基与氧原子连结后的生成基团，其中芳基是指任何从芳香环衍生出的官能基。
在本发明的一实施方式中，当R1及/或R2为烷氧基时，其是甲氧基(Methoxy group)。甲氧基具有-O-CH3的结构，在以下化学式中以「OMe」表示。
在本发明的一实施方式中，当R1及/或R2为烯氧基时，其是烯丙氧基(Alloxy group)。烯丙氧基具有
Figure PCTCN2015076381-appb-000016
的结构，在以下化学式中以「OAll」表示。
在本发明的一实施方式中，当R1及/或R2为芳氧基时，其是苄氧基(Benzyloxy group)。苄氧基具有
Figure PCTCN2015076381-appb-000017
的结构，在以下化学式中以「OBn」表不。
在本发明的一实施方式中，当R3及/或R4为烃氧基时，其是独立选自烯氧基或芳氧基。
在本发明的一实施方式中，当R3及/或R4为烯氧基时，其是烯丙氧基。
在本发明的一实施方式中，当R3及/或R4为芳氧基时，其是苄氧基。
在本发明的一实施方式中，当R3及/或R4为酰氧基时，其是乙酰氧基(Acetoxy group)。乙酰氧基具有
Figure PCTCN2015076381-appb-000018
的结构，在以下化学式中以「OAc」表示。
在本发明的一实施方式中，R3及R4是相同的取代基。
在本发明的一实施方式中，当R5为酰氧基时，其是乙酰氧基。
在本发明的一实施方式中，R5是相同的取代基。
在本发明的一实施方式中，异类叶升麻苷衍生物是选自以下的结构：
Figure PCTCN2015076381-appb-000019
Figure PCTCN2015076381-appb-000020
本发明的另一方面是提供一种药物用于治疗或预防类淀粉胜肽相关疾病的用途，其中药物包含前述的异类叶升麻苷衍生物。
根据本发明的一实施方式，类淀粉胜肽是β-类淀粉胜肽(β-amyloid peptide，Aβ)。
β-类淀粉胜肽是类淀粉前驱蛋白(Amyloid precursor protein，APP)经由不同分泌酵素(Secretase)的作用，从原始含770个氨基酸的蛋白质到含37～49个氨基酸的β-类淀粉胜肽。常见的β-类淀粉胜肽Aβ40与Aβ42，其中Aβ42因具有较高的疏水性(hydrophobic)，故比起Aβ40更容易形成β-类淀粉斑(β-amyloid plaque)。沉积的Aβ纤维具有神经毒性，可引发连串的复杂反应，例如：突触的变化、Tau蛋白磷酸化、神经传导物质减少、神经胶细胞增生以及发炎反应等。这些反应可导致一系列病理损伤，例如斑块形成、神经原纤维缠积，从而造成神经元的变性和功能障碍，甚至死亡，最终造成神经退化性疾病；β-类淀粉斑堆积被认为是造成阿兹海默氏症的原因之一，因此目前许多针对治疗或预防阿兹海默症的发生与减缓症状的恶化的相关药物的开发均以干扰β-类淀粉胜肽的生产途径为主，其是藉由减少β-类淀粉胜肽的生成、抑制β-类淀粉胜肽于胞外的累积、以及抑制β-类淀粉胜肽的聚集，从而防止β-类淀粉斑的形成。本发明的异类叶升麻苷衍生物可抑制β-类淀粉胜肽的聚集，进而具有神经保护及治疗神经退化性疾病等功效。
本发明的又一方面是提供一种药物用于预防眼部病变的用途，其中药物包含前述的异类叶升麻苷衍生物。
随着个体年龄增长，自由基对细胞中DNA、蛋白质、脂類及其它胞内大分子物质的氧化伤害的累积会导致衰老，其中视网膜及中枢神经是统的退化被认为与氧化压力伤害具有高度相关性。视网膜色素上皮细胞(Retinal Pigment Epidermis，RPE)是位于视网膜神经上皮层和脉络膜之间，司多种生理功能， 例如：视网膜屏障、吞噬功能、参与视循环代谢、抗氧化功能及分泌生长因子等。视网膜色素上皮细胞与其它组织相同，容易受氧化压力伤害，使细胞死亡进而造成视网膜病变、视觉功能异常，严重者会导致视觉功能丧失。因此，视网膜色素上皮细胞常被用于研究视网膜病变疾病的细胞模式，例如：糖尿病视网膜病变或老年性黄斑病变。本发明的异类叶升麻苷衍生物可减少自由基，避免氧化压力伤害，进而具有抗氧化、保护视网膜细胞及预防眼部病变等功效。
本发明的异类叶升麻苷衍生物是藉由改变异类叶升麻苷的化学结构，以达到治疗或预防与类淀粉胜肽相关疾病、神经保护、治疗神经退化性疾病、以及预防眼部病变等功效。
以下列举数个实施例以更详尽阐述本发明的方法，然其仅为例示说明的用，并非用以限定本发明，本发明的保护范围当以后附的申请专利范围所界定者为准。
异类叶升麻苷衍生物的合成
以下利用步骤A～G合成实施例的异类叶升麻苷衍生物。
步骤A包含以下步骤：
1.在室温(Room temperature，r.t)下，添加5毫摩尔(mmol)的三氟化硼-乙醚络合物(Boron trifluoride-diethyl etherate，BF3Et2O)溶液至25毫升(mL)的二氯甲烷(Dichloromethane，DCM)溶液中，其包含5mmol的β-D-葡萄糖五乙酸酯(β-D-glucose pentaacetate)，且分别包含10mmol的具有式(II)的结构的化合物1a～1f。式(II)的结构为：
Figure PCTCN2015076381-appb-000021
其中化合物1a的R1为氢，R2为氢；化合物1b的R1为氢，R2为氯；化合物1c的R1为甲氧基(OMe)，R2为甲氧基；化合物1d的R1为氢，R2为苄氧基(OBn)；化合物1e的R1为苄氧基，R2为苄氧基；以及化合物1f的R1为苄氧基，R2为氢。
2.将步骤1的反应混合物搅拌6小时，随后与饱和碳酸氢钠水溶液剧烈搅拌30分钟。
3.将步骤2的萃取物干燥、过滤并蒸发。接着，将残余物以硅胶管柱层析用乙酸乙酯/正己烷混合溶剂(EA∶n-hexane＝1∶4，v/v)进行纯化，以得到具有式(III)的结构的化合物2a～2f，其中化合物2a的产率为45％。式(III)的结构为：
Figure PCTCN2015076381-appb-000022
其中化合物2a的R1为氢，R2为氢；化合物2b的R1为氢，R2为氯；化合物2c的R1为甲氧基，R2为甲氧基；化合物2d的R1为氢，R2为苄氧基；化合物2e的R1为苄氧基，R2为苄氧基；以及化合物2f的R1为苄氧基，R2为氢。
步骤A的反应过程如下：
Figure PCTCN2015076381-appb-000023
步骤B包含以下步骤：
1.分别将2mmol的化合物2d～2f、10％的钯碳(Palladium on carbon，Pd/C)催化剂以及甲醇(methanol，MeOH)的混合物于室温下通入氢气搅拌6小时。接着，藉由过滤移除催化剂，并将滤液在真空下(in vacuo)浓缩，以得到黄色残余物。将残余物以硅胶管柱层析用乙酸乙酯/正己烷混合溶剂(EA∶n-hexane＝1∶1，v/v)进行纯化，以得到中间产物。
2.将步骤1的中间产物与碳酸钾(Potassium carbonate，K2CO3)、烯丙基溴(allyl bromide)以及丙酮(acetone)混合。混合物用氯化钙干燥管(CaCl2)在硅油浴中回流10小时，随后冷却至室温。
3.将步骤2的不溶性盐类过滤，并以DCM洗涤。接着，将滤液以及DCM蒸发。
4.将步骤3的粗产物于10％的氢氧化钾-甲醇(KOH-MeOH)溶液中搅拌30分钟。接着，将混合物于减压下蒸发。将残余物以硅胶管柱层析用乙酸乙酯/正己烷混合溶剂(EA∶n-hexane＝1∶2，v/v)进行纯化，以得到具有式(IV-1)的结构的化合物3d～3f。式(IV-1)的结构为：
Figure PCTCN2015076381-appb-000024
其中化合物3d的R1为氢，R2为烯丙氧基(OAll)；化合物3e的R1为烯丙氧基，R2为烯丙氧基；以及化合物3f的R1为烯丙氧基，R2为氢。
步骤B的反应过程如下：
Figure PCTCN2015076381-appb-000025
步骤C包含以下步骤：
1.将1.5mmol的甲醇钠(Sodium methoxide，NaOMe)加至分别含有3 mmol的化合物2a～2f的15mL甲醇溶液中。将混合物于室温下搅拌30分钟。
2.将步骤1的混合物于减压下蒸发。将残余物以硅胶管柱层析用乙酸乙酯/正己烷混合溶剂(EA∶n-hexane＝1∶4，v/v)进行纯化，以得到具有式(IV-1)的结构的化合物3a～3c以及3g～3i，其中化合物3a的产率为85％。化合物3a的R1为氢，R2为氢；化合物3b的R1为氢，R2为氯；化合物3c的R1为甲氧基，R2为甲氧基；化合物3g的R1为氢，R2为苄氧基；化合物3h的R1为苄氧基，R2为苄氧基；以及化合物3i的R1为苄氧基，R2为氢。
步骤C的反应过程如下：
Figure PCTCN2015076381-appb-000026
步骤D包含以下步骤：
1.将0.01mL的乙酰氯(acetyl chloride)分别加至含有2mmol的化合物2a或2b以及1∶1的甲醇-二氯甲烷(MeOH/DCM)的10mL溶液中。将混合物于室温下搅拌48小时，在时间结束时以薄层层析(Thin layer chromatography，TLC)指示反应完成。
2.将步骤1的混合物以三乙醇胺(Triethanolamine，TEA)中和。接着，将反应混合物浓缩，并将残余物以硅胶管柱层析用乙酸乙酯(EA)作为冲提液进行纯化，以得到具有式(IV-2)的结构的化合物3j以及3k。式(IV-2)的结构为：
Figure PCTCN2015076381-appb-000027
其中化合物3j的R1为氢，R2为氢；以及化合物3k的R1为氢，R2为氯。
步骤D的反应过程如下：
Figure PCTCN2015076381-appb-000028
步骤E包含以下步骤：
1.根据不同的化合物，步骤E的步骤1可分为以下三种条件：
a.将2mmol的化合物3a～3c、3g、3h、3j以及3k在0℃下分别加至含有2.2mmol的二-邻-乙酰基阿魏酸氯化物(di-O-acetylferulic acid chloride)、二氯甲烷以及1.5mL的吡啶(pyridine)的溶液中。
b.将2mmol的化合物3d～3f在0℃下分别加至含有2.2mmol的二-邻-烯丙基阿魏酸氯化物(di-O-allylferulic acid chloride)、二氯甲烷以及1.5mL的吡啶的溶液中。
c.将2mmol的化合物3a、3c、3g以及3i在0℃下分别加至含有2.2mmol的二-邻-苄基阿魏酸氯化物(di-O-benzylferulic acid chloride)、二氯甲烷以及1.5mL的吡啶的溶液中。接着，将混合物于10℃下搅拌10小时。将溶剂挥发，并将残余物溶解于乙酸乙酯中。
2.将步骤1的中间产物的有机层依次用水和盐水洗涤，以MgSO4干燥并蒸发。将残余物以硅胶管柱层析用乙酸乙酯/正己烷混合溶剂(EA∶n-hexane＝1∶1，v/v)进行纯化，以得到具有式(V-1)的结构的化合物4d～4f、具有式(V-2)的结构的化合物4a～4c、4g与4h、具有式(V-3)的结构的化合物4i与4m～4o以及具有式(V-4)的结构的化合物4j与4k，其中化合物4a的产率为43％。
式(V-1)的结构为：
Figure PCTCN2015076381-appb-000029
其中化合物4d的R1为氢，R2为烯丙氧基；化合物4e的R1为烯丙氧基，R2为烯丙氧基；以及化合物4f的R1为烯丙氧基，R2为氢。
式(V-2)的结构为：
Figure PCTCN2015076381-appb-000030
其中化合物4a的R1为氢，R2为氢；化合物4b的R1为氢，R2为氯；化合物4c的R1为甲氧基，R2为甲氧基；化合物4g的R1为氢，R2为芐氧基；以及化合物4h的R1为苄氧基，R2为苄氧基。
式(V-3)的结构为：
Figure PCTCN2015076381-appb-000031
其中化合物4i的R1为苄氧基，R2为氢；化合物4m的R1为氢，R2为苄氧基；化合物4n的R1为氢，R2为氢；以及化合物4o的R1为甲氧基，R2为甲氧基。
式(V-4)的结构为：
Figure PCTCN2015076381-appb-000032
其中化合物4j的R1为氢，R2为氢；以及化合物4k的R1为氢，R2为氯。
步骤E的形成化合物4d～4f的反应过程如下：
Figure PCTCN2015076381-appb-000033
；形成化合物4a～4c、4g与4h的反应过程如下：
Figure PCTCN2015076381-appb-000034
；形成化合物4i与4m～4o的反应过程如下：
Figure PCTCN2015076381-appb-000035
；以及形成化合物4j与4k的反应过程如下：
Figure PCTCN2015076381-appb-000036
步骤F包含以下步骤：
1.分别将化合物4d～4e与氯化亚铜(CuCl)及氯化钯(PdCl2)于甲醇及水的混合物在室温下强烈搅拌，以得到具有式(VI-1)的结构的化合物5d～5f。式(VI-1)的结构为：
Figure PCTCN2015076381-appb-000037
其中化合物5d的R1为氢，R2为羟基；化合物5e的R1为羟基，R2为羟基；以及化合物5f的R1为羟基，R2为氢。
操作F的形成化合物5d～5f的反应过程如下：
Figure PCTCN2015076381-appb-000038
步骤G包含以下步骤：
1.分别于含有2mmol的化合物4a～4c、4g、4h、4j与4k的二氯甲烷溶液在10℃下加入40％的含1mL甲胺的甲醇。
2.将步骤1的反应混合物搅拌20分钟后，在真空下浓缩。将残余物以硅胶管柱层析用甲醇/二氯甲烷混合溶剂(1∶20，v/v)进行纯化，以得到具有式(VI-1)的结构的化合物5a～5c、5g与5h以及具有式(VI-2)的结构的化合物5j与5k，其中化合物5a的产率为90％。化合物5a的R1为氢，R2为氢；化合物5b的R1为氢，R2为氯；化合物5c的R1为甲氧基，R2为甲氧基；化合物5g的R1为氢，R2为苄氧基；以及化合物5h的R1为苄氧基，R2为苄氧基。式(VI-2)的结构为：
Figure PCTCN2015076381-appb-000039
其中化合物5j的R1为氢，R2为氢；以及化合物5k的R1为氢，R2为氯。
步骤G的形成化合物5a～5c、5g与5h的反应过程如下：
Figure PCTCN2015076381-appb-000040
；以及形成化合物5j与5k的反应过程如下：
Figure PCTCN2015076381-appb-000041
以下实验例是利用将下表一所列的样品1～9配制成不同浓度的溶液，进行后续各种实验。
表一
Figure PCTCN2015076381-appb-000042
4o1HNMR(CDCl3)δ7.60(d，1H，J＝18)，δ7.43-7.21(m，15H)，δ7.31-7.25(m，3H)δ6.77(d，1H，J＝9)，δ6.29(d，1H，J＝18)，δ5.16(d，4H，J＝9)，δ4.64-4.40(m，1H)，δ4.36-4.11(m，2H)，δ3.76-3.35(m，6H)，δ2.94(t，2H，J＝6)。
4i1HNMR(CDCl3)δ7.67(d，1H，J＝16)，δ7.45-7.29(m，3H)，δ7.24(d，1H，J＝12)，δ6.8(s，1H)，δ7.76(s，2H)，δ6.53(d，1H，J＝16)，δ4.45-3.90(m，3H)，δ3.90-3.81(m，1H)，δ3.78-3.74(m，8H)，δ3.73-3.70(m，1H)，δ3.60-2.86(m，5H)。
4m1HNMR(CDCl3)δ7.60(d，1H，J＝18)，δ7.43-6.84(m，22H)，δ6.29(d，1H，J＝18)，δ5.13(s，4H)，δ4.97(s，2H)，δ4.68-4.63(m，1H)，δ4.37-4.06(m，2H)，δ3.73-3.36(m，6H)，δ2.94(t，2H，J＝6)。
4n1HNMR(CDCl3)δ7.60(d，1H，J＝18)，δ7.43-7.01(m，15H)，δ7.41-7.45(m，3H)δ6.87(d，1H，J＝9)，δ6.29(d，1H，J＝18)，δ5.16(d，4H，J＝9)，δ4.64-4.40(m，1H)，δ4.36-4.11(m，2H)，δ3.76-3.35(m，6H)，δ2.94(t，2H，J＝6)。
5a1HNMR(CDCl3)δ7.34-7.21(m，7H)，δ6.80-6.53(m，3H)，δ5.02(s，2H)，δ4.38-4.05(m，3H)，δ3.73-4.3.70(m，1H)，δ3.22-2.92(m，3H)，δ2.91-2.62(m，7H)，δ2.62(t，2H，J＝6)
首先，分别精称表一的编号样品1～9，以二甲基亚砜(Dimethyl sulfoxide，DMSO)为溶剂配制成浓度为10mM的储备溶液。储备溶液所包含的样品的分子量、重量以及溶剂体积示于下表二。
表二
  样品分子量 样品重量(mg) 溶剂体积(mL)
样品1 656.6 10.1 1.53
样品2 691.6 11.2 1.62
样品3 626.4 10.8 1.72
样品4 446.5 11.2 2.50
样品5 732.4 11.8 1.61
样品6 732.4 10.5 1.43
样品7 686.7 11 1.60
样品8 552.5 10.2 1.85
样品9 478.45 20 4.18
接着，将浓度为10mM的储备溶液配制成不同样品浓度的溶液。将储备溶液配制为浓度5μM是取0.5μL的储备溶液定量至1mL；配制为浓度10μM是取 1μL的储备溶液定量至1mL；配制为浓度20μM是取2μL的储备溶液定量至1mL；配制为浓度50μM是取5μL的储备溶液定量至1mL；配制为浓度100μM是取1μL的储备溶液定量至0.1mL；以及配制为浓度200μM是取2μL的储备溶液定量至0.1mL。
实验例一至实验例四是藉由下列三个方向评估本发明的异类叶升麻苷衍生物的药效，包含：1)是否能降低细胞生成β-类淀粉胜肽(Aβ)；2)是否能透过促进细胞负责清除β-类淀粉胜肽酵素的活性、进而增加其清除β-类淀粉胜肽的效率；以及3)是否能抑制Aβ40与Aβ42的聚集。
实验例一、抑制Aβ40累积的试验(I)
本实验例分为两阶段：第一阶段使用较低样品浓度做初步筛选；第二阶段则是根据第一阶段的结果，进一步选取有效的样品，提高样品的试验浓度，在不影响细胞毒性的状况下，以取得试验样品抑制Aβ40累积的最佳浓度与最佳效果。
实验方法：表现瑞典型突变人类APP695的人类神经瘤母细胞(SH-SY5Y-APP695)培养于3.5cm培养皿至细胞浓度达90％满，于分盘(passage)时在24-孔盘中于每个孔洞(well)种4 x 105个细胞。隔天将培养液置换为300μL的化学成分确定的培养基(chemical-defined medium)，其是DMEM/F12培养基，包含5mM的Hepes缓冲液、0.6％的葡萄糖、3mM的NaHCO3、2.5μM的谷氨酰胺(glutamine)、100μg/mL的transferrin、20nM的黄体素(progesterone)、60μM的腐胺(putrescine)、30nM的亚硒酸钠(sodium selenite)以及2μg/mL的肝素(heparin)。于每个孔洞加入3μL的测试样品，每个浓度样品各4组。将细胞置于培养箱中(37℃，5％CO2)以样品处理24小时后，收集培养液并以免疫分析试剂(Human Aβ1-40Immunoassay kits，Cat.KHB3482，Life Technologies)分析样品处理后培养基中的Aβ40含量，并利用细胞存活率分析(MTT assay)评估经过样品处理对细胞造成的毒性。
样品1的试验浓度为5μM与10μM，样品2～8的试验浓度为10μM与20μM。以未加入任何试验样品的SH-SY5Y-APP695细胞培养基中的Aβ40含量定为对照组并设定为100％，经各测试样品处理后的培养基中Aβ40含量则与对照组相比较，并以百分比表示。另以β-分泌酶抑制剂(β-secretase inhibitor，β-SI)作为 正对照组。实验结果显示于图1A～图1D，其中图1A及图1C是样品抑制Aβ40累积的试验结果图，图1B及图1D是经样品处理后的细胞存活率分析结果图。图1A～图1D的结果为四个实验组的平均值±标准误差(n＝4)，而对照组与测试样品组间以杜纳多重比较检定(Dunnett’s multiple comparison test)分析其统计差异，其中以“*”表示p＜0.05；以“**”表示p＜0.01；以“***”表示p＜0.001；并以“****”表示p＜0.0001。
实验例一是先使用较低浓度的样品做初步筛选，根据图1A～图1D的结果，样品3、4以及6具有抑制Aβ40累积的效果，其中以样品3对于细胞中Aβ40累积的抑制效果最佳，其抑制Aβ40累积的效果是随着样品浓度的上升而增加。
实验例二、抑制Aβ40累积的试验(II)
实验例二是依据实验例一的试验结果，选出具有明显抑制Aβ40累积效果的样品3、4以及6，提高样品的试验浓度至20、50、与100μM，并以实验例一的方法进行试验。每个浓度的样品各4组，其实验结果是显示如图2A～图2B。图2A是样品抑制Aβ40累积的试验结果图，图2B是经样品处理后的细胞存活率分析结果图。图2A～图2B的结果为四个实验组的平均值±标准误差(n＝4)，而对照组与测试样品组间以杜纳多重比较检定分析其统计差异，其中以“*”表示p＜0.05；以“**”表示p＜0.01；以“***”表示p＜0.001；并以“****”表示p＜0.0001。
实验例二是增加样品3、4以及6的样品浓度，在不影响细胞毒性的状况下，以取得试验样品抑制Aβ40累积的最佳浓度与最佳效果。根据图2A～图2B的结果显示，在不造成细胞毒性的状况下，样品3在20μM可抑制约20％的累积，且样品6在50μM可抑制约40％的累积，而提高样品浓度则未能进一步促进样品4抑制Aβ累积的效果。
因此，根据实验例一及实验例二的结果可知，本发明的异类叶升麻苷衍生物确实具有抑制Aβ40累积的功效。
实验例三、抑制Aβ聚集的试验(I)
实验例三是验证样品抑制Aβ40聚集与抑制Aβ42寡聚的功效。
Aβ40聚集：将Aβ40溶液以DMSO回溶至10mg/mL。每组反应包含0.5μl的10mg/mL的Aβ40与4.5μL的以杜氏磷酸盐缓冲生理盐水(Dulbecco′s Phosphate-Buffered Saline，D-PBS)稀释后的试验样品。样品1的浓度为10μM与100μM，样品2～8的浓度为20μM与200μM。反应总体积为5μL，每个浓度各6组，于37℃的培养箱反应4小时后，加入200μL的硫代黄素T工作溶液(Thioflavin T working solution，ThT working solution)，充分混合，其中硫代黄素T为10μM的硫代黄素T溶于磷酸钾缓冲液(Potassium phosphate buffer，pH6.0)。取200μL的混合液置于黑色透明底的96孔盘，并由盘子底部测量硫代黄素T的荧光强度(Ex：440nm，Em：482nm)，藉以判定Aβ40的聚集程度。本实验例是以硫代黄素T试验(thioflavin T assay，ThT assay)测量Aβ聚集的程度。由于ThT与刚果红(Congo red)类衍生物可与聚合型态的A)β蛋白形成键结，因此Aβ聚集程度越高，键结的ThT数量则越多。藉由侦测ThT的含量变化，便可推估Aβ聚集的改变程度。以未加入任何试验样品反应的读值(即只含有0.5μL的Aβ40与4.5μL的D-PBS，其中Aβ40的最终浓度为1mg/mL)作为对照组，将其读值设定为100％，并以异类叶升麻苷(IsoA)正对照组。样品的读值则与对照组做比较并以百分比表示。实验结果如图3A～图3B，其是显示六个实验组的平均值±标准误差(n＝6)，而对照组与测试样品组间以杜纳多重比较检定分析其统计差异，其中以“*”表示p＜0.05；以“**”表示p＜0.01；以“***”表示p＜0.001；并以“****”表示p＜0.0001。
图3A是显示低浓度样品1～9抑制Aβ40聚集的试验结果。样品1的浓度为10μM，样品2～9的浓度为20μM，而IsoA的浓度为10μg/mL。以未加入任何样品的对照组所测得的读值设为100％，并将其余数值作相对应调整。图3B是显示高浓度样品1～9抑制Aβ40聚集的试验结果。样品1的浓度为100μM，样品2～9的浓度为200μM，而IsoA的浓度为100μg/mL。以未加入任何样品的对照组所测得的读值设为100％，并将其余数值作相对应调整。根据图3A的试验结果显示，在低浓度时，样品2在20μM的浓度可抑制约20％的Aβ40聚集，而在同样的浓度，样品4与样品8可抑制约30％的Aβ40聚集。根据图3B的提高试验样品浓度并重复试验的结果显示，在200μM的浓度，样品2与样品4可抑制约50％的Aβ40聚集，而样品8有抑制高达70％的Aβ40聚集的效果。
实验例四、抑制Aβ聚集的试验(II)
实验例四是提高样品浓度，验证样品抑制Aβ42聚集的功效。
实验方法：Aβ42以DMSO回溶至2.5mg/mL，样品1～9则以D-PBS稀释至适当浓度。样品1的浓度为10μM与100μM，样品1～9的浓度为20μM与200μM，而IsoA的浓度为10μg/mL与100μg/mL。每组反应包含1μL的Aβ42(最终浓度：0.25mg/mL)与9μL的试验样品，每个浓度的样品各8组，混合均匀后置于37℃下反应30分钟。反应完毕后加入200μL的硫代黄素T工作溶液充分混合，取200μL的混合液置于透明底的黑色96孔盘并由盘子底部测量硫代黄素T的荧光强度(Ex：440nm，Em：482nm)，藉以判定Aβ42的聚集程度。以未加入任何试验样品反应(即只含有1μL的Aβ42与9μL的D-PBS，其中Aβ42的最终浓度为0.25mg/mL)作为对照组，将其读值设定为100％，并以异类叶升麻苷(IsoA)正对照组。样品的读值则与对照组做比较并以百分比表示。实验结果示于图4A～图4B，其是显示九个实验组的平均值±标准误差(n＝8)，而对照组与测试样品组间以杜纳多重比较检定分析其统计差异，其中以“*”表示p＜0.05；以“**”表示p＜0.01；以“***”表示p＜0.001；并以“****”表示p＜0.0001。
图4A是显示低浓度样品1～9抑制Aβ42聚集的试验结果。样品1的浓度为10μM，样品2～9的浓度为20μM，而IsoA的浓度为10μg/mL。Aβ42的聚集量是以硫代黄素T试验测量，以未加入任何样品的对照组所测得的读值设为100％，并将其余数值作相对应调整。图4B是显示高浓度样品1～9抑制Aβ42聚集的试验结果。样品1的浓度为100μM，样品2～9的浓度为200μM，而IsoA的浓度为100μg/mL。Aβ42的聚集量是以硫代黄素T试验测量，以未加入任何样品的对照组所测得的读值设为100％，并将其余数值作相对应调整。
根据图4A的试验结果显示，在低样品浓度时，样品2在20μM的浓度可抑制约20％的Aβ42聚集，而样品4与样品8在20μM的浓度各别可抑制约50％与60％的Aβ42聚集。根据图4B的试验结果显示，提高样品浓度后，样品2在200μM的浓度可抑制约40％的Aβ42聚集，样品4在200μM的浓度也可抑制约70％的Aβ42聚集，而样品8在200μM的浓度可完全抑制任何的Aβ42聚集。
因此，根据实验例三及实验例四的结果可知，本发明的异类叶升麻苷衍生物确实具有抑制Aβ40聚集与抑制Aβ42聚集的功效。
综上所述，根据实验例一至实验例四的结果可知，样品3、4以及6能降低细胞生成Aβ40，其中以样品6的效果最佳。而在评估样品是否能抑制Aβ聚集的 部份，以硫代黄素T试验测量Aβ聚集的程度。试验结果显示样品2、4以及8可有效抑制Aβ40与Aβ42的聚集，其中以样品8的效果最佳，其在200μM的浓度可抑制约70％的Aβ40聚集与约100％的Aβ42聚集。因此，本发明的异类叶升麻苷衍生物确实具有抑制类淀粉胜肽聚集的功效。
除了β-类淀粉胜肽外，本发明亦利用不同氧化压力对于视网膜上皮细胞的影响，观察本发明的异类叶升麻苷衍生物对于视网膜色素上皮细胞的保护作用。
实验例五、Aβ降解的试验
实验例五是验证样品活化细胞外(extracellular)分解Aβ40酵素活性的药物，提高酵素分解Aβ40的能力、降低细胞外Aβ40含量的功效。
实验方法：将2x107个老鼠神经母细胞(Neuro-2a)贴于一T175培养皿，隔夜后换以30mL的化学成分确定的培养基再培养24小时，24小时后取出与细胞培养过的化学成分确定的培养基，称为条件培养基(conditioned medium)，以13,000rpm离心五分钟后取上清液。于300μl条件培养基中加入10ng Aβ40与试验药物，使其于37℃反应24小时后，用免疫分析试剂(Human Aβ1-40Immunoassay kits，Cat.KHB3482，Life Technologies)来测量各反应中所残留的Aβ量，来检视试验药物是否可促进培养基中酵素降解Aβ的活性。以未加入任何试验药物(仅含10ng Aβ)设定为对照组，其所测得的Aβ含量设为100％，经药物处理后所得到的Aβ含量则与对照组做比对，并以百分比表示。实验结果示于图5，其是显示四个实验组的平均值±标准误差(n＝4)，而对照组与测试样品组间以杜纳多重比较检定(Dunnett’s multiple comparison test)分析其统计差异，其中以“*”表示p＜0.05；以“**”表示p＜0.01；以“***”表示p＜0.001；并以“****”表示p＜0.0001。
根据图5的试验结果显示，以未加入任何药物(C)所得的Aβ40含量值设为100％，其余数值则作相对应的调整，结果以百分比表示的。样品9在50μM及100μM的浓度下均可有效促进细胞外Aβ40的分解。
因此，根据实验例五的结果可知，本发明的异类叶升麻苷衍生物确实具有Aβ降解的功效。
实验例六、预防眼睛病变的试验
人类视网膜色素上皮细胞(Human retinal pigment epithelium cell，ARPE-19)培养在含10％胎牛血清(fetal bovine serum，FBS)的DMEM/F12细胞培养液(Life Techonlogies)中，达90％满时分盘于96孔盘中，每个孔洞种4.5X103个细胞。隔天将样品2及8以二甲基亚砜(Dimethyl sulfoxide，DMSO)稀释至试验浓度的200倍，再加入适量含5％胎牛血清的DMEM/F 12培养液，稀释至试验浓度的2倍，再与0.2mM叔丁基过氧化氢(terr-butyl hydroperoxide，tBHP，Sigma)等比例混合至试验浓度(含0.5％二甲基亚砜)加入含细胞的孔盘中，置于细胞培养箱反应24小时后，利用MTT溶液进行测量细胞存活率。以波长570nm测定吸光值，将未经药物处理的细胞设为控制组，其所取得的吸光值取平均值并设定为100％，经药物处理的细胞所取得的吸光值则依据下列公式计算细胞存活率：
细胞存活率：(实验组吸光值/控制组平均吸光值)*100％。
叔丁基过氧化氢是一种有机过氧化氢，会经由自由基代谢，接者引起脂质氧化与细胞分子产生共价键结而对细胞造成损伤，因此常被广泛应用于氧化压力造成细胞损伤的研究中。
实验结果示于图6A～图6B，其是显示六个实验组的平均值±标准误差(n＝6)，而伤害组(接受伤害药物，无保护药物)和测试药物组及控制组(Control；含0.5％DMSO)间以杜纳多重比较检定分析其统计差异，结果表示：“*”表示p＜0.05；“**”表示p＜0.01；“***”表示p＜0.001；“****”表示p＜0.0001。
根据图6A～图6B的试验结果显示，人类视网膜色素上皮细胞在tBHP存在下会造成30％～40％细胞死亡，但分别加入样品2(浓度分别为6.25M、12.5M、以及25M)及样品8(浓度分别为8.75M、17.5M、以及35M)可明显抑制人类视网膜色素上皮细胞死亡，甚至存活率优于控制组。
因此，根据实验例六的结果可知，本发明的异类叶升麻苷衍生物对于叔丁基过氧化氢所造成的人类视网膜色素上皮细胞的氧化压力损伤确具有保护作用。
综上所述，本发明提供一种异类叶升麻苷衍生物，包含异类叶升麻苷衍生物的药物具有抑制类淀粉胜肽聚集、预防眼部病变等功效。
虽然本发明已以实施方式揭露如上，然其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视后附的申请专利范围所界定者为准。

Claims (23)

  1. 一种异类叶升麻苷衍生物，具有式(I)的结构：
    Figure PCTCN2015076381-appb-100001
    在该式(I)中，R1及R2是独立选自氢、卤素、羟基、或烃氧基，R3及R4是独立选自羟基、烃氧基或酰氧基，R5是独立选自羟基或酰氧基。
  2. 如权利要求1所述的异类叶升麻苷衍生物，其中当R1及/或R2为烃氧基时，其是独立选自烷氧基、烯氧基或芳氧基。
  3. 如权利要求2所述的异类叶升麻苷衍生物，其中当R1及/或R2为烷氧基时，其是甲氧基。
  4. 如权利要求2所述的异类叶升麻苷衍生物，其中当R1及/或R2为烯氧基时，其是烯丙氧基。
  5. 如权利要求2所述的异类叶升麻苷衍生物，其中当R1及/或R2为芳氧基时，其是苄氧基。
  6. 如权利要求1所述的异类叶升麻苷衍生物，其中当R3及/或R4为烃氧基时，其是独立选自烯氧基或芳氧基。
  7. 如权利要求6所述的异类叶升麻苷衍生物，其中当R3及/或R4为烯氧基时，其是烯丙氧基。
  8. 如权利要求6所述的异类叶升麻苷衍生物，其中当R3及/或R4为芳氧基时，其是苄氧基。
  9. 如权利要求1所述的异类叶升麻苷衍生物，其中当R3及/或R4为酰氧基时，其是乙酰氧基。
  10. 如权利要求1所述的异类叶升麻苷衍生物，其中R3及R4是相同的取代基。
  11. 如权利要求1所述的异类叶升麻苷衍生物，其中当R5为酰氧基时，其是乙酰氧基。
  12. 如权利要求1所述的异类叶升麻苷衍生物，其中R5是相同的取代基。
  13. 如权利要求1所述的异类叶升麻苷衍生物，其中该异类叶升麻苷衍生物是选自以下的结构：
    Figure PCTCN2015076381-appb-100002
  14. 一种如权利要求1所述的异类叶升麻苷衍生物的用途，其是用于制备治疗或预防与类淀粉胜肽相关疾病的药物。
  15. 如权利要求14所述的用途，其中该与类淀粉胜肽相关疾病为神经退化性疾病。
  16. 如权利要求14所述的用途，其中该与类淀粉胜肽相关疾病为阿兹海默氏病、轻度认知障碍、路易体性痴呆、唐氏症候群、遗传性脑出血并发淀粉样变性病(荷兰型)；关岛帕金森痴呆复合症、进行性核上麻痹、多发性硬化、库贾氏病、帕金森氏病、额颞叶型失智症、皮克病、肌萎缩侧索硬化症、包涵体肌炎、成人发病的糖尿病、老年性心肌淀粉样变性病或内分泌肿瘤。
  17. 如权利要求14所述的用途，其中该与类淀粉胜肽相关疾病为眼部病变。
  18. 如权利要求14所述的用途，其中该与类淀粉胜肽相关疾病为神经元退化、视觉皮质缺损、青光眼、白内障、眼部淀粉样变性病、黄斑部病变性、视神经玻璃疣、视神经病变、视神经炎或网格状营养不良。
  19. 如权利要求14所述的用途，其中该类淀粉胜肽是β-类淀粉胜肽。
  20. 一种异类叶升麻苷衍生物的制造方法，包含：
    将具有式(II)的结构的化合物与β-D-葡萄糖五乙酸酯反应，以形成具有式(III)的结构的化合物，其中该式(II)为：
    Figure PCTCN2015076381-appb-100003
    该式(II)为：
    Figure PCTCN2015076381-appb-100004
    在该式(II)以及该式(III)中，R1及R2是独立选自氢、氯、甲氧基或苄氧基；
    (1)将该具有式(III)的结构的该化合物与钯碳以及甲醇的混合物反应，移除该钯碳并纯化后，与碳酸钾、烯丙基溴以及丙酮混合，经回流后，于氢氧化钾-甲醇溶液中反应，以形成具有式(IV-1)的结构的化合物，其中该式(IV-1)为：
    Figure PCTCN2015076381-appb-100005
    在该式(IV-1)中，R3及R4是独立选自氢或烯丙氧基，(2)将该具有式(III)的结构的该化合物溶于甲醇中，并与甲醇钠混合，以形成具有该式(IV-1)的结构的化合物，其中R3及R4是独立选自氢、氯、甲氧基或苄氧基，或(3)将该具有式(III)的结构的该化合物与乙酰氯以及甲醇-二氯甲烷反应，以形成具有式(IV-2)的结构的化合物，其中该式(IV-2)为：
    Figure PCTCN2015076381-appb-100006
    在该式(IV-2)中，R5及R6是独立选自氢或氯；以及
    将该具有式(IV-1)的结构的该化合物或该具有式(IV-2)的结构的该化合物与二-邻-乙酰基阿魏酸氯化物、二-邻-烯丙基阿魏酸氯化物或二-邻-苄基阿魏酸氯化物于含有二氯甲烷及吡啶的溶液中反应，以形成具有式(V-1)～(V-4)任一结构的化合物，其中该式(V-1)为：
    Figure PCTCN2015076381-appb-100007
    在该式(V-1)中，R7及R8是独立选自氢或烯丙氧基，该式(V-2)为：
    Figure PCTCN2015076381-appb-100008
    在该式(V-2)中，R9及R10是独立选自氢、甲氧基或苄氧基，该式(V-3)为：
    Figure PCTCN2015076381-appb-100009
    在该式(V-3)中，R11及R12是独立选自氢、甲氧基或苄氧基，该式(V-4)为：
    Figure PCTCN2015076381-appb-100010
    在该式(V-4)中，R13及R14是独立选自氢或氯。
  21. 如权利要求20所述的制造方法，更包含将该具有式(V-1)的结构的该化合物与氯化亚铜及氯化钯于甲醇及水的混合物中反应，以形成具有式(VI-1)的结构的化合物，其中该式(VI-1)为：
    Figure PCTCN2015076381-appb-100011
    在该式(VI-1)中，R15及R16是独立选自氢或羟基。
  22. 如权利要求20所述的制造方法，更包含将该具有式(V-2)的结构的该化合物与含甲胺的甲醇反应，以形成具有式(VI-1)的结构的化合物，其中该式(VI-1)为：
    Figure PCTCN2015076381-appb-100012
    在该式(VI-1)中，R15及R16是独立选自氢、氯、甲氧基或苄氧基。
  23. 如权利要求20所述的制造方法，更包含将该具有式(V-4)的结构的该化合物与含甲胺的甲醇反应，以形成具有式(VI-2)的结构的化合物，其中该式(VI-2)为：
    Figure PCTCN2015076381-appb-100013
    在该式(VI-2)中，R17及R18是独立选自氢或氯。
PCT/CN2015/076381 2014-04-10 2015-04-10 异类叶升麻苷衍生物及其制造方法与用途 Ceased WO2015154721A1 (zh)

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