WO2011082617A1 - (e)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethylene and preparation method and use thereof - Google Patents

Abstract

Disclosed are new (E)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl) ethylene with following structure formula (I) and its preparation method and use in the manufacture of a medicament for preventing and/or treating neurodegenerative diseases. It is proved by test that the compound has great protective effect on neurons and can markedly improve the learning and memory disorder in mice induced by scopolamine. Therefore, the compound has likely preventing and/or treating effect on neurodegenerative diseases and has potential use in the fields of pharmaceutics.

Description

 (-1-(3,5-Dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)acetamidine and its preparation brigade and use

 Technical field

 The invention belongs to the technical field of pharmaceutical application, and particularly relates to a novel compound ^)-1 -(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl) Ethylene (English name: (E)-l-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxyl-4-methoxyphenyl)ethylene) and its preparation method and in the preparation of a preventive and/or therapeutic nerve Use in drugs for degenerative diseases.

 Background technique

Neurodegenerative diseases (ND) are a group of chronic, degenerative nervous system diseases based on primary neuronal degeneration. Although the lesions and causes of these diseases vary, neurodegeneration is a common denominator. Such diseases mainly include Alzheimer's disease (AD), Parkinson's (PD), Huntington's disease (Huntington disease) and the like. AD and PD are two common neurodegenerative diseases whose onset and age are closely related. With the aging of the world's population, the incidence of AD is increasing. Scientists point out that there are an estimated 22 million AD patients worldwide in 2025, and the number of patients will reach 45 million by 2050. In Europe and the United States, 4%-12% of the elderly over 60 years old have dementia, more than half of them are AD ^[1] ; AD has become the fourth largest killer in the elderly after relaying cardiovascular disease, cancer and stroke in the elderly. ". The incidence of dementia in China is close to that in Europe and the United States. There are more than 5 million elderly patients with dementia, of which AD accounts for 2/3 ^[2 ' ^3] . In addition, PD also accounts for a large proportion of the elderly with neurodegenerative diseases. Neurodegenerative diseases such as AD and PD will bring a heavy mental and economic burden to society and families.

The causes of AD and PD are very complicated, and it is a multi-pathogenic, multi-gene, multi-link comprehensive Syndrome, its exact molecular mechanism of action is still unclear, which brings great difficulties in finding the ideal drug for prevention and treatment. In recent years, more and more studies have shown that oxidative stress plays a very important role in the pathogenesis of neurodegenerative diseases ^[4] . Excessive production of reactive oxygen species, hydroxyl radicals, superoxide anions, and hydrogen peroxide can be observed in most neurodegenerative diseases, and reactive oxygen species exert their toxic effects mainly through three pathways: lipid peroxidation, protein Oxidation and

DNA oxidation. Usually, an antioxidant system is present in the body against the excessive production of reactive oxygen species, including antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT:), glutathione peroxidase (GSH). -Px), as well as some small molecule antioxidants - vitamin E, vitamin 0, etc. ^[5] . The study found that the central nervous system of patients with neurodegenerative diseases is very sensitive to oxidative stress, and its antioxidant enzymes against oxidative stress are inferior to other tissues ^[6] . Although it is unclear whether oxidative stress is the cause of these neurodegenerative diseases, studies have suggested that oxidative stress accelerates the disease process of the disease ^[7] .

The typical pathological features of AD are senile plaques and neurofibrillary tangles ^[8] , in which the main component of senile plaques is β-amyloid (Αβ), and the main component of neurofibrillary tangles is tau. A large number of ensemble and ex vivo studies have confirmed that both Αβ and tau have significant neurocytotoxicity. Studies have found that the toxic effects of Aβ are mediated by cell membrane binding, which in turn aggregates and ages, eventually leading to the accumulation of reactive oxygen species in the cells ^[9] . Oxidative stress also further leads to the accumulation of Αβ ^[10] and tau protein, which ultimately accelerates the pathogenesis of AD. In the whole animal, it was also confirmed that the injection of Αβ into the brain of rats can induce spatial learning and memory impairment. In addition, the study found that Αβ is present in the brain mitochondria of AD patients and ΑΡΡ transgenic animals ^[12 , ^13] . A large number of in vivo and in vitro studies suggest that Αβ is closely related to mitochondrial function defects. Αβ treatment leads to decreased mitochondrial membrane potential, ROS production, oxygen oligosaccharide and morphological changes in PC12 cells ^[14] , leading to neurons or cell lines. Mitochondrial work Can damage ^[15 _ ^17] . Severe mitochondrial metabolic dysfunction is also prevalent in the brains of AD patients and precedes the appearance of clinicopathological features ^[18] .

The main symptoms of PD are slow exercise, unstable posture and tremor. In patients with PD, irreversible nigrostriatal dopaminergic neuron loss occurs in the inner side of the brain ^[19] . The exact mechanism of bowel I death from PD neurons is still unclear, but studies suggest that cerebral oxidative stress, mitochondrial dysfunction, and excitotoxicity may be the cause of PD and mediate its pathogenesis ^[2Q] . Studies have found that dopamine oxidizes to form a semiquinone, which accelerates the oxidation of dopamine by monoamine oxidase B, and generates reactive oxygen species such as hydrogen peroxide, superoxide anion, and hydroxyl radicals ^[21] , which ultimately accelerates the pathogenesis of PD. In addition, the activity of the mitochondrial electron-respiratory chain complex enzyme Complex I was found to be decreased in PD patients [ ^22] , and the decrease in Complex I activity led to an increase in the level of active oxygen in the body ^[23] . Therefore, studies suggest that mitochondria may play an important role in the pathogenesis of PD. In fact, a large number of researchers have developed effective drugs for PD against oxidative stress and mitochondrial function, including MitoQ, a drug targeting mitochondria, which has begun Phase II clinical studies [24]. These studies suggest that oxidative stress and mitochondrial function play an important role in the pathogenesis of neurodegenerative diseases such as AD and PD. Therefore, starting from the antioxidant activity of the compound, it is possible to develop a novel and targeted drug or drug candidate for treating a neurodegenerative disease. China has a long history of Chinese herbal medicine use, and the experience of modern drug development has also proved that natural products play a vital role in the discovery and development of modern drugs ^[25] . Therefore, studies on the chemical constituents and biological activities of Chinese herbal medicines are expected to find new and effective, low-toxic innovative drugs for the prevention and/or treatment of neurodegenerative diseases. Summary of the invention In the past four years, the inventors have screened a large number of plant extracts and natural monomer compounds to find a new compound with relatively simple structure and good neuroprotection.

(^)-1-(3,5-Dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene.

 So far, no structure of ^(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl:) ethylene has been found, preparation method and use thereof Report. The present invention relates to (^)-1-(3,5-dimethoxyphenyl:)-2-(3,5-dihydroxy-4-methoxyphenyl:)ethene as a novel compound. In the biological experiment of neuronal cell protection, it was found for the first time that it has strong neuroprotective effects in vivo and in vitro, and can significantly improve scopolamine-induced learning and memory impairment in mice, which may prevent neurodegenerative diseases and/or Or therapeutic effect, thus having potential use in the pharmaceutical field.

Accordingly, it is an object of the present invention to provide a novel compound (^)-1- 3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methyl) represented by the following structural formula I Oxyphenyl) ethylene:

 Another object of the present invention is to provide a preparation of the compound of the present invention, ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene. method. The preparation method mainly comprises the steps of: pulverizing the fruit of the branch of the genus Mesquite (Me/ azedarach Linn.) or the palm family of the palm family (Palmae family), and extracting with 95% ethanol. The extract was subjected to silica gel column chromatography to obtain a crude compound of the title compound, and then purified by reverse-phase silica gel column chromatography, and finally purified by a dextran gel (LH-20) to obtain a pure target compound.

A further object of the present invention is to provide a compound of the invention ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene in the preparation In the prevention and/or treatment of drugs for neurodegenerative diseases Use.

 The invention still further provides a pharmaceutical composition for preventing and/or treating a neurodegenerative disease, the pharmaceutical composition comprising a therapeutically effective amount of (-1-(3,5-dimethoxybenzene) having the structure of formula I Base:) 2-(3,5-Dihydroxy-4-methoxyphenyl:)ethylene and a pharmaceutically acceptable carrier.

 "Safe, effective dose" means that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective dose of the compound is determined by the specific circumstances such as the age, condition, and course of treatment of the subject. The compositions of the present invention preferably comprise from 1% by weight to 96% by weight of a compound of formula I, most preferably from 10% to 85% by weight of a compound of formula I and from 15% to 90% by weight of a pharmaceutically acceptable carrier .

 "Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermixing with the compounds of the invention and with respect to each other without significantly reducing the potency of the compound. Examples of pharmaceutically acceptable carriers include sugars (such as glucose, sucrose, lactose, etc.), starch (such as corn starch, potato starch, etc.), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose). Sodium, cellulose acetate, etc., gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as Propylene glycol, glycerin, mannitol, sorbitol, etc., emulsifiers (eg Tween®), wetting agents (eg sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, none Pyrogen water, etc. The choice of the carrier of the composition of the present invention depends on the mode of administration of the compound, and can be formulated into general pharmaceutical tablets, granules, capsules, oral liquid preparations and general pharmaceutical preparations.

Beneficial effect The test data of the examples show that the compound of the present invention, ^)-l-(3,5-dimethoxyphenyl:)-2-(3,5-dihydroxy-4-methoxyphenyl:)ethene has The following advantages: (1) in vitro hydrogen peroxide induced in vitro cell model ^[26] results show that it has shown significant cytoprotection at 0.1 μΜ concentration, and hydrogen peroxide at a concentration of 5 μΜ, 10 μΜ The resulting nerve cell damage is almost completely reversed, and this protection also presents a good dose-dependent relationship. It is the strongest neuroprotective agent we have ever discovered; (2) Pharmacodynamics using AD animal models Evaluation results show that (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene can significantly improve memory impairment; (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl:)ethene is derived from traditional Chinese medicines, which are long-term Clinical application has shown that the compound has no obvious toxicity, and our preliminary study also found (-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl) Ethylene at high dose (1 g/kg) for mice No obvious toxic effects; (4) (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethylene has a novel structure, Different from the existing anti-oxidation nerve cell protectants, it has a simple structure, a small molecular weight, and is relatively easy to pass the blood-brain barrier. Based on (^)-1-(3,5-dimethoxyphenyl)-2 -(3,5-Dihydroxy-4-methoxyphenyl)ethene has advantages in chemical structure, efficacy, toxicity, etc., and has good development prospects, and is expected to develop into a novel preventive and/or therapeutic structure. Drugs for neurodegenerative diseases.

 Figure 1 is a flow diagram showing the preparation of the compounds of the present invention.

2 is a normal control group, a H ₂ 0 ₂ damage model group, and a compound (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4) using the present invention. Bar graph of the survival rate of SH-SY5Y cells induced by ₃ -methoxyphenyl) ethylene, where *p < 0.05 compared to the normal control group, **p < 0.05, ^## p < 0.01 compared to Hydrogen peroxide group. Figure 3 shows the normal control group, the H ₂ 0 ₂ injury model group, and the compound (^)-1-(3,5- using the present invention. Dimethoxyphenyl) -2- post (3,5-dihydroxy-4-methoxyphenyl) vinyl ¾0 ₂ induced cell morphology in FIG SH-SY5Y cells: (A) control group; (B) Hydrogen peroxide group; (C) hydrogen peroxide + 10 μΜ compound of the invention; (D) hydrogen peroxide + 5 μΜ of the compound of the invention; (Ε) hydrogen peroxide + 1 μΜ of the compound of the invention; (F) hydrogen peroxide +0.5 μΜ of the compound of the invention. Figure 4 is a graph showing the induction of oxygen deficiency in the compound (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene of the present invention. A protective effect of SH-SY5Y cell viability decreased, where ^## ? < 0.01 compared to the normal control group, ** Ρ < 0.01 compared to the oxygen oligosaccharide-deficient group. Figure 5 is a graph showing that the compound (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene of the present invention is induced by scopolamine A picture of the improvement in learning and memory impairment in rats, where ^## Ρ

< 0.01 compared to the normal control group, *Ρ < 0.05 compared to the scopolamine group.

Figure 6 is a graph showing that the compound (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene of the present invention is induced by scopolamine A graph of the effect of MDA levels on lipid peroxidation products in the rat cerebral cortex, where ^## Ρ<0.01 compared to the normal control group, **Ρ<0.05 compared to the scopolamine group.

 Specific form

 The compound of the present invention))-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene can be obtained from the genus 楝azedarach Linn.) or Palmae family Palm flower (^ww chinensis R. Brown) is prepared by general chemical extraction and separation methods. The separation process is shown in Figure 1.

Pu H (Me/ <2 ozeiforac z Linn.) Wo neutron ί: from each South Island; W-( ivistona chinensis R. Brown) leaves from Hainan Island; 95% ethanol (analytically pure, Shanghai Chemical Reagent Company) Ethyl acetate (analytical grade, Shanghai Chemical Reagent Company); Dichloromethane (analytical grade, Shanghai Chemical Reagent Company); Methanol (analytical grade, Shanghai Chemical Reagent Company); Petroleum ether (60-90 °C, analytical grade) , Shanghai Chemical Reagent Company: Acetone (analytical grade, Shanghai Chemical Reagent Company: Silica gel and GF254 silica gel prefabricated board (green Island Marine Institute); C18 reverse silica gel (150-200 mesh, Merck); MCI (CHP20P, 75-150 μ, Mitsubishi Chemical Industries Ltd.); Sephadex LH-20, 20-150 μ , Amersham).

 Preparation Example 1: Separation of (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-) having the structure of formula I from 楝(vto chinensis R. Brown) 4-methoxyphenyl)ethylene

 10 Kg dried lychee leaves, crushed and extracted with 95% ethanol at room temperature for 3 times, evaporated to ethanol to obtain 440 g of extract, the extract was partitioned with ethyl acetate and water (1:1), and the organic phase was evaporated to dryness at MCI. The column was eluted with a gradient of 30% -50% methanol, and the fractions were separated by silica gel column chromatography (dichloromethane/methanol = 50: 1-30: 1) using a reversed phase silica gel (RP-18, 60). (Methanol) eluted to give (^)-1-(3,5-dimethoxyphenyl-2-(3,5-dihydroxy-4-methoxyphenylethylene) as a crude sample, then use The sugar gel (sephadex LH-20, methanol (100%)) was purified to give 120 mg of the final compound.

 Preparation Example 2: Separation of (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-) having the structure of formula I from sylvestris (Me/ azedarach Linn.) 4-methoxyphenyl)ethylene

5.0 Kg of dried palmetto fruit, crushed and extracted with 95% ethanol at room temperature for 3 times, evaporated to ethanol to obtain 400 g of extract, partitioned with ethyl acetate and water (1:1), and evaporated to dryness to give 70 g of acetic acid. The ethyl ester fraction and the ethyl acetate fraction were eluted with a petroleum ether/acetone system on a silica gel column to give -1-(3,5-dimethoxyphenyl)-2-(3,5-di The pure product of hydroxy-4-methoxyphenylethylene was used as a control sample, and was detected by silica gel thin layer chromatography (developing agent: methylene chloride/methanol = 30:1) as the basis for collecting the fractions. Part of the AF, the main compound of part F is ^) -1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene, further Elution with reverse phase silica gel (RP-18, 60% methanol:) gave ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxy A crude sample of phenylethylene was then purified using a dextran gel (sephadex LH-20, methanol (100%)) to give 200 mg of the final compound. Identification of the compounds prepared in the preparation examples

 Instrument: Infrared (Perkin-Elmer 577 spectrometer, KBr tablet); Nuclear magnetic (Bruker AM-400 spectrometer); Mass spectrometer (Finnigan MAT 95 mass spectrometer).

 The structure of the compound was confirmed by 1D NMR, MS, IR 1) and 2D NMR (Formula 2) data.

 Equation 2: Key HMBC coupling (; H→C)

High-resolution mass spectrometry showed that the molecular ion peak was mh 302.1151, indicating that its molecular formula is C ₁₇ H ₁₈ 0 ₅ (calculated value: 302.1154) and has 9 degrees of unsaturation. Consistent with the molecular formula, ¹³ C NMR (Table 1, DEPT-containing data:) indicates that the molecule has a 3,5-dimethoxy-substituted phenyl group (benzene ring: 5 _C 161.7, 161.7, 104.8, 104.8, 127.9, 100.0; methoxy: 5 _C 55.3, 55.3), a 3,5-dihydroxy-4-methoxy substituted phenyl (benzene ring: 5 _C 151.1, 151.1, 136.0, 129.6, 106.3, 106.3; methoxy Base: S _c 60.2) and a disubstituted double bond (S _c 133.6, 140.2). On HMBC (Formula 2), the coupling between the proton on MeO corresponding to 5 _C 55.3 and the olefinic carbon of S _c 161.7, 104.8, 100.0 further demonstrates the presence of a 3,5-dimethoxy substituted phenyl group; Coupling between protons on MeO of S _c 60.2 and olefins of S _c 151.1 _: 136.0, and coupling between protons on CH corresponding to S _c 106.3 and olefins of S _c 151.1, 136.0, 106.3, further proof The presence of a 3,5-dihydroxy-4-methoxy substituted phenyl group; the coupling of a proton on the last disubstituted double bond with an olefin of S _c 127.9, 129.6, demonstrates this disubstituted double bond and two A link between substituted phenyl groups. At the same time, the large coupling constant between the protons of the disubstituted double bond (J = 26.6 Hz) clearly indicates that the double bond adopts the E-type configuration.

该化合物的部分物理性质及理化数据见表 表 1 : ^)-1-(3,5-二甲氧基苯基 )-2-(3,5-二羟基 -4-甲氧基苯基)乙烯的 理化数据 (丙酮中 -6¾ By the above identification, it was confirmed that the compound prepared by the present invention is a novel compound represented by the following formula I (^)-1-(3,5-Dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene:

The physical properties and physical and chemical data of the compound are shown in Table 1: ^)-1-(3,5-Dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl) Physical and chemical data of ethylene (Acetone - 63⁄4

Straight 5 _C (DEPT) IR (KBr, cm" ¹ ) MS (ret. %)

1 133.6 (CH) 3448 302 (100)

2 140.2 (CH) 2964 269 (52)

 Γ 127.9 (C) 2841 255 (5)

 2' 104.8 (CH) 1593 241 (26)

 161.7 (C) 1508 226 (6)

 4' 100.0 (CH) 1463 211 (7)

 5' 161.7 (C) 1367 198 (5)

 6' 104.8 (CH) 1207

 1" 129.6 (C) 1155

 2" 106.3 (CH) 1055

 3" 151.1 (C) 978

 4" 136.0 (C) 837

 5" 151.1 (C)

 6" 106.3 (CH)

3 '-OMe 55.3 (CH ₃ )

5'-OMe 55.3 (CH ₃ )

4"-OMe 60.2 (CH ₃ )

Test Example 1: In vitro pharmacological test 1 (effect of test compound on H ₂ 0 ₂ induced SH-SY5Y cell damage)

SH-SY5Y (ATCC) cells were cultured in a 37 ° C incubator at 5% CO ₂ . The culture medium was MEM/F12 (l :i >, and 10% FBS was added. The culture medium was changed every 3-4 days. Cell fusion rate At about 80%, it was digested with 0.125% trypsin and passaged at a ratio of 1:2. Stably grown SH-SY5Y cells were seeded at a density of l x lO ⁵ / ml in a 96-well plate at 100 μΐ/well and cultured in a 37 ° C incubator at 5% C0 ₂ for approximately 24 h. Replace the fresh medium and divide the cells into normal control group, 3⁄40 ₂ damage model group, (E)-l-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy- 4-methoxyphenyl) ethylene pretreatment group. The test compound group was pretreated with Ο.ΙμΜ, 1μΜ, 5μΜ, 10 μΜ for 2 h, then H ₂ 0 ₂ (final concentration 100 μΜ), and the control group was added the same concentration of 3⁄40 ₂ culture in the same medium. hour. Then, the cell viability of each group was evaluated according to the sputum method, that is, MTT (final concentration 0.5 mg/ml) was added after 24 h, and culture was continued for 4 h at 37 °C, and the solution was added, and DMSO (100%) was added, 100 μΐ/well. The crystal dye was sufficiently dissolved by shaking for 5 min, and the absorbance was measured by a microplate reader (measurement wavelength 490 nm). Note: The viable cells can undergo a redox reaction with MTT to produce formazan (blue-violet crystal:), and the amount of the latter is proportional to the number of living cells, thereby determining cell viability. The crystals were dissolved in DMSO and detected at a wavelength of 490 nm.

 The results of cell viability are shown in Figure 2. It was found by the study of Fig. 2 that the compound (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene of the present invention can be significantly improved. The cell viability damage caused by hydrogen peroxide almost restored the cell viability to normal level at 5 μΜ, 10 μΜ, which showed a good cytoprotective effect. As shown in Figure 3, normal cells are mostly adherent, synaptic and evenly distributed in the culture plate. The number of cells damaged by hydrogen peroxide is significantly reduced, some cells shrink and the synapses disappear. The cells protected by ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl:)ethene are both morphologically and quantitatively Significant improvements have occurred, especially in the ΙΟμΜ treatment group.

Test Example 2: In vitro pharmacological test 2 (effect of test compound on induction of SH-SY5Y cell damage by oxygen glucose deficiency model) SH-SY5Y (ATCC) cells were cultured in a 37 ° C incubator at 5% CO ₂ . The culture medium was MEM/F12 (1:1) and 10% FBS was added. The culture medium was changed every 3-4 days. When the cell fusion rate was about 80%, it was digested with 0.125% trypsin and passaged at a ratio of 1:2. Stably grow SH-SY5Y cells, inoculate them in a 96-well plate at a density of l × lO ⁵ / ml, 100 μΐ / well, and culture in a constant temperature incubator at 37 ° C in 5% C0 ₂ to divide the cells into normal controls. Group, oxygen deficiency model group, test compound group. The normal control group was washed once with EBSS (sugar containing:) buffer and replaced with DMEM (low sugar) medium; OGD (oxygen sugar deficiency treatment) injury model group and test compound group were washed with EBSS (no sugar:), and changed. Into DMEM (; sugar free) medium. Add the test compound (final concentration ΙμΜ, 5μΜ, 10 μΜ), put it into OGD special sealed box, and mix 95% N ₂ , 5 % C0 ₂ mixed gas, 37 °0 constant temperature incubator, hypoxia and sugar deficiency After 1 hour, the normal culture conditions were resumed for another 24 hours; during the normal control, the cells were still cultured under normal culture conditions. Then, the cell viability of each group was evaluated according to the MTT method. After 24 hours of gP, MTT (final concentration 0.5 mg/ml) was added, and incubation was continued at 37 °C for 4 h. The solution was discarded, DMSO (100%) was added, and the crystal was fully dissolved by shaking for 5 min. Dye, microplate reader to measure absorbance (measuring wavelength 490 nm Note: Surviving cells can produce hyperthyroidism with MTT (blue-violet crystal:), the latter is proportional to the number of living cells, which can Cell viability was determined. The crystals were dissolved in DMSO and detected at a wavelength of 490 nm.

 The cell viability results are shown in Fig. 4, and it was found that 10 μΜ of the compound of the present invention can significantly improve cell damage induced by oxygen deficiency.

 Test example Overall animal pharmacology test

 ( ), Dimethoxyphenyl, Dihydroxymethoxyphenyl) Ethylene Improves Memory Retention in Animals and Antioxidant Activity at Animal Level

In this study, a cholinergic receptor antagonist, scopolamine, was used to induce learning and memory impairment in mice. Channel-based maze evaluates learning and memory, and promotes (E)-l-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene Pharmacodynamics was evaluated.

 ICR mice (male and female, weighing 18-22 g, provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences:) were randomized. The mice were trained in learning and memory using a channel water maze. There were four non-export channels (blind ends) in the water maze and a platform at the end. The mice were lightly placed into the water from the starting point toward the wall, and at the same time, the time to travel the mouse to the platform and the number of times the blind end was reached before reaching the platform was recorded. If the mouse does not reach the platform within 60 s during training, use a ruler to introduce it into the correct path to the platform, and stay on the platform for 30 so. Train twice a day for 4 days for slow learning. Appropriate intensive training. On the fourth day of training, the last 3 times of the mice that reached the platform time less than 30 s and entered the blind end less than 4 times were randomly selected. On the 5th day, the drug was administered orally, and 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, and 70 mg/kg of different doses of the compound were administered orally (control and model groups were given the same amount). Solvent (edible oil:), 40 minutes later, intraperitoneal injection of scopolamine 4.5 mg / kg (distilled water in the control group), 20 minutes after the determination of water maze results, to the time of landing to the platform (ie latency, more than 60 The second is calculated in 60 seconds, as shown in Fig. 5A:) and the number of errors (the number of times of entering the blind end, as shown in Fig. 5B:) as an evaluation index.

 The study found that the compounds of the present invention significantly reduced the latency of the mouse to find the platform (Fig. 5A) and reduced the number of memory errors (Fig. 5B) with significant memory improvement.

 Effect of (-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene on the level of malondialdehyde (MDA) in animal models

In addition, the level of malondialdehyde (MDA), the product of oxidative stress, was also detected in this animal model. The mouse cortex was isolated, homogenized with PBS, and then fully referenced to the experimental procedure on the MDA kit developed in Nanjing. experiment. The specific operation is: O.lmol/L phosphate buffer in mouse cerebral cortex Make a tissue homogenate. According to the MDA kit instructions, the groups are divided into standard tubes, standard blank tubes, assay tubes, and assay blank tubes. According to different groups, the standard reagent, absolute ethanol, test sample, glacial acetic acid and the three reagents provided in the kit itself are selectively added. After mixing the vortex, the bath was centrifuged at 95 ° C for 40 minutes, then at 3500-4000 rpm, and centrifuged for 10 minutes. The supernatant was taken and the absorbance values of the tubes were measured at 532 nm. The protein content of each sample was also determined by Coomassie Brilliant Blue method.

 As a result, it was confirmed that the compound of the present invention can significantly reduce the level of MDA in the mouse cerebral cortex, suggesting that the compound of the present invention also has an anti-oxidative stress effect at the overall animal level (Fig. 6).

 Thus, the above ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene has strong antioxidant activity and learning and memory. Improves the effect, and has low toxicity (no toxicity at lg/kg:), comprehensive pharmacological, chemical, and safety data, suggesting ^)-1-(3,5-dimethoxyphenyl)-2- (3,5-Dihydroxy-4-methoxyphenyl)ethene is highly medicinal and will be expected to be a new generation of preventive and/or therapeutic neurodegenerative effects through further pharmacodynamics, safety and pharmacokinetic evaluation. Drug candidate for disease. The present invention relates to (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene and its antioxidant activity and memory improvement The role has not been reported, and its excellent neuroprotective activity is of particular importance.

 The pharmaceutical composition of the present invention, -1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene, is a general pharmaceutical tablet. The preparation method of the preparation agent, the granule, the capsule and the oral liquid preparation. The proportions of the compositions of the following examples are all in weight percent.

Medicinal tablets (3.5%): The following amounts of (-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl:) ethylene were used. Mix well with the additive, use 10% pre-gelatinized starch slurry as binder, wet granulation, drying, add appropriate amount of magnesium stearate and mix to make tablets. ( -1-(3,5-dimethyl Oxyphenyl: 7.2 g of 2-(3,5-dihydroxy-4-methoxyphenyl)ethene; hydroxypropylcellulose 120 g; microsilica gel 30 g _; pregelatinized starch 20 g; magnesium stearate in an appropriate amount; made into 1000 tablets.

 Medicinal tablets (10%): (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene 20 g; hydroxy Propylene cellulose 120g; Micronized silica gel 30g; Pregelatinized starch 20g; Magnesium stearate amount; Mix evenly, use 10% pregelatinized starch slurry as binder, wet granulation, drying, add appropriate amount of magnesium stearate Mix and compress into 1000 pieces.

Granules (6%): (^)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene 28 g; sucrose 250 g _; Hydroxypropyl cellulose 120g _; micro-silica gel 30g; citric acid, sodium bicarbonate amount; the above composition is mixed evenly, with appropriate amount of 70% ethanol as a wetting agent, wet granulation, sieving, drying, sub-packaging 200 Bag granules.

Capsule (20%): (-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethylene 38.7g _; hydroxypropyl cellulose 120g _; micro-powder silica gel 30g; citric acid, sodium bicarbonate amount; mix the above composition evenly, use appropriate amount of 70% ethanol as humectant, wet granulation, sieving, drying, adding appropriate amount of magnesium stearate, mixing evenly , divided into 1000 hard capsules.

 Capsule (80%): added in 150g (^)-l-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl:) ethylene The citric acid, sodium bicarbonate and magnesium stearate are mixed evenly, milled into 300-400 mesh granules, and packaged into 500 hard capsules.

 Oral solution (10%): Take the appropriate amount of distilled water, add 12g of polysorbate-80 to make a solution, then add ^)-1-(3,5-dimethoxyphenyl)-2-(3,5-two 100 g of hydroxy-4-methoxyphenyl)ethene was stirred and dissolved while heating. Further, 15 g of sucrose and a preservative were dissolved in distilled water, and slowly added to the above solution under stirring, and distilled water was added to 1000 mL, and mixed. Cool, filter, dispense 100 parts, sterilize, and make 10% oral solution.

Oral solution (60%): Take the appropriate amount of distilled water, add 50g of polysorbate-80 to make a solution, plus Into ^) -1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methoxyphenyl)ethene 600g while stirring to dissolve, in addition, sucrose 80g, a proper amount of preservative dissolved in distilled water, slowly added to the above solution under stirring, add distilled water to 1000 mL, mix, cool, filter, dispense into 100, sterilize, to obtain 60% oral solution.

 The preparation steps, the identification process and the pharmacological experiment process of the compound of the present invention are exemplified above by way of specific examples, but various modifications and changes can be made thereto without departing from the spirit and scope of the present invention. In the case of the appended claims, all such modifications are within the scope of the invention.

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Claims

Rights request 1. A compound of the formula (I)-1-(3,5-dimethoxyphenyl)-2-(3,5-dihydroxy-4-methylphenyl)ethene:

 2. A method of preparing a compound according to claim 1, characterized in that the method comprises the steps of: pulverizing the fruit of the leaves of the genus Amaranthus or the palm of the genus Palmetto, using 95% ethanol After extraction, the obtained extract is subjected to silica gel column chromatography to obtain a crude compound of the target compound, and then purified by reverse-phase silica gel column chromatography, and finally purified by a glucan gel to obtain a pure target compound.  3. Use of a compound according to claim 1 for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease.  A pharmaceutical composition for preventing and/or treating a neurodegenerative disease comprising from 1% by weight to 96% by weight of the compound of claim 1 and the balance of a pharmaceutically acceptable carrier.  The pharmaceutical composition for preventing and/or treating a neurodegenerative disease according to claim 4, which comprises 10% by weight to 85% by weight of the compound according to claim 1 and 15% by weight to 90% by weight of pharmaceutically Acceptable carrier.  The pharmaceutical composition for preventing and/or treating a neurodegenerative disease according to claim 4 or 5, which is formulated into a tablet, a capsule, a granule or an oral solution. The pharmaceutical composition for preventing and/or treating a neurodegenerative disease according to claim 4 or 5, wherein the pharmaceutically acceptable carrier comprises sugar, starch, cellulose And its derivatives, gelatin, talc, solid lubricants, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives and pyrogen-free water.