WO2023202477A1 - 二氢杨梅素四氢吡咯复合物及其制备方法和应用 - Google Patents

二氢杨梅素四氢吡咯复合物及其制备方法和应用 Download PDF

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WO2023202477A1
WO2023202477A1 PCT/CN2023/088270 CN2023088270W WO2023202477A1 WO 2023202477 A1 WO2023202477 A1 WO 2023202477A1 CN 2023088270 W CN2023088270 W CN 2023088270W WO 2023202477 A1 WO2023202477 A1 WO 2023202477A1
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dihydromyricetin
tetrahydropyrrole
complex
anhydrous methanol
dhmp
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French (fr)
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梁京
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北京佳福瑞生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/20—Hypnotics; Sedatives
    • 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
    • A61P25/22—Anxiolytics
    • 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
    • A61P25/24—Antidepressants
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/02—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements
    • C07D295/027—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements containing only one hetero ring

Definitions

  • the invention relates to the field of medical technology, and specifically relates to a dihydromyricetin tetrahydropyrrole complex and its preparation method and application.
  • DHM Dihydromyricetin
  • DHM Alzheimer's disease
  • AD Alzheimer's disease
  • other neurodegenerative disease treatment drugs have many limitations, and they also face many difficulties in formulation development, which affects formulation production. Therefore, maintaining the original efficacy of DHM and reducing its above-mentioned defects to obtain better efficacy is the key to developing new dosage forms of DHM.
  • the structure of the complex was characterized using ultraviolet spectrum, infrared spectrum, mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy, the physical and chemical properties of the complex were determined, and the antioxidant activity of the complex was investigated.
  • C3-OH, C5-OH, C7-OH, C3′-OH, C5′-OH in the dihydromyricetin molecule are combined with the active hydrogen of theanine to form a complex through hydrogen bonding; complex removal ⁇
  • the ability of OH free radicals is stronger than that of dihydromyricetin and Vc, and its ability to scavenge O2- ⁇ free radicals and DPPH free radicals is equivalent to that of dihydromyricetin and stronger than Vc.
  • the combination of dihydromyricetin and theanine not only improves its water solubility, but also plays a strengthening role in antioxidant activity.
  • dihydromyricetin-theanine complex and dihydromyricetin phospholipid complex have achieved certain results in terms of water solubility and antioxidant activity, the actual biological efficacy when applied to medicine is still far from ideal, especially when achieving sustainable development. Industrial production and synthesis.
  • the technical problem to be solved by the present invention is to provide a dihydromyricetin-tetrahydropyrrole complex, which has improved solubility and stability, improved biological efficacy, and can be applied in the field of medical technology.
  • the technical solution adopted by the present invention is the dihydromyricetin tetrahydropyrrole complex.
  • the molecular formula of the dihydromyricetin tetrahydropyrrole complex is as shown in formula (I):
  • dihydromyricetin DHM By modifying dihydromyricetin DHM, the solubility and stability have been improved, and the biological efficacy has been improved, thereby achieving industrial production and synthesis and future usability for patients, and can be used in the field of medical technology; water solubility (Solubility) and stability are essential physical and chemical properties of organic small molecule drugs and are also very important issues in drug invention. Improved aqueous solubility generally results in better drug potency and a more satisfactory pharmacokinetic profile. It should be emphasized that there are many ways to form dihydromyricetin DHM into salts, but the key is to not change the drug-forming properties of DHM that have been discovered.
  • Another problem to be solved by the present invention is to provide a preparation method of dihydromyricetin tetrahydropyrrole complex.
  • the preparation method includes the following steps:
  • step S3 react the dihydromyricetin anhydrous methanol solution obtained in step S1 and the tetrahydropyrrole anhydrous methanol solution obtained in step S2 at room temperature to obtain a reactant;
  • step S4 Filter the reactant obtained in step S3, wash the filter cake with anhydrous methanol, and drain it under reduced pressure to obtain the dihydromyricetin tetrahydropyrrole complex.
  • the inert gas in step S2 is argon.
  • the concentration of dihydromyricetin anhydrous methanol solution in step S1 is 150-350mmol/L;
  • the concentration of the tetrahydropyrrole anhydrous methanol solution in step S2 is 1-10 mol/L.
  • the molar ratio of dihydromyricetin and tetrahydropyrrole in step S3 is 1:0.7-1.3.
  • the molar ratio of dihydromyricetin and tetrahydropyrrole in step S3 is 1:1.
  • step S1 add anhydrous methanol to dihydromyricetin and then move it to an ice-water bath and stir for 30 minutes; in step S3, add 2 drops of tetrahydropyrrole anhydrous methanol solution/ After adding it to the dihydromyricetin anhydrous methanol solution in the ice-water bath at a speed of 2 seconds, the reaction was carried out under room temperature conditions.
  • the preparation method of dihydromyricetin in step S1 is:
  • step S1-2 Under the protection of inert gas, mix the 2-hydroxy-4,6-dimethoxymethyleneoxyacetophenone obtained in step S1-1 with anhydrous tetrahydrofuran; then add sodium hydride and Chloromethyl methyl ether; after the reaction is completed, 2,4,6-trimethoxymethyleneoxyacetophenone is obtained;
  • step S1-3 Under the protection of inert gas, mix the 2,4,6-trimethoxymethyleneoxyacetophenone obtained in step S1-2 with THF and water; then add sodium hydroxide and 3, 4,5-trihydroxybenzaldehyde; after the reaction is completed, (E)-3-trihydroxybenzyl-1-(2,4,6-trimethoxymethyleneoxyphenyl)-vinylketone is obtained;
  • step S1-4 Mix (E)-3-trihydroxybenzene-1-(2,4,6-trimethoxymethyleneoxyphenyl)-vinyl ketone obtained in step S1-3 with 30% Mix hydrogen peroxide; after the reaction is completed, add saturated sodium sulfite solution to quench the reaction, then extract with ethyl acetate, spin off the solvent under reduced pressure, then dissolve the solvent-removed residue in methanol, and add dilute hydrochloric acid. After the reaction is completed, Spin off the solvent under reduced pressure to obtain the dihydromyricetin tetrahydropyrrole complex.
  • Another problem to be solved by the present invention is the application of the dihydromyricetin-tetrahydropyrrole complex of the present invention in the treatment of neurological dysfunction diseases.
  • the neurological dysfunction disease is sleep disorder, anxiety, depression, post-traumatic stress disorder, Alzheimer's disease, dementia, Parkinson's disease, stroke, epilepsy, autism, and alcohol use disorder.
  • the dihydromyricetin tetrahydropyrrole complex provided by the invention maintains the biological ability of dihydromyricetin (DHM), improves its medicinal efficacy, improves water solubility, and enhances the effect on GABA A Rs (low to 0.1nM), which is 1000 times that of natural DHM. It can replicate the effect of DHM and restore the expression of gephyrin level in experimental animals. It can improve the recognition/memory impairment of experimental animals, improve behavioral impairments such as anxiety, depression, and sleep disorders, and reduce seizures and brain damage caused by stroke. It can be used as a method to improve brain functions related to motor function after stroke. treatment approach. DHMP showed similar potency in activating GABA AR and was 1000 times more potent than DHM. Indications for DHM application include sleep disorders, anxiety, depression, and early Alzheimer's disease. Mid-stage dementia, Parkinson's disease, other neurodegenerative diseases, stroke, epilepsy, autism, alcohol use disorders, and addiction to benzodiazepines and other drugs.
  • DHMP Dihydromyricetin tetrahydropyrrole complex
  • Figure 1 is a flow chart for the preparation of dihydromyricetin tetrahydropyrrole complex (DHMP) of the present invention
  • Figures 2 and 3 are the nuclear magnetic resonance spectra of the dihydromyricetin tetrahydropyrrole complex (DHMP) prepared by the present invention, and Figures 4 and 5 are the nuclear magnetic resonance spectra of the dihydromyricetin (DHM);
  • DHMP dihydromyricetin tetrahydropyrrole complex
  • Figures 6-9 are whole-cell patch clamp current comparison diagrams of mouse brain slices that are positively regulated by GABA A R by the dihydromyricetin-tetrahydropyrrole complex (DHMP) of the present invention.
  • DHMP dihydromyricetin-tetrahydropyrrole complex
  • FIGS 10 and 11 are comparative diagrams of the effects of the dihydromyricetin-tetrahydropyrrole complex (DHMP) of the present invention on the level expression of mouse guidance support proteins;
  • DHMP dihydromyricetin-tetrahydropyrrole complex
  • Figure 12 Figure 13 and Figure 14 are comparative diagrams of the improvement effect of dihydromyricetin tetrahydropyrrole complex (DHMP) of the present invention on cognitive/memory disorders in mice;
  • DHMP dihydromyricetin tetrahydropyrrole complex
  • FIGS 15-20 are comparative diagrams of the effects of the dihydromyricetin-tetrahydropyrrole complex (DHMP) of the present invention on improving the locomotor activity of mice and reducing anxiety and epileptic seizures;
  • DHMP dihydromyricetin-tetrahydropyrrole complex
  • FIGS 21-23 are comparative diagrams of the effect of the dihydromyricetin-tetrahydropyrrole complex (DHMP) of the present invention on improving stroke-induced brain damage in mice;
  • DHMP dihydromyricetin-tetrahydropyrrole complex
  • FIGS 24-29 are diagrams showing the effect of the dihydromyricetin tetrahydropyrrole complex (DHMP) of the present invention on reducing brain damage caused by stroke by inhibiting excessive release of glutamate;
  • DHMP dihydromyricetin tetrahydropyrrole complex
  • Figure 30 is a diagram showing the enhancing effect of dihydromyricetin-tetrahydropyrrole complex (DHMP) on GABA AR according to the present invention.
  • DHMP dihydromyricetin-tetrahydropyrrole complex
  • Figures 31 to 40 are graphs showing the non-toxic safety test results of the dihydromyricetin tetrahydropyrrole complex (DHMP) of the present invention.
  • the dihydromyricetin tetrahydropyrrole complex of the present invention has a molecular formula as shown in formula (I):
  • the preparation method of dihydromyricetin tetrahydropyrrole complex includes the following steps:
  • step S3 react the dihydromyricetin anhydrous methanol solution obtained in step S1 and the tetrahydropyrrole anhydrous methanol solution obtained in step S2 at room temperature to obtain a reactant;
  • step S4 Filter the reactant obtained in step S3, wash the filter cake with anhydrous methanol, and drain it under reduced pressure to obtain the dihydromyricetin tetrahydropyrrole complex.
  • the inert gas in step S2 is argon.
  • the concentration of the dihydromyricetin anhydrous methanol solution in step S1 is 150-350mmol/L, preferably 250mmol/L; the concentration of the tetrahydropyrrole anhydrous methanol solution in step S2 is 1-10mol/L. L, preferably 1 mol/L.
  • the molar ratio of dihydromyricetin and tetrahydropyrrole in step S3 is 1:0.7-1.3, and the preferred molar ratio of dihydromyricetin and tetrahydropyrrole is 1:1.
  • step S1 add anhydrous methanol to dihydromyricetin and then move it to an ice water bath and stir for 30 minutes; in step S3, add tetrahydropyrrole anhydrous methanol solution at a rate of 2 drops/second. After adding to the anhydrous methanol solution of dihydromyricetin in an ice-water bath, the reaction was carried out at room temperature.
  • the preparation method of dihydromyricetin in step S1 is:
  • step S1-2 Under the protection of inert gas, mix the 2-hydroxy-4,6-dimethoxymethyleneoxyacetophenone obtained in step S1-1 with anhydrous tetrahydrofuran; then add sodium hydride and Chloromethyl methyl ether; after the reaction is completed, 2,4,6-trimethoxymethyleneoxyacetophenone is obtained;
  • step S1-3 Under the protection of inert gas, mix the 2,4,6-trimethoxymethyleneoxyacetophenone obtained in step S1-2 with THF and water; then add sodium hydroxide and 3, 4,5-trihydroxybenzaldehyde; after the reaction is completed, (E)-3-trihydroxybenzaldehyde is obtained Benzene-1-(2,4,6-trimethoxymethyleneoxyphenyl)-vinylketone;
  • step S1-4 Mix (E)-3-trihydroxybenzene-1-(2,4,6-trimethoxymethyleneoxyphenyl)-vinyl ketone obtained in step S1-3 with 30% Mix hydrogen peroxide; after the reaction is completed, add saturated sodium sulfite solution to quench the reaction, then extract with ethyl acetate, spin off the solvent under reduced pressure, then dissolve the solvent-removed residue in methanol, and add dilute hydrochloric acid. After the reaction is completed, Spin off the solvent under reduced pressure to obtain the dihydromyricetin tetrahydropyrrole complex.
  • DHMP dihydromyricetin tetrahydropyrrole complex
  • compound 1 is 2,4,6-trihydroxyacetophenone and compound 2 is 2-hydroxy- 4,6-dimethoxymethyleneoxyacetophenone
  • compound 3 is 2,4,6-trimethoxymethyleneoxyacetophenone
  • compound 4 is 3,4,5-trihydroxybenzaldehyde
  • Compound 5 is (E)-3-trihydroxybenzene-1-(2,4,6-trimethoxymethyleneoxyphenyl)-vinyl ketone
  • compound 6 is dihydromyricetin;
  • the neurological dysfunction diseases are sleep disorders, anxiety, depression, post-traumatic stress disorder, and Alzheimer's disease , dementia, Parkinson's disease, stroke, epilepsy, autism, alcohol use disorder.
  • Wild type Wild type (Wild type, referred to as Wt mouse) Wild type (Wt., C57BL/6, Charles River Lab, USA) and transgenic mice (Transgenetic(TG)-SwDI, referred to as TG mice, Transgenetic(TG)-SwDI (Jacson Lab, USA)) are both male and 20 months old.
  • TG mouse model of Alzheimer's disease AD has behavioral deficits, such as lack of exploratory/motor activity, increased anxiety, and susceptibility to epilepsy; (2) TG mice lose cognitive memory. These abnormal behavioral changes are consistent with human studies and are commonly seen in people with Alzheimer's disease. So in this example we used the same genetic mouse.
  • Transverse slices 400 ⁇ m thick brain slices of the dorsal hippocampus were obtained from Wt mice and TG-SwDI mice (Jacson Lab, USA, male, 20 months old) using a vibratome (VT 100; International Technology Products). Ensure activity of all at least three layers of neurons within the brain slice. (Note: Equivalent to a piece of living brain tissue) Continuously perfuse the slices with artificial cerebrospinal fluid (ACSF). Artificial cerebrospinal fluid (ACSF) consists of the following substances (in mM): 125NaCl, 2.5KCl, 2CaCl 2 , 2MgCl 2 , 26NaHCO 3 and 10D-glucose.
  • ACSF was continuously input into 95% O 2 /5% CO 2 to ensure that the slices were fully oxygenated, with a pH of 7.4, and maintained at 34 ⁇ 0.5°C for perfusion to ensure the activity of neurons in the brain slices (Note: During the entire experiment, the brain Nerves can maintain physiological functions).
  • TTX tetrodotoxin calcium channel blocker 0.5 ⁇ M and D(-)-2-amino-5-phosphonopentanoate (APV) (40 ⁇ M ), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 ⁇ M and CGP54626 (1 ⁇ M, GAB A BR antagonist) were added to ACSF to pharmacologically isolate GABA AR -mediated mini-inhibitory postsynaptic currents (mIPSCs).
  • mIPSCs mini-inhibitory postsynaptic currents
  • the patch electrode was filled with an internal solution containing the following (in mM): 137CsCl, 2MgCl2 , 1CaCl2 , 11EGTA, 10HEPES, and 3ATP, with the pH adjusted to 7.30 with CsOH.
  • In vivo cranial nerve current recording was performed on dentate gyrus granule cells (DGC) in hippocampal slices.
  • EPM elevated plus maze test
  • Table 1 is a summary of Figures 6-9.
  • DHM starts to produce a concentration-dependent enhancement of GABAergic Itonic from 0.1 ⁇ M
  • DHMP starts to produce a significant and concentration-dependent enhancement of GABAergic Itonic from 0.001 ⁇ M.
  • DHMP shows 100 times greater efficacy.
  • DHMP starting at 0.001 ⁇ M, produced a significant concentration-dependent enhancement of mIPSCs.
  • DHM only begins to produce intentional enhancement at 0.3 ⁇ M. The results show that DHMP can maintain the biological activity of DHM, increase its efficacy and improve water solubility.
  • FIG. 6-9 and Table 1 need to be read together.
  • Table 1 is a comparison of the values in Figure 7 and Figure 9 above. It can be clearly seen that the concentration that can cause I current changes in the DHMP group is 0.001, and significant current changes begin. At the same time, The principle can cause significant changes in the protrusion current at 0.001, while DHM starts to change at 0.1 (DHMP has a more obvious effect because of its fast absorption).
  • mice C57BL/6, Charles River Lab, USA
  • TG-SwDI Jacson Lab, USA mice
  • mice Divide the two types of mice into three groups, namely the control group, the AD group, and the AD+DHMP group; measure the amount of Gephyrin in the control group, AD group, and AD+DHMP group respectively; then take out all the mice.
  • the brains were analyzed, that is, SDS-PAGE and Western blot analysis were performed respectively; specifically: proteins were separated on SDS-polyacrylamide gels (Sigma) using Bio-Rad Mini-Protean 3 cell system; proteins were transferred to polyacrylamide gels (Sigma).
  • DHM restores impaired GABAergic inhibitory transmission in TG mouse models by restoring Gephyrin, a postsynaptic GABA A R anchor protein that can regulate the formation and plasticity of GABAergic synapses in TG mice;
  • Gephyrin protein levels were tested in the hippocampus (left) and cortex (right) of mice was repeated to test the effects of DHMP.
  • Gephyrin is a key scaffolding protein that organizes inhibitory postsynaptic receptor density and is involved in the accumulation of GABA AR at postsynaptic sites. Gephyrin not only has structural functions at GABAergic synaptic sites but also plays a crucial role in synaptic dynamics. Therefore, we compared TG mice with Wt mice and tested Gephyrin protein levels in the hippocampus (left) and cortex (right) of TG mice and DHMP-treated TG mice, respectively. From Western blot analysis, As shown in Figure 10, compared with Wt mice, TG mice have reduced Gephyrin levels, that is, application of DHMP can reversibly reduce Gephyrin levels in Alzheimer's disease AD animals.
  • mice were given an oral dose of DHMP of 0.5 mg/kg per day for three months; the oral dose of DHMP here was only 1/4 of the dose of DHM.
  • the reason why a lower dose was not selected is because it needs to be confirmed: 1) whether DHMP and DHM have the same effect; 2) whether DHMP is more effective than natural DHM.
  • Gephyrin levels partially recovered after 1 month of treatment with DHMP (0.5 mg/kg, 1/4 dose of DHM, daily orally) in mice, and after 3 months of DHMP treatment in mice, Gephyrin levels returned to the normal range, orally
  • a small dose (1/4 of the dose of DHM) of DHMP can reverse the level of pontin in a short time, as shown in Figure 11, indicating that DHMP can quickly reach the brain through the blood-brain barrier and reverse the effect of pontin, further indicating that the modified DHM (ie DHMP) is quickly absorbed into the blood circulation when taken orally into the body, and quickly enters the brain to exert its medicinal effects.
  • TG mice may lead to reduced synaptic clustering of GABA AR , which may play a role in impaired GABAergic inhibitory neurotransmission.
  • GABA AR GABAergic inhibitory neurotransmission
  • TG mouse models exhibit cognitive and memory loss, lack of exploratory/motor activity, increased anxiety, and susceptibility to epilepsy.
  • the results show that DHMP treatment can restore Gephyrin levels, which is a potential mechanism for the behavioral changes and improvement of learning and cognitive functions in the TG mouse animal model of the AD group treated with DHMP.
  • DHMP has the same effect as DHM and is more effective than natural DHM (DHMP is 1/4 the dose of DHM, i.e. the same effect can be achieved with a smaller dose).
  • mice C57BL/6, Charles River Lab, USA
  • TG-SwDI mice Jacson Lab, USA mice
  • mice were divided into four groups: (1) C57BL/6 Wt male mice were orally administered with 2% sucrose; (2) C57Bl/6 Wt male mice were orally administered with 2% of 0.5 mg/kg DHMP (1/4 dose of DHM) Sucrose; (3) TG-SwDI mice were orally administered 2% sucrose; (4) TG-SwDI mice were orally administered 2% sucrose at 0.5 mg/kg DHMP (1/4 dose of DHM).
  • Days 1-3 Habituation (once a day).
  • Open-top containers (open air) are used with cameras to record animal behavior. Place the animal into a container without objects for 5 minutes. Every animal has the exact same background.
  • Toys Two identical objects (toys (defined as FO): familiar objects) are placed into specific locations in the container. The animal was then placed in the container to explore the object for 5 minutes. Retention test 1.5 hours: Each animal is placed in its home cage for 1.5 hours after familiarization. One of the toys was replaced by a new toy (a novel object, very different from the FO). We then placed an animal into the container with the object for 3 minutes and videotaped it for offline scoring. Scores are based on how long the animal spends exploring each toy. The pairs of objects in one set had been tested previously to avoid the mice's natural preference for shape or light reflection; we used a total of 4 sets of objects in this test.
  • ORI object recognition index
  • New context recognition (NCR, B in Figure 12): Days 1-3 are habituation (once a day), and two contexts (containers) A and B with similar field areas are very different in shape.
  • Context A is a rectangle and Context B is a circular container. The top of the container is open with bedding to minimize stress and a video camera is mounted on top to record the animal's behavior.
  • Each animal was placed in Environment A without toys for 5 min and then returned to its home cage for 30 min. The animals were then placed in environment B without toys for 5 minutes.
  • Day 4 is for familiarization. Two different sets of toys were used as familiar objects, referred to as FO1 and FO2. Each group consists of two phases They are composed of the same toys, but the shapes of FO1 and FO2 are very different.
  • FO1's two toys were placed at specific locations in situation A. Each animal was placed in Context A and allowed to explore FO1 for 5 min; the animal was then returned to its home cage for 30 min. The two toys of FO2 are placed at specific locations in background B. Each animal was placed in context B and allowed to explore FO2 for 5 min. Day 5 (24 hours after familiarization): A memory test was conducted to determine each animal's memory retention of familiar objects. A toy in FO1 in context A is exchanged with a toy in FO2. Each animal was then allowed to explore the object in context A for 3 min. The test was videotaped and analyzed offline. The time spent exploring familiar objects (FO1) and exchange objects (FO2) was calculated, where exploration equals touching the object with the nose or paws, or sniffing within 1.5 cm of the object.
  • FO1 familiar objects
  • FO2 exchange objects
  • TG mice show signs of reduced cognitive memory. These abnormal behavioral changes are consistent with human studies and are commonly seen in AD patients.
  • mice C57BL/6, Charles River Lab, USA
  • TG-SwDI mice Jacson Lab, USA mice
  • mice were divided into three groups: (1) Control group: C57BL/6Wt male mice (2% sucrose, oral); (2) AD group: TG mice (2% sucrose, oral); (3) AD+ DHMP group: TG mice were given DHMP (0.5 mg/kg (1/4 dose of DHM) in 2% sucrose).
  • locomotor activity To measure locomotor activity, the total number of entries per animal was measured; statistical differences were determined using analysis of variance; the effect of DHMP on anxiety levels was determined through these experiments.
  • the open field test was used to determine the effect of DHMP on Alzheimer's disease (AD) symptom-like behaviors in aged TG2576 mice.
  • Running distance is one of the parameters that quantify athletic activity (Figure 15).
  • Weight control mice ran 922 ⁇ 75 cm in 10 minutes.
  • TG mice ran a much shorter distance (235 ⁇ 16 cm), while the AD+DHMP group (TG mice treated with DHMP) increased the distance to 682 ⁇ 109 cm.
  • Control Wt mice showed frequent rearing (23.3 ⁇ 3.4 times), explored the center of the empty field 2.0 ⁇ 0.6 times, and stayed in the center for 0.14 ⁇ 0.1 min.
  • AD group TG-mice showed rearing 12.1 ⁇ 2.1 times, explored the center of the open field (0.2 ⁇ 0.1 times), and stayed in the center for only 0.02 ⁇ 0.02 minutes.
  • DHMP treatment of TG mice in the AD+DHMP group increased rearing (19.5 ⁇ 2.0 times), time to explore the center (1.4 ⁇ 1.4 times), and time to stay in the center (0.13 ⁇ 0.05 minutes) (see Figure 16-18 for details) shown).
  • the results showed that TG mice in the AD group had reduced exploratory/locomotor activities, and daily oral DHMP (1/4 dose of DHM) treatment of mice in the AD+DHMP group improved locomotor activities and increased exploratory activities, which is an important characteristic of animals. kind of instinct. Measuring anxiety using EPM.
  • mice spent 48.5 ⁇ 7.5% of the total time in the open arm and 33.5 ⁇ 3.2% in the closed arm.
  • TG-mice with AD spent significantly less time in the open arm and longer in the closed arm compared to control Wt mouse controls (statistically significant vs. Wt mouse controls; e.g. Figure 19), while TG- mice in the AD+DHMP group spent a similar amount of time in each arm.
  • Pentylenetetrazole (PTZ) is a non-competitor for the GAB A receptor complex Sexual antagonist. PTZ (42mg/kg) was used to induce epileptic seizures in mice.
  • SPD rats Male, adult rats weighing 200 ⁇ 20g, Charles River Lab, USA.
  • Stroke is a leading cause of limb dysfunction, but currently there are no pharmacological treatments available to promote functional recovery. Recent research shows that the brain has a limited ability to repair after a stroke. Neural repair after stroke involves remapping cognitive function in tissues adjacent to or associated with stroke. Therefore, in the first time limit the release of extra valleys by excitatory neurons Neurotoxic damage caused by amino acids is important.
  • Figures 21-23 show the results measured on day 10. In the 5-minute grid walking test ( Figure 21), stroke rats could only perform for 0.8 ⁇ 0.85 minutes and often fell off the grid. Rats in the experimental group (stroke + DHMP treatment) could perform for nearly 3.6 ⁇ 0.5 minutes, compared with 3.8 ⁇ 0.3 minutes in the control group. In the open-air test (Fig. 22), stroke rats in the control group could only run a distance of ⁇ 270 cm. However, mice in the experimental group with stroke + DHMP treatment could run ⁇ 700 cm. Stroke rats were unable to enter the center of the open field (Fig. 23).
  • Figure 24 is the recording results of the brain slices in the control group;
  • Figure 25 is the comparison of the post-synaptic micro excitatory postsynaptic current (mEPSC) peak values obtained in the brain slices of the control group when DHMP was administered at different doses;
  • Figure 26 is the dose dependence of DHMP The mEPSC recorded in the brain slices of stroke mice was significantly reduced;
  • Figure 27 is a comparison of the mEPSC peak values obtained in the brain slices of stroke mice when different doses of DHMP were administered.
  • mEPSC post-synaptic micro excitatory postsynaptic current
  • DHMP could reduce mEPSC frequency to a value similar to that of the control group ( Figure 28); in addition, DHMP reduced the total charge transfer of mEPSC to a level similar to the control level in the ischemia group ( Figure 29).
  • the results indicate that DHM can inhibit excessive glutamate release, thereby reducing glutamate-induced neurotoxicity.
  • oral administration of a small dose of DHMP (1/4 the dose of DHM) can show that DHMP can quickly reach the brain through the blood-brain barrier and quickly inhibit the excessive release of glutamate to reduce brain damage caused by stroke, thereby limiting brain damage caused by stroke. damage, shorten the course of stroke, and restore brain function to regulate motor activities after stroke; it is proven that modified DHM (DHMP) orally administered into the body can be quickly absorbed into the blood circulation and quickly enter the brain to exert its medicinal effect.
  • DHMP modified DHM
  • DHMP dihydromyricetin tetrahydropyrrole complex
  • mice Male, weight 200 ⁇ 20g; female, weight 190 ⁇ 20g.
  • n 5/group/sex, Charles River Lab, USA).
  • Animal group The control group received 5% sucrose (SUC.2ml/100g body weight), DHMP 1mg/kg group, DHMP 10mg/kg group and DHMP 100mg/kg group.
  • T in Table 2 above represents temperature, the unit is °C; P represents pulse, the unit is times/minute; B represents breathing, the unit is times/minute.

Abstract

本发明公开了一种二氢杨梅素四氢吡咯复合物及其制备方法和应用，二氢杨梅素四氢吡咯复合物，所述的二氢杨梅素四氢吡咯复合物的分子式如式(I)所示；二氢杨梅素四氢吡咯复合物的制备方法，包括以下步骤：S1：将无水甲醇加入到二氢杨梅素中获得二氢杨梅素无水甲醇溶液；S2：在惰性气体保护下，将无水甲醇加入到四氢吡咯中，获得四氢吡咯无水甲醇溶液；S3：将步骤S1获得的二氢杨梅素无水甲醇溶液和步骤S2获得的四氢吡咯无水甲醇溶液室温下进行反应；S4：将步骤S3获得的反应残留物过滤，无水甲醇洗涤滤饼，并减压抽干即可得到二氢杨梅素四氢吡咯复合物。二氢杨梅素四氢吡咯复合物在神经***功能障碍疾病治疗药物中的应用。

Description

二氢杨梅素四氢吡咯复合物及其制备方法和应用
相关申请
本申请主张于2022年4月19日提交的、名称为“二氢杨梅素四氢吡咯复合物及其制备方法和应用”的中国发明专利申请：202210408113.X的优先权。
技术领域
本发明涉及医药技术领域，具体涉及一种二氢杨梅素四氢吡咯复合物及其制备方法和应用。
背景技术
二氢杨梅素(Dihydromyricetin，以下简称DHM)是一种从枳椇属和藤茶中提取出的天然类黄酮化合物，由于其具有各种健康益处已被广泛应用于补品市场。
由于DHM是一种天然类黄酮化合物，其具有一些缺点，例如在氧气、光和高温暴露环境中易于降解和氧化，而且具有低的溶解性，将其开发为阿尔茨海默病(以下简称AD)和其它神经退行性疾病治疗药物存在许多限制，在制剂研发上也面临许多困难，使制剂生产受到影响。因此，保持DHM的原始功效，减少其上述缺陷以获得更加优秀的效能是研发DHM新剂型的关键所在。
在中国《现代食品科技》期刊，2014年第10期，曹敏惠等人发表有“藤茶二氢杨梅素茶氨酸复合物的制备及抗氧化活性研究”，公开了二氢杨梅素在天然藤茶中含量高达25.6％，具有多种生理功能，受到广泛关注。但二氢杨梅素常温水溶性差，生物利用度较低，限制了其应用范围，为了提高二氢杨梅素的水溶性，对二氢杨梅素进行分子修饰，制备了二氢杨梅素-茶氨酸复合物，利用紫外光谱、红外光谱、质谱和核磁共振氢谱对复合物进行了结构表征，测定了复合物的理化性质，并考察了复合物的抗氧化活性。通过结构分析，二氢杨梅素分子中C3-OH、C5-OH、C7-OH、C3′-OH、C5′-OH与茶氨酸的活泼氢通过氢键结合形成复合物；复合物清除·OH自由基的能力均强于二氢杨梅素和Vc，清除O2-·自由基和DPPH自由基的能力和二氢杨梅素相当，比Vc强。二氢杨梅素和茶氨酸的复合，不仅提高其水溶性，在抗氧化活性方面也起到了强化的作用。
在中国《中成药》期刊，2021年12月第43卷第12期，魏永鸽等人发表有“二氢杨梅素磷脂复合物及其滴丸的制备及其体内药动学比较”，公开了制备二氢杨梅素磷脂复合物及其滴丸，并比较其体内药动学。滴丸可改善二氢杨梅素磷脂复合物的累积溶出度及口服生物利用度。
虽然上述二氢杨梅素-茶氨酸复合物、二氢杨梅素磷脂复合物在水溶性以及抗氧化活性方面取得一定的效果，但是实际生物效能上应用于医药还是很不理想，尤其是实现可工业化生产合成。
发明内容
本发明要解决的技术问题是，提供一种二氢杨梅素四氢吡咯复合物，在溶解性、稳定性得到改进，生物效能得到提高，并能将其应用于医药技术领域。
为解决上述技术问题，本发明采用的技术方案是，该二氢杨梅素四氢吡咯复合物，所述的二氢杨梅素四氢吡咯复合物的分子式如式(I)所示：
通过对二氢杨梅素DHM的修饰改变，在溶解性、稳定性得到改进，生物效能得到了提高，从而实现可工业化生产合成和未来对患者的可使用性，可应用于医药技术领域；水溶性(溶解性)和稳定性是有机小分子药物必不可少的物理化学性质，也是药物发明中非常重要的问题。改善的水溶性通常导致更好的药物效力和更令人满意的药代动力学曲线。需要强调的是，二氢杨梅素DHM成盐方法很多，但是能够不改变已经发现的DHM的成药性是关键。
本发明要解决的另一个问题是提供一种二氢杨梅素四氢吡咯复合物的制备方法，所述的制备方法包括以下步骤：
S1：将无水甲醇加入到二氢杨梅素中获得二氢杨梅素无水甲醇溶液；
S2：在惰性气体保护下，将无水甲醇加入到四氢吡咯中，获得四氢吡咯无水甲醇溶液；
S3：将所述步骤S1获得的二氢杨梅素无水甲醇溶液和所述步骤S2获得的四氢吡咯无水甲醇溶液室温下进行反应，获得反应物；
S4：将所述步骤S3获得的反应物过滤，无水甲醇洗涤滤饼，并减压抽干即可得到二氢杨梅素四氢吡咯复合物。
优选的，所述步骤S2中的惰性气体为氩气。
优选的，所述步骤S1中的二氢杨梅素无水甲醇溶液的浓度为150-350mmol/L；所述步 骤S2中的四氢吡咯无水甲醇溶液的浓度为1-10mol/L。
优选的，所述步骤S3中的二氢杨梅素和四氢吡咯的摩尔比为1:0.7-1.3。
优选的，所述步骤S3中的二氢杨梅素和四氢吡咯的摩尔比为1：1。
优选的，在所述步骤S1中，将无水甲醇加入到二氢杨梅素中后移到冰水浴中进行搅拌30分钟；在所述步骤S3中，将四氢吡咯无水甲醇溶液2滴/秒的速度加到在冰水浴中的二氢杨梅素无水甲醇溶液后，再升至室温条件下进行反应。
优选的，所述步骤S1中的二氢杨梅素制备方法为：
S1-1：在2,4,6-三羟基苯乙酮中加入无水二氯甲和N,N-二异丙基乙基胺，再以1滴/秒的速度加入氯甲基甲基醚，反应完成后，得2-羟基-4,6-二甲氧基亚甲氧基苯乙酮；
S1-2：在惰性气体保护下，将所述步骤S1-1得到的2-羟基-4,6-二甲氧基亚甲氧基苯乙酮与无水四氢呋喃混合；再先后加入氢化钠和氯甲基甲基醚；待反应完成后，得2,4,6-三甲氧基亚甲氧基苯乙酮；
S1-3：在惰性气体保护下，将所述步骤S1-2得到的2,4,6-三甲氧基亚甲氧基苯乙酮与THF和水混合；再先后加入氢氧化钠和3,4,5-三羟基苯甲醛；待反应完成后，得(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮；
S1-4：将所述步骤S1-3得到的(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮与30％的双氧水混合；反应完成后，加入饱和亚硫酸钠溶液淬灭反应，而后用乙酸乙酯萃取，减压旋去溶剂，再将去掉溶剂的残留物溶于甲醇中，并加入稀盐酸，待反应完成后，减压旋去溶剂，即可得二氢杨梅素四氢吡咯复合物。
本发明要解决的另一个问题是将本发明的二氢杨梅素四氢吡咯复合物在神经***功能障碍疾病治疗药物中的应用。
优选的，所述神经***功能障碍疾病为睡眠障碍、焦虑、抑郁、创伤后应激障碍、阿兹海默症、痴呆症、帕金森病、中风、癫痫、自闭症、酒精使用障碍。
本发明提供的二氢杨梅素四氢吡咯复合物(DHMP)保持了二氢杨梅素(DHM)的生物能力，提高了其药效，改善了水溶性，增强了对于GABAARs的作用(低至0.1nM)，是天然DHM的1000倍，其可以复制DHM的功效，恢复实验动物导向支撑蛋白水平(gephyrin level)的表达。可以改善实验动物的识别/记忆损伤，改善行为损伤，例如焦虑、抑郁、睡眠障碍，并减少发作，降低中风导致的脑部损伤，可以作为中风后产生的和运动功能相关的脑部功能改善的治疗手段。DHMP显示了在活化GABAAR的相似效能，并高于DHM效能的1000倍。DHM应用的适应症包括睡眠障碍、焦虑、抑郁，阿兹海默症早 中期阶段、痴呆症、帕金森病，其它神经退化性疾病，中风、癫痫、自闭症、酒精使用障碍，以及苯二氮卓类(Benzodiazepines)及其它药物成瘾性。
二氢杨梅素四氢吡咯复合物(DHMP)具有生物安全性。每日口服剂量为1,10,和100mg/kg时，在两周的功能观察组合实验(functional observational battery，FOB)研究中，实验大鼠未发现任何的负性改变。在六个月的1,10,和500mg/kg用药的长期评价中，没有毒副作用，没有代谢率的改变。
附图说明
下面结合附图和本发明的实施方式进一步详细说明：
图1是本发明的二氢杨梅素四氢吡咯复合物(DHMP)的制备流程图；
图2、3是本发明制备得到的二氢杨梅素四氢吡咯复合物(DHMP)核磁共振图谱，图4、5是二氢杨梅素(DHM)核磁共振图谱；
图6-9是本发明的二氢杨梅素四氢吡咯复合物(DHMP)对GABAAR的正向调节小鼠脑切片的全细胞膜片钳电流比较图；
图10和图11是本发明的二氢杨梅素四氢吡咯复合物(DHMP)对小鼠导向支撑蛋白水平表达的影响比较图；
图12、图13和图14是本发明的二氢杨梅素四氢吡咯复合物(DHMP)对小鼠认知/记忆障碍的改善作用比较图；
图15-20是本发明的二氢杨梅素四氢吡咯复合物(DHMP)改善小鼠运动活动，减少焦虑及癫痫发作的效果比较图；
图21-23是本发明的二氢杨梅素四氢吡咯复合物(DHMP)改善小鼠中风诱发的脑损伤效果比较图；
图24-29是本发明的二氢杨梅素四氢吡咯复合物(DHMP)通过抑制过量释放的谷氨酸来减少中风引起的脑损伤效果图；
图30是本发明的二氢杨梅素四氢吡咯复合物(DHMP)对GABAAR的增强作用图；
图31-图40是本发明的二氢杨梅素四氢吡咯复合物(DHMP)的无毒安全性试验结果图。
具体实施方式
本发明的二氢杨梅素四氢吡咯复合物，分子式如式(I)所示：
二氢杨梅素四氢吡咯复合物的制备方法，包括以下步骤：
S1：将无水甲醇加入到二氢杨梅素中获得二氢杨梅素无水甲醇溶液；
S2：在惰性气体保护下，将无水甲醇加入到四氢吡咯中，获得四氢吡咯无水甲醇溶液；
S3：将所述步骤S1获得的二氢杨梅素无水甲醇溶液和所述步骤S2获得的四氢吡咯无水甲醇溶液室温下进行反应，获得反应物；
S4：将所述步骤S3获得的反应物过滤，无水甲醇洗涤滤饼，并减压抽干即可得到二氢杨梅素四氢吡咯复合物。
所述步骤S2中的惰性气体为氩气。
所述步骤S1中的二氢杨梅素无水甲醇溶液的浓度为150-350mmol/L，优选的是250mmol/L；所述步骤S2中的四氢吡咯无水甲醇溶液的浓度为1-10mol/L，优选的是1mol/L。
所述步骤S3中的二氢杨梅素和四氢吡咯的摩尔比为1:0.7-1.3，优选的二氢杨梅素和四氢吡咯的摩尔比为1：1。
在所述步骤S1中，将无水甲醇加入到二氢杨梅素中后移到冰水浴中进行搅拌30分钟；在所述步骤S3中，将四氢吡咯无水甲醇溶液2滴/秒的速度加到在冰水浴中的二氢杨梅素无水甲醇溶液后，再升至室温条件下进行反应。
所述步骤S1中的二氢杨梅素制备方法为：
S1-1：在2,4,6-三羟基苯乙酮中加入无水二氯甲和N,N-二异丙基乙基胺，再以1滴/秒的速度加入氯甲基甲基醚，反应完成后，得2-羟基-4,6-二甲氧基亚甲氧基苯乙酮；
S1-2：在惰性气体保护下，将所述步骤S1-1得到的2-羟基-4,6-二甲氧基亚甲氧基苯乙酮与无水四氢呋喃混合；再先后加入氢化钠和氯甲基甲基醚；待反应完成后，得2,4,6-三甲氧基亚甲氧基苯乙酮；
S1-3：在惰性气体保护下，将所述步骤S1-2得到的2,4,6-三甲氧基亚甲氧基苯乙酮与THF和水混合；再先后加入氢氧化钠和3,4,5-三羟基苯甲醛；待反应完成后，得(E)-3-三羟基 苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮；
S1-4：将所述步骤S1-3得到的(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮与30％的双氧水混合；反应完成后，加入饱和亚硫酸钠溶液淬灭反应，而后用乙酸乙酯萃取，减压旋去溶剂，再将去掉溶剂的残留物溶于甲醇中，并加入稀盐酸，待反应完成后，减压旋去溶剂，即可得二氢杨梅素四氢吡咯复合物。
具体的制备实施例如下：
如图1所示，是本实施例的二氢杨梅素四氢吡咯复合物(DHMP)的制备流程，其中化合物1为2,4,6-三羟基苯乙酮，化合物2为2-羟基-4,6-二甲氧基亚甲氧基苯乙酮，化合物3为2,4,6-三甲氧基亚甲氧基苯乙酮，化合物4为3,4,5-三羟基苯甲醛，化合物5为(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮，化合物6为二氢杨梅素；
化合物2的制备：在氩气保护下，将化合物1(10.0mmol)加入到含有搅拌子的100mL的圆底烧瓶中，抽换气后，在室温下向该烧瓶中加入20mL的无水二氯甲烷(Dichloromethane，DCM)和N,N-二异丙基乙基胺(N,N-Diisopropylethylamine,DIPEA,30mmol)。而后，在0℃下缓慢加入氯甲基甲基醚(Chloromethyl Methyl Ether,MOMCl,30mmol)。反应完成后，减压旋去溶剂，将去掉溶剂的残留物采用柱层析法即可得化合物2；
化合物3的制备：在氩气保护下，将化合物2(8.0mmol)加入到含有搅拌子的100mL的圆底烧瓶中，抽换气后，在室温下向该圆底烧瓶中加入20mL的无水四氢呋喃(Tetrahydrofuran，THF)。而后，在0℃下先后加入氢化钠(NaH,Sodium hydride,9.6mmol)和MOMCl(9.6mmol)。待反应完成后，减压旋去溶剂，将去掉溶剂的残留物采用柱层析法即可得化合物3；
化合物5的制备：在在氩气保护下，将化合物3(6.0mmol)加入到含有搅拌子的100mL的圆底烧瓶中，抽换气后，在室温下向该圆底烧瓶中加入15mL的THF和10mL水。而后，在室温下先后加入氢氧化钠(NaOH,Sodium hydroxide,12.0mmol)和化合物4(12.0mmol)。待反应完成后，用乙酸乙酯萃取3次，合并有机相，减压旋去溶剂，将去掉溶剂的残留物采用柱层析法即可得化合物5；
化合物6的制备：将化合物5(6.0mmol)和NaOH溶液(5M，60mL)加入到含有搅拌子和50mL甲醇的100mL的圆底烧瓶中。在室温下，向上述溶液中加入30％的双氧水(hydrogen peroxide,H2O2,1.8mL,18.0mmol)。反应完成后，加入饱和亚硫酸钠溶液淬灭反应，而后用乙酸乙酯萃取三次，合并有机相，减压旋去溶剂，将去掉溶剂的残留物溶于30mL甲醇中，并加入2N稀盐酸(hydrochloric acid,HCl,5mL)，将反应溶液加热至55℃ 直至反应完全。待反应完成后，减压旋去溶剂，柱层析即可得化合物6。
二氢杨梅素四氢吡咯复合物的制备：在氩气保护下，将制得的化合物6二氢杨梅素(DHM，dihydromyricetin，1.6g，5.0mmol)加入到含有搅拌子的100mL的圆底烧瓶中，抽换气后，在室温下向该圆底烧瓶中加入20mL的无水甲醇；在室温下搅拌30分钟后，将圆底烧瓶移到冰水浴中并继续搅拌30分钟，在氩气保护下，10mL无水甲醇制成四氢吡咯(化合物纯度≥98％)的四氢吡咯无水甲醇溶液；而后，将配制好的四氢吡咯无水甲醇溶液以2滴/秒的速度加到冰水浴中的含有DHM的烧瓶中，并使其自然升至室温进行反应。反应完成后，减压旋去溶剂，并将去掉溶剂后反应物的残留物过滤，获得滤饼，将滤饼用甲醇洗三次后减压抽干即可得到目标产物，即本发明的浅褐色二氢杨梅素四氢吡咯复合物。
得到的二氢杨梅素四氢吡咯复合物的核磁共振图谱如图2、3所示，具体为：1H NMR(400MHz,D2O):δ1.94-1.97(m,4H,2CH2),3.22-3.26(m,4H,2CH2),4.52-4.56(m,1H,CH),4.84(d,1H,J＝11.6,CH),5.64(s,1H,ArH),5.71(m,1H,ArH),6.62(s,1H,ArH)；13C NMR(100MHz,CD3OD):δ23.6,45.4,71.2,82.9,97.7,98.6,107.71,107.75,127.8,133.9,145.6,162.3,162.9,178.4,193.7；
化合物6的二氢杨梅素的核磁共振图谱如图4、5所示，具体为：1H NMR(400MHz,CD3OD):δ4.46(d,1H,J＝11.2Hz,CH),4.84(d,1H,J＝11.2Hz,CH),5.88(d,1H,J＝1.6Hz,ArH),5.92(d,1H,J＝1.6HZ,ArH),6.53(s,1H,ArH)；13C NMR(100MHz,CD3OD):δ73.7,85.3,96.2,97.3,108.0,129.1,134.9,146.9,164.5,165.3,168.7,198.3；
由核磁共振图谱，对比图4、5和图2、3可知，二氢杨梅素与四氢吡咯形成了复合物，即本发明的二氢杨梅素四氢吡咯复合物，分子式如式(I)所示。
本发明的二氢杨梅素四氢吡咯复合物在神经***功能障碍疾病治疗药物中的应用；所述神经***功能障碍疾病为睡眠障碍、焦虑、抑郁、创伤后应激障碍、阿兹海默症、痴呆症、帕金森病、中风、癫痫、自闭症、酒精使用障碍。
针对本发明的二氢杨梅素四氢吡咯复合物的应用，本发明进行了以下的应用实施例，具体的：
应用实施例1：二氢杨梅素四氢吡咯复合物(DHMP)的功效性评价在本实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物和二氢杨梅素的功效性对比，具体实验过程如下：
1、实验材料
野生小鼠(Wild type，简称Wt小鼠)Wild type(Wt.，C57BL/6,CharlesRiver Lab,USA)) 和转基因小鼠(Transgenetic(TG)-SwDI，简称TG小鼠，Transgenetic(TG)-SwDI(Jacson Lab,USA))均为雄性，20个月大。
2、实验方法
小鼠脑切片的全细胞膜片钳记录：
我们已经证明了(1)阿尔茨海默病AD的TG小鼠模型具有行为缺陷，例如缺乏探索/运动活动、焦虑增加和癫痫易感性；(2)TG小鼠失去认知记忆。这些异常行为变化与人类研究一致，并且通常见于阿尔兹海默病患者。所以在本实施例中我们采用相同基因鼠。
使用振动切片机(VT 100；国际技术产品)从Wt小鼠和TG-SwDI小鼠(Jacson Lab,USA，雄性，20个月大)获得背侧海马的横向切片(400μm厚脑切片)，已确保脑切片内所有至少三层神经元的活性。(注：相当于一块活的大脑组织)用人工脑脊液(ACSF)连续灌注切片，人工脑脊液(ACSF)由以下物质组成(以mM为单位)：125NaCl、2.5KCl、2CaCl2、2MgCl2、26NaHCO3和10D-葡萄糖。ACSF连续输入95％O2/5％CO2以确保切片充分充氧，pH为7.4，并保持在34±0.5℃进行灌流，以确保脑片中神经元的活性(注：整个实验过程中脑神经均能保持生理功能)。将河豚毒(tetrodotoxin，TTX)钙通道阻断剂0.5μM、D(-)-2-氨基-5-膦酰基戊酸脂(D(-)-2-amino-5-phosphonopentanoate,APV)(40μM)、6-氰基-7-硝基喹啉-2,3-二酮(6-cyano-7-nitroquinoxaline-2,3-dione，CNQX)10μM和CGP54626(1μM，GABABR拮抗剂)添加到ACSF以药理学分离GABAAR介导的微型抑制性突触后电流(mIPSC)。贴片电极填充了含有以下物质(以mM为单位)的内部溶液：137CsCl、2MgCl2、1CaCl2、11EGTA、10HEPES和3ATP，用CsOH将pH值调节至7.30。记录针对海马切片的齿状回颗粒细胞(DGC)进行活体脑神经电流记录。
使用膜片钳放大器进行电压钳全细胞记录，在高架十字迷宫试验(elevated plus maze test，EPM)上测试所有动物的焦虑水平。将动物放在迷宫的中心区域，测试5分钟并记录视频。对以下测量进行评分：进入张开双臂、闭合双臂或中心平台的次数以及在这些区域中花费的时间。数据报告为武器条目数的百分比、在不同条目中花费的时间百分比和总条目数。
3、实验结果
为了评估DHMP是否仍能增强GABAAR，对脑切片中海马回中的DGC的全细胞用电压钳位记录技术进行了测试，以测试DHMP和DHM对GABAergic(抑制神经能)的突触外张力电流(Itonic)和突触后微型抑制性突触后电流(mIPSC)的影响进行比较，如图6-9所示，我们以μM为单位增加了DHMP和DHM的剂量：分别为0.001、0.003、0.01、 0.03、0.1、0.3、1.0和30μM。与DHM增强GABAergic Itonic和mIPSCs相比，如表1所示。结果表明，DHMP可以保持DHM的生物活性，增加其功效，提高水溶性。
表1是对图6-9的小结。DHM开始从0.1μM，浓度依赖性的增强GABAergic Itonic，而DHMP是在0.001μM开始产生显著性及浓度依赖性的增强GABAergic Itonic。比较于DHM，DHMP显示出100倍强功效。在对于微型抑制性突触后电流(mIPSC)的影响，DHMP是在0.001μM开始，产生对于微型抑制性突触后电流(mIPSC)的浓度依赖性的显著性增强。而DHM是在0.3μM才开始产生有意增强作用。结果表明，DHMP可以保持DHM的生物活性，增加其功效，提高水溶性。
表1 DHMP与DHM的效果对照
图6-9和表1需结合一起看，表1是对于上图7和图9中数值进行比较，可以清楚看到DHMP组可以引起I电流变化的浓度是0.001，开始有显著电流变化，同理可以在0.001引起突出电流的显著变化，而DHM是在0.1开始产生变化(DHMP因为吸收快产生的效果更明显)。
应用实施例2：二氢杨梅素四氢吡咯复合物(DHMP)对小鼠导向支撑蛋白水平(gephyrin level)表达的影响
在本应用实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物对小鼠Gephyrin水平表达的影响，具体实验过程如下：
1、实验材料：如应用实施例1所用动物分别为Wt小鼠(C57BL/6,CharlesRiver Lab,USA)和TG-SwDI(Jacson Lab,USA)小鼠，均为雄性，20个月大。
2、实验方法：将两种小鼠均分为三组，分别为对照组、AD组、AD+DHMP组；分别测定对照组、AD组、AD+DHMP组的Gephyrin量；再取出所有小鼠的大脑并进行分析，即分别进行SDS-PAGE和蛋白质印迹分析；具体为：使用Bio-Rad Mini-Protean 3细胞***在SDS-聚丙烯酰胺凝胶(Sigma)上分离蛋白质；将蛋白质转移到聚偏二氟乙烯(PDVF)膜(Sigma)上，并用4％脱脂奶粉封闭；将印迹与兔抗gephyrin(Santa Cruz,1:200)和小鼠抗β-肌动蛋白(Sigma,1:1000)一起在4℃下孵育过夜，然后使用HRP偶联二抗(1:5000)进行两次室温下数小时(3-5小时)，使用ECL检测试剂盒(Sigma)检测条带 并暴露于X射线胶片；使用ImageQuant5.2(Molecular Dynamics)通过光密度分析条带，归一化为相应的β-肌动蛋白信号并进行比较。
3、实验结果
曾有相关文献报道DHM通过恢复Gephyrin来恢复TG小鼠模型中受损的GABAergic抑制传递，这是一种突触后GABAAR锚蛋白，可调节TG小鼠中GABAergic突触的形成和可塑性；在此，重复了以前的方法(即测试了小鼠的海马(左)和皮质(右)中的Gephyrin蛋白质水平)来测试DHMP的效果。
Gephyrin是组织抑制性突触后受体密度的关键支架蛋白，涉及GABAAR在突触后位点的聚集。Gephyrin不仅在GABAergic突触部位具有结构功能，而且在突触动力学中起着至关重要的作用。因此，我们对TG小鼠与Wt小鼠进行了对比，分别测试了TG小鼠和DHMP处理的TG小鼠的海马(左)和皮质(右)中的Gephyrin蛋白质水平，从蛋白质印迹分析显示，如图10所示，与Wt小鼠相比，TG小鼠的Gephyrin水平降低，即应用DHMP可逆转的降低阿尔茨海默病AD动物体内Gephyrin水平。
实验中，小鼠每天DHMP的口服用量为0.5mg/kg，持续三个月；此处DHMP口服用量仅是DHM的1/4剂量。之所以没有选择更低的剂量，是因为需要确认：1)DHMP与DHM是否具有相同的效果；2)DHMP是否比天然DHM更有效。
小鼠DHMP(0.5mg/kg，DHM的1/4剂量，每日口服)治疗1个月后，Gephyrin水平部分恢复，而小鼠DHMP治疗3个月后，Gephyrin水平恢复到正常水平范围，口服小剂量(DHM的1/4剂量)DHMP可以在短时间内逆转桥蛋白水平，如图11所示，说明DHMP可以通过血脑屏障迅速到达大脑起到逆转桥蛋白的作用，进一步说明经过修饰的DHM(即DHMP)口服进入体内能够被迅速吸收进入血液循环，并快速进入大脑发挥药效。TG小鼠中Gephyrin水平的降低可能导致GABAAR的突触聚集减少，这可能在GABA能抑制性神经传递受损中发挥作用。因此，TG小鼠模型表现出认知和记忆丧失、缺乏探索/运动活动、焦虑增加和癫痫易感性。结果表明，DHMP治疗可以恢复Gephyrin水平，这是DHMP治疗AD组的TG小鼠动物模型的行为改变和学习和认知功能改善的潜在机制。DHMP具有与DHM相同的效果，且比天然DHM更有效(DHMP是DHM的1/4剂量，即使用更小的剂量可以达到相同的效果)。
应用实施例3：二氢杨梅素四氢吡咯复合物(DHMP)对小鼠认知/记忆障碍的改善作用
在本实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物对小鼠认知/记忆 障碍的改善作用，具体实验过程如下：
1、实验材料：如应用实施例1所用动物，Wt小鼠(C57BL/6,CharlesRiver Lab,USA)和TG-SwDI(Jacson Lab,USA)小鼠，均为雄性，20个月大。
2、实验动物分组
将小鼠分为四组：(1)C57BL/6 Wt雄性小鼠口服2％蔗糖；(2)C57Bl/6 Wt雄性小鼠口服0.5mg/kg DHMP(1/4剂量的DHM)的2％蔗糖；(3)TG-SwDI小鼠口服2％蔗糖；(4)TG-SwDI小鼠口服0.5mg/kg DHMP(1/4剂量的DHM)的2％蔗糖。
3、实验方法
目标识别测试(novel object recognition，NOR)：
第1-3天：习惯(每天一次)。
顶部开口容器(露天)与摄像机一起使用以记录动物行为。将动物放入没有物体的容器中5分钟。每只动物的背景都完全相同。
第4天：熟悉。
两个相同的物体(玩具(定义为FO)：熟悉的物体)被放入容器的特定位置。然后将动物放入容器中探索物体5分钟。保留试验1.5小时：每只动物在熟悉后被放置在其家笼中1.5小时。其中一个玩具被一个新玩具取代(新颖的物体，与FO非常不同)。然后我们将一只动物放入装有物体的容器中3分钟，并对其进行录像以进行离线评分。分数基于动物探索每个玩具的时间。一组中的这对对象之前已经过测试，以避免小鼠对形状或光反射的自然偏好；我们在这个测试中总共使用了4组对象。
第5天：24小时保留试验。
我们在容器中放置了另一个新玩具(NO)和一个原始对象(FO)。然后我们将动物放入容器中并测试3分钟。我们根据动物探索每个物体的时间长短对动物的行为进行离线评分。计算对象识别指数(object recognition index，ORI)，使得ORI＝(tn-tf)/(tn+tf)，其中tf和tn代表探索熟悉和新颖的时间对象。
新的上下文识别(new context recognition,NCR，图12的B)：第1-3天是习惯(每天一次)，具有相似字段区域的两个上下文(容器)A和B在形状上非常不同。上下文A是一个矩形，上下文B是一个圆形容器。容器顶部打开，带有垫料以最大程度地减少压力，并在顶部装有摄像机以记录动物的行为。每只动物在没有玩具的情况下被放置在环境A中5分钟，然后回到家笼中30分钟。然后将动物放置在没有玩具的环境B中5分钟。第4天是为了熟悉。两组不同的玩具被用作熟悉的对象，分别称为FO1和FO2。每组由两个相 同的玩具组成，而FO1和FO2的形状非常不同。FO1的两个玩具分别被放置在情境A中的特定位置。每只动物被放置在情境A中并允许探索FO1 5分钟；然后动物回到家笼中30分钟。FO2的两个玩具分别放置在背景B中的特定位置。每只动物被放置在背景B中并允许探索FO2 5分钟。第5天(熟悉后24小时)：进行记忆试验以确定每只动物对熟悉对象的记忆保留。上下文A中FO1的一个玩具与FO2中的一个玩具交换。然后每只动物被允许探索上下文A中的对象3分钟。该测试被录像并离线分析。计算探索熟悉物体(FO1)和交换物体(FO2)所花费的时间，其中探索等于用鼻子或爪子触摸物体，或在物体1.5厘米范围内嗅探。
识别指数(recognition index,RI)用公式计算；Index＝(tn-tf/(tn+tf)，其中tf表示在同一上下文中探索先前遇到的熟悉对象的时间，tn表示在不同上下文中探索该对象的时间。增加探索中呈现的对象先前在相同上下文中遇到的对象的不同上下文被解释为增加了上下文记忆的形成。
4、实验结果
曾有研究证明TG小鼠表现出认知记忆降低的迹象。这些异常的行为变化与人类研究一致，常见于AD患者。在这项评估研究中，我们用NOR测试评估小鼠的认知记忆(图12和图13)，治疗3个月后，Wt小鼠花费更多时间探索新物体(ORI＝68.8±9.8％)。在TG小鼠中，ORI降低至49.8±4.7％。用DHMP处理的Wt小鼠(即DHMP组的Wt小鼠)表现出与Wt小鼠对照组相似的识别；结果表明，DHMP显著改善了TG小鼠的NOR(ORI＝68.6±5.8％)。
然后进行评估NCR(图12和图14)，计算了RI。将TG小鼠与Wt小鼠对照相比，TG小鼠表现出降低的RI(49.9±2.6％)；DHMP显著改善了TG小鼠的RI(RI＝66.6±5.8％)。DHMP治疗逆转了TG小鼠的RI，并显示出显著的情境记忆改善。结果表明，每日口服DHMP(1/4剂量的DHM)不仅可以复制DHM效应，而且DHMP可以在短时间内改善患有阿尔兹海默症TG小鼠的认知记忆，说明DHMP可以通过血脑屏障迅速到达大脑起到逆转桥蛋白的作用，进一步说明经过修饰的DHM(即DHMP)口服进入体内能够被迅速吸收进入血液循环，并快速进入大脑发挥药效。(注：佐证)
应用实施例4：二氢杨梅素四氢吡咯复合物(DHMP)对小鼠行为障碍和癫痫发作的改善作用
在本实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物对小鼠行为障碍和癫痫发作的改善作用，具体实验过程如下：
1、实验材料：如应用实施例1所用动物，Wt小鼠(C57BL/6,CharlesRiver Lab,USA)和TG-SwDI(Jacson Lab,USA)小鼠，均为雄性，20个月大的老年TG2576小鼠。
2、实验分组
将小鼠分为三组：(1)对照组：C57BL/6Wt雄性小鼠(2％蔗糖，口服)；(2)AD组：TG小鼠(2％蔗糖，口服)；(3)AD+DHMP组：TG小鼠给予DHMP(0.5mg/kg(1/4剂量的DHM)在2％蔗糖中)。
3、实验方法
(1)旷场运动试验
为了测量运动活性，测量每只动物的总进入次数；使用方差分析确定统计差异；通过这些实验确定DHMP对焦虑水平的影响。
(2)高架十字迷宫试验(EPM)
在安静、黑暗的房间中进行，只有低功率的红灯；将大鼠置于迷宫中心区域，记录其行为视频5分钟。在离线分析期间对进入张开臂、闭合臂或中心平台的次数以及在这些区域中的每一个中花费的时间进行评分。数据报告为各组条目数的百分比、在不同组中花费的时间百分比和总条目数。
4、实验结果
用旷场试验测定DHMP对老年TG2576小鼠的阿尔茨海默病(AD)症状样行为的影响。跑步距离是量化运动活动的参数之一(图15)。重量对照小鼠在10分钟内跑了922±75厘米。TG小鼠跑的距离要短得多(235±16cm)，而AD+DHMP组(用DHMP处理的TG小鼠)跑的距离增加到682±109cm。对照组Wt小鼠表现出频繁饲养(23.3±3.4次)，探索空场中心2.0±0.6次，并在中心停留0.14±0.1分钟。AD组TG-小鼠显示饲养12.1±2.1倍，探索开放场的中心(0.2±0.1倍)，并在中心停留仅0.02±0.02分钟。AD+DHMP组的TG小鼠的DHMP治疗增加了饲养(19.5±2.0倍)、探索中心的时间(1.4±1.4倍)和停留在中心的时间(0.13±0.05分钟)(详见图16-18所示)。结果表明，AD组的TG小鼠降低了探索/运动活动，AD+DHMP组小鼠的每日口服DHMP(1/4剂量的DHM)治疗可改善运动活动并增加探索活动，这是动物的一种本能。用EPM测定焦虑。与对照组Wt小鼠对照在开放臂中花费了总时间的48.5±7.5％，在闭合臂中花费了33.5±3.2％。与对照组Wt小鼠对照相比，患有AD的TG-小鼠在张开臂中花费的时间明显更短，在闭合臂中花费的时间更长(统计显著性与Wt小鼠对照；如图19所示)，而AD+DHMP组TG-小鼠花费了相似的时间在每个手臂。戊四唑(PTZ)是GABAA受体复合物的非竞争 性拮抗剂。使用PTZ(42mg/kg)诱导小鼠癫痫发作。与Wt小鼠对照的癫痫发作持续时间为1.4±0.6分钟，患有AD的TG-小鼠的癫痫发作持续时间显著增加(4.9±0.8分钟)，而AD+DHMP治疗的小鼠的癫痫发作持续时间显著减少(1.8±0.3分钟)(如图20所示)。这些结果表明患有AD的TG小鼠表现出冷漠样行为缺陷、焦虑和癫痫敏感性，每天进行DHMP治疗可以改善和预防这些症状。这些结果与行为DHMP行为涉及GABAARs以及GABAergic***有助于AD行为变化的概念是一致的。
应用实施例5：二氢杨梅素四氢吡咯复合物(DHMP)对小鼠中风引起的脑损伤的改善作用
在本实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物对小鼠中风引起的脑损伤的改善作用，具体实验过程如下：
1、实验材料：SPD级大鼠(Sprague Dawley，简称SD)Sprague Dawley(SD)大鼠(雄性，体重200±20g成年鼠，CharlesRiver Lab,USA)。
2、实验方法
(1)中风鼠模型
在第1天，我们通过左颈动脉注射硅胶，通过手术使左侧脑中风。将左侧脑中风大鼠分为两组，一组为实验组(中风+DHMP治疗)，即从第2天开始，我们开始每天灌胃一次DHMP 0.5mg/kg(1/4剂量的DHM)；另一组为对照组，手术后的大鼠接受生理盐水2mL/kg，进行灌胃治疗作为药物对照；在治疗天(D)10、20和30进行网格行走和露天行走。
(2)单侧颈动脉夹闭诱发小鼠中风模型
使用单侧颈动脉夹闭诱发中风动物模型来测试DHMP效应。我们夹住了左颈动脉，以手术方式使左侧脑卒中。10分钟后，快速取出大脑并准备好海马切片进行电生理记录；使用Axopatch 200B放大器(Molecular Devices)将海马神经元全细胞电压钳位在-70mV的保持电位。在电气补偿之前，接入电阻(<25M)。细胞内信号在3kHz进行低通滤波，并以20kHz的采样频率进行数字化。记录药理学上分离的微型兴奋性突触后电流(mEPSCs)。
3、实验结果
中风是导致肢体功能障碍的主要原因，但目前没有可用于促进肢体功能康复的药物疗法。最近的研究表明，大脑在中风后的修复能力有限。中风后的神经修复涉及重新映射邻近或与中风相关的组织中的认知功能。因此，在第一时间限制由兴奋性神经元释放额外谷 氨酸引起的神经毒性损伤很重要。图21-23显示了第10天测量的结果。在5分钟网格行走测试(图21)中，中风大鼠只能执行0.8±0.85分钟，并且经常从网格中掉下来。实验组(中风+DHMP治疗)的大鼠可以进行近3.6±0.5分钟，而对照组为3.8±0.3分钟。在露天试验(图22)中，对照组的中风大鼠只能跑距离～270厘米。而实验组中风+DHMP治疗的小鼠可以跑～700厘米。中风大鼠无法进入开阔场(图23)的中心。
为了了解中风诱发脑损伤的潜在机制，我们还使用单侧颈动脉夹闭诱发中风动物模型来测试DHMP效应。图24是对照组的脑片记录结果；图25是不同计量DHMP给药时在对照组的脑片获得的突触后微型兴奋性突触后电流(mEPSC)峰值比较，图26是DHMP剂量依赖性地显著降低中风鼠脑片记录mEPSC；图27是不同计量DHMP给药时在中风鼠脑片获得的mEPSC峰值比较。DHMP可以将mEPSC频率降低到与对照组相似的值(图28)；此外，DHMP降低了mEPSC的总电荷转移，使其相似于缺血组的对照水平(图29)。结果表明DHM可以抑制过量的谷氨酸释放，从而降低谷氨酸诱导的神经毒性。同时结果表明口服小剂量DHMP(DHM的1/4剂量)可以说明，DHMP可以通过血脑屏障迅速到达大脑，迅速抑制过度释放的谷氨酸来减少中风引起的脑损伤，从而限制中风引起的脑损伤，缩短中风病程，恢复脑功能以调节中风后的运动活动；证明经过修饰的DHM(即DHMP)口服进入体内能够被迅速吸收进入血液循环，并快速进入大脑发挥药效。
应用实施例6：二氢杨梅素四氢吡咯复合物(DHMP)对GABAAR的增强作用效果探究
在本实施例中考察制备实施例中制备的二氢杨梅素四氢吡咯复合物对GABAAR增强作用的实验探究，具体实验过程如下：
1、实验材料：人***(奥森教授细胞株库赠与)。
2、实验方法：在***上表达GABAA5，然后记录DHMP增强GABA与DHM进行对比。
3、实验结果：DHM可从0.3到300μM的剂量增强GABAA5，而DHMP对GABAA5的增强作用表现出1000倍的效力(剂量从0.003μM起)，如图30所示。
应用实施例7：二氢杨梅素四氢吡咯复合物(DHMP)的安全性实验探究
在本实施例中考察二氢杨梅素四氢吡咯复合物(DHMP)的安全性试验，具体实验过程如下：
1、实验材料：Sprague Dawley(SD)大鼠(雄性，体重200±20g；雌性，体重190±20g。n＝5/组/性别，CharlesRiver Lab,USA)。
Sprague Dawley成年大鼠，雄性，体重从175±5g开始，这项研究是在符合GLP的设施中进行的。
2、实验分组：
动物组：对照组接受5％蔗糖(SUC.2ml/100g体重)、DHMP 1mg/kg组、DHM P 10mg/kg组和DHMP 100mg/kg组。
3、实验方法：
(1)对上述动物分组进行为期两周的大鼠功能观察，记录FOB参数(体温、心跳、呼吸比)，并分别以1mg/kg、10mg/kg和100mg/kg的剂量每天口服DHMP。
(2)对上述动物分组进行为期6个月的长期评估研究中，并分别以1mg/kg、10mg/kg和500mg/kg的剂量每天口服DHMP，并记录FOB参数(体温、心跳、呼吸比)。
4、实验结果：
单剂SUC或DHMP后，在治疗后连续观察动物4小时，主要关注动物的心理行为、自主活动、毛发、腺体分泌、粪便、死亡等是否有任何负面变化。我们发现这些评估没有显著变化；在为期两周的大鼠功能观察(FOB)研究中，以1mg/kg、10mg/kg和100mg/kg的剂量每天口服DHMP，未发现任何显著的负面变化。
在两周内，我们检查了动物的皮毛、对处理的反应等；所有的老鼠都没有表现出毛皮损失或对处理的负面反应。在实验过程中没有观察到死亡或受伤，并且我们还确保在每次治疗后连续观察动物4小时。主要关注动物的心理行为、自主活动、毛发、粪便、腺体分泌、死亡等方面的任何负面变化。FOB参数，包括大鼠的体温、心跳和呼吸比，如表2所示。我们发现这些评估没有显著变化，结果表明DHMP是安全的化合物。
表2各组的FOB参数
上述表2中的T表示温度，单位是℃；P表示脉冲，单位是次/分钟；B表示呼吸，单位是次/分钟。
为了确定DHMP是否显示出毒性迹象，测试了慢性DHMP给药对大鼠的影响，以评 估代谢率的变化。测量了体重(图31、图32)、食物摄入量(图33、图34)、水摄入量(图35、图36)、粪便***量(图37、图38)和尿液***量(图39、图40)作为代谢率参数；并比较了所有组的代谢率。
在一项为期6个月的长期评估研究中，以1mg/kg、10mg/kg和500mg/kg的剂量每天口服DHMP，结果表明DHMP不会改变代谢率，即该结果表明DHMP对于日常口服应用是安全的(图11a-图11j所示)。
以上显示和描述了本发明的基本原理、主要特征及优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

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  1. 一种二氢杨梅素四氢吡咯复合物，其特征在于，所述的二氢杨梅素四氢吡咯复合物的分子式如式(I)所示：
  2. 一种权利要求1所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述的制备方法包括以下步骤：
    S1：将无水甲醇加入到二氢杨梅素中获得二氢杨梅素无水甲醇溶液；
    S2：在惰性气体保护下，将无水甲醇加入到四氢吡咯中，获得四氢吡咯无水甲醇溶液；
    S3：将所述步骤S1获得的二氢杨梅素无水甲醇溶液和所述步骤S2获得的四氢吡咯无水甲醇溶液室温下进行反应，获得反应物；
    S4：将所述步骤S3获得的反应物过滤，无水甲醇洗涤滤饼，并减压抽干即可得到二氢杨梅素四氢吡咯复合物。
  3. 根据权利要求2所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述步骤S2中的惰性气体为氩气。
  4. 根据权利要求2所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述步骤S1中的二氢杨梅素无水甲醇溶液的浓度为150-350mmol/L；所述步骤S2中的四氢吡咯无水甲醇溶液的浓度为1-10mol/L。
  5. 根据权利要求2所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述步骤S3中的二氢杨梅素和四氢吡咯的摩尔比为1:0.7-1.3。
  6. 根据权利要求5所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述步骤S3中的二氢杨梅素和四氢吡咯的摩尔比为1：1。
  7. 根据权利要求2所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，在所述步骤S1中，将无水甲醇加入到二氢杨梅素中后移到冰水浴中进行搅拌30分钟；在所述步骤S3中，将四氢吡咯无水甲醇溶液2滴/秒的速度加到在冰水浴中的二氢杨梅素无水甲醇溶液后，再升至室温条件下进行反应。
  8. 根据权利要求2所述的二氢杨梅素四氢吡咯复合物的制备方法，其特征在于，所述步骤S1中的二氢杨梅素制备方法为：
    S1-1：在2,4,6-三羟基苯乙酮中加入无水二氯甲和N,N-二异丙基乙基胺，再以1滴/秒的速度加入氯甲基甲基醚，反应完成后，得2-羟基-4,6-二甲氧基亚甲氧基苯乙酮；
    S1-2：在惰性气体保护下，将所述步骤S1-1得到的2-羟基-4,6-二甲氧基亚甲氧基苯乙酮与无水四氢呋喃混合；再先后加入氢化钠和氯甲基甲基醚；待反应完成后，得2,4,6-三甲氧基亚甲氧基苯乙酮；
    S1-3：在惰性气体保护下，将所述步骤S1-2得到的2,4,6-三甲氧基亚甲氧基苯乙酮与THF和水混合；再先后加入氢氧化钠和3,4,5-三羟基苯甲醛；待反应完成后，得(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮；
    S1-4：将所述步骤S1-3得到的(E)-3-三羟基苯-1-(2,4,6-三甲氧基亚甲氧基苯基)-乙烯基酮与30％的双氧水混合；反应完成后，加入饱和亚硫酸钠溶液淬灭反应，而后用乙酸乙酯萃取，减压旋去溶剂，再将去掉溶剂的残留物溶于甲醇中，并加入稀盐酸，待反应完成后，减压旋去溶剂，即可得二氢杨梅素四氢吡咯复合物。
  9. 一种如权利要求1所述的二氢杨梅素四氢吡咯复合物在神经***功能障碍疾病治疗药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述神经***功能障碍疾病为睡眠障碍、焦虑、抑郁、创伤后应激障碍、阿兹海默症、痴呆症、帕金森病、中风、癫痫、自闭症、酒精使用障碍。
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