阅读说明：本技术 酰基间苯三酚低聚体其制备方法和应用 (Acylphenol oligomer, preparation method and application thereof ) 是由 倪伟 严欢 秦徐杰 刘晖 孟庆红 刘海洋 于 2021-07-26 设计创作，主要内容包括：本发明属于植物化学和药物学技术领域,提供了酰基间苯三酚低聚体、含有酰基间苯三酚低聚体的药物组合物、酰基间苯三酚低聚体的制备方法及其应用。本发明酰基间苯三酚低聚体主要包括6种酰基间苯三酚三聚体和1种酰基间苯三酚二聚体偶联单萜,这类化合物对人源乙酰胆碱酯酶具有明显的抑制活性,能够用于治疗阿尔茨海默病及其并发症。(The invention belongs to the technical field of phytochemistry and pharmacology, and provides an acyl phloroglucinol oligomer, a pharmaceutical composition containing the acyl phloroglucinol oligomer, a preparation method of the acyl phloroglucinol oligomer and application of the acyl phloroglucinol oligomer. The acyl phloroglucinol oligomer mainly comprises 6 acyl phloroglucinol tripolymers and 1 acyl phloroglucinol dipolymer coupling monoterpene, and the compounds have obvious inhibitory activity on human acetylcholinesterase and can be used for treating Alzheimer disease and complications thereof.)

1. An acylphloroglucinol oligomer, characterized by having one of the following structural formulae:

acylphloroglucinol trimer myrtucommulone D (1a), gallistemonotrimer A (1b), gallistemonotrimer B (2a), gallistemonotrimer C (2b), gallistenone D (3a) and gallistemonotrimer D (3b) and acylphloroglucinol dimer coupling monoterpene gallistarone G (4),

2. a pharmaceutical composition comprising as an active pharmaceutical ingredient any one or more of the acylphloroglucinol oligomers of claim 1 or a pharmaceutically acceptable prodrug, derivative thereof, together with a pharmaceutically acceptable carrier, adjuvant or vehicle.

3. Use of the acylphloroglucinol oligomer of claim 1, or a pharmaceutically acceptable prodrug, derivative thereof, for the manufacture of a medicament, pharmaceutical composition or functional food for the treatment of Alzheimer's Disease (AD) and its complications.

4. An acetylcholinesterase (AChE) inhibitor, wherein said inhibitor is selected from the group consisting of the acylphloroglucinol oligomer or derivative of claim 1.

5. A process for preparing an acylphloroglucinol oligomer of claim 1, comprising the steps of:

1) optionally drying and pulverizing plant material of Callistemon (Callistemon R.Br.), extracting with organic solvent, and desolventizing to obtain extract of Callistemon;

2) and sequentially carrying out column chromatography, crystallization and recrystallization on the callistemon plant extract to obtain the acyl phloroglucinol oligomer.

6. The method according to claim 5, wherein the cajeput plant material is branches and leaves, fruits, seeds or mixtures thereof of cajeput plants, preferably cajeput plants are cajeput (C.Salignus DC.), cajeput (C.rigidus R.Br.) or cajeput (C.viminalis (Soland.) Chemicals).

7. The method according to claim 5, wherein in step 1) the volume ratio of organic solvent to the plant material of the genus cajeput is from 1:1 to 10:1, preferably 5: 1.

8. The preparation method according to claim 5, wherein in step 1), the organic solvent comprises at least one of petroleum ether, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, and formic acid; preferably, the organic solvent is acetone.

9. The method of claim 5, wherein in step 2), the column chromatography comprises normal phase silica gel column chromatography, reverse phase silica gel column chromatography, medium pressure chromatography separation gel, preparative or semi-preparative High Performance Liquid Chromatography (HPLC), chiral HPLC; preferably, the column chromatography comprises normal phase silica gel column chromatography, reverse phase RP-18 column chromatography, semi-preparative High Performance Liquid Chromatography (HPLC) and chiral HPLC.

10. The preparation method according to claim 5, wherein in the step 2), the eluent for column chromatography comprises at least one of petroleum ether, n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, isopropanol, acetonitrile, water and formic acid; preferably, the normal phase silica gel column chromatography is carried out by adopting petroleum ether and ethyl acetate according to the volume ratio of 100:1 to 1:1 for gradient elution; performing reversed-phase RP-18 column chromatography, and performing gradient elution by using methanol and water according to the volume ratio of 85:15 to 100: 0; eluting by adopting chloroform and methanol according to the volume ratio of 1:1 in gel column chromatography; performing gradient elution on medium-pressure chromatographic separation gel (MCI) by using methanol and water according to a volume ratio of 5:5 to 9: 1; preparative or semi-preparative HPLC is eluted by gradient from 88:12 to 100:0 by volume ratio of acetonitrile and water; chiral HPLC was gradient eluted with n-hexane and isopropanol from 99:1 to 92:8 by volume ratio.

11. The method according to claim 5, wherein the solvent system used for the crystallization and recrystallization in step 2) is at least one of methanol, ethanol, acetone, ethyl acetate, chloroform, and dichloromethane; preferably, methanol/acetone (3:1, v/v) is used.

Technical Field

The invention belongs to the technical field of phytochemistry and pharmacology, and particularly relates to an acyl phloroglucinol oligomer, a pharmaceutical composition containing the acyl phloroglucinol oligomer, a preparation method of the acyl phloroglucinol oligomer and application of the acyl phloroglucinol oligomer in preparation of the pharmaceutical composition for treating Alzheimer disease.

Background

Alzheimer's Disease (AD), known by the general public as "senile dementia", is one of the most common neurodegenerative diseases of the brain and is clinically characterized by hypomnesis, progressive and irreversible cognitive impairment, language deterioration, severe behavioral abnormalities and the like. A statistical study (JIAL, DUY, CHU L, et al, PREVANCE, risk factors, and management of both dementia and fine cognitive impact in additives 60 years or in The word a cross-sectional study [ J ]. The Lancet Public Health,2020,5(12): e-e 671.) published in Lancet-Public Health, 12.2020, shows about 1507 million patients among The elderly in China, and 983 million patients among The Alzheimer's disease patients. There are approximately 5000 million patients with Alzheimer's Disease worldwide, and the number of morbidities is increasing, doubling every 20 years, and is expected to reach 1.31 million by 2050 (Alzheimer's Disease International. world Alzheimer report 2015: the global impact of the division, https:// www.alz.co.uk/research/world Alzheimer report 2015).

The pathogenesis of alzheimer disease is complex, and there are many hypothesis mechanisms, and a great deal of clinical research mainly depends on different hypotheses to find a scheme for treating or relieving the disease. Currently, drugs for treating AD at home and abroad are mainly human acetylcholinesterase inhibitors (hAChEIs) capable of promoting and maintaining the function of residual cholinergic neurons, including donepezil (donepezil), galantamine (galanthamine), rivastigmine (rivastigmine), huperzine a (huperzine a), etc., and memantine hydrochloride (memantine hydrochloride), which is an N-methyl-D-aspartate receptor (NMDA) antagonist. Recently marketed drugs, such as adakanumab (adducanumab) produced by bohai key (Biogen) approved by FDA at month 6 of 2021, which depends on the amyloid beta hypothesis, and low-molecular acid oligosaccharide compound GV-971 (ninth phase one) approved by NMPA at month 11 of 2019, remodel the intestinal flora balance by targeting the brain-intestinal axis, inhibit abnormal increase of specific metabolites of the intestinal flora, reduce peripheral and central inflammation, reduce beta amyloid deposition and Tau protein hyperphosphorylation. However, the current medicines can only delay the progress of the disease and can not cure the disease, and the curative effect is controversial. Therefore, the search for new and highly effective drugs for the prevention and treatment of AD remains a problem to be solved.

In recent years, researches on prevention and treatment of AD in traditional Chinese medicine have attracted wide attention, and are expected to bring new hopes for patients. Therefore, finding acetylcholinesterase (AChE) inhibitors from medicinal plants (or traditional Chinese medicines) is one of the important ways to develop anti-AD drugs.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel natural product, namely an acylphloroglucinol oligomer, which is a highly efficient novel acetylcholinesterase inhibitor, and provides a reasonable and effective novel drug molecule and treatment scheme for developing and developing drugs for treating alzheimer disease and other neurodegenerative diseases.

The purpose of the invention is realized by the following technical scheme.

In a first aspect, the present invention provides an acylphloroglucinol oligomer having one of the following structural formulae:

acylphloroglucinol trimer myrtucommulone D (1a), gallistemonotrimer A (1B), gallistemonotrimer B (2a), gallistemonotrimer C (2B), gallistenone D (3a) and gallistemonotrimer D (3B) and acylphloroglucinol dimer coupling monoterpenes gallistrinone G (4),

in a second aspect, the present invention provides a pharmaceutical composition comprising an acylphloroglucinol oligomer as shown above or a pharmaceutically acceptable prodrug, derivative thereof as an active pharmaceutical ingredient, together with a pharmaceutically acceptable carrier, adjuvant or vehicle.

In a third aspect, the present invention relates to the use of the above-mentioned acylphloroglucinol oligomers or their pharmaceutically acceptable prodrugs, derivatives for the preparation of a medicament or a pharmaceutical composition or a functional food for the treatment of alzheimer's disease and its complications.

In a fourth aspect, the present invention provides an acetylcholinesterase (AChE) inhibitor which is an acylphloroglucinol oligomer or derivative thereof as described above.

In a fifth aspect, the present invention provides a process for the preparation of an acylphloroglucinol oligomer as described above, comprising the steps of:

1) optionally drying and pulverizing plant material of Callistemon (Callistemon R.Br.), extracting with organic solvent, and desolventizing to obtain extract of Callistemon;

2) and sequentially carrying out column chromatography, crystallization and recrystallization on the callistemon extract to obtain the acyl phloroglucinol oligomer.

In a preferred embodiment, the organic solvent is at least one of petroleum ether, n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, isopropanol, acetonitrile, water, and formic acid.

Acetylcholine helps to transfer information between nerve cells and to maintain memory, and acetylcholinesterase (AChE) is a specific cholinesterase that hydrolyzes acetylcholine. The human acetylcholinesterase inhibitor (hAChEIs) can effectively reduce and prevent the decomposition of acetylcholinesterase on acetylcholine, and is one of effective ways for developing the medicines for treating neurodegenerative diseases. The hAChE inhibitory activity test performed in vitro shows that the acyl phloroglucinol oligomer has better inhibitory activity to hAChE and IC of the acyl phloroglucinol oligomer₅₀The value is between 1.22 and 16.76. mu.M, and can be used for treating Alzheimer's disease and complications thereof, so that the acylphloroglucinol oligomers of the present invention provide a reliable option for treating Alzheimer's disease and complications thereof.

Drawings

FIG. 1 is a schematic structural diagram of an acylphloroglucinol oligomer according to the present invention.

FIG. 2 is a crystal structure diagram of a myrtucommulone D (1 a)/callystemontrimer A (1b) racemate which is an acylphloroglucinol oligomer according to the present invention.

FIG. 3 shows the molecular docking results of the acylphloroglucinol oligomers myrtucommulone D (1a), gallistemonotrimer A (1B), gallistemonotrimer B (2a), gallistemonotrimer C (2B), gallistenone D (3a), gallistemonotrimer D (3B) and gallisterone G (4) of the present invention with hAChE (PDB ID 4EY 6).

Detailed Description

In order that the invention may be more fully understood, a preferred embodiment of the invention will now be described. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

According to a first aspect of the present invention, there is provided an acylphloroglucinol oligomer comprising acylphloroglucinol trimer myrtucommulone D (1a), gallistemontrimer A (1B), gallistemontrimer B (2a), gallistemontrimer C (2B), gallistetenone D (3a) and gallistemontrimer D (3B) and acylphloroglucinol dimer coupling monoterpene gallistarone G (4) having the formula:

according to a second aspect of the present invention, there is provided a pharmaceutical composition comprising an acylphloroglucinol oligomer or a pharmaceutically acceptable prodrug, derivative thereof as an active pharmaceutical ingredient, together with pharmaceutically acceptable carriers, adjuvants and excipients.

In an embodiment of the present invention, the acylphloroglucinol oligomers of the present invention may be present in an optically pure form or in the form of a racemate.

It will be appreciated by those skilled in the art that pharmaceutically acceptable derivatives of the acylphloroglucinol oligomers of the present invention, such as salts, esters, solvates or hydrates of the acylphloroglucinol oligomers, may also be used in the pharmaceutical compositions of the present invention.

Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutically acceptable carriers or excipients are one or more of solid, semi-solid, and liquid diluents, fillers, and pharmaceutical adjuvants, including but not limited to fillers (diluents), lubricants (glidants or anti-adherents), dispersants, wetting agents, binders, solubilizers, antioxidants, bacteriostats, emulsifiers, disintegrants, and the like. The binder comprises syrup, acacia, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethylcellulose, ethyl cellulose or hydroxypropyl methylcellulose), gelatin slurry, syrup, starch slurry or polyvinylpyrrolidone; the filler comprises lactose, sugar powder, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricant comprises superfine silica gel powder, magnesium stearate, talcum powder, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol and the like; the disintegrating agent comprises starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium lauryl sulfate, water or alcohols, and the like; the antioxidant comprises sodium sulfite, sodium bisulfite, sodium pyrosulfite, dibutylbenzoic acid, etc.; the bacteriostatic agent comprises 0.5% of phenol, 0.3% of cresol, 0.5% of chlorobutanol and the like; the emulsifier comprises polysorbate-80, sorbitan fatty acid, lecithin, soybean lecithin, etc.; the solubilizer comprises Tween-80, bile, glycerol, etc.

When the acylphloroglucinol oligomer or the pharmaceutically acceptable prodrug or derivative thereof of the present invention is used as a medicament, it may be administered directly or in the form of a pharmaceutical composition. In the pharmaceutical composition of the present invention, the pharmaceutical composition may contain 0.1 to 99%, preferably 0.5 to 90%, of the acylphloroglucinol oligomer or its derivative, based on the total weight of the pharmaceutical composition.

A therapeutically effective amount of an acylphloroglucinol oligomer, or a pharmaceutically acceptable prodrug, derivative thereof, refers to an administration level or amount that is directed to, without causing significant negative or adverse side effects to the recipient: (1) delaying or preventing the onset of a targeted pathological condition or disorder; (2) slowing or stopping the progression, exacerbation, or worsening of one or more symptoms of the targeted pathological condition or disorder; (3) alleviating symptoms of the targeted pathological state or disorder; (4) improving the severity or incidence of the targeted pathological condition or disorder; or (5) cure targeted pathological conditions or disorders. A therapeutically effective amount may be administered prior to the onset of the targeted pathological condition or disorder for prophylactic or defensive action. Alternatively or additionally, a therapeutically effective amount of the acylphloroglucinol oligomer may be administered after the onset of the targeted pathological condition or disorder for therapeutic effect. It will be appreciated that the effective dose will depend on the age, sex, health and weight of the recipient. Generally, an effective amount is determined by the person administering the treatment, e.g., the treating physician.

The pharmaceutical composition of the present invention may be administered in the form of a dose per unit body weight. All the pharmaceutical compositions taking the acylphloroglucinol oligomer or the pharmaceutically acceptable prodrug and derivative thereof as the active ingredient can be prepared into various dosage forms by adopting a method generally recognized in the pharmaceutical and food fields, such as liquid preparations (injection, suspension, emulsion, solution, syrup and the like), solid preparations (tablets, capsules, granules, medicinal granules and the like), sprays, aerosols and the like. The pharmaceutical composition can be administered by injection (intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection), oral administration, sublingual administration, mucosal dialysis and the like, so as to treat Alzheimer's disease and complications thereof.

According to a third aspect of the present invention, there is provided the use of an acylphloroglucinol oligomer or a pharmaceutically acceptable prodrug, derivative thereof for the manufacture of a medicament or a pharmaceutical composition or a functional food for the treatment of alzheimer's disease and its complications.

According to a fourth aspect of the present invention, there is provided a novel class of acetylcholinesterase inhibitors (AChEI) which are the above-mentioned acylphloroglucinol oligomers or their derivatives, and which are effective in alleviating and preventing the decomposition of acetylcholine by acetylcholinesterase, thereby promoting and maintaining the function of the remaining cholinergic neurons, thereby achieving the purpose of treating alzheimer's disease and its complications.

According to a fifth aspect of the present invention, there is provided a process for preparing an acylphloroglucinol oligomer, comprising the steps of:

1) optionally drying and pulverizing plant material of Callistemon (Callistemon R.Br.), extracting with organic solvent, and desolventizing to obtain extract of Callistemon;

2) and sequentially carrying out column chromatography, crystallization and recrystallization on the callistemon extract to obtain the acyl phloroglucinol oligomer.

In an embodiment of the invention, the plant of the melaleuca alternifolia is a plant of the Myrtaceae (Myrtaceae), melaleuca (calilstemon r.br.), including, but not limited to, melaleuca serrulata (c.viminalis (Soland.) Cheel.), melaleuca serrulata (c.salignus), melaleuca rubra (c.rigidus), melaleuca americana (c.citrinus), melaleuca rubra (c.phoenicis), melaleuca rubra (c.polandii), melaleuca multiflora (c.spicifolius), melaleuca lineare (c.linearifolius), melaleuca longus (c.pearsonii), melaleuca purpurea (c.cv.pure patchoulus), melaleuca pinifolia japonica (c.pinifolia), and the like. In a preferred embodiment of the invention, the plants of cajeput are cajeput willow, cajeput or cajeput pendula. In a further preferred embodiment, the plants of the cajeput species are cajeput willow leaves.

In a particular embodiment of the invention, the cajeput material may be branches and leaves, fruits, seeds or mixtures thereof of the cajeput plant. For fresh red globe plant material or those having a high moisture content, e.g., greater than 15%, the material may be dried and then pulverized or ground to facilitate extraction. Drying, pulverizing or grinding can be carried out by means of a conventional technique in the art, such as natural sun drying, oven heat drying, pulverizer pulverizing, and mill pulverizing. In a further preferred embodiment, the cajeput plant material is seed of cajeput willow. In a preferred embodiment, the branches, leaves, seeds of the plants of the cajeput species are dried.

In an embodiment of the present invention, the organic solvent used in step 1) may be at least one of petroleum ether, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, and formic acid; preferably, the organic solvent is acetone.

In an embodiment of the invention, the extraction may be performed at least 1 time, such as 2, 3, 4, 5 times, preferably 3 times, with an organic solvent and the extracts combined, each extraction may be performed for about 60-84 hours, such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 39, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 hours, preferably 70-74 hours, further preferably 72 hours.

In embodiments of the invention, the volume ratio of organic solvent to melaleuca plant material may be from 1:1 to 10:1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and ratios between any two of the foregoing, such as 1.5:1, 2.5:1, 3.5:1, 4.5:1, 5.5:1, and the like, preferably 5: 1.

In a particular embodiment of the invention, step 2) comprises in particular:

in step 2), the column chromatography comprises normal phase silica gel column chromatography, reverse phase silica gel column chromatography, medium pressure chromatography separation of gel, preparative or semi-preparative High Performance Liquid Chromatography (HPLC), chiral HPLC; preferably, the column chromatography comprises normal phase silica gel column chromatography, reverse phase RP-18 column chromatography, semi-preparative High Performance Liquid Chromatography (HPLC) and chiral HPLC.

In step 2), the eluent for column chromatography comprises at least one of petroleum ether, n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, isopropanol, acetonitrile, water and formic acid, such as a combination of two or more, such as a combination of any two of the above organic solvents, a combination of any three, a combination of any four, and the like; preferably, the normal phase silica gel column chromatography is carried out by adopting petroleum ether and ethyl acetate according to the volume ratio of 100:1 to 1:1 for gradient elution; performing reversed-phase RP-18 column chromatography, and performing gradient elution by using methanol and water according to the volume ratio of 85:15 to 100: 0; eluting by adopting chloroform and methanol according to the volume ratio of 1:1 in gel column chromatography; performing gradient elution on medium-pressure chromatographic separation gel (MCI) by using methanol and water according to a volume ratio of 5:5 to 9: 1; preparative or semi-preparative HPLC is eluted by gradient from 88:12 to 100:0 by volume ratio of acetonitrile and water; chiral HPLC was gradient eluted with n-hexane and isopropanol from 99:1 to 92:8 by volume ratio.

The solvent system used for crystallization and recrystallization in step 2) is at least one of methanol, ethanol, acetone, ethyl acetate, chloroform and dichloromethane, such as a combination of two or more, such as any one of the above organic solvents, or a combination of any two, a combination of any three, a combination of any four, and the like; preferably, methanol/acetone (3:1, v/v) is used. Crystallization is a conventional technique in the art, such as a slow solvent evaporation method, in which a sample is heated to dissolve, and then placed in a refrigerator at 4 ℃ to slowly evaporate the solvent to form crystals. In a specific embodiment, the specific temperature of heating is not particularly limited as long as the sample can be dissolved.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and substitutions may be made by those skilled in the art without departing from the spirit and scope of the invention, and all such modifications and substitutions are intended to be within the scope of the claims.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, the optical rotation was measured on a JASCO P-1020 polarimeter; high resolution mass spectrometry (HRESIMS) was determined by an Agilent 1290 UPLC/6540Q-TOF mass spectrometer;¹H,¹³c NMR and 2D NMR spectra were measured on a Bruker Avance III-600 NMR spectrometer using deuterated chloroform as solvent; ECD was measured on an Applied Photophysics instrument; compound single crystal data were determined on Bruker D8 Quest; normal phase silica gel (200-mesh and 300-mesh) for column chromatography and a thin layer chromatography plate are all products of Qingdao ocean chemical industry factories; the sephadex Saphadex LH-20 is a product of GE Healthcare; the reverse phase material RP-18 and RP-18 thin layer plates are products of Germany Merck company; semi-preparative HPLC was performed on Agilent 1260 or Agilent 1100 chromatographs, respectively, using Angilent ZORBAX SB-C18 (9.4X 250mm,5.0 μm) or Daicel CHIRALPAK IC (10X 250mm,5.0 μm) columns, respectively.

Example 1: preparation method of acylphloroglucinol oligomer

This example provides the preparation of seven compounds, Myrtucommulone D (1a), gallistemonotrimer A (1B), gallistemonotrimer B (2a), gallistemonotrimer C (2B), gallistenone D (3a), gallistemonotrimer D (3B), acylphloroglucinol dimer coupled monoterpene gallistrilone G (4).

(1) Drying and crushing seeds of the melaleuca alternifolia to obtain plant materials;

(2) extracting the plant materials at room temperature with acetone as organic solvent at a volume ratio of 5:1 for 3 times (72 hr each time), mixing extractive solutions, filtering, and concentrating the filtrate under reduced pressure to obtain extract of pasty seed of Melaleuca alternifolia;

(3) performing normal-phase silica gel column chromatography on the plant extract (the mass ratio of the silica gel amount to the extract is 10:1), and performing gradient elution by using petroleum ether/ethyl acetate as an eluent (each gradient has 3-5 column volumes) to obtain six parts: fr.1 (petroleum ether: ethyl acetate: 100:1, v/v), fr.2 (petroleum ether: ethyl acetate: 60:1, v/v), fr.3 (petroleum ether: ethyl acetate: 20:1, v/v), fr.4 (petroleum ether: ethyl acetate: 10:1, v/v), fr.5 (petroleum ether: ethyl acetate: 5:1, v/v); fr.6 (petroleum ether: ethyl acetate ═ 1:1, v/v);

(4) and (3) carrying out reverse phase silica gel column chromatography (Rp-18) on the collected Fr.5 part, carrying out gradient elution (each gradient is 3-5 column volumes) by using methanol/water as an eluent, and collecting eight parts in a segmented manner: fr.5-1 (methanol: water: 85:15, v/v), fr.5-2 (methanol: water: 87:13, v/v), fr.5-3 (methanol: water: 89:11, v/v), fr.5-4 (methanol: water: 91:9, v/v) and fr.5-5 (methanol: water: 93:7, v/v); fr.5-6 (methanol: water ═ 95:5, v/v); fr.5-7 (methanol: water: 97:3, v/v); fr.5-8 (methanol: water ═ 100:0, v/v);

(5) fr.5-3 collected in the above step was subjected to semi-preparative HPLC (column: angiolent ZORBAX SB-C18; mobile phase: acetonitrile-water 88:12 → 100: 0; flow rate: 6.0mL/min) to give a mixture/racemate of 2a/2b and 3a/3b and acylphloroglucinol dimer-coupled monoterpene G (4); heating Fr.5-4 part with methanol-acetone (3:1, v/v) to dissolve completely, cooling, standing in refrigerator at 4 deg.C for crystallization, filtering, washing after crystal (light yellow crystal) is formed, and repeating the above steps for recrystallization to obtain 1a/1b mixture/racemate.

(6) The three pairs of mixtures/racemates 1a/1B, 2a/2B and 3a/3B were resolved by chiral HPLC (column: Daicel CHIRALPAK IC; mobile phase: n-hexane-isopropanol, 99:1 → 92: 8; flow rate: 2.5mL/min) to give optically pure Myrtucomm Mulone D (1a), gallistemontrimer A (1B), gallistemontrimer B (2a), gallistemontrimer C (2B), gallistetenone D (3a), gallistemontrimer D (3B) six acylphloroglucinol trimers, respectively.

Physical constants and spectral data for Myrtucommulone D (1a) and gallistemontrimer A (1 b): pale yellow crystals (methanol-acetone, 3:1, v/v); UV (CH)₃OH)λ_max(log ε)205(4.58),223(4.48),273(4.40),297(4.46),354(3.64) nm; specific optical rotation of 1aSpecific optical rotation of 1b 1a, ECD (MeOH,. DELTA.. di-chromatic) 213(-9.44),233(+1.96),261(-9.84),298(+6.59),315(+3.57),333(+4.79) nm; 1b Electron circular dichroism ECD (MeOH,. DELTA.. di-elect cons.) 213(+9.49),233(-1.97),261(+9.88),298(-6.62),315(-3.59),333(-4.81) nm; IR (KBr) v_max3445,2970,1720,1660cm^-1；¹³C(CDCl₃150MHz) and¹H(CDCl₃600MHz) NMR data are shown in Table 1; (+) -HRESIMS M/z651.3540[ M + H]⁺(calculation value C)₃₈H₅₁O₉,651.3533)。

Physical constants and spectral data for Callistemontrmers B (2a) and C (2 b): a light yellow gum; UV (CH)₃OH)λ_max(log ε)206(4.61),231(4.43),274(4.38),301(4.51),360(3.59) nm; specific optical rotation of 2aSpecific optical rotation of 2b2a electron circular dichroism ECD (MeOH,. DELTA.. epsilon.) 262(-13.33),291(+7.58) nm; 2b electron circular dichroism ECD (MeOH,. DELTA.. epsilon.) 262(+13.32),291(-7.58) nm; IR (KBr) v_max3448,2969,1718,1659cm^-1；¹³C(CDCl₃150MHz) and¹H(CDCl₃600MHz) NMR data are shown in Table 1; (+) -HRESIMS M/z651.3533[ M + H]⁺(calculation value C)₃₈H₅₁O₉，651.3528)。

Physical constants and spectral data for Callistenone D (3a) and callastomer D (3 b): a light yellow gum; UV (CH)₃OH)λ_max(log ε)206(4.61),231(4.43),274(4.38),301(4.51),360(3.59) nm; specific optical rotation of 3a 3b specific optical rotation3a Electron circular dichroism ECD (MeOH,. DELTA.. epsilon.) 257(-13.33),290(+7.58) nm; electron circular dichroism ECD (MeOH,. DELTA.. epsilon.) 257(+13.32) of 3b, 290(-7.58) nm; IR (KBr) v_max3448,2969,1718,1659cm^-1；¹³C(CDCl₃150MHz) and¹H(CDCl₃600MHz) NMR data are shown in Table 1; (+) -HRESIMSm/z 651.3533[ M + H]⁺(calculation value C)₃₈H₅₁O₉,651.3528)。

Physical constants and spectral data for Callistrione G (4): a colorless oil; UV (CH)₃OH)λ_max(log ε)206(4.39),238(4.16),280(4.38),356(3.49) nm; 4 specific optical rotationIR(KBr)ν_max3432,2971,2864,1715,1620,1602,1473,1415,1381,1134cm^-1；¹³C(CDCl₃150MHz) and¹H(CDCl₃600MHz) NMR data are shown in Table 1; (+) -HRESIMSm/z581.3478[ M + H]⁺(calculation value C)₃₅H₄₉O₇，581.3473)。

TABLE 1 of Callistemontrimer sA-D and gallilone G¹³C (150MHz) and¹h (600MHz) NMR Nuclear magnetic resonance Spectroscopy data (CDCl)₃)。

Example 2: preparation of acylphloroglucinol oligomers

This example provides the preparation of seven compounds, Myrtucommulone D (1a), gallistemonotrimer A (1B), gallistemonotrimer B (2a), gallistemonotrimer C (2B), gallistenone D (3a), gallistemonotrimer D (3B), acylphloroglucinol dimer coupled monoterpene gallistrilone G (4).

(1) Drying and crushing branches and leaves of the willow leaf and the red globe amaranth to obtain plant materials;

(2) extracting the plant material with petroleum ether/ethyl acetate (1:1, v/v) at room temperature as organic solvent at a volume ratio of 5:1 for 3 times (60 hr each time), mixing extractive solutions, filtering, and concentrating the filtrate under reduced pressure to obtain extract of branch and leaf of cajeput willow leaf;

(3) performing normal-phase silica gel column chromatography on the plant extract (the mass ratio of the silica gel amount to the extract is 5:1), and performing gradient elution by using petroleum ether/acetone as an eluent (each gradient has 3-5 column volumes) to obtain six parts: fr.1 (petroleum ether: acetone ═ 0:1, v/v), fr.2 (petroleum ether: acetone ═ 100:1, v/v), fr.3 (petroleum ether: acetone ═ 50:1, v/v), fr.4 (petroleum ether: acetone ═ 25:1, v/v); fr.5 (petroleum ether: acetone: 10:1, v/v), fr.6 (petroleum ether: acetone: 5:1, v/v);

(4) and (3) performing gradient elution (each gradient is 3-5 column volumes) on the collected Fr.5 part by adopting medium-pressure chromatographic separation gel MCI and using methanol/water as an eluent, and collecting three parts in a sectional manner: fr.5-1 (methanol: water ═ 5:5, v/v), fr.5-2 (methanol: water ═ 7:3, v/v), fr.5-3 (methanol: water ═ 9:1, v/v);

(5) performing reverse phase silica gel column chromatography (Rp-18) on the Fr.5-2 collected in the step, performing gradient elution (each gradient has 3-5 column volumes) by using acetonitrile/water as an eluent, and collecting five parts by sections: fr.5-2-1 (acetonitrile: water ═ 6:4, v/v), fr.5-2-2 (acetonitrile: water ═ 7:3, v/v), fr.5-2-3 (acetonitrile: water ═ 8:2, v/v), fr.5-2-4 (acetonitrile: water ═ 9:1, v/v) and fr.5-2-5 (acetonitrile: water ═ 1:0, v/v);

(6) performing semi-preparative High Performance Liquid Chromatography (HPLC) on the Fr.5-2-3 collected in the step, taking acetonitrile/water as a mobile phase, and performing gradient elution (the concentration of the acetonitrile is 70-100%, v/v) to obtain a mixture/racemate of 1a/1b, 2a/2b and 3a/3b and an acylphloroglucinol dimer coupling monoterpene G (4);

(7) the three pairs of mixtures/racemates 1a/1B, 2a/2B and 3a/3B were resolved by chiral HPLC (column: Daicel CHIRALPAK IC; mobile phase: n-hexane-isopropanol, 99:1 → 92: 8; flow rate: 2.5mL/min) to give optically pure Myrtucomm Mulone D (1a), gallistemontrimer A (1B), gallistemontrimer B (2a), gallistemontrimer C (2B), gallistetenone D (3a), gallistemontrimer D (3B) six acylphloroglucinol trimers, respectively.

Example 3: preparation method of acylphloroglucinol oligomer

This example provides the preparation of seven compounds, Myrtucommulone D (1a), gallistemonotrimer A (1B), gallistemonotrimer B (2a), gallistemonotrimer C (2B), gallistenone D (3a), gallistemonotrimer D (3B), acylphloroglucinol dimer coupled monoterpene gallistrilone G (4).

(1) Drying and crushing the mixture of branches and leaves of the callistemon pendula and fruits to obtain plant materials;

(2) extracting the plant materials with methanol at room temperature at a volume ratio of 10:1 at a ratio of organic solvent to plant materials of 10:1 for 3 times (24 hr each time), mixing extractive solutions, filtering, and concentrating the filtrate under reduced pressure to obtain extract of pasty seed of Melaleuca alternifolia;

(3) performing normal-phase silica gel column chromatography on the plant extract (the mass ratio of the silica gel amount to the extract is 5:1), and performing gradient elution by using chloroform/methanol as an eluent (each gradient has 3-5 column volumes) to obtain five parts: fr.1 (chloroform: methanol: 0:1, v/v), fr.2 (chloroform: methanol: 200:1, v/v), fr.3 (chloroform: methanol: 100:1, v/v), fr.4 (chloroform: methanol: 50:1, v/v); fr.5 (chloroform: methanol-25: 1, v/v);

(4) and (3) performing gradient elution (each gradient is 3-5 column volumes) on the collected Fr.4 part by adopting medium-pressure chromatographic separation gel MCI and using methanol/water as an eluent, and collecting four parts in sections: fr.4-1 (methanol: water ═ 4:6, v/v), fr.4-2 (methanol: water ═ 6:4, v/v), fr.4-3 (methanol: water ═ 8:2, v/v) and fr.4-4 (methanol: water ═ 9:1, v/v);

(5) the Fr.4-3 fraction collected in the above step was purified by gel column chromatography (Sephadex LH-20) with chloroform: methanol (1:1, v/v) is used as eluent, and four parts are obtained by sectional collection: fr.4-3-1, Fr.4-3-2, Fr.4-3-3 and Fr.4-3-4;

(6) performing semi-preparative High Performance Liquid Chromatography (HPLC) on the Fr.4-3-2 collected in the step, taking acetonitrile/water as a mobile phase, and performing gradient elution (the concentration of acetonitrile is 70-100%, v/v) for 0-30 min to obtain the acyl phloroglucinol dimer coupling monoterpene G (4);

(7) and heating the Fr.4-3-4 part collected in the step by using methanol-acetone (3:1, v/v) to fully dissolve, cooling, placing in a refrigerator at 4 ℃ for crystallization, filtering and washing after crystals (light yellow crystals) are formed, and repeating the step for recrystallization to obtain the mixture/racemate of 1a/1 b. The mother liquor remaining after crystallization was filtered, and a mixture/racemate of 2a/2b and 3a/3b was obtained by semi-preparative HPLC (column: angiont ZORBAX SB-C18; mobile phase: acetonitrile-water 88:12 → 100: 0; flow rate: 6.0 mL/min). The three pairs of mixtures/racemates 1a/1B, 2a/2B and 3a/3B were resolved by chiral HPLC (column: Daicel CHIRALPAK IC; mobile phase: n-hexane-isopropanol, 99:1 → 92: 8; flow rate: 2.5mL/min) to give optically pure Myrtucomm Mulone D (1a), gallistemontrimer A (1B), gallistemontrimer B (2a), gallistemontrimer C (2B), gallistetenone D (3a), gallistemontrimer D (3B) six acylphloroglucinol trimers, respectively.

Example 4: inhibitory Activity of Acylphloroglucinol oligomers on hAChE

In this example, the inhibitory activity of 7 compounds (1a, 1b, 2a, 2b, 3a, 3b, 4) on Human acetylcholinesterase (hAChE) was verified.

Acetylcholine is an important neurotransmitter and, when its content is reduced, it causes neurodegenerative diseases such as senile dementia. The human acetylcholinesterase is specific cholinesterase for hydrolyzing acetylcholine, so the screening of the high-efficiency hAChE inhibitor is one of effective ways for developing the medicine for treating neurodegenerative diseases.

1. Principle of experiment

hAChE can catalyze the degradation of substrate analogue of thioacetylcholine iodide to generate thiocholine and acetic acid, the reaction product reacts with color-developing agent DTNB to generate a yellow substance, and specific light absorption is carried out at 405 nm. If the compound has inhibition effect on hAChE, the amount of the hAChE for catalyzing the degradation of the iodinated thioacetylcholine is reduced, and the yellow compound generated by the reaction with DTNB is correspondingly reduced, namely the light absorption value at 405nm is reduced, so that the compound with the inhibition activity can be screened.

2. Reagent instrument

Reagent:

7 compounds prepared in the examples; na (Na)₂HPO₄；NaH₂PO₄(ii) a Human acetylcholinesterase (hAChE), thioacetylcholine iodide, DTNB, galantamine.

The instrument comprises the following steps: MultiskanFC, Thermo corporation.

3. Experimental methods

3.1. Using phosphate buffer (0.1 MNa per 100mL phosphate buffer)₂HPO₄94.7mL of solution; 0.1M NaH₂PO₄5.3mL of solution, pH adjusted to 8.0) dilute hAChE to 0.1U/mL working solution;

3.2. preparing 6.25mM solution (working solution) of thioacetyl choline iodide and DTNB by using phosphate buffer solution;

3.3. test compounds were diluted with DMSO to form a concentration gradient. The positive control is galanthamine, and is diluted into a concentration gradient by DMSO; the negative control group (NC group) was a 2% DMSO solvent control.

3.4. The reaction was performed in a 96 well plate, plating was performed in 200. mu.L/system, and 3 replicates were performed for each sample;

(a) setting of test compound and positive control concentration gradients (diluted with 1% DMSO):

(b) plate paving: 200 μ L/system, final DMSO concentration in each well was 0.1%, and 3 duplicate wells were made for each sample

5. The absorbance at 405nm was measured every 30 seconds for 1 hour after addition of the developer and substrate.

6. The absorbance of the sample at about 1 for the average absorbance of the NC group was selected, the average absorbance of the test compound was calculated (test compound measurement-background), and the hAChE inhibition of the test compound was calculated as (NC-test compound absorbance average)/NC × 100%.

The results show that all tested compounds have significant inhibitory effect on hAChE (Table 2), IC₅₀The value is between 1.22 and 16.76. mu.M. Further molecular docking experiments show that all test compounds can enter the elongated hydrophobic aromatic pocket of hAChE well and bind to amino acid residues through hydrogen bonding, pi-pi interactions and pi-sigma interactions.

TABLE 2.7 test Compounds (1a, 1b, 2a, 2b, 3a, 3b, 4) for human acetylcholinesterase inhibitory Activity