Pyrrolizidine phthalide isoquinoline alkaloids and preparation and use thereof

Abstract

The present application relates to a kind of tetrahydropyrrolizino [2, 3-c] quinoline alkaloid compound and derivative with novel structure and preparation method and purposes thereof.The new compound bungeanoline G provided in the present application is extracted and separated from Papaveraceae Corydalis bungeana Turcz.The present application also proposes a method for artificially synthesizing bungeanoline G, and a series of derivatives of bungeanoline G are prepared.Biological activity experiments show that the compounds have acetylcholinesterase and butyrylcholinesterase inhibitory activity, and can be applied in the preparation of drugs for treating or improving various neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel tetrahydropyrrolizino[2,3-c]quinoline alkaloid compound and a preparation method thereof, and a use as an acetylcholinesterase and / or butyrylcholinesterase inhibitor for treating or improving diseases associated with neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis and the like. BACKGROUND

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease that seriously affects the health of the elderly and has become a global public health problem. According to the World Health Organization (WHO), there are currently more than 55 million people worldwide with dementia, and AD is the most common form of dementia, accounting for 60-70% of all cases. Natural alkaloids are an important source of anti-AD drug research and development. Among the four anti-AD drugs approved by the US FDA, the skeletal structures of two of them, rivastigmine and galantamine, are derived from natural alkaloids. In recent years, the discovery of new anti-AD drugs from cholinesterase inhibitors has become a hot spot in AD drug research and development. Recent studies have shown that dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) have better efficacy and fewer side effects than AChE inhibitors alone in the treatment of AD. One of the important reasons is that in the brains of patients with advanced AD, BChE plays a crucial role in regulating acetylcholine levels. The current development of anti-AD drugs relies too much on the structure of known bioactive compounds, and although some target compounds have been discovered in the development of anti-AD lead compounds, they are often simple structural modifications of existing cholinesterase inhibitors. Therefore, the discovery of novel dual cholinesterase inhibitors with unique carrier scaffolds from natural alkaloids has important research significance and value for the treatment of AD.

[0003] Corydalis bungeana Turcz. is the dried whole herb with roots of Corydalis bungeana Turcz. of the family Papaveraceae, also known as Diding, Kuding, and Xiaojicai. It has a bitter taste and a cold nature, and has the effects of clearing heat and resolving toxicity, resolving and detumescence, and is mainly used for treating epidemic febrile disease, sore throat and swollen pain. Modern studies have shown that C. bungeana has the effects of inhibiting immune function, anti-inflammatory, sedation and hypnotic, etc. Alkaloids are the main bioactive components in C. bungeana. It has been reported that alkaloids from C. bungeana have the effects of analgesic and anti-inflammatory, sedation and hypnotic, antiviral, antibacterial, inhibition of immune function, cytotoxicity, etc.

[0004] The tetrahydropyrrolizino[2,3-c]quinoline alkaloid compound (bungeanoline G) in the present application is a new compound which is extracted and separated from bungea glabrescens for the first time, and has not been reported in other natural sources, and there is no report about the compound and its pharmacological activity in the existing literature. Because bungeanoline G has a unique 6 / 6 / 5 / 5 tetra-cyclic fused tetrahydropyrrolizino[2,3-c]quinoline skeleton, the bungeanoline G is chemically synthesized and its derivatives are prepared in the present application. The pharmacological activity test shows that the compound and its derivatives have significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, and can be applied in the preparation of drugs for treating or improving Alzheimer's disease, vascular dementia, myasthenia gravis and the like. SUMMARY

[0005] One of the purposes of the present application is to provide a tetrahydropyrrolizino[2,3-c]quinoline alkaloid compound with a novel structure and a preparation method thereof. These compounds have significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, and have application prospects for the development of therapeutic drugs for the currently highly concerned neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis and the like.

[0006] To achieve the above purpose, the present application proposes a series of tetrahydropyrrolizino[2,3-c]quinoline alkaloid derivatives, and pharmaceutically acceptable salts, solvates, hydrates or crystal forms thereof, whose general formula (I) is as follows:

[0007]

[0008] Among them,

[0009] R1-R4 are each independently one or two or more of H, halogen, C 1-3 alkyl, hydroxyl, C 1-3 alkoxy, and the like;

[0010] n is 1, 2, or 3.

[0011] In some embodiments, R1-R4 of the compound I are each independently one or two or more of H, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, hydroxyl, methoxy, ethoxy, propoxy, isopropoxy; further, R1-R4 are each independently one or two or more of H, F, Br, methyl, ethyl, propyl, hydroxyl, methoxy, ethoxy, propoxy; preferably, R1-R4 are each independently one or two or more of H, Br, methyl, ethyl, methoxy, ethoxy.

[0012] In some embodiments, n of compound I is independently 1, 2, or 3; preferably, n is independently 1 or 2.

[0013] In some embodiments, compound I is a racemate, or one or both of 7aR or 7aS, as shown in the following formulae I-achiral, I-7aS and I-7aR:

[0014]

[0015] In some embodiments, preferred compounds include one or more of the following:

[0016]

[0017]

[0018] In some embodiments, compound I forms a salt with a pharmaceutically acceptable acid, which includes one or more of HCl, HBr, trifluoroacetic acid, sulfuric acid, formic acid, acetic acid, succinic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, citric acid, and the general formula is shown in I-X:

[0019]

[0020] Another object of the present application is to provide a composition of any one or more of the compounds of the present application with a pharmaceutically acceptable adjuvant, carrier, diluent and / or other active compound. They can be used for the preparation of a pharmaceutical preparation containing at least one of the compounds of the present application.

[0021] The present application also relates to the use of any of the compounds I or a pharmaceutical composition thereof as an effective ingredient in the preparation of a medicament for the purpose of treating or ameliorating diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis, etc. by inhibiting acetylcholinesterase or / and butyrylcholinesterase.

[0022] The compounds according to the present application, or salts with a pharmaceutically acceptable acid, can be prepared in a suitable galenic form, for example oral, injectable, sprayable compositions, etc. according to acceptable pharmaceutical procedures. The pharmaceutical compositions according to the present application comprise an effective amount of the compounds of the present application, and a suitable pharmaceutically acceptable carrier or diluent, which are well known in the art. The carrier can be any inert material, organic or inorganic, suitable for enteral, transdermal or parenteral administration, such as water, gelatin, gum arabic, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide, etc. The composition can also contain other pharmaceutically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavorings, buffers, etc.

[0023] The composition according to the present application can be formulated into solid or liquid dosage forms for oral administration, such as tablets, capsules, powders, syrups; into sterile solution, suspension or emulsion dosage forms for parenteral administration; into dry powder formulations, sterile solution, suspension or emulsion dosage forms for spray administration.

[0024] It is another object of the present application to provide the use of the compounds of the present application in the treatment of various neurodegenerative diseases. The dosage of the specific compound will vary depending on its potency, mode of administration, age and weight of the patient, and severity of the condition being treated.

[0025] The present application relates to a novel tetrahydropyrrolizino[2,3-c]quinoline alkaloid compound and derivatives thereof, and a preparation method and use thereof. The novel compound bungeanoline G provided by the present application is extracted and separated from Corydalis bungeana Turcz. of the Papaveraceae family. The present application also provides a method for artificially synthesizing bungeanoline G, and a series of derivatives of bungeanoline G. Bioactivity experiments show that the compounds have acetylcholinesterase and butyrylcholinesterase inhibitory activity, and can be used in the preparation of drugs for treating or improving various neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 , characterization of BChE inhibitory activity of representative compound I-1 of the natural compound I; 1 H- 1 H COSY and key HMBC;

[0027] Figure 2 , characterization of BChE inhibitory activity of representative compound I of the natural compound I;

[0028] Figure 3 , characterization of AChE inhibitory activity of representative compound I of the natural compound I;

[0029] Figure 4 , structural schematic diagram of the compound I of the preferred embodiment of the present application. DETAILED DESCRIPTION

[0030] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, so that the implementation process of how the present application applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented.

[0031] It should be understood, when used in this specification and the appended claims, the terms "comprise", "comprising", "include", "including", "contain", "containing" and the like, indicate the presence of the described features, integers, steps, operations, elements, and / or components but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0032] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present embodiments.

[0033] The present embodiments also report a method for preparing the above-mentioned compound I. With reference to similar literature reports or the specific examples shown below, an experienced researcher can easily select appropriate reaction conditions for the chemical reactions. The necessary starting materials for such reactions can be purchased or prepared by conventional methods.

[0034] According to the conditions disclosed in the present embodiments, the following methods are provided:

[0035] Natural product extraction method

[0036] The present embodiments provide a method for preparing the natural compound bungeanoline G (I-1), which comprises the following steps: grinding dry root-bearing whole plants of Kudou to obtain medicinal material powder, heating extraction at 60-70°C for 6-24 hours using 50-90% (by volume) ethanol, filtering the extraction liquid using a 80-mesh filter screen, and concentrating the extraction liquid under reduced pressure to obtain an extract; dissolving the extract using 0.01-0.2 mol / L sulfuric acid, adjusting the pH to 2-3, adding petroleum ether in a volume ratio of 1:1-1:2 to the extract, and taking the water layer. Adjusting the pH of the obtained water layer to 9-10 using 0.01-2 mol / L sodium hydroxide, adding dichloromethane in a volume ratio of 1:1-1:2 to the water layer, and obtaining the dichloromethane layer. Separating the dichloromethane layer through multiple steps of high-performance preparative liquid chromatography to obtain the compound bungeanoline G (I-1).

[0037] The present embodiments also provide a method for artificially synthesizing the above-mentioned compound I or a pharmaceutically acceptable salt thereof, which comprises the following synthesis method:

[0038] Synthesis method 1

[0039]

[0040] In the reaction formula, R1-R4 and n are the same as defined in the above claims 1-4.

[0041] Reaction a

[0042] Substrate 1 (1 mmol, 1.0 mole equivalent) was dissolved in THF (10 ml), then substrate 2 (1.5 mole equivalent relative to substrate 1), Pd(PPh3)2Cl2 (0.03 mole equivalent relative to substrate 1), CuI (0.03 mole equivalent relative to substrate 1), PPh3 (0.03 mole equivalent relative to substrate 1) and triethylamine (2 mole equivalent relative to substrate 1) were added, and the solution was stirred at room temperature. After the reaction system was replaced with nitrogen atmosphere, it was heated to 60°C and stirred for 12 hours. After the reaction was completed as monitored by LC-MS, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography to obtain product 3 (as substrate 3).

[0043] Reaction b

[0044] Substrate 3 (1 mmol, 1.0 mole equivalent) was dissolved in acetone (15 ml), and water (5 ml), mercury sulfate (1.5 mole equivalent relative to substrate 3) and concentrated sulfuric acid (2 mole equivalent relative to substrate 3) were added in sequence with stirring. The mixture was stirred at 30°C for 72 hours. After the reaction was completed as monitored by LC-MS, saturated sodium carbonate solution was added to the reaction solution to neutralize the sulfuric acid to pH 8. The mixture was extracted with ethyl acetate (10 ml each time) for 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the corresponding product 4 (as substrate 4).

[0045] Reaction c

[0046] Substrate 4 (1 mmol, 1.0 mole equivalent) was dissolved in methanol (10 ml), and acetic acid (4 mole equivalent relative to substrate 4) and benzylamine (4 mole equivalent relative to substrate 4) were added. After the mixture was stirred at 25°C for 8 hours, sodium cyanoborohydride (4 mole equivalent relative to substrate 4) was added, and the mixture was stirred at 25°C for 16 hours. Water (10 ml) was added to the reaction solution, and the mixture was stirred for 0.5 hours. The mixture was extracted with ethyl acetate (10 ml each time) for 3 times. The organic phase was discarded, and concentrated hydrochloric acid (38% by mass) was added dropwise to the aqueous phase to adjust the pH to 2. Palladium-carbon (10 wt%, 0.2 mass equivalent relative to substrate 4) was added, and the mixture was stirred at 25°C under normal pressure in a hydrogen atmosphere for 16 hours. Saturated sodium carbonate solution was added to the reaction solution to adjust the pH to 8. The mixture was extracted with ethyl acetate (10 ml each time) for 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the target product I.

[0047] Reaction d

[0048] Substrate 4 (1 mmol, 1.0 molar equivalent) was dissolved in methanol (10 ml), acetic acid (4 molar equivalent relative to substrate 4) and p-methoxybenzylamine (4 molar equivalent relative to substrate 4) were added, and the mixture was stirred at 25°C for 2 hours. Sodium cyanoborohydride (4 molar equivalent relative to substrate 4) was added, and the mixture was stirred at 25°C for 16 hours. The methanol was removed under reduced pressure, saturated sodium carbonate solution was added to bring the pH to 8, and the mixture was extracted with ethyl acetate (10 ml each time) three times. The organic phase was dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the corresponding product 5 (as substrate 5).

[0049] Reaction e

[0050] Substrate 5 (1 mmol, 1.0 molar equivalent) was dissolved in acetonitrile (5 ml) and water (20 ml), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ, 1.5 molar equivalent relative to substrate 5) was added, and the mixture was heated to 70°C and stirred for 12 hours. The acetonitrile was removed under reduced pressure, 2N hydrochloric acid (5 ml) and an equal volume of ethyl acetate were added, and the mixture was separated. The organic phase was extracted with 2N hydrochloric acid (5 ml each time) three times, and the organic phase was discarded. The acid aqueous phase was combined and adjusted to pH 8 with saturated sodium carbonate solution, and extracted with ethyl acetate (5 ml each time) three times. The organic phase was dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the target product I.

[0051] When R1-R4 in compound I are not all Br, and at least one is H, compound I derivatives can be further synthesized using synthesis method 2, as shown below:

[0052] Synthesis method 2

[0053]

[0054] Br in compound I-a can be substituted at any one or two of R2-R4, and R refers to other R1-R4 substituents except for Br under reaction condition f. R' in I-b refers to the group after the Br group is replaced under reaction condition g; "alkyl boronic acid or boronic acid ester" refers to C 1-3 alkyl boronic acid or boronic acid ester; both compound I-a and I-b are within the scope of compound I.

[0055] Reaction f

[0056] Compound I (substrate I) (1 mmol, 1.0 molar equivalent) was dissolved in acetic acid (10 ml), N-bromosuccinimide (NBS, 1.2 molar equivalent relative to substrate I) was added under ice bath, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was quenched by adding saturated sodium sulfite solution. The reaction solution was concentrated under reduced pressure, saturated sodium carbonate solution was added to make the solution alkaline (pH 8), and the solution was extracted with dichloromethane (10 ml each time) for 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the target product I-a (as compound I-a).

[0057] Reaction g

[0058] Compound I-a (1 mmol, 1.0 molar equivalent) was dissolved in dioxane (10 ml), water (2 ml), C 1-3 alkylboronic acid or boronic ester substrate (3 molar equivalents relative to substrate I-a), Pd(PPh3)4 (0.05 molar equivalents relative to substrate I-a), and potassium carbonate (3 molar equivalents relative to substrate I-a) were added under nitrogen protection, and the reaction solution was stirred at 90°C for 16 hours. The reaction solution was cooled to room temperature, filtered, the filter cake was washed with ethyl acetate, the filtrate was washed with saturated brine (10 ml each time) for 3 times, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the target product I-b.

[0059] When compound I is a chiral monomer, the racemate can be separated by chiral chromatography:

[0060] Separation method

[0061] The racemic compound I was dissolved in methanol, and separated and purified by supercritical fluid chromatography through BH (chiral chromatographic column) with mobile phase A being CO2 and B being (0.1% (volume concentration) triethylamine-methanol) under gradient elution conditions of 0-12 min: 80% A. The chiral monomers I-S and I-R were collected according to the chromatographic peaks.

[0062] When compound I forms a salt with a pharmaceutically acceptable acid (HX) (these acids refer to one or more of HCl, HBr, trifluoroacetic acid, sulfuric acid, formic acid, acetic acid, succinic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and citric acid), compound I can be mixed with an appropriate amount of HX:

[0063]

[0064] Reaction h

[0065] Compound I (1 mmol, 1.0 molar equivalent) was dissolved in dioxane or tetrahydrofuran or dichloromethane or ethyl acetate (5 ml), HX (1.2 molar equivalent relative to substrate I) was added, stirred at room temperature for 1 hour, concentrated under reduced pressure, the solid was precipitated, filtered and dried to obtain the target product I-X.

[0066] The following examples are intended to illustrate the present application and not to further limit it, which can be implemented in any way described in the summary of invention.

[0067] Preparation example of natural compound I-1 of the present application:

[0068] Compound preparation and structure identification:

[0069] In the following preparation examples, the preparation system includes Waters Alliance, including e2695 separation unit, 2998 PDA detector, data processing by empow er 3; Waters AutoP automatic purification system, including 2545 separation unit, 2767 sample manager, 2489 dual wavelength detector; Novasep HPLC industrial grade high pressure liquid preparation chromatography and HIPERSEP software. Reagents include chromatographic grade methanol and acetonitrile purchased from Fisher Scientific (Loughborough, UK), chromatographic grade formic acid, acetic acid, ammonia, triethylamine, ammonium formate, sodium monohydrogen phosphate, sodium dihydrogen phosphate purchased from Biotang (Hebei, China), laboratory water from Milli-Q ultra-pure water purification system (Billerica, MA, USA), preparation grade methanol purchased from Shanghai Xingke High-purity Solvent Co., Ltd. (Shanghai, China). Preparation column and semi-preparation column: C18HCE (15 μm, 100 mm x 250 mm), C18HCE (10 μm, 100 mm x 325 mm), C18CE (7 μm, 50 mm x 250 mm), FC8HL (3.5 μm, 30 mm x 150 mm), C18HCE (5 μm, 10 mm x 150 mm) and BH (5 μm, 4.6 mm x 150 mm) (Dalian Sipu Jinggong Co., Ltd.).

[0070] The nuclear magnetic resonance spectrum used for structure identification was measured by Bruker AVIII-600 nuclear magnetic resonance spectrometer (Bruker, German), and the compounds were dissolved in deuterated methanol (MeOD) or deuterated chloroform (CDCl3). Mass spectrometry was performed by Agilent 1290 Infinity LC / 6540 Q-TOF MS liquid chromatography-mass spectrometry system for sample separation and analysis.

[0071] The preparation and compound identification steps are as follows:

[0072] (1) Medicinal material extraction: 100 kg of dried root and whole grass of Kudou- ding was taken and powdered. 1000 liters of 70% ethanol was heated to 70 °C and extracted for 6 hours. The extract was filtered through an 80 mesh filter and the solvent was removed by rotary evaporation to obtain an extract. The extract was dissolved in 0.01 mol / L sulfuric acid, and the pH was adjusted to 2 with 2% sulfuric acid solution. Petroleum ether was added in a 1:1 volume ratio to extract the water layer. The obtained water layer was adjusted to pH 10 with 2 mol / L sodium hydroxide, and dichloromethane was added in a 1:1 volume ratio to extract the dichloromethane layer, which was the crude base.

[0073] (2) The crude base obtained in step (1) was subjected to first-dimensional separation and purification using C18HCE (15um, 100x250mm, reverse phase column), with a flow rate of 320 mL / min, mobile phase A being (0.1% by volume) formic acid-methanol, and B being (0.1% by volume) formic acid-water, with gradient elution conditions of 0-8 min: 25% A (volume ratio, same below), 8-23 min: 40% A, 23-38 min: 80% A, 38-55 min: 100% A, and the sample was taken at elution time 4.2-26.0 min, and concentrated to obtain the total alkaloid sample of Kudou-ding.

[0074] (3) The total alkaloid of Kudou-ding obtained in step (2) was subjected to second- dimensional separation and purification using C18HCE (10um, 100x325mm, reverse phase column), with a flow rate of 320 mL / min, mobile phase A being (0.1% by volume) formic acid-methanol, and B being (0.1% by volume) formic acid-water, with gradient elution conditions of 0-5 min: 20% A, 5-50 min: 20-45% A, 50-60 min: 95% A, and 11 sub-fractions F1 (5.7-10.7 min), F2 (10.7-17.1 min), F3 (17.1-20.9 min), F4 (20.9-25.7 min), F5 (25.7-29.2 min), F6 (29.2-33.0 min), F7 (33.0-36.3 min), F8 (36.3-40.4 min), F9 (40.4-44.4 min), F10 (44.4-55.6 min), and F11 (55.6-60 min) were collected according to the corresponding elution time.

[0075] (4) The sub-fraction F4 obtained in step (3) was subjected to third dimensional separation and purification using C8CE (7 μm, 50 x 250 mm, reversed-phase chromatographic column) with mobile phase A of (0.1% by volume) 25% ammonia water-methanol and B of (0.1% by volume) 25% ammonia water-water, and gradient elution conditions of 0-5 min: 5% A, 5-50 min, 5-95% A, 50-60 min: 95% A, to collect 15 sub-fractions F4-1 (4.0-7.8 min), F4-2 (7.8-10.1 min), F4-3 (10.1-13.4 min), F4-4 (13.4-18.1 min), F4-5 (18.1-21.3 min), F4-6 (21.3-23.7 min), F4-7 (23.7-28.5 min), F4-8 (28.5-30.7 min), F4-9 (30.7-33.6 min), F4-10 (33.6-35.0 min), F4-11 (35.0-37.6 min), F4-12 (37.6-39.5 min), F4-13 (39.5-43.9 min), F4-14 (43.9-46.1 min), F4-15 (46.1-57.0 min) according to the elution time of the corresponding fractions.

[0076] (5) The sub-fraction F4-13 obtained in step (4) was subjected to fourth dimensional separation and purification using FC8HL (3.5 μm, 30 x 100 mm, reversed-phase chromatographic column) with mobile phase A of (20 mM) ammonium formate-95% methanol / water (by volume) and B of (20 mM) ammonium formate-water, and gradient elution conditions of 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, to collect 10 sub-fractions F4-13-1 (1.5-2.5 min), F4-13-2 (9.1-9.6 min), F4-13-3 (9.6-11.0 min), F4-13-4 (11.0-11.6 min), F4-13-5 (11.6-12.4 min), F4-13-6 (12.4-13.6 min), F4-13-7 (13.6-14.5 min), F4-13-8 (14.5-16.2 min), F4-13-9 (16.2-16.9 min), F4-13-10 (16.9-19.2 min) according to the elution time of the corresponding fractions.

[0077] (6) The sub-fraction F4-13-10 obtained in step (5) was subjected to fifth dimensional separation and purification by C18HCE (5 μm, 10×150 mm, reversed-phase chromatographic column) with mobile phase A of (volume concentration 0.1%) formic acid-methanol and B of (volume concentration 0.1%) formic acid-water under gradient elution condition of 0-5 min: 25% A, 5-15 min: 25-45% A, 15-20 min: 45% A, to obtain compound I-1 (t R = 6.83 min), named as bungeanoline G.

[0078] (7) The compound I-1 obtained in step (6) was subjected to separation and purification by supercritical fluid chromatography by BH (5 μm, 10×250 mm, chiral chromatographic column) with mobile phase A of CO2 and B of (volume concentration 0.1%) triethylamine-methanol under gradient elution condition of 0-12 min: 80% A, to obtain compounds I-1a (t R = 5.0 min) and I-1b (t R = 6.9 min), named as (-)-(R)-bungeanoline G and (+)-(S)-bungeanoline G, respectively.

[0079] The above compound has the following physicochemical properties and spectroscopy characteristics:

[0080] Bungeanoline G: yellow powder; 1 H (600 MHz) and 13 The C (150 MHz) NMR data are shown in Table 1; HRESIMS m / z 211.1234 [M+H] + (calcd for C 14 H 15 N2, 211.1235); 1a: -31 (c 0.42, MeOH); 1b: 37 (c 0.78, MeOH); ECD (MeOH) 1a: λ max (Δε) 255 (-2.74), 349 (0.04); 1b: λ max (Δε) 255 (4.52), 347 (-0.05).

[0081] Artificial synthesis method of compound I of the present application

[0082] The physicochemical properties and spectroscopy characteristics of all compounds are shown in Table 1. 1H-NMR spectra were collected on a Brucker AVANCE III 400 MHz instrument, and the resulting spectra were referenced to tetraethylsilane (TMS) as an internal standard. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on a Waters Alliance e2695-ZQ2000 system, and m / z values are reported. Unless otherwise noted, all solvents were used directly, all ratios of mixed solvents refer to volume ratios (v / v), and all temperatures are in degrees Celsius (°C).

[0083] The following non-limiting examples and pharmacological experiments will further illustrate the present application. Unless otherwise specified, the compounds are racemates or mixtures of diastereomers.

[0084] Example 1

[0085]

[0086] 1.1 Synthesis of 4-chloro-3-(5-chloropent-1-yn-1-yl)quinoline

[0087] The target product was obtained in 85% yield according to the procedure and conditions described in reaction a of General Compound Synthesis Method 1 above, using 4-chloro-3-bromo-quinoline and 5-chloropent-1-yn as the starting materials. 1 H-NMR (400 MHz, CDC13) δ 8.82 (s, 1H), 8.22 (dd, J = 8.4, 0.9 Hz, 1H), 8.09 (dd, J = 8.4, 0.5 Hz, 1H), 7.74 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.65 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 3.80 (t, J = 6.3 Hz, 2H), 2.77 (t, J = 6.8 Hz, 2H), 2.15 (p, J = 6.6 Hz, 2H). LCMS [M+H] + m / z 264.06.

[0088] 1.2 Synthesis of 5-chloro-1-(4-chloroquinolin-3-yl)pentan-2-one

[0089] The target product was obtained in 57% yield according to the procedure and conditions described in reaction b of General Compound Synthesis Method 1 above, using 4-chloro-3-(5-chloropent-1-yn-1-yl)quinoline as the starting material. 1H-NMR (400 MHz, CDC13) δ 8.73 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.78 (t, J = 7.0 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 4.11 (s, 2H), 3.60 (s, 2H), 2.82 (t, J = 6.2 Hz, 2H), 2.21 - 2.04 (m, 2H). LCMS [M+H] + m / z 282.00.

[0090] 1.3 Synthesis of 7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (Bungeanoline G, 1-1)

[0091] Following the procedure and conditions described in step c of General compound synthesis method 1, the target product 1-1 was obtained from 5-chloro-l-(4-chloroquinolin-3-yl)pentan-2-one in 27% yield. 1 H-NMR (400 MHz, CDC13) δ 8.73 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.78 (t, J = 7.0 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 4.11 (s, 2H), 3.60 (s, 2H), 2.82 (t, J = 6.2 Hz, 2H), 2.21 - 2.04 (m, 2H). LCMS [M+H] 13 C-NMR (101 MHz, CDC13) δ 156.60, 148.06, 145.44, 128.44, 127.69, 123.28, 122.06, 119.16, 117.24, 65.69, 51.05, 30.67, 25.85. HRMS (ESI): calcd for C 14 H 14 N2[M+H] + m / z 211.1235, found 211.1186.

[0092] Example 2

[0093]

[0094] 2.1 Synthesis of 4-chloro-3-(5-chloropent-1-yn-1-yl)-6-methoxyquinoline

[0095] Following the procedure and conditions described in reaction a of the general compound synthesis method 1, the target product was obtained in 69% yield, using 3-bromo-4-chloro-6-methoxyquinoline and 5-chloropent-1-yn as the substrates. 1 H-NMR (400 MHz, CDC13) δ 8.68 (s, 1H), 7.97 (d, J = 9.1 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.38 (d, J = 2.8 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 3.98 (s, 3H), 3.80 (t, J = 6.3 Hz, 2H), 2.77 (t, J = 6.8 Hz, 2H), 2.20 - 2.10 (m, 2H). [M+H] + m / z 264.06.

[0096] 2.2 Synthesis of 5-chloro-1-(4-chloro-6-methoxyquinolin-3-yl)pentan-2-one

[0097] Following the procedure and conditions described in reaction b of the general compound synthesis method 1, the target product was obtained in 35% yield, using 4-chloro-3-(5-chloropent-1-yn-1-yl)-6-methoxyquinoline as the substrate. 1 H-NMR (400 MHz, CDC13) δ 8.58 (s, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H), 7.41 (dd, J = 9.1, 2.8 Hz, 1H), 4.08 (s, 2H), 3.98 (s, 3H), 3.60 (t, J = 6.2 Hz, 2H), 2.80 (t, J = 6.9 Hz, 2H), 2.16 - 2.06 (m, 2H). LCMS [M+H] + m / z 312.09.

[0098] 2.3 Synthesis of 4-chloro-6-methoxy-3-((1-(4-methoxybenzyl)pyrrolidin-2-yl)methyl)quinoline

[0099] Following the procedure and conditions described in reaction d of the general compound synthesis method 1, the target product was obtained in 32% yield, using 5-chloro-1-(4-chloroquinolin-3-yl)pentan-2-one as the substrate. 1H-NMR (400 MHz, CDC13) δ 8.63 (s, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.46 (d, J = 2.7 Hz, 1H), 7.36 (dd, J = 9.2, 2.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 6.88 - 6.83 (m, 2H), 4.09 (d, J = 12.8 Hz, 1H), 3.98 (s, 3H), 3.82 - 3.77 (m, 3H), 3.38 (dd, J = 14.5, 6.0 Hz, 2H), 3.07 - 2.83 (m, 3H), 2.33 - 2.18 (m, 1H), 1.77 - 1.65 (m, 4H). LCMS [M+H] + m / z 397.25.

[0100] 2.4 Synthesis of 2-methoxy-7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (I-2)

[0101] Following the procedure and conditions described in reaction e of the general compound synthesis method 1 above, using 5-chloro-l-(4-chloroquinolin-3-yl)pentan-2-one as the starting material, the target product I-2 was obtained in 27% yield. 1 H-NMR (400 MHz, CDC13) δ 8.63 (s, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.46 (d, J = 2.7 Hz, 1H), 7.36 (dd, J = 9.2, 2.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 6.88 - 6.83 (m, 2H), 4.09 (d, J = 12.8 Hz, 1H), 3.98 (s, 3H), 3.82 - 3.77 (m, 3H), 3.38 (dd, J = 14.5, 6.0 Hz, 2H), 3.07 - 2.83 (m, 3H), 2.33 - 2.18 (m, 1H), 1.77 - 1.65 (m, 4H). LCMS [M+H] 13 C-NMR (101 MHz, CDC13) δ 155.94, 155.22, 143.48, 142.90, 129.51, 120.17, 119.61, 117.78, 100.22, 65.57, 54.43, 50.82, 30.87, 25.82. HRMS (ESI): calcd for C 15 H 16 N2O [M+H] + m / z 241.1341, found 241.1286.

[0102] Example 3

[0103]

[0104] 3.1 Synthesis of 4-chloro-3-(6-chlorohex-1-yn-1-yl)quinoline

[0105] Following the procedure and conditions described in reaction a of the general compound synthesis method 1, the target product was obtained in 78% yield using 3-bromo-4-chloro-quinoline and 6-chlorohex-1-yn as the starting materials. 1 H-NMR (400 MHz, CDC13) δ 8.83 (s, 1H), 8.24 (dd, J = 8.4, 0.9 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.75 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.66 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 3.65 (t, J = 6.5 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H), 2.05 (tt, J = 13.1, 6.7 Hz, 2H), 1.87 (dt, J = 9.6, 7.0 Hz, 2H). [M+H] + m / z 278.06.

[0106] 3.26-chloro-1-(4-chloro-6-methoxyquinolin-3-yl)hexan-2-one

[0107] Following the procedure and conditions described in reaction b of the general compound synthesis method 1, the target product was obtained in 75% yield using 4-chloro-3-(5-chloropent-1-yn-1-yl)-6-methoxyquinoline as the starting material. 1 H-NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 8.24 (dd, J = 8.4, 0.7 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.76 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.72 - 7.61 (m, 1H), 4.07 (s, 2H), 3.62 - 3.48 (m, 2H), 2.73 - 2.56 (m, 2H), 1.87 - 1.73 (m, 4H). LCMS [M+H] + m / z 296.06.

[0108] 3.3 Synthesis of 4-chloro-3-((1-(4-methoxybenzyl)piperidin-2-yl)methyl)quinoline

[0109] Following the procedure and conditions described in reaction d of the general compound synthesis method 1, the target product was obtained in 53% yield using 6-chloro-1-(4-chloro-6-methoxyquinolin-3-yl)hexan-2-one as the starting material. 1H-NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 8.22 (dd, J = 8.4, 0.9 Hz, 1H), 8.08 (dd, J = 8.4, 0.6 Hz, 1H), 7.71 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.62 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.27 - 7.25 (m, 2H), 6.88 - 6.76 (m, 2H), 4.03 (d, J = 13.4 Hz, 1H), 3.79 (s, 3H), 3.57 (d, J = 13.4 Hz, 1H), 3.47 (dd, J = 13.0, 4.0 Hz, 1H), 3.09 - 2.99 (m, 1H), 2.95 (d, J = 2.0 Hz, 1H), 2.85 (dt, J = 11.3, 4.4 Hz, 1H), 2.39 - 2.28 (m, 1H), 1.76 - 1.66 (m, 1H), 1.61 - 1.50 (m, 3H), 1.44 - 1.34 (m, 2H). LCMS [M+H] + m / z 381.30.

[0110] 3.4 Synthesis of 7,7a,8,9,10,11-Hexahydroindolizino[2,3-c]quinoline (I-3)

[0111] Following the procedure described in reaction e of the general compound synthesis method 1 above, using 4-chloro-3-((1-(4-methoxybenzyl)piperidin-2-yl)methyl)quinoline as the starting material, the target product I-3 was obtained in 52% yield. 1 H-NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 8.22 (dd, J = 8.4, 0.9 Hz, 1H), 8.08 (dd, J = 8.4, 0.6 Hz, 1H), 7.71 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.62 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.27 - 7.25 (m, 2H), 6.88 - 6.76 (m, 2H), 4.03 (d, J = 13.4 Hz, 1H), 3.79 (s, 3H), 3.57 (d, J = 13.4 Hz, 1H), 3.47 (dd, J = 13.0, 4.0 Hz, 1H), 3.09 - 2.99 (m, 1H), 2.95 (d, J = 2.0 Hz, 1H), 2.85 (dt, J = 11.3, 4.4 Hz, 1H), 2.39 - 2.28 (m, 1H), 1.76 - 1.66 (m, 1H), 1.61 - 1.50 (m, 3H), 1.44 - 1.34 (m, 2H). LCMS [M+H] 13 C-NMR (101 MHz, CDC13) δ 154.89, 145.61, 140.23, 130.43, 126.27, 124.41, 122.84, 118.04, 114.82, 65.53, 47.75, 32.35, 32.23, 25.65, 23.46. HRMS (ESI): calcd for C15 H 16 N2[M+H] + m / z 225.1392, found 225.1342.

[0112] Example 4

[0113]

[0114] 4.1 Synthesis of 4-chloro-3-(6-chlorohex-1-yn-1-yl)-6-methoxyquinoline

[0115] The title compound was obtained from 3-bromo-4-chloro-6-methoxyquinoline and 6-chlorohex-1-yn according to the procedure and conditions described in Reaction a, General procedure for compound synthesis 1, in 75% yield. 1 H-NMR (400 MHz, CDC13) δ 8.68 (s, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.37 (dd, J = 9.1, 2.8 Hz, 1H), 3.98 (s, 3H), 3.65 (t, J = 6.5 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H), 2.05 (dt, J = 14.6, 6.7 Hz, 2H), 1.91 - 1.80 (m, 2H). LCMS [M+H] + m / z 308.10.

[0116] 4.2 Synthesis of 4-chloro-1-(4-chloro-6-methoxyquinolin-3-yl)hexan-2-one

[0117] The title compound was obtained from 4-chloro-3-(6-chlorohex-1-yn-1-yl)-6- methoxyquinoline according to the procedure and conditions described in Reaction b, General procedure for compound synthesis 1, in 59% yield. 1 H-NMR (400 MHz, CDC13) δ 8.56 (s, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H), 7.41 (dd, J = 9.1, 2.8 Hz, 1H), 4.05 (s, 2H), 3.98 (s, 3H), 3.58 - 3.49 (m, 2H), 2.66 - 2.56 (m, 2H), 1.84 - 1.75 (m, 4H). LCMS [M+H] + m / z 326.11.

[0118] 4.3 Synthesis of 4-chloro-6-methoxy-3-((1-(4-methoxybenzyl)piperidin-2-yl)methyl)quinoline

[0119] The reaction was carried out with 6-chloro-1-(4-chloro-6-methoxyquinolin-3-yl)hexan-2-one according to the method described in step d of reaction d of the general compound synthesis method 1. The target product was obtained in a yield of 32%. 1 H-NMR (400 MHz, CDC13) δ 8.54 (s, 1H), 7.96 (d, J = 9.1 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 7.35 (dd, J = 9.1, 2.8 Hz, 1H), 7.27 (d, J = 4.2 Hz, 2H), 6.85 - 6.80 (m, 2H), 4.02 (d, J = 13.4 Hz, 1H), 3.98 (d, J = 4.9 Hz, 3H), 3.79 (s, 3H), 3.56 (d, J = 13.4 Hz, 1H), 3.44 (dd, J = 12.7, 3.7 Hz, 1H), 3.06 - 2.89 (m, 2H), 2.87 - 2.77 (m, 1H), 2.39 - 2.27 (m, 1H), 1.70 (dq, J = 9.7, 6.1 Hz, 1H), 1.55 (dd, J = 10.9, 5.3 Hz, 3H), 1.43 - 1.32 (m, 2H). LCMS [M+H] + m / z 411.24.

[0120] 4. Synthesis of 4-methoxy-7,7a,8,9,10,11-hexahydroindolizino[2,3-c]quinoline (I-4)

[0121] The reaction was carried out with 4-chloro-6-methoxy-3-((1-(4-methoxybenzyl)piperidin-2-yl)methyl)quinoline according to the method described in step e of reaction e of the general compound synthesis method 1. The target product I-4 was obtained in a yield of 56%. 1 H-NMR (400 MHz, CDC13) δ 8.54 (s, 1H), 7.96 (d, J = 9.1 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 7.35 (dd, J = 9.1, 2.8 Hz, 1H), 7.27 (d, J = 4.2 Hz, 2H), 6.85 - 6.80 (m, 2H), 4.02 (d, J = 13.4 Hz, 1H), 3.98 (d, J = 4.9 Hz, 3H), 3.79 (s, 3H), 3.56 (d, J = 13.4 Hz, 1H), 3.44 (dd, J = 12.7, 3.7 Hz, 1H), 3.06 - 2.89 (m, 2H), 2.87 - 2.77 (m, 1H), 2.39 - 2.27 (m, 1H), 1.70 (dq, J = 9.7, 6.1 Hz, 1H), 1.55 (dd, J = 10.9, 5.3 Hz, 3H), 1.43 - 1.32 (m, 2H). LCMS [M+H] 13C-NMR (101 MHz, CDCI3) δ 156.74, 155.98, 136.61, 132.93, 123.88, 123.67, 117.56, 114.10, 103.03, 65.46, 55.84, 47.53, 32.85, 31.50, 25.72, 23.09. HRMS (ESI): calcd for C 16 H 18 N2O[M+H] + m / z 255.1497, found 225.1440.

[0122] Example 5

[0123]

[0124] The reaction was carried out according to the general compound synthesis method 2, reaction f, using I-1 and appropriate amount of NBS as substrates, to give the target products I-5, I-6, I-7, I-8, with the yield of 68%, 23%, 12%, 10%, respectively.

[0125] 5.1 2-bromo-7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (I-5):

[0126] 1 H-NMR (400 MHz, CDCI3) δ 8.46 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H), 7.64 (dd, J = 9.1, 2.1 Hz, 1H), 4.26 (dt, J = 10.4, 5.3 Hz, 1H), 3.95 - 3.85 (m, 1H), 3.47 (dd, J = 16.9, 9.4 Hz, 1H), 3.30 (dd, J = 16.1, 10.4 Hz, 1H), 3.16 - 3.10 (m, 1H), 2.13 - 1.99 (m, 3H), 1.43 (dt, J = 10.9, 3.5 Hz, 1H). 13 C-NMR (101 MHz, CDCI3) δ 156.88, 147.27, 146.23, 132.29, 130.88, 125.41, 121.21, 119.18, 117.84, 66.90, 51.79, 31.70, 31.45, 26.89. HRMS (ESI): calcd for C 14 H 13 BrN2[M+H] + m / z 289.0340, found 289.0275.

[0127] 5.2 3-bromo-7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (I-6):

[0128] 1 H-NMR (400 MHz, CDC13) δ 8.42 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.66 (dd, J = 9.1, 2.1 Hz, 1H), 4.36 - 4.23 (m, 1H), 3.96 - 3.85 (m, 1H), 3.58 - 3.45 (m, 1H), 3.29 (dd, J = 16.2, 10.5 Hz, 1H), 3.12 (dd, J = 16.2, 4.9 Hz, 1H), 2.18 - 2.09 (m, 3H), 1.44 (dt, J = 11.0, 7.7 Hz, 1H). 13 C-NMR (101 MHz, CDC13) δ 157.60, 145.07, 143.58, 133.40, 128.68, 125.54, 121.46, 118.25, 118.22, 67.41, 51.01, 31.51, 30.91, 26.95. HRMS (ESI): calcd for C 14 H 13 BrN2[M+H] + m / z 289.0340, found 289.0260.

[0129] 5.3 4-bromo-7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (I-7):

[0130] 1 H-NMR (400 MHz, CDC13) δ 8.42 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.66 (dd, J = 9.1, 2.1 Hz, 1H), 4.36 - 4.23 (m, 1H), 3.96 - 3.85 (m, 1H), 3.58 - 3.45 (m, 1H), 3.29 (dd, J = 16.2, 10.5 Hz, 1H), 3.12 (dd, J = 16.2, 4.9 Hz, 1H), 2.18 - 2.09 (m, 3H), 1.44 (dt, J = 11.0, 7.7 Hz, 1H). 13C-NMR (101 MHz, CDC13) δ 157.98, 147.21, 146.31, 132.43, 124.92, 124.28, 123.16, 121.46, 119.31, 66.98, 52.17, 31.72, 31.36, 26.89. HRMS (ESI): calcd for C 14 H 13 BrN2[M+H] + m / z 289.0340, found 289.0257.

[0131] 5.4 2,4-Dibromo-7a,8,9,10-tetrahydro-7H-pyrrolizino[2,3-c]quinoline (I-8):

[0132] 1 H-NMR (400 MHz, CDC13) δ 8.46 (s, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 2.1 Hz, 1H), 4.16 (dq, J = 15.6, 5.0 Hz, 1H), 3.83 - 3.70 (m, 1H), 3.29 (tt, J = 12.5, 6.2 Hz, 1H), 3.20 (dd, J = 16.3, 10.4 Hz, 1H), 3.05 (dd, J = 16.3, 4.5 Hz, 1H), 2.05 - 1.90 (m, 3H), 1.31 (qd, J = 11.1, 7.3 Hz, 1H). 13 C-NMR (101 MHz, CDC13) δ 157.04, 147.33, 145.09, 134.95, 126.04, 125.34, 122.39, 119.75, 116.52, 67.07, 52.00, 31.70, 31.29, 26.89. HRMS (ESI): calcd for C 14 H 12 Br2N2[M+H] + m / z 368.9425, found 368.9347.

[0133] Example 6

[0134]

[0135] The target product I-9 was obtained by following the procedures and conditions described in reaction g of General compound synthesis method 2 above, using I-5 and methyl boronic acid as starting materials, in 39% yield. 1H-NMR (400 MHz, CDC13) δ 8.43 (s, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.68 (s, 1H), 7.41 (d, J = 8.6 Hz, 1H), 4.30 - 4.15 (m, 1H), 3.94 - 3.84 (m, 1H), 3.48 (dd, J = 17.7, 8.9 Hz, 1H), 3.28 (dd, J = 15.9, 10.3 Hz, 1H), 3.13 (dd, J = 15.8, 4.2 Hz, 1H), 2.50 (s, 3H), 2.05 (ddt, J = 20.1, 11.4, 4.0 Hz, 3H), 1.49 - 1.38 (m, 1H). 13 C-NMR (101 MHz, Acetone) δ 157.04, 147.87, 146.09, 133.97, 130.81, 129.37, 121.84, 120.22, 118.42, 66.55, 52.36, 31.85, 26.83, 21.83. HRMS (ESI): calcd for C 15 H 16 N2[M+H] + m / z 225.1392 found 225.1339.

[0136] Example 7

[0137] The target product I-9-HCOOH was obtained by following the procedures and conditions described in Reaction h of General Compound Synthesis Method 3 above, using I-9 and formic acid as the starting materials in 95% yield. 1 H-NMR (400 MHz, CDC13) δ 8.43 (s, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.68 (s, 1H), 7.41 (d, J = 8.6 Hz, 1H), 4.30 - 4.15 (m, 1H), 3.94 - 3.84 (m, 1H), 3.48 (dd, J = 17.7, 8.9 Hz, 1H), 3.28 (dd, J = 15.9, 10.3 Hz, 1H), 3.13 (dd, J = 15.8, 4.2 Hz, 1H), 2.50 (s, 3H), 2.05 (ddt, J = 20.1, 11.4, 4.0 Hz, 3H), 1.49 - 1.38 (m, 1H). 13C-NMR (101 MHz, Acetone) δ 162.89, 157.72, 145.58, 142.99, 134.28, 131.66, 126.89, 122.32, 120.73, 117.36, 67.06, 51.12, 31.30, 30.73, 26.63, 20.72. HRMS (ESI): calcd for C 15 H 16 N2[M+H] + m / z 225.1392 found 225.1339.

[0138] Example 8

[0139]

[0140] The reaction was carried out according to the procedure described in reaction g of the general compound synthesis method 2 above, using I-5 and ethylboronic acid as starting materials. The target product I-9 was obtained in 43% yield. 1 H-NMR (400 MHz, CDC13) δ 8.37 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.63 (s, 1H), 7.40 (dd, J = 8.7, 1.8 Hz, 1H), 4.25 - 4.12 (m, 1H), 3.91 - 3.80 (m, 1H), 3.50 - 3.41 (m, 1H), 3.23 (dd, J = 15.9, 10.3 Hz, 1H), 3.07 (dd, J = 15.9, 4.3 Hz, 1H), 2.73 (q, J = 7.6 Hz, 2H), 2.03 - 1.91 (m, 3H), 1.38 (dt, J = 10.5, 6.7 Hz, 1H), 1.25 (t, J = 7.6 Hz, 3H). 13 C-NMR (101 MHz, Acetone) δ 157.55, 147.11, 144.98, 140.52, 130.26, 128.65, 120.59, 120.23, 118.06, 66.77, 52.00, 31.70, 29.06, 26.87, 15.49. HRMS (ESI): calcd for C 16 H 18 N2[M+H] + m / z 239.1548 found 239.1494.

[0141] Example 9

[0142]

[0143] The reaction was carried out according to the procedure described in reaction h of the general compound synthesis method 3. The target product I-10-HCOOH was obtained in 93% yield using I-10 and formic acid as the starting materials. 1 H-NMR (400 MHz, CDC13) δ 8.36 (s, 1H), 8.32 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.87 (s, 1H), 7.60 (dd, J = 8.7, 1.9 Hz, 1H), 4.41 - 4.37 (m, 1H), 4.04 (ddd, J = 10.6, 8.3, 2.3 Hz, 1H), 3.65 (td, J = 9.5, 7.3 Hz, 1H), 3.40 - 3.27 (m, 1H), 3.19 (dd, J = 16.1, 5.0 Hz, 1H), 2.83 (q, J = 7.6 Hz, 2H), 2.19 - 2.08 (m, 3H), 1.60 - 1.47 (m, 1H), 1.31 (t, J = 7.6 Hz, 3H). 13 C-NMR (101 MHz, Acetone) δ 163.58, 158.31, 144.69, 141.73, 140.88, 131.24, 125.92, 121.28, 120.89, 116.95, 67.36, 50.72, 31.19, 30.45, 28.53, 26.69, 15.01. HRMS (ESI): calcd for C 16 H 18 N2[M+H] + m / z 239.1548 found 239.1494.

[0144] The natural product I-1 1 H-NMR and 13 C-NMR data are shown in Table 1, and the key 2D NMR information of the compounds are shown in Figure 1 .

[0145] Table 1, The natural product I-1 1 H-NMR and 13 C-NMR data (δ H and δ C , MeOD)

[0146]

[0147]

[0148] Activity test examples:

[0149] Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were purchased from Sigma-Aldrich. Physostigmine, galantamine, iodide thioacetylcholine (ATCI) and iodide thio butyrylcholine (BTCI) were purchased from Aladdin Reagent Co., Ltd. 5,5-dimercapto-bis-2-nitrobenzoic acid (DTNB) was purchased from Shanghai Mirer Biochemical Technology Co., Ltd. Ethylenediaminetetraacetic acid disodium salt (EDTA) and sodium dodecyl sulfate (SDS) were purchased from Solabio Technology Co., Ltd. Bovine serum albumin (BSA) was purchased from Bovogen Laboratories Pty Ltd. Na2HPO4 and NaH2PO4 were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. The buffers were prepared as follows: phosphate buffer A (0.1 M, pH = 7.4, containing 1% BSA), phosphate buffer B (0.1 M, pH = 7.4, containing 1 mM EDTA), phosphate buffer C (0.1 M, pH = 7.4).

[0150] For the AChE and BChE inhibitory activity of compound I, the modified Ellman method was used for detection. The principle of the detection is that AChE or BChE hydrolyzes the substrate ATCI or BTCI to generate product thiocholine, which reacts with the developer DTNB to generate a yellow compound. The amount of product thiocholine is determined indirectly by colorimetry, so that the amount of hydrolysis product thiocholine reflects the activity of AChE or BChE. Physostigmine (PSM) and galantamine (GLM) were used as positive controls. The specific detection operation is as follows: first, 12.5 μL of 0.50 U / mL AChE solution or 0.10 U / mL BChE solution (dissolved in phosphate buffer A) was added to a 96-well plate, and 12.5 μL of phosphate buffer A was added to the experimental control group. Second, 15 μL of phosphate buffer B (blank group), physostigmine or galantamine (positive control), compound I (experimental group) were added respectively, all samples were dissolved in phosphate buffer B; 15 μL of phosphate buffer B was added to the experimental control group. The highest final concentration of physostigmine and galantamine was 10 μM, 4-fold dilution, 7 concentration points (10 μM, 2.5 μM, 0.625 μM, 0.1563 μM, 0.0391 μM, 0.001 μM, 0.0025 μM); the highest final concentration of compound I was 100 μM, 4-fold gradient dilution, 7 concentration points (100 μM, 25 μM, 6.25 μM, 1.563 μM, 0.391 μM, 0.098 μM, 0.024 μM). Third, after shaking uniformly, 25 μl of ATCI or BTCI solution with a concentration of 2 mM (dissolved in phosphate buffer C) was added, and after mixing, it was placed in a 37℃ constant temperature incubator for incubation for 20 min. Fourth, 25 μl of DTNB solution with a concentration of 6 mM (dissolved in phosphate buffer C, containing 2% SDS by mass concentration) was added, and finally 22.5 μl of phosphate buffer B was added. After shaking uniformly, the absorbance value (A) was measured at 412 nm wavelength, and the enzyme inhibition rate was calculated. Enzyme activity inhibition rate (%) = [(A 空白组 -A 阳性控制 )-(A 实验组 -A 实验控制组 )] / (A 空白组 -A 阳性控制 )×100.

[0151] Table II, AChE and BChE inhibitory activity data of compound I

[0152]

[0153] Note: a indicates that the compound is not detected due to solubility problems.

[0154] The AChE and BChE inhibitory activity data of compounds I are shown in Table II, and the dose-dependent inhibition curves of AChE and BChE are shown in Figures 1 and 2, respectively. The test results show that six compounds have both AChE and BChE dual inhibitory activity, among which compounds I-1 and I-7 have the most significant activity (the inhibitory activity against AChE and BChE is less than 1 μM); in addition, five compounds have significant AChE inhibitory activity. Therefore, the compounds of the present application can be used to develop lead compounds for treating or ameliorating neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis, etc. Figure 2 and Figure 3 The AChE and BChE inhibitory activity data of compounds I are shown in Table II, and the dose-dependent inhibition curves of AChE and BChE are shown in Figures 1 and 2, respectively. The test results show that six compounds have both AChE and BChE dual inhibitory activity, among which compounds I-1 and I-7 have the most significant activity (the inhibitory activity against AChE and BChE is less than 1 μM); in addition, five compounds have significant AChE inhibitory activity. Therefore, the compounds of the present application can be used to develop lead compounds for treating or ameliorating neurodegenerative diseases such as Alzheimer's disease, vascular dementia, myasthenia gravis, etc.

Claims

1. A tetrahydropyrrolizino[2,3-c]quinoline alkaloid compound, a compound represented by the general structural formula I or a pharmaceutically acceptable salt thereof: wherein, R1-R4 are each independently H, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, hydroxyl, methoxyl, ethoxyl, propoxyl, isopropoxyl; n is 1, 2 or 3. ； 2. The compound of claim 1, wherein R1-R4 are each independently H, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, hydroxyl, methoxyl, ethoxyl, propoxyl, isopropoxyl. R1-R4 are each independently H, bromo, chloro, fluoro, iodo, C 1-3 alkyl, hydroxy, C 1-3 alkoxy; 3. The compound of claim 1, wherein n is independently 1, 2 or 3.

2. The compound of claim 1, wherein 4. The compound of claim 1, wherein R1-R4 are each independently H, F, Br, methyl, ethyl, propyl, hydroxyl, methoxyl, ethoxyl, propoxyl.

5. The compound of claim 1, wherein n is independently 1 or 2.

3. The compound of claim 2, wherein 6. The compound of claim 1, wherein R1-R4 are each independently H, Br, methyl, ethyl, methoxyl, ethoxyl.

7. The compound is one of the following:

4. The compound of claim 3, wherein 8. Use of the compound of any one of claims 1-7 as an active ingredient for the preparation of a medicament for treating or ameliorating a neurodegenerative disease, or as an active ingredient for the preparation of a cholinesterase inhibitor.

5. The compound of claim 1, wherein Compound I is one of racemic I-achiral, or 7a R or 7a S as shown in the following formulae I-achiral, I-7a S and I-7a R . 。 6. The compound of claim 1, wherein 9. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof as an active ingredient, in combination with any pharmaceutically acceptable adjuvant and / or other active compounds. 。 7. The compound of any one of claims 1-5, wherein 10. The pharmaceutical composition of claim 9, wherein the neurodegenerative disease is selected from Alzheimer's disease, vascular dementia or myasthenia gravis.

11. A method for treating or ameliorating a neurodegenerative disease, comprising the following steps: (1) extracting medicinal materials: taking dry whole grass with roots of Kudou- ding, crushing to obtain medicinal powder; extracting with 50-90% volume concentration of ethanol at 60-70℃ for 6-24 hours, filtering the extract with a 70-80 mesh filter screen, and then removing the solvent from the filtrate by rotary evaporation to obtain an extract; dissolving the extract with 0.01-0.2 mol / L sulfuric acid, adjusting the pH to 2-3 with 2% sulfuric acid solution, and then extracting with petroleum ether in a volume ratio of 1:1-1:2, and taking the water layer; adjusting the pH of the obtained water layer to 9-10 with 0.01-2 mol / L sodium hydroxide, extracting with dichloromethane in a volume ratio of 1:1-1:2, and obtaining the dichloromethane layer as a crude base; (2) purifying the crude base obtained in step (1) by first-dimensional separation with a C18HCE reverse-phase chromatographic column, using 0.1%-2% formic acid-methanol as the mobile phase A and 0.1%-2% formic acid-water as the mobile phase B, gradient elution conditions of 0-8 min: 25% A, 8-23 min: 40% A, 23-38 min: 80% A, 38-55 min: 100% A, taking the sample from 4.2-26 min, and concentrating to obtain a total alkaloid sample of Kudou-ding.

10. Use of a pharmaceutical composition according to claim 9, characterized in that: ​ 11. A method for preparing the compounds I-1, I-1a and I-1b according to claim 6 from the traditional Chinese medicinal material Kuding, characterized in that, ​ ​ ​ (3) The total alkaloids obtained in step (2) are subjected to second dimension separation and purification by C18HCE reverse phase chromatographic column, mobile phase A is 0.1%-2% formic acid-methanol by volume concentration, B is 0.1%-2% formic acid-water by volume concentration, gradient elution conditions are as follows: 0-5 min: 20% A, 5-50 min: 20-45% A, 50-60 min: 95% A, 11 sub-fractions F1: 5.7-10.7 min, F2: 10.7-17.1 min, F3: 17.1-20.9 min, F4: 20.9-25.7 min, F5: 25.7-29.2 min, F6: 29.2-33.0 min, F7: 33.0-36.3 min, F8: 36.3-40.4 min, F9: 40.4-44.4 min, F10: 44.4-55.6 min and F11: 55.6-60 min are collected in sequence according to the corresponding effluent time; (4) The sub-fraction F4 obtained in step (3) is subjected to third dimension separation and purification by C8CE reverse phase chromatographic column, mobile phase A is 20-25% ammonia water-methanol by mass concentration, B is 20-25% ammonia water-water by mass concentration, gradient elution conditions are as follows: 0-5 min: 5% A, 5-50 min: 5-95% A, 50-60 min: 95% A, 15 sub-fractions F4-1: 4.0-7.8 min, F4-2: 7.8-10.1 min, F4-3: 10.1-13.4 min, F4-4: 13.4-18.1 min, F4-5: 18.1-21.3 min, F4-6: 21.3-23.7 min, F4-7: 23.7-28.5 min, F4-8: 28.5-30.7 min, F4-9: 30.7-33.6 min, F4-10: 33.6-35.0 min, F4-11: 35.0-37.6 min, F4-12: 37.6-39.5 min, F4-13: 39.5-43.9 min, F4-14: 43.9-46.1 min, F4-15: 46.1-57.0 min are collected in sequence according to the corresponding effluent time; (5) The sub-fraction F4-13 obtained in step (4) is subjected to fourth dimensional separation and purification using an FC8HL reverse phase chromatographic column, the mobile phase A is 5-50 mM ammonium formate-95% methanol / water, B is 5-50 mM ammonium formate-water, the gradient elution conditions are 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 10 sub-fractions are collected according to the chromatographic peaks in turn as F4-13-1: 1.5-2.5 min, F4-13-2: 9.1-9.6 min, F4-13-3: 9.6-11.0 min, F4-13-4: 11.0-11.6 min, F4-13-5: 11.6-12.4 min, F4-13-6: 12.4-13.6 min, F4-13-7: 13.6-14.5 min, F4-13-8: 14.5-16.2 min, F4-13-9: 16.2-16.9 min, F4-13-10: 16.9-19.2 min; (6) The sub-fraction F4-13-10 obtained in step (5) is subjected to fifth dimensional separation and purification by C18HCE, the mobile phase A is formic acid-methanol with a volume concentration of 0.1% to 2%, B is formic acid-water with a volume concentration of 0.1% to 2%, the gradient elution condition is 0-5 min: 25% A, 5-15 min: 25-35% A, and natural product I-1 is collected according to the chromatographic peak t R =6.58~7.07min; (7) The natural product I-1 obtained in step (6) is separated and purified by supercritical fluid chromatography through a BH chiral column, a mobile phase A is CO2, B is triethylamine-methanol with a volume concentration of 0.1% to 2%, and gradient elution conditions are 0-12 min: 80% A, and the natural product I-1a, t R =4.0~6.5min and I-1b, t R =6.5~8.0min.

12. A method of synthesizing a compound I according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, characterized in that, The synthesis method is as shown below: Synthesis method 1 ； In the reaction formula, R1-R4 and n are the same as defined in any one of claims 1-4 above. Reaction a 1 mmol, 1.0 molar equivalent of substrate 1 is dissolved in 10-100 ml of THF, then 1-3 molar equivalents of substrate 2 relative to substrate 1, 0.01-0.1 molar equivalents of Pd(PPh3)2Cl2 relative to substrate 1, 0.01-0.1 molar equivalents of CuI relative to substrate 1, 0.01-0.1 molar equivalents of PPh3 relative to substrate 1, and 1-5 molar equivalents of triethylamine relative to substrate 1 are added, and the solution is stirred at room temperature; after the reaction system is replaced with a nitrogen atmosphere, it is heated to 30-60°C and continues to be stirred for 5-24 hours; after the reaction is completely monitored by LC-MS or thin layer chromatography plate TLC, the reaction liquid is reduced to room temperature, filtered, the filtrate is concentrated by rotary evaporation, and the concentrate is purified by silica gel column chromatography to obtain product 3 as substrate 3; Reaction b 1 mmol, 1.0 molar equivalent of substrate 3 is dissolved in 10-50 ml of acetone, and 1-10 ml of water, 1-3 molar equivalents of mercury sulfate relative to substrate 3, and 1-3 molar equivalents of 98% concentrated sulfuric acid relative to substrate 3 are added in sequence under stirring, and stirred at 25-40°C for 24-72 hours; after the reaction is completely monitored by LC-MS or TLC, the reaction liquid is neutralized with saturated sodium carbonate solution and sulfuric acid to pH 8-9, and extracted with 10-50 ml of ethyl acetate 2-5 times each time, the organic phase is dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain the corresponding product 4 as substrate 4; Reaction c Dissolve 1 mmol, 1.0 molar equivalent of substrate 4 in 2-20 ml of methanol, add 1-5 molar equivalent of acetic acid and 1-5 molar equivalent of benzylamine relative to substrate 4, stir at 25 °C for 1-12 hours, then add 1-5 molar equivalent of sodium cyanoborohydride relative to substrate 4, stir at 25 °C for 2-24 hours; add 2-20 ml of water to the reaction solution and stir for 0.5-1 hour, wash 2-5 times with 2-20 ml of ethyl acetate each time, discard the organic phase, add concentrated hydrochloric acid with a mass concentration of 38% to the aqueous phase to adjust the pH to 2-3, add 0.1-2 mass equivalent of 10 wt% palladium on carbon relative to substrate 4, stir at 25 °C under normal pressure for 3-24 hours under hydrogen; add saturated sodium carbonate solution to the reaction solution until the pH is 8-9, extract 2-5 times with 2-20 ml of ethyl acetate each time, dry the organic phase over anhydrous sodium sulfate, concentrate by rotary evaporation, and purify by silica gel column chromatography to obtain the target product I; Reaction d Dissolve 1 mmol, 1.0 molar equivalent of substrate 4 in 2-20 ml of methanol, add 1-5 molar equivalent of acetic acid and 1-5 molar equivalent of p-methoxybenzylamine relative to substrate 4, stir at 25 °C for 1-5 hours, then add 1-5 molar equivalent of sodium cyanoborohydride relative to substrate 4, stir at 25 °C for 2-24 hours; remove methanol from the reaction solution under reduced pressure, add saturated sodium carbonate solution to the reaction solution until the pH is 8-9, extract 2-5 times with 2-20 ml of ethyl acetate each time, dry the organic phase over anhydrous sodium sulfate, concentrate by rotary evaporation, and purify by silica gel column chromatography to obtain the corresponding product 5 as a substrate 5; Reaction e Dissolve 1 mmol, 1.0 molar equivalent of substrate 5 in 2-20 ml of acetonitrile and 8-80 ml of water, add 1-3 molar equivalent of 2,3-dichloro-5,6-dicyano-p-benzoquinone relative to substrate 5, heat to 70 °C and stir for 1-12 hours, remove acetonitrile from the reaction solution under reduced pressure, add 2-5 ml of 2N hydrochloric acid and an equal volume of ethyl acetate, separate the layers, extract the organic phase with 1-5 ml of 2N hydrochloric acid 2-5 times, discard the organic phase, combine the acid aqueous phases, and adjust the pH to 8-9 with saturated sodium carbonate solution, extract 2-5 times with 2-20 ml of ethyl acetate each time, dry the organic phase over anhydrous sodium sulfate, concentrate by rotary evaporation, and purify by silica gel column chromatography to obtain the target product I; Alternatively, when R1-R4 in compound I are all not Br and at least one is H, compound I can be further modified using synthesis method 2, as shown below: Synthesis method 2 ； Br in compound I-a can be substituted at any one or both of R2-R4, R refers to the other R1-R4 substituent(s) in addition to Br which is substituted under reaction condition f, R' in I-b refers to the group after substitution of the Br group under reaction condition g; "alkyl boronic acid or boronic ester" refers to C 1-3 alkyl boronic acid or boronic ester; both compounds I-a and I-b are within the scope of the definition of compound I; Reaction f 1 mmol, 1.0 molar equivalent of substrate I was dissolved in 1-10 ml of acetic acid, 1-5 molar equivalents of N-bromosuccinimide relative to substrate I was added under ice bath, stirred at room temperature for 0.5-1 hour, saturated sodium sulfite solution was added to quench the reaction; the reaction solution was concentrated under reduced pressure, saturated sodium carbonate solution was added to the pH range 8-9, 2-20 ml of dichloromethane was used to extract 2-5 times, the organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and then purified by silica gel column chromatography to obtain the target product I-a as compound I-a; Reaction g Dissolve 1 mmol, 1.0 mole equivalent of compound I-a in 1-10 ml of dioxane, add 1-5 ml of water, 1-5 mole equivalent of C 1-3 alkyl boronic acid or borate substrate, 0.01-0.1 mole equivalent of Pd(PPh3)4 relative to substrate I-a, and 1-5 mole equivalent of potassium carbonate relative to substrate I-a, nitrogen protection, 50-100°C stirring for 8-24 hours; the reaction solution is reduced to room temperature, filtered, the filter cake is washed with ethyl acetate, 2-20 ml each time, the filtrate is washed with saturated brine 2-5 times, dried with anhydrous sodium sulfate, concentrated by rotary evaporation, and then purified by silica gel column chromatography to obtain the target product I-b.

13. The synthesis method according to claim 12, characterized in that, When compound I is a chiral monomer, racemate can be resolved by chiral chromatography: Resolution method The racemic compound I is dissolved in methanol and purified by supercritical fluid chromatography over a BH chiral column, using as mobile phase A CO2 and B 0.1-2% triethylamine in methanol, gradient elution conditions 0-12 min: 80% A, and collecting the chromatographic peaks to obtain the chiral monomers I- S and I- R ; and / or, when compound I forms a salt with a pharmaceutically acceptable acid HX, these acids refer to one of HCl, HBr, trifluoroacetic acid, sulfuric acid, formic acid, acetic acid, succinic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, citric acid, and the synthesis method is as follows: Synthesis method 3 ； Reaction h 1 mmol, 1.0 molar equivalent of compound I was dissolved in 1-10 ml of dioxane or tetrahydrofuran or dichloromethane or ethyl acetate, 1-3 molar equivalents of HX relative to substrate I was added, stirred at room temperature for 0.5-2 hours, concentrated under reduced pressure, and the solid was precipitated as the target product I-X.

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