CN114230582B - Novel securinine dimer and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of medicines, and in particular relates to application of a novel securinine dimer or pharmaceutically acceptable salt thereof in preparation of a medicine for treating Alzheimer disease. The novel securinine dimer has a structural formula shown in a general formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof:

in the formula: the linking group Linker is selected from:

R ¹ is a hydrogen atom; x is selected from

Or

Description

Novel securinine dimer and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, and relates to a medicine for treating Alzheimer disease. In particular to application of a novel securinine dimer in preparing a medicament for treating Alzheimer disease.

Background

Alzheimer's Disease (AD), commonly known as senile dementia, is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and memory impairment, and the common clinical symptoms are progressive memory disorder, cognitive disorder, personality disorder, etc., and the pathological features of the brain are mainly a large number of neuronal loss, senile plaques composed of beta amyloid (Α β), and neurotangles composed of Tau protein. AD is the most common type of senile dementia, accounting for 60% -80% of all dementia patients, and has now become the third leading cause of death in the elderly following cardiovascular disease and cancer. Epidemiological investigations have shown that the worldwide incidence of AD continues to rise, with the expectation that by 2050, the number of patients will exceed 1.5 billion, which is certainly a public health and wellness problem that is not overlooked by all humans. However, since the pathophysiological changes of AD involve multiple factors, no specific therapeutic drugs have been available so far.

Currently, there are two major classes of anti-AD drugs approved by the FDA for marketing, the first class being acetylcholinesterase inhibitors (achei), such as donepezil, galantamine, tacrine, rivastigmine, and the like. The medicine can recover the acetylcholine level in the brain of AD patients to a certain extent by inhibiting the acetylcholine hydrolysis, temporarily delay the course of disease and improve the cognitive function. The second class is N-methyl-D-aspartic acid (NMDA) receptor antagonists, representing the drug as memantine. The medicine can relieve neuron damage caused by overexcitation of glutamatergic system, improve learning and memory functions to a certain extent, and relieve symptoms of middle and late AD patients (Cummings JL, et al, alzheimer Res Ther,2014,6 (4): 37). However, the therapeutic strategies of these drugs focus mainly on the compensation of neurotransmitters and the inhibition of specific enzymes or receptors, which are mainly used to alleviate the symptoms of AD, and cannot reverse the phenomena of severe loss of synapses and neuronal damage occurring in the late stage of the disease process, thereby fundamentally preventing or reversing the progress of the disease. A large number of studies indicate that in the course of AD, early processes such as loss or dysfunction of the neural synapses can gradually progress to overall damage and apoptosis of neurons, resulting in destruction of neural circuits that function as cognition, while restoration of neural function requires remodeling of the neural synapses and reconstruction of related neural circuits. Recently, the development of AD drugs for antagonizing traditional pathogenic factors such as a β protein and Tau protein has been successively proved to fail, and recently, the effect of improving cognition of adacanumab, which is a monoclonal antibody drug for clearing a β, is still controversial although FDA approved. Among these, in part, it is believed that simple clearance of harmful substances such as A β protein or Tau protein in the middle-late phase of AD has failed to repair the damaged synapses and the neural circuits that they make up (Ferreira ST, et al, front Cell Neurosci,2015, 191, overk CR, et al, biochem Pharmacol,2014,88 (4): 508-516). Therefore, the development of drugs or intervention methods for promoting or protecting the neurosynaptic function is an important new approach for the development of AD drugs. The biosynthesis of proteins determines the precise expression of proteins in various physiological activities of cells and is a key regulatory site for adaptation and survival of cells under stress conditions. Since neurons are specialized cells with high demand for energy metabolism, protein synthesis needs to dynamically occur in dendrites or synapses distant from the cell body, which determines the precise occurrence of synapse formation and plasticity, and maintains the brain's functions of learning and memory. When protein synthesis is decreased, synthesis of important synaptic proteins such as glutamate receptors, synaptophysins, etc. is hindered, affecting the structure and function of the synapse, leading to a variety of neurological dysfunctions (Tom DS, et al, neuron,2014,81 (4): 958-958, buffington SA, et al, annu Rev neurosci,2014, 37. In recent years, abnormal or sustained inhibition of protein synthesis has become one of the generally recognized pathological mechanisms of neurological diseases such as AD. The level of protein synthesis in brain tissue samples from AD patients and model animals is significantly reduced and protein homeostasis is disrupted, resulting in insufficient levels of important synaptoproteins. In animal models of AD, it has been demonstrated that neurodegenerative disorders in model animals can be effectively alleviated or reversed by restoring or partially restoring the level of protein synthesis in neurons (Hernandez-Ortega K, et al, brain Pathol,2016,26 (5): 593-605 Yang WZ, et al, neurobiol aging,2016, 41. Therefore, by restoring the protein synthesis level of neurons, it is expected to increase the expression of synaptoprotein in the brain of AD patients and improve the function of neurosynapses, thereby becoming a new strategy for effectively treating AD.

However, the compounds are metabolized and eliminated in animals quickly, which influences the good therapeutic effect of the compounds in the animals. Chinese patent CN104761572B discloses a securinega suffruticosa type alkaloid dimer compound or a pharmaceutically acceptable salt thereof which can be applied to the preparation of drugs for treating neurodevelopment, nerve injury, neurodegenerative diseases and learning and memory disorders. Some simple types of securinine dimer compounds (e.g., SN 3-L6) can significantly up-regulate the level of neuronal protein synthesis, while having the effects of promoting neuronal differentiation and enhancing neurosynaptic function (Tang G, et al, ACS Chem Neurosci,2016,7 1442-1451 liao y, et al, front pharmacol,2018, 290), but these compounds are metabolized faster in animals, affecting their good therapeutic effects in vivo.

Therefore, it is necessary to develop a drug for improving the effect of the securinega suffruticosa alkaloid dimer compound in treating AD, which can solve the above technical problems.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel securinine dimer compound for treating Alzheimer disease.

The invention is realized by the following technical scheme:

a novel securinine dimer having a structural formula shown in formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof:

in the formula:

the linking group Linker is selected from:

r1 is a hydrogen atom;

x is selected from

Or

n is 1, 2, 3 or 4.

In a preferred embodiment of the present invention, the compounds represented by formula II include, but are not limited to:

in another preferred embodiment of the present invention, the compound represented by the general formula I, wherein Linker = B, has the structural formula represented by the general formula III:

in a preferred embodiment of the present invention, the compounds of formula III include, but are not limited to:

the invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of securinine dimer shown in general formulas I, II and III, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or prodrug molecule thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention relates to application of securinine dimer shown in general formulas I, II and III, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or prodrug molecule thereof, or pharmaceutical composition thereof containing therapeutically effective dose in preparing a medicament for treating Alzheimer disease.

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

"prodrug" means a prodrug that is converted in vivo to the structure of the compounds referred to herein and pharmaceutically acceptable salts thereof.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

Synthesis method of compound in the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the first scheme comprises the following steps:

the preparation method of the compound shown in the general formula II comprises the following steps:

at room temperature, securinine (1.0 equiv.) is dissolved in a stirred dichloromethane solvent, and trimethylsilyl azide (5.0 equiv.), acetic acid (5.0 equiv.) and DBU (0.05 equiv.) are added successively. Heating to 35 ℃, reacting for 8 hours, and concentrating the organic solvent under reduced pressure to obtain a crude product. The crude product is separated and purified by silica gel column chromatography to obtain a pair of diastereoisomer azide compounds N10a and N10b. Subsequently, azide compound N10a or N10b (1.0 equiv.) was dissolved in the stirred DMSO solvent, followed by the addition of copper (2 equiv.), copper sulfate pentahydrate (0.05 equiv.) and diyne (0.27 equiv.). After the reaction was carried out for 5 hours in the dark, ethyl acetate was added, and the filtrate was obtained by filtration. The filtrate was washed with water 3 times, with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. And separating and purifying the obtained crude product by silica gel column chromatography to obtain a compound II with a general formula.

X is as defined in claim 1.

Scheme two is as follows:

the preparation method of the compound shown in the general formula III comprises the following steps:

azide compound N10a or N10b (1.0 equiv.) was dissolved in stirred DMSO solvent, followed by the addition of copper (2.0 equiv.), copper sulfate pentahydrate (0.05 equiv.) and compound III-1 (0.27 equiv.) of formula (la). After 5 hours of light-shielding reaction, ethyl acetate was added and the filtrate was filtered. The filtrate was washed with water 3 times, with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. And separating and purifying the obtained crude product by silica gel column chromatography to obtain a compound III with a general formula.

The invention has the beneficial effects that:

the invention develops novel securinine dimer compounds taking triazole as a connecting group by changing the connecting group (Linker), the novel securinine dimer compounds show more excellent activities of inducing neural differentiation and promoting protein synthesis than simple securinine dimer compounds (such as SN 3-L6), and have better stability in cells and animal bodies, the cognitive function of AD model mice is obviously improved after administration, and the novel securinine dimer compounds are expected to be developed into effective treatment drugs for AD.

Drawings

FIG. 1 shows the effect of novel securinine dimer compound (25. Mu.M) in promoting the synthesis of neo-2 a cell neogenesis protein

FIG. 2 Effect of Compound 3 on promoting Neogenin Synthesis in Neuro-2a cells at Low concentration

FIG. 3 improvement of memory cognitive function in New object recognition test in AD model mice by Compound 3

FIG. 4 Compound 3 improves spatial learning and memory function in Morris water maze test in AD model mice

Detailed Description

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shifts (. Delta.) are given as 10-6 (ppm). NMR was measured using Bruker AVANCE-300 and Bruker AVANCE-400 nuclear magnetic resonance spectrometers in deuterated chloroform (CDCl 3) as solvent and Tetramethylsilane (TMS) as internal standard.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: thermo, model: finnigan LCQ advantage MAX).

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

Known starting materials for the present invention can be synthesized by or according to methods known in the art, or can be purchased from Acros Organics, aldrich Chemical Company, shao Yuan Chemical technology (Accela ChemBio Inc), carbofuran, annagi, darril Chemicals, and the like.

The reactions were carried out under an argon atmosphere or a nitrogen atmosphere, unless otherwise specified in the examples.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

In the examples, the solution means an aqueous solution without specific indication.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, an eluent system for column chromatography for separation and purification of the compound, and a developing solvent system for thin layer chromatography including: a: a dichloromethane/methanol system; b: n-hexane/ethyl acetate systems; c: petroleum ether/ethyl acetate systems; d: acetone; e: a dichloromethane/acetone system; f: ethyl acetate/dichloromethane systems; g: ethyl acetate/dichloromethane/n-hexane; h: ethyl acetate/dichloromethane/acetone. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can be added for adjustment.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The specific compounds prepared in the following examples include, but are not limited to, those shown in tables 1-10 above.

EXAMPLE 1 general preparation of Compounds N1 to N6

At room temperature, securinega suffruticosa alkaloid (217mg, 1mmol) was dissolved in a stirred mixed solvent of dichloromethane (5 mL) and methanol (0.5 mL), and potassium phosphate (K) was added successively ₃ PO ₄ 64mg,0.3 mmol) and the corresponding primary amine (R-NH) ₂ 5 mmol). After the temperature was raised to 35 ℃, the reaction was carried out for 8 hours. The organic solvent was concentrated under reduced pressure to give the crude product. And separating and purifying the obtained crude product by silica gel column chromatography to obtain corresponding compounds N1-N6.

EXAMPLE 2 preparation of Compound 1

Securinine (2.17g, 10mmol) was dissolved in a stirred dichloromethane (50 mL) solvent at room temperature, and trimethylsilyl azide (6.58mL, 50mmol), acetic acid (2.86mL, 50mmol) and DBU (75L, 0.5mol) were added successively. Heating to 35 ℃, reacting for 8 hours, and concentrating the organic solvent under reduced pressure to obtain a crude product. The crude product is separated and purified by silica gel column chromatography to obtain a pair of diastereoisomer azide compounds N10a and N10b. Subsequently, azide N10a was dissolved in a stirred DMSO (2 mL) solvent, and copper (127mg, 2mmol), copper sulfate pentahydrate (13mg, 0.05mmol) and 1, 5-hexadiyne (26. Mu.L, 0.27 mmol) were added successively. After 5 hours in the dark, ethyl acetate (10 mL) was added and the mixture was filtered to obtain a filtrate. The filtrate was washed 3 times with water (3X 10 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The obtained crude product was purified by silica gel column chromatography to obtain compound 1 as a white solid (total yield: 51%).

1H NMR(300MHz,CDCl3)δ7.55(s,2H),5.68(s,2H),4.70(t,J＝8.3Hz,2H),3.34(d,J＝6.1Hz,2H),3.20(d,J＝7.4Hz,4H),3.04(s,5H),2.79(dt,J＝11.0,9.6Hz,4H),2.35(d,J＝13.5Hz,2H),1.64–1.48(m,4H),1.39–1.11(m,10H)；13C NMR(75MHz,CDCl3)δ172.3,169.9,147.1,119.2,111.8,90.1,64.5,63.3,60.6,49.5,35.8,29.2,25.9,25.4,23.9,21.2；HR-ESI-MS m/z 599.3086[M+H]+。

EXAMPLE 3 preparation of Compound 2

Referring to the synthesis method of example 2, using azide compound N10b as a starting material, compound 2 was obtained as a white solid (yield 47%).

1H NMR(300MHz,CDCl3)δ7.30(d,J＝10.2Hz,2H),5.78(d,J＝22.4Hz,2H),4.81(s,2H),3.58(s,2H),3.38(s,4H),3.03(s,9H),2.43(dd,J＝11.1,5.7Hz,2H),1.86(s,2H),1.70–1.13(m,13H)；13CNMR(75MHz,CDCl3)δ172.3,171.1,146.9,120.4,111.5,90.4,64.1,60.1,48.7,32.3,28.1,25.9,25.0,23.9,21.7；HR-ESI-MS m/z 599.3088[M+H]+。

EXAMPLE 4 preparation of Compound 3

Referring to the synthesis method of example 2, azide compound N10a as a starting material was reacted with 1, 6-heptadiyne to obtain compound 3 as a white solid (yield: 69%).

1H NMR(300MHz,CDCl3)δ7.55(s,2H),5.71(s,2H),5.26(d,J＝5.7Hz,1H),4.73(t,J＝8.2Hz,2H),3.40(d,J＝6.0Hz,2H),3.18(dd,J＝32.4,6.3Hz,6H),2.93–2.67(m,8H),2.45(d,J＝13.4Hz,2H),2.08–1.95(m,2H),1.61(t,J＝9.5Hz,4H),1.44–1.11(m,10H),0.82(s,1H)；13C NMR(75MHz,CDCl3)δ172.2,170.0,147.4,119.0,111.6,90.2,64.8,63.2,61.1,49.9,36.3,29.2,26.4,25.1,23.6,21.1；HR-ESI-MS m/z 613.3245[M+H]+。

EXAMPLE 5 preparation of Compound 4

Referring to the synthesis procedure of example 2, azide compound N10b as a starting material was reacted with 1, 6-heptadiyne to obtain compound 4 as a white solid (yield: 54%).

1H NMR(300MHz,CDCl3)δ7.28(d,J＝8.8Hz,2H),5.83(s,2H),5.28(s,1H),4.78(d,J＝27.5Hz,2H),3.60(s,2H),3.42(d,J＝13.5Hz,4H),3.03(s,6H),2.70(t,J＝7.4Hz,4H),2.47(dd,J＝11.3,5.9Hz,2H),2.04–1.94(m,2H),1.88(s,1H),1.61(s,2H),1.50–1.41(m,5H),1.38(d,J＝8.5Hz,4H),1.23(s,1H),0.85(t,J＝6.7Hz,1H)；13C NMR(75MHz,CDCl3)δ172.5,171.2,147.6,120.1,111.5,90.2,64.2,60.1,48.7,32.5,28.8,28.2,26.1,24.7,24.0,21.7；HR-ESI-MS m/z 613.3245[M+H]+。

EXAMPLE 6 preparation of Compound 5

Referring to the synthesis procedure of example 2, azide compound N10a as a starting material was reacted with 1, 7-octadiyne to obtain compound 5 as a white solid (yield 75%).

1H NMR(300MHz,CDCl3)δ7.50(s,2H),5.71(s,2H),4.73(t,J＝8.3Hz,2H),3.40(d,J＝5.9Hz,2H),3.32–3.06(m,6H),2.90–2.78(m,3H),2.70(s,4H),2.45(d,J＝13.5Hz,2H),1.75–1.55(m,8H),1.41–1.16(m,9H),0.84(d,J＝5.6Hz,1H).13C NMR(75MHz,CDCl3)δ172.5,169.9,148.4,118.6,112.1,90.4,64.7,63.8,61.0,49.9,36.3,29.3,28.9,26.0,25.4,23.9,21.3；HR-ESI-MS m/z627.3390[M+H]+。

EXAMPLE 7 preparation of Compound 6

Referring to the synthesis method of example 2, 1, 7-octadiyne was reacted with azide compound N10b as a starting material to obtain compound 6 as a white solid (yield 60%).

1H NMR(300MHz,CDCl3)δ5.78(s,2H),5.24(s,1H),4.77(s,2H),3.55(s,2H),3.43–3.21(m,4H),2.98(s,6H),2.62(s,4H),2.40(dd,J＝10.6,5.6Hz,2H),1.82(s,2H),1.60(d,J＝17.4Hz,4H),1.52(s,2H),1.46–1.13(m,11H)；13C NMR(75MHz,CDCl3)δ172.2,171.0,147.6,119.8,111.3,90.3,63.9,60.2,60.0,48.5,32.2,28.4,27.9,26.0,25.0,23.8,21.5；HR-ESI-MS m/z 627.3382[M+H]+。

EXAMPLE 8 preparation of Compound 7

Referring to the synthesis of example 2, azide compound N10a as a starting material was reacted with propargyl ether to obtain compound 7 as a white solid (yield 57%).

1H NMR(300MHz,CDCl3)δ7.76(s,2H),5.62(s,2H),4.77–4.40(m,6H),3.40–3.00(m,8H),2.80–2.70(m,4H),2.35(s,2H),1.55–1.45(m,4H),1.30–1.00(m,10H)；13C NMR(75MHz,CDCl3)δ171.9,169.7,144.1,120.7,111.3,89.8,64.2,63.2,63.0,60.4,53.4,49.2,35.5,28.9,25.6,23.5,20.9；HR-ESI-MS m/z 615.3038[M+H]+。

EXAMPLE 9 preparation of Compound 8

Referring to the synthesis of example 2, azide compound N10b as a starting material was reacted with propargyl ether to give compound 8 as a white solid (yield 57%).

1H NMR(300MHz,CDCl3)δ7.58(d,J＝3.8Hz,2H),5.80(s,2H),5.25(s,1H),4.90–4.55(m,6H),3.65–3.27(m,6H),3.01(s,6H),2.43(dd,J＝10.6,5.5Hz,2H),1.48–1.24(m,11H)；13C NMR(75MHz,CDCl3)δ172.2,171.0,144.5,122.1,111.5,90.4,64.0,63.4,48.6,32.3,29.6,28.0,25.9,23.8,21.4.；HR-ESI-MS m/z 615.3038[M+H]+。

EXAMPLE 10 preparation of Compound 9

Referring to the synthesis method of example 2, compound N4 was reacted with azide compound N10a as a starting material to obtain compound 9 as a white solid (yield 56%).

1H NMR(300MHz,CDCl3)7.67(s,1H),5.74(s,1H),5.58(s,1H),4.77(t,J＝8.4Hz,1H),3.89–3.78(m,2H),3.45(d,J＝6.5Hz,1H),3.33–3.20(m,3H),3.18–3.09(m,2H),2.92–2.81(m,6H),2.61(d,J＝16.4Hz,1H),2.48(d,J＝10.9Hz,2H),2.14(d,J＝1.1Hz,1H),1.92(d,J＝10.5Hz,2H),2.00–1.70(m,3H),1.65(d,J＝11.1Hz,1H),1.63–1.53(m,2H),1.43–1.22(m,8H)；13C NMR(75MHz,CDCl3)174.2,173.0,172.2,169.7,146.6,119.6,111.8,111.0,91.3,90.1,64.6,63.6,62.8,60.8,59.8,58.9,49.7,48.7,42.7,36.0,32.3,30.1,29.4,26.0,25.7,23.9,23.7,21.4,21.2；HR-ESI-MS m/z 533.2873[M+H]+。

EXAMPLE 11 preparation of Compound 10

Referring to the synthesis method of example 2, azide compound N10b as a starting material was reacted with compound N4 to obtain compound 10 as a white solid (yield: 64%).

1H NMR(300MHz,CDCl3)7.42(s,1H),5.82(s,1H),5.58(s,1H),4.85–4.80(m,1H),3.86–3.74(m,2H),3.61–3.58(m,1H),3.39(d,J＝3.4Hz,1H),3.26–3.23(m,1H),3.12–3.00(m,4H),2.92–2.80(m,4H),2.64–2.60(m,3H),2.50–2.43(m,1H),2.15(s,1H),1.92–1.85(m,4H),1.59–1.28(m,10H)；13C NMR(75MHz,CDCl3)174.2,173.1,172.3,171.0,146.5,120.7,111.6,111.0,91.3,90.5,64.1,62.8,60.6,60.1,59.8,59.2,48.8,48.7,42.6,41.0,32.5,32.4,30.1,28.1,26.1,25.8,24.0,23.8,22.6,21.7,21.5；HR-ESI-MS m/z 533.2865[M+H]+。

Example 12 novel securinine dimer Compound neural differentiation promoting action

(1) The experimental method comprises the following steps: after recovery of Neuro-2a cells (purchased from American type culture collection cell bank), they were cultured in a growth medium (MEM +10% FBS +100U/mL of penicillin and 100. Mu.g/mL of streptomycin), plated in a 100mm dish, and cultured in a constant temperature incubator containing 5% CO2 at 37 ℃. And (3) carrying out passage when the cells grow to 60-70%, sucking out the culture solution in a culture dish, adding a proper amount of PBS (phosphate buffer solution), washing, digesting for 45s by using 0.25% trypsin, adding a growth culture medium after the adherent cells are spherical, stopping digestion, uniformly mixing, and carrying out passage by 1. When the differentiation of the neural cell line was induced, the cell density was 2X 104 cells/35 mm dish or 1X 104 cells/well (12-well plate), the culture medium was changed to the differentiation medium (MEM +0.5% FBS +100U/mL penicillin and 100. Mu.g/mL streptomycin) after culturing for 24 hours, and the mixture was treated with 25. Mu.M of the securinine dimer compound for 48 hours. The differentiation morphology and neurite of the nerve cell strain are observed by an immunofluorescence staining method, and the method comprises the following specific steps:

(1) washing the cells with PBS (containing Ca2+/Mg2 +) for 1 time, adding 800 mu L of 4% paraformaldehyde/4% sucrose, and fixing for 20-30 min at room temperature;

(2) washing with PBS for 3 times, adding blocking solution (PBS containing 1% bovine serum albumin, 4% sheep serum albumin and 0.4% Triton X-100) and blocking for 20min;

(3) the blocking solution was discarded, a primary antibody dilution (PBS containing 1% bovine serum albumin, 1% goat serum albumin and 0.4% Triton X-100) containing the β -tubulin III antibody (1;

(4) washed 3 times with PBS, diluted with a fluorescent secondary antibody Alexa Fluor-488 coat anti-mouse IgG antibody (1;

(5) the plate was washed 3 times with PBS and mounted.

Photographs were taken with an upright fluorescence microscope and statistically analyzed using Metamorph software. Cells with neurite length greater than 20 μm are defined as differentiated neural cells. There are two main parameters for measurement analysis: 1) The rate of cell differentiation; 2) Total neurite length per differentiated cell.

The experimental results are as follows: as shown in Table 1, compounds 1-10 with triazole as a connecting group have the activity of promoting neural differentiation at 25 mu M; wherein, the activity of the triazole compound 3 in the two aspects of promoting the differentiation of nerve cells and promoting the growth of neurite is obviously superior to that of a simple securinine dimer compound SN3-L6. As shown in Table 2, compound 3 still had a pro-neural differentiation activity at a low dose (1. Mu.M), whereas SN3-L6 was inactive at this concentration. Therefore, the securinine dimer compound SN3-L6 with the simpler compound 3 shows stronger effect of promoting neural differentiation.

TABLE 1 Activity of novel securinine dimer compound for promoting Neuro-2a cell differentiation at 25. Mu.M dose

The data of the experimental results are expressed as mean ± s.e.m (standard error of the mean) with three separate experiments, each group analyzing at least 100 cells and being statistically processed using an unpaired t-test method,. P <0.05,. P <0.01,. P <0.001.

TABLE 2 Activity of Compound 3 to promote differentiation of Neuro-2a cells at a dose of 1. Mu.M

The data of the experimental results are expressed as mean ± s.e.m (standard error of the mean) with three independent experiments, each group analyzing at least 100 cells and being statistically processed using an unpaired t-test method,. P <0.05,. P <0.01.

Example 13 novel securinine dimer Compound promoting Synthesis of neogenetic protein in nerve cells

The experimental method comprises the following steps: the synthesis amount of the neogenetic protein of the nerve cells treated by the securinine dimer compound is determined by a puromycin (puromycin) labeling method. Neuro-2a cells were seeded at a density of 6X 105 cells/35 mm dish in a 35mm dish, cultured overnight in a constant temperature incubator containing 5% CO2 at 37 ℃ and then mixed with securinine dimer compound to a final concentration of 25. Mu.M, and DMSO was added to the same volume as that of the control group to incubate for 1 hour in a constant temperature incubator containing 5% CO2 at 37 ℃. Then, 1. Mu.M puromycin was added to the medium, and the mixture was further cultured in a constant temperature incubator containing 5% CO2 at 37 ℃ for 0.5 hour. Subsequently, the cells were washed twice with ice D-PBS, and the cell-disrupted proteins were collected with RIPA lysate (containing protease inhibitor), followed by Western blot analysis to detect the content of puromycin-labeled nascent proteins with an antibody to puromycin.

The experimental results are as follows: as shown in figure 1, compounds 1-10 all had significant effect of promoting nerve cell protein synthesis at 25 μ M dose. As shown in FIG. 2, compound 3 still has the effect of promoting protein synthesis at low doses of 10. Mu.M and 1. Mu.M, while simple securinine dimer analog SN3-L6 is inactive at 1. Mu.M, indicating that compound 3 shows a stronger effect of promoting protein synthesis in nerve cells than the simple securinine dimer analog SN3-L6.

The figure 1 is a bar chart of Western blot for detecting the content of puromycin marked nascent proteins. Neuro-2a cells were treated with 25. Mu.M compound for 1h, followed by 1. Mu.M puromycin for 0.5h, and DMSO group was used as a control group. The data of the experimental results are expressed as mean ± s.e.m (standard error of the mean), with three independent experiments and statistical treatment using One-way ANOVA test with P <0.01 and P <0.001.

The figure 2 is a bar chart of Western blot for detecting the content of puromycin-labeled nascent protein. Neuro-2a cells were treated with 1. Mu.M or 10. Mu.M compound for 1h, followed by 1. Mu.M puromycin for 0.5h, and DMSO group was used as a control group. The data for the experimental results are expressed as mean ± s.e.m (standard error of the mean), with three independent experiments and statistical treatment using One-way ANOVA test, P <0.05, differential components vs DMSO.

EXAMPLE 14 determination of the intracellular enrichment Capacity of Compound 3

Earlier researches find that the in vivo metabolism of the simple securinine dimer compound SN3-L6 is faster, so we firstly test the intracellular enrichment capacity of the triazole compound 3 and compare the intracellular enrichment capacity with the SN3-L6.

(1) The experimental method comprises the following steps: after 6X 105 Neuro-2a cells were plated on a 35mm dish and placed overnight in a constant temperature incubator containing 5% CO2 at 37 ℃, 25. Mu.M of Compound 3 and SN3-L6 were added to the growth medium, as follows:

(1) after 1h and 24h incubation with compound addition, dishes were washed 3 times with ice D-PBS, 396 μ L of saline was added to each dish, cells were scraped and transferred to 1.5mL EP tubes;

(2) after ultrasonic cracking on ice for 30min, adding 4 μ L of internal standard compound to make its final concentration 50ng/mL;

(3) adding 600 μ L ethyl acetate (analytically pure), vortexing for 10min, standing at room temperature for 10min until two liquid phases are separated, centrifuging at 10000rpm at 4 deg.C for 10min, collecting 500 μ L upper organic phase, and repeatedly extracting for 3 times;

(4) volatilizing the organic extraction solvent at 30 ℃ by a vacuum centrifugal concentrator;

(5) adding 400 μ L of 80% methanol into the sample for redissolution, performing ultrasonic treatment in room temperature water bath for 30min to completely dissolve the sample, and then vortexing for 2min; (6) centrifuging at 10000rpm for 10min at room temperature, taking 100 μ L of supernatant solution to a liquid phase analysis bottle, and detecting content by using a high resolution liquid phase mass spectrometer.

And calculating the content of the intracellular compound according to the formula f '= fi/fs = (Mi/Ai)/(Ms/As) and (Ai/As) × f' = Ci/Cs and a recovery formula. Intracellular compound concentration profiles at different time of action were then fitted by Graph prism 5.0 software.

The experimental results are as follows: as shown in Table 3, after incubation for 1h and 24h, the content of Compound 3 in Neuro-2a cells was higher than that of SN3-L6, indicating that Compounds 3 and 13 better enter and enrich in cells than the simple securinine dimer compound SN3-L6.

TABLE 3 determination of the amount of Compound 3 in Neuro-2a cells after different periods of action

Data for the experimental results are expressed as mean ± s.e.m (standard error of the mean) with three sets of parallel samples.

EXAMPLE 15 in vivo Metabolic analysis of Compound 3

According to the experimental results of examples 21 to 23, compound 3 has the best activity and the highest ability to enter cells, so that compound 3 is selected as a representative compound to study the metabolic condition in animals.

The experimental method comprises the following steps: compound 3 or SN3-L6 was administered to C57BL/6 mice at a dose of 25mg/kg by gavage, eyeball bleeding was performed at 4 time points of 5min, 30min, 60min and 120min after administration, respectively, and brain tissue was harvested, 3 mice per group. The method comprises the following specific steps:

injecting sodium pentobarbital into abdominal cavity of mouse, after anesthesia, picking eyeball to collect blood, collecting in 1.5mL centrifuge tube containing anticoagulant, and mixing by gently inverting. Taking 500 mu L of blood; centrifuging at 4 ℃ for 3800rpm and 20min; collecting upper layer plasma 300 μ L, adding 1mL of extractant (ethyl acetate), and vortexing for 5min;

perfusing the mouse with PBS, taking out the whole brain, weighing, adding 200 μ L deionized water, homogenizing on ice, adding 1mL ethyl acetate, and vortexing for 5min;

adding an extracting agent into blood and brain tissue respectively, vortexing, standing at normal temperature for 15-30min, centrifuging at 4 ℃ at 12000rpm for 20min; taking an upper organic phase, and reserving the upper organic phase in a new pipe; extracting the lower layer homogenate for 2 times by the same method; mixing the two extracts, and volatilizing in a nitrogen blowing instrument; dissolving with 100-200 μ L mobile phase (methanol or acetonitrile), and detecting content with high resolution liquid phase mass spectrometer.

The experimental results are as follows: as shown in Table 4, the content of compound 3 in plasma and brain tissue was significantly higher than that of simple securinine dimer compound SN3-L6 at 5min, 30min, 60min and 120min, which indicates that the metabolic stability of compound 3 in mouse blood is significantly better than that of SN3-L6.

TABLE 4 determination of the content of Compound 3 and SN3-L6 in the plasma of mice after different times of gastric gavage

Data for experimental results are expressed as mean ± s.e.m (standard error of the mean) with 3 mice per group;

TABLE 5 determination of the content of Compound 3 and SN3-L6 in the brain tissue of mice after different times of gavage

Data for experimental results are expressed as mean ± s.e.m (standard error of the mean) with 3 mice per group; BLQ Below limit of quantity.

Example 16 Compound 3 improves memory cognitive function in AD model mice in a novel object recognition assay

The experimental method comprises the following steps: the new object identification test is a learning memory test method established by utilizing the principle that rodents have exploration tendency to new objects in nature, and is used for measuring the memory and cognitive functions of the animals in a short time or a long time. This example uses APP1/PS1 double transgenic mice, classical AD model mice, that begin to develop cognitive impairment at 4-5 months of age and gradually become worse. Experimental Male APP1/PS1 mice were used, randomized into groups (10 mice per group), dosed with Compound 3, SN3-L6 or solvent, and gavage at 7 months of age, once daily for 4 weeks, with three doses (6.25 mg/kg, 12.5mg/kg and 25 mg/kg) of Compound 3 and SN3-L6 each set, the solvent being physiological saline (5% Tween-80 was added for solubilization), and the corresponding volume of drug or solvent per mouse was gavaged at 10. Mu.L/g. In addition, a group of same age male Wild (WT) mice was set and given solvent. After the administration is finished, a new object identification test is carried out, and the experiment mainly comprises an adaptation stage, a training stage and a test stage:

an adaptation stage: the mice are sequentially placed in an experimental device (a white plastic box of 40 multiplied by 40 cm) without any object, so that the mice can be freely explored for 5min to adapt to the experimental environment, and the interference of the new environment explored by the animals on the experiment is reduced;

a training stage: two identical objects (a white plastic bottle cap, 4cm in diameter and 2.8cm in height) were glued to the bottom plate of the experimental setup with double-sided adhesive tape, the mice were placed in the experimental setup with the two objects facing away, allowed to explore freely for 10min and recorded with a video system, after which the animals were taken out and placed back in the rearing cage. After 1h of interval, carrying out a test period experiment;

and (3) a testing stage: one of the objects is changed into an object with a different shape (a square building block, 4.6 multiplied by 2.2 multiplied by 3.5 cm), the original object is called an old object or a familiar object, the new changed object is called a new object or a novel object, the mouse is placed into the experimental device in a mode of back to back of the two objects, the mouse is freely explored for 10min and recorded by a video system, and the animal is taken out and placed back into the rearing cage after the operation is finished;

the total distance of movement of the mice and the time to sniff different objects were analyzed using Topscan 3.0 software.

The experimental results are as follows: as shown in fig. 3, in the solvent group, the WT mice had significant difference in the sniffing time for the new and old objects, while the AD model mice had no difference in the sniffing time for the new and old objects, indicating that the memory cognitive function of the AD mice was impaired; after the administration of 12.5 or 25mg/kg SN3-L6 or compound 3 for 4 weeks continuously, the AD mice show obvious difference on the sniffing time of new and old objects; however, at the 6.25mg/kg dose, compound 3 was still effective in restoring cognitive levels in AD mice, with no effect from SN3-L6. This indicates that the effective dose of compound 3 is lower than that of SN3-L6, and the memory cognitive function of AD model mice in a new object recognition test can be better improved.

The figure 3 shows the ability of different groups of mice to recognize new objects during the test period. new is the time for sniffing new objects, old is the time for sniffing old objects, recognition index = time for sniffing new or old objects/total time for sniffing new and old objects. For each group of 12 mice, the data of the experimental results were expressed as mean ± s.e.m (standard error of the mean) and statistically processed by One-way ANOVA test with P <0.05, P <0.01, P <0.0001, ns, no signature.

Example 17 compound 3 improves spatial learning memory function in Morris water maze test in AD model mice experimental approach: the Morris water maze test judges the spatial learning and memory function of rodents by searching for a platform in water and analyzing the time and the path taken by the rodents to search for the platform. The subjects, grouping protocol and mode of administration were in accordance with example 16. The experimental process mainly comprises a training stage and a testing stage:

a training stage: a round water jar (120 cm diameter, 45cm depth) was filled with water containing titanium dioxide (to reduce underwater visibility) and the temperature was maintained at 25 ℃. Two mutually perpendicular main axes are designated, the pool is divided into four equal quadrants, and the intersections of these axes with the pool edges are designated north (N), south (S), east (E) and west (W). A circular platform (with the diameter of 10cm and the height of 30 cm) is placed in the center of a certain quadrant (target quadrant), and different patterns are attached to walls corresponding to different quadrants to serve as a maze clue for space learning. The training lasts for 7 days, 3 times per day (90 s each group), with 30min intervals between each training. The round platform was placed on the water surface on day 1. During the experiment, the mice are placed into water facing the pool wall, the positions (N, NE and E) of the mice are different during 3 times of training, if the platform is not found within 90s, the platform needs to be introduced, the latency period is recorded as 90s, and the experimental process is recorded by a video system. On days 2-7, the test was the same as the procedure on day 1, except that the platform was submerged 0.5cm below the water surface;

and (3) a testing stage: on day 8 (24 hours after the last test), the platform was removed, the mouse was placed in water facing the pool wall, and the video system was used to record the movement within 90 s;

the residence time and swimming speed of the mice in different quadrants were analyzed using Topscan 3.0 software.

The experimental results are as follows: as shown in fig. 4, after 4 weeks of continuous gavage, the retention time of the AD mice in the solvent control group in the target quadrant and other quadrants was not different, indicating that the spatial learning and memory function of the AD mice in the Morris water maze test was impaired; after 25mg/kg SN3-L6 or compound 3 is given, the stay time of the AD mouse in the target quadrant is the longest, and the stay time of the AD mouse in other quadrants shows statistical difference; however, compound 3 was effective in restoring cognitive levels in AD mice at 6.25 or 12.5mg/kg doses, with no effect from SN3-L6. The effective dosage of the compound 3 is lower, and the spatial learning and memory functions of AD mice in the Morris water maze test can be better improved.

The figure 4 shows the residence time of different groups of mice in the target and other quadrants, respectively, during the test phase. Target is the residence time of the mouse in the Target quadrant, and Average of others is the residence time of the mouse in other quadrants. For each group of 12 mice, the data of the experimental results were represented by mean ± s.e.m (standard error of the mean) and statistically processed by One-way ANOVA test with P <0.05, P <0.01, P <0.0001, ns, no signature.

It should be noted that the above-mentioned embodiments of the present invention are described in detail, but the present invention is only exemplary, and the present invention is not limited to the above-mentioned embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (7)

1. A securinine dimer compound having a structure shown in formula I:

in the formula:

the linking group Linker is selected from:

x is selected from

Or alternatively

n is 1, 2, 3 or 4;

when the Linker = A, the securinine dimer compound has a structural formula shown in formula II:

when the Linker = B, the securinine dimer compound has a structural formula shown in formula III:

2. the method of claim 1, wherein the dimer-based compound is selected from the group consisting of:

。

3. a process for preparing a compound of formula II according to claim 1, which process comprises:

at normal temperature, dissolving securinine in a stirred dichloromethane solvent, sequentially adding trimethylsilyl azide, acetic acid and DBU, heating to 35 ℃, reacting for 8 hours, concentrating an organic solvent under reduced pressure to obtain a crude product, and separating and purifying the obtained crude product by silica gel column chromatography to obtain a pair of diastereoisomer azide compounds N10a and N10b; then, dissolving the azide compound N10a or N10b in a stirred DMSO solvent, and sequentially adding copper, copper sulfate pentahydrate and a diyne compound; reacting for 5 hours in a dark place, adding ethyl acetate, and filtering to obtain filtrate; washing the filtrate with water for 3 times, washing with saturated salt water, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain crude product; separating and purifying the obtained crude product by silica gel column chromatography to obtain a compound II with a general formula; x is as defined in claim 1.

4. A process for the preparation of a compound of formula III according to claim 1, which process comprises:

dissolving an azide compound N10a or N10b in a stirred DMSO solvent, sequentially adding copper, copper sulfate pentahydrate and a compound III-1 in a general formula, reacting for 5 hours in a dark place, adding ethyl acetate, filtering to obtain a filtrate, washing the filtrate with water for 3 times, washing with saturated salt, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain a crude product; and separating and purifying the obtained crude product by silica gel column chromatography to obtain a compound III with a general formula.

5. The pharmaceutical composition of the securinega suffruticosa alkaloid dimer shown in general formulas I, II and III, or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-2, and one or more pharmaceutically acceptable carriers, diluents or excipients.

6. Use of securinine dimer represented by general formulae I, II and III, or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-2 for the preparation of a medicament for the treatment of alzheimer's disease.

7. Use of a pharmaceutical composition according to claim 5 in the manufacture of a medicament for the treatment of alzheimer's disease.

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