CN114230582A - 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:

R¹is 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, the common clinical symptoms are progressive memory disorder, cognitive disorder, personality disorder and the like, and the pathological features of the brain are mainly massive neuronal loss, senile plaques consisting of beta amyloid (Α β) and neuro-tangles consisting 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 an estimated number of patients exceeding 1.5 million by 2050, 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 mainly focus on the compensation of neurotransmitters and the inhibition of specific enzymes or receptors, which are mainly used for alleviating the symptoms of AD, and cannot reverse the phenomena of severe loss of nerve synapses and neuronal damage occurring in the middle and late stages of the disease, so that the development of the disease cannot be fundamentally reversed or prevented. 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. This is due, in part, to the inability to repair damaged synapses and their constituent neural circuits by simple clearance of harmful substances such as A β protein or Tau protein in the middle and late stages of AD (Ferreira ST, et al, Front Cell Neurosci,2015,9: 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 synaptoproteins such as glutamate receptors, synaptophysin, etc. is hindered, affecting the structure and function of the neurosynaptic, leading to a variety of neurological dysfunctions (Tom DS, et al, Neuron,2014,81(4): 958-. 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 AD animal models, it has been demonstrated that neurodegeneration 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: 19-24). Therefore, by restoring the protein synthesis level of neurons, it is expected to improve the expression of critical 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 more quickly, and the good therapeutic effect of the compounds in the animals is influenced. 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 (such as SN3-L6) can significantly up-regulate the synthesis level of neuronal proteins, and have the effects of promoting neuronal differentiation and enhancing neurosynaptic function (TangG, et al, ACS Chem Neurosci,2016,7: 1442-1451; Liao Y, et al, Front Pharmacol,2018,9:290), but the compounds are metabolized and eliminated in animals, so that the compounds exert good therapeutic effects in the animals.

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 when Linker ═ B, has a 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 as general formulas I, II and III, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or prodrug molecule thereof, or a 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 in admixture with other chemical components, as well as other components such as physiologically/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 is as follows:

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

at room temperature, securinerine suffruticosa (1.0equiv.) was dissolved in a stirred dichloromethane solvent, followed by the addition of trimethylsilyl azide (5.0equiv.), acetic acid (5.0equiv.) and DBU (0.05 equiv.). Heating to 35 ℃, reacting for 8 hours, and concentrating the organic solvent under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to give a pair of diastereomer azides N10a and N10 b. Subsequently, the azide compound N10a or N10b (1.0equiv.) was dissolved in the stirred DMSO solvent, and copper (2equiv.), copper sulfate pentahydrate (0.05equiv.) and the diyne compound (0.27equiv.) were added in succession. 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 II:

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

azide compound N10a or N10b (1.0equiv.) was dissolved in stirred DMSO solvent, followed by the addition of copper (2.0equiv.), copper sulfate pentahydrate (0.05equiv.) and compound III-1 of formula (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 III with a general formula.

The invention has the beneficial effects that:

the invention develops novel securinine dimer compounds with 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 SN3-L6), and have better stability in cells and animal bodies, the cognitive function of an AD model mouse 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-protein in Neuro-2a cells

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 in 10-6 (ppm). NMR was measured on Bruker AVANCE-300 and Bruker AVANCE-400 NMR spectrometers in deuterated chloroform (CDCl3) as the solvent and Tetramethylsilane (TMS) as the 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 of 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, Annage, Darrill Chemicals, and the like.

In the examples, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

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 unless otherwise specified.

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), the developing solvent used for the reaction, the eluent system for column chromatography for the isolation and purification of the compound and the 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. 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.

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-N6

At room temperature, securinine (217mg,1mmol) was dissolved in a stirred mixed solvent of dichloromethane (5mL) and methanol (0.5mL), and potassium phosphate (K) was added successively₃PO₄64mg,0.3mmol) and the corresponding primary amine (R-NH)₂5 mmol). After the temperature was raised to 35 ℃, the reaction was carried out for 8 hours. The organic solvent is concentrated under reduced pressure to obtain a 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 (50mL) solvent at room temperature and trimethylsilyl azide (6.58mL,50mmol), acetic acid (2.86mL,50mmol) and DBU (75L,0.5mol) were added sequentially. Heating to 35 ℃, reacting for 8 hours, and concentrating the organic solvent under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to give a pair of diastereomeric azide compounds N10a and N10 b. The azide N10a was then dissolved in a stirred DMSO (2mL) solvent, and copper (127mg,2mmol), copper sulfate pentahydrate (13mg,0.05mmol) and 1, 5-hexadiyne (26. mu.L, 0.27mmol) were added sequentially. After 5 hours in the absence of light, ethyl acetate (10mL) was added and the mixture was filtered to obtain a filtrate. The filtrate was washed 3 times with water (3X 10mL), 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 procedure of example 2, starting from azide compound N10b, compound 2 was obtained as a white solid (47% yield).

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 procedure of example 2, azide compound N10a 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 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 was used as a starting material to react 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 procedure of example 2, azide compound N10b was used as a starting material to react with 1, 7-octadiyne 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 was reacted with propargyl ether starting from it to give compound 7 as a white solid (57% yield).

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 was reacted with propargyl ether starting from azide compound N10 to give compound 8 as a white solid (57% yield).

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, azide compound N10a was reacted with compound N4 to obtain compound 9 as a white solid (56% yield).

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 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: neuro-2a cells (purchased from American type culture collection cell bank) were recovered, cultured in growth medium (MEM + 10% FBS +100U/mL penicillin and 100. mu.g/mL 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, carrying out passage according to a ratio of 1:10, and carrying out passage for one day in three days. 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 medium was cultured for 24 hours in a growth medium and then changed to a differentiation medium (MEM + 0.5% FBS +100U/mL penicillin and 100. mu.g/mL streptomycin), and a securinine dimer compound was added at a concentration of 25. mu.M and treated 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:

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

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 20 min;

thirdly, removing the blocking solution, adding primary antibody diluent (containing 1 percent of bovine serum albumin, 1 percent of sheep serum albumin and 0.4 percent of Triton X-100 PBS) containing the beta-tubulin III antibody (1:3000), and incubating for 1h at room temperature or incubating overnight at 4 ℃;

washing the mixture for 3 times by using PBS, adding a diluent (PBS containing 1 percent of bovine serum albumin and 0.4 percent of Triton X-100) containing a fluorescent secondary antibody Alexa Fluor-488goat anti-mouse IgG antibody (1:5000), and incubating the mixture for 1 hour at room temperature;

washing with PBS for 3 times, and sealing.

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 taking triazole as a connecting group have the neural differentiation promoting activity at 25 mu M; wherein, the activity of triazole compound 3 in promoting nerve cell differentiation and promoting neurite growth is obviously superior to that of simple securinine dimer compound SN 3-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 simpler securinine dimer compound SN3-L6 of the 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 independent experiments, each group analyzing at least 100 cells and statistically processed using an unpaired t-test method with 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 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 plated in 35mm dishes at a density of 6X 105 cells/35 mm dish, cultured overnight in a constant temperature incubator containing 5% CO2 at 37 ℃ and then added with securinerine foline dimer compound to a final concentration of 25. mu.M, added with DMSO of the same volume as that of the control group, and cultured 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. Then, the cells were washed twice with ice-D-PBS and then cell-disrupted proteins were collected with RIPA lysate (containing protease inhibitor), followed by Western blot assay to detect the content of puromycin-labeled nascent proteins with puromycin antibody.

The experimental results are as follows: as shown in figure 1, compounds 1-10 all have obvious effect of promoting nerve cell protein synthesis under 25 μ M dosage. 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-based compound SN3-L6 is inactive at 1. mu.M, indicating that compound 3 shows a stronger effect of promoting protein synthesis in nerve cells than simple securinine dimer-based compound SN 3-L6.

FIG. 1 is a histogram of Western blot for detecting puromycin-labeled nascent protein content. 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.

FIG. 2 is a histogram of Western blot for detecting puromycin-labeled nascent protein content. Neuro-2a cells were treated with 1. mu.M or 10. mu.M compound for 1h, and then treated with 1. mu.M puromycin for 0.5h, with DMSO as a control. 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

Early studies found that simple securinine dimer compound SN3-L6 is metabolized in vivo faster, so we first tested the intracellular enrichment capacity of triazole compound 3 and compared with SN 3-L6.

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

adding a compound, incubating for 1h and 24h, washing the culture dish for 3 times by using ice D-PBS, adding 396 mu L of physiological saline into each culture dish, scraping cells and transferring the cells into a 1.5mL EP tube;

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

③ adding 600 mu L ethyl acetate (analytically pure), whirling for 10min, standing for 10min at room temperature until two liquid phases are layered, then centrifuging for 10min at 10000rpm and 4 ℃, taking 500 mu L of upper organic phase, and repeatedly extracting for 3 times;

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

adding 400 mu L of 80% methanol into the sample for redissolving, carrying out ultrasonic treatment in a room-temperature water bath for 30min to completely dissolve the sample, and then vortexing for 2 min; sixthly, centrifuging at room temperature of 10000rpm for 10min, taking 100 mu L of supernatant solution to a liquid phase analysis bottle, and carrying out content detection by using a high-resolution liquid phase mass spectrometer.

The content of intracellular compounds was calculated according to the formula f '═ fi/fs ═ (Mi/Ai)/(Ms/As) and (Ai/As) × f' ═ Ci/Cs and the 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 SN3-L6, indicating that Compounds 3 and 13 entered and enriched cells better than the simple securinega suffruticosa alkaloid dimer class compound SN 3-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 21-23, the compound 3 has the best activity and the strongest cell entering ability, so that the compound 3 is selected as a representative compound to study the metabolic condition of the compound in an animal body.

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

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

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 5 min;

adding an extracting agent into blood and brain tissue respectively, vortexing, standing at normal temperature for 15-30 min, and centrifuging at 4 ℃ and 12000rpm for 20 min; 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 the mixture by using 100-200 mu L of mobile phase (methanol or acetonitrile), and detecting the content by using a 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 is significantly higher than that of simple securinega suffruticosa alkaloid 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 SN 3-L6.

TABLE 4 determination of the content of Compound 3 and SN3-L6 in the plasma 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;

TABLE 5 determination of the content of Compound 3 and SN3-L6 in the brain tissue of mice after various 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 a New object recognition test in AD model mice

The experimental method comprises the following steps: the new object identification test is a learning and memory test method established by utilizing the principle that rodents have a tendency to explore 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 (6.25mg/kg, 12.5mg/kg and 25mg/kg) of compound 3 and SN3-L6, in physiological saline (5% tween-80 was added to aid dissolution), and each mouse was gavaged with a corresponding volume of drug or solvent at 10 μ 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) are adhered to a bottom plate of the experimental device by using double-sided adhesive tape, a mouse is placed into the experimental device in a mode of facing back to 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 a rearing cage after the process is finished. After 1h interval, carrying out 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 3.5cm), 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 12.5 or 25mg/kg SN3-L6 or compound 3 continuous gavage for 4 weeks, the AD mice show obvious difference on the sniffing time of new and old objects; however, compound 3 was still effective in restoring cognitive levels in AD mice at the 6.25mg/kg dose, with no effect from SN 3-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, and the recognition index is the total time for sniffing new or old objects/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 is mainly divided into 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 30cm) is placed at 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 spatial 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 in water facing the pool wall, the positions of the mice are different during 3 times of training (N, NE and E), if the platform is not found within 90s, the platform needs to be led to the platform, the latency period is recorded as 90s, and the experimental process is recorded by a video system. On days 2-7, the test is the same as the step on day 1, except that the platform is submerged at a position 0.5cm below the water surface;

and (3) a testing stage: on day 8 (24 hours after the last experiment), the platform was removed, the mouse was placed in the 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; AD mice had the longest residence time in the target quadrant after administration of 25mg/kg SN3-L6 or compound 3, showing statistical differences from the residence times in the other quadrants; however, compound 3 was effective in restoring cognitive levels in AD mice at doses of 6.25 or 12.5mg/kg, with no effect from SN 3-L6. The fact that the effective dose of the compound 3 is lower can better improve the spatial learning and memory function of AD mice in the Morris water maze test.

The figure 4 shows the residence time of the 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 the other quadrants. The data for the experimental results are 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 sign.

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 (9)

1. A novel securinine dimer compound having a structure 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:

x is selected from

Or

n is 1, 2, 3 or 4.

2. The novel securinine dimer according to claim 1, wherein said dimer compound has a structural formula shown in formula II or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof:

wherein: x is selected from

Or

n is 1, 2, 3 or 4.

3. Novel securinine dimer according to claim 2, said dimer-like compound being selected from:

4. the novel securinine dimer according to claim 1, wherein said dimer compound has a structural formula shown in formula III or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof when Linker ═ B:

5. novel securinine dimer according to claim 4, said dimer-like compound being selected from:

6. a method for preparing a securinega suffruticosa alkaloid dimer compound of general formula II according to claim 2, comprising:

at normal temperature, securinerine suffruticosa (1.0equiv.) is dissolved in a stirred dichloromethane solvent, and trimethylsilyl azide (5.0equiv.), acetic acid (5.0equiv.) and DBU (0.05equiv.) are added successively; heating to 35 ℃, reacting for 8 hours, and concentrating the organic solvent under reduced pressure to obtain a crude product; separating and purifying the obtained crude product by silica gel column chromatography to obtain a pair of diastereoisomer azide compounds N10a and N10 b; subsequently, azide compound N10a or N10b (1.0equiv.) was dissolved in the stirred DMSO solvent, followed by the addition of copper (2equiv.), copper sulfate pentahydrate (0.05equiv.) and diyne compound (0.27 equiv.); after the reaction is carried out for 5 hours in a dark place, ethyl acetate is added, and the filtrate is obtained through filtration; 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.

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

azide compound N10a or N10b (1.0equiv.) was dissolved in stirred DMSO solvent, copper (2.0equiv.), copper sulfate pentahydrate (0.05equiv.) and compound III-1 of general formula (0.27equiv.) were added sequentially; after the reaction is carried out for 5 hours in a dark place, ethyl acetate is added, and the filtrate is obtained through filtration; 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; and separating and purifying the obtained crude product by silica gel column chromatography to obtain a compound III with a general formula.

8. A pharmaceutical composition comprising the securinine dimer represented by the general formulae I, II and III according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

9. Use of securinega suffruticosa alkaloid dimer represented by general formulae I, II and III according to any one of claims 1-7, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or prodrug molecule thereof, or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment of Alzheimer's disease.

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