CN113603684B - 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product and preparation method and application thereof - Google Patents

1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product and preparation method and application thereof Download PDF

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CN113603684B
CN113603684B CN202110798812.5A CN202110798812A CN113603684B CN 113603684 B CN113603684 B CN 113603684B CN 202110798812 A CN202110798812 A CN 202110798812A CN 113603684 B CN113603684 B CN 113603684B
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oxadiazole
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pinge
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tac
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CN113603684A (en
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孙昊鹏
李琦
王园园
林宏智
乔玉婷
冯锋
柳文媛
曲玮
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China Pharmaceutical University
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Abstract

The invention discloses a 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product, a preparation method and application thereof, and the structure of the compound is as follows. The invention uses acetylcholinesterase inhibition activity, nrf2 activation activity and selectivity screening and Morris water maze experiment as a carrier to evaluate the compounds shown in the general formulas I, II and III to treat Alzheimer's disease (especially moderate and severe Alzheimer's disease), and finds that the compounds have good in vitro and in vivo activity and extremely high selectivity, and can be used as a precursor substance for further developing the effect of resisting Alzheimer's disease by selectively inhibiting acetylcholinesterase and activating Nrf 2.

Description

1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product and preparation method and application thereof
Technical Field
The invention relates to chemical medicine, in particular to a 1,2, 4-oxadiazole Nrf2 activator-tack Lin Pinge product, a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is the most common cause of dementia, accounting for 60% -70% of global cases. Of the population older than 65, about 10% are considered to have AD; in the > 85 year old population, this number rises to 32%, with an estimated annual incidence of AD of 6.48%. The memory and cognitive functions of AD patients are gradually lost, involving the fields of language, visual space and execution. The pathological features of Alzheimer's disease in the brain are amyloid (Abeta) plaques and abnormal tau tangles. Based on the temporary appearance of amyloid and tau lesions and evidence that aβ overproduction leads to AD, an amyloid cascade hypothesis was proposed that aβ accumulation is the major event leading to a general-type effect that ultimately leads to neuronal damage. However, there is growing evidence that the amyloid cascade alone cannot explain most of the pathogenesis of AD, suggesting that other pathological processes are also involved. In particular, inflammation has become a critical factor after the discovery of elevated levels of markers of inflammation in AD patients and AD risk genes associated with innate immune function.
Neuroinflammation generally refers to an inflammatory response within the central nervous system that can be caused by a variety of pathological lesions, including infection, trauma, ischemia, and toxins. This process is marked by the production of a variety of factors including pro-inflammatory cytokines (including IL-1 beta, IL-6, IL-18 and TNF), chemokines (such as CCL1, CCL5 and CXCL 1), small molecule messengers (including prostaglandins and nitric oxide), and the production of reactive oxygen species by innate immune cells. The innate immune cells involved in this process are mainly microglia and astrocytes, but capillary endothelial cells and infiltrated blood cells also cause neuroinflammation, especially when the blood-brain barrier (BBB) is subject to biochemical or mechanical damage. The release of pro-inflammatory molecules may lead to synaptic dysfunction, neuronal death and neurogenesis inhibition. IL-1β induces synaptic loss by increasing prostaglandin E2 production, resulting in the release of presynaptic glutamate and activation of postsynaptic N-methyl-d-aspartate receptors, and when the NF- κB pathway is inhibited, tumor necrosis factor causes neuronal death by activating tumor necrosis factor receptor 1 (Tumor Necrosis Factor Receptor 1, TNFR 1) and recruiting caspase 8. In addition, the complement system may be activated, promoting phagocytic function of microglial cells, which may lead to improper synaptic pruning. Anti-inflammatory cytokines are also produced during neuroinflammation, possibly as part of a complex mechanism that prevents excessive neuroinflammation. However, in the context of neurodegenerative diseases, neuroinflammation is often a chronic process that cannot be solved by itself, and is considered to be an important driving factor for the disease.
Neuroinflammation may be an important component of the pathological initiation of AD. Epidemiological studies have shown that dementia is positively correlated with previous reports on infection, smoking and diabetes. The potential protective effects of non-steroidal anti-inflammatory drugs (Nonsteroidal Antiinflammatory Drugs, NSAIDs) on AD have also been proposed, although the efficacy of these drugs is still controversial. Systemic inflammation caused by the above risk factors can induce central nervous system inflammation through periventricular organs, vagus nerve, active transport or BBB disruption. Autopsy studies on young patients experiencing systemic inflammation have found that microglial morphological and immunological changes are similar to those observed in elderly or demented patients. Traumatic brain injury, suspected of increasing the risk of AD, has also been found to trigger persistent neuroinflammation. Microglial activation at the pre-plaque stage was observed in animal models of AD, and a human neuroimaging study reported an increase in microglial activation in patients with mild cognitive impairment (mild cognitive impairment, MCI) in the absence of amyloid tracers. Both findings indicate that neuritis is an early event in alzheimer's disease. Further studies have found that injection of aβ alone to the brain is insufficient to induce amyloid pathology in primate models, but that combined injection of lipopolysaccharide and aβ or aβ in animal models of chronic systemic inflammation may lead to the formation of amyloid plaques. More compelling evidence comes from a human necropsy study that found that microglial cells were not associated with morphological changes in asymptomatic individuals of elevated amyloid burden at different ages, strongly suggesting that neuroinflammation is a prerequisite for AD pathogenesis, except for aβ deposition. Evidence of a genome-wide association study, which found that mutations in microglial or innate immunity genes such as CD33, TREM2 and complement receptor type 1 are associated with increased incidence of AD in humans, also supports the onset of neuroinflammation in AD. There is growing evidence that microglial initiation occurs in the context of AD, meaning that chronic low-level stimuli (including systemic inflammation and aging) cause naive microglial cells to take a process of altered state that can lead to exaggerated or inappropriate inflammatory responses when these microglial cells are exposed to repeated pathological stimuli.
To date, only five drugs have been approved by the FDA in the united states for use in the treatment of AD. Including rivastigmine, galantamine, donepezil, memantine, and tacrine which has been subsequently removed for hepatotoxicity and the like. In China, in addition to the four drugs mentioned above, which are still marketed in the United states, huperzine A has also been approved for the treatment of AD. The above drugs are acetylcholinesterase inhibitors except memantine. These drugs can only temporarily delay the course of the disease in the early stages of the disease and the therapeutic effects vary widely among patients. Because AD is a systemic disease with complex etiology, it is difficult to achieve therapeutic goals only for individual links in the course of numerous diseases. Thus, the simultaneous design of multi-target drug molecules (multiple-target directed ligands, MTDLs) for their multiple targets has become a new hotspot in drug development for the treatment of AD. Compared with the traditional single drug single target (one-target) scheme, the strategy is more suitable for the complex pathological environment of AD.
Nrf2, as a transcription factor with broad activity, can exert positive effects on multiple links of AD at the same time. By activating the downstream genes, the abnormal and serious oxidative stress state in brain tissue cells of AD patients can be relieved, and the effect of resisting neuroinflammation is also achieved. Also, activation of Nrf2 may promote the cell to expel excess metal ions out of the cell. In addition, nrf2 also has an important effect on energy metabolism and can regulate mitochondrial function. Thus, activation of Nrf2 can systematically provide relief and protection against damage to brain cells in AD patients. Another common molecular target for multi-target anti-AD drugs is Acetylcholinesterase (AChE). Inhibition of AChE activates the cholinergic system of the patient's brain, improving the cognitive ability of the patient. Currently available drugs for the treatment of AD are also inhibitors of this target. Furthermore, AChE has also been reported to accelerate the aggregation process of aβ through the outer Zhou Yin ion region (peripheral anionic site, PAS) in its catalytic pocket.
Disclosure of Invention
The invention aims to: the invention aims to provide a 1,2, 4-oxadiazole Nrf2 activator-tacrine split product with good in-vitro and in-vivo activities, extremely high selectivity and other effects.
It is another object of the present invention to provide a process for the preparation and use of the above compounds.
The technical scheme is as follows: the invention provides a 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product or pharmaceutically acceptable salt thereof, the structure of which is shown as the general formulas I, II and III:
wherein,
r represents hydrogen, optionally substituted halogen, C1-C4 alkyl or C1-C4 alkoxy.
Further, the compound is any one of the following:
further, the pharmaceutically acceptable salt is selected from hydrochloride, maleate or citrate.
The preparation method comprises the following steps:
a preparation method of a compound with Alzheimer disease resistance shown in a general formula I comprises the following steps: to be used forIs used as a starting material and is protected by benzyl to obtain +.>Using 2-methyl-5-cyanopyridine as starting materialReflux-extracting with hydroxylamine hydrochloride and triethylamine in ethanol to obtain intermediate ∈10->Will->Dissolving N, N '-carbonyldiimidazole in N, N' -dimethylformamide, stirring at room temperature, and adding +.>Adding into a reaction bottle to obtain a 1,2, 4-oxadiazole ring system intermediate, and removing benzyl for protection to obtain +. >Taking methyl anthranilate as a raw material, and obtaining the +.>And (3) withThe target compound with the structure of the general formula I is obtained through Williamson reaction. The reaction formula is as follows:
(a)i：BnBr，K 2 CO 3 ，CH 3 CN，reflux，8h；ii：2N NaOH(aq)，MeOH，105℃，6h；(b)Hydroxylamine hydrochloride，K 2 CO 3 ，EtOH，reflux，10h；(c)i：CDI，DMF，110℃，5h；ii：HCl(aq)，EtOH，reflux，6h；(d)NaOH，H 2 O，r.t.，12h；(e)6-Amino-1-hexanol，POCl 3 ，reflux，3h；(f)i：6-Amino-1-hexanol，1-pentanol，150℃，24h；ii：CCl 4 ，Ph 3 P，DCM，r.t.，24h；(g)K 2 CO 3 ，DMF，90℃，36h.
having the formula IIA method for preparing an anti-alzheimer's disease compound comprising: to be used forIs taken as a raw material, is reacted with bromopropyne after esterification, and is hydrolyzed to obtain the +.>And->The cyclization is carried out to obtain->The preparation method comprises the steps of taking methyl anthranilate as a raw material, hydrolyzing, cyclizing, aminating, then ammonolyzing by ethanolamine, carrying out acylation reaction by using p-toluenesulfonyl chloride, and then reacting with TMSN3 to obtain +.>And->And obtaining the target compound with the structure shown in the general formula II through click reaction. The reaction is as follows
(a)i：H 2 SO 4 ，MeOH，reflux，6h；ii：3-Bromopropyne，K 2 CO 3 ，Me 2 CO，reflux，6h；iii：2NNaOH(aq)，MeOH，100℃，6h；(b)Hydroxylamine hydrochloride，K 2 CO 3 ，EtOH，reflux，10h；(c)CDI，DMF，110℃，5h；(d)NaOH，H 2 O，r.t.，12h；(e)Cyclohexanone，POCl 3 ，reflux，3h；(f)i：ethanolamine，1-pentanol，150℃，24h；ii：TsCl，TEA，DCM，60℃，18h；(g)TMSN3，DMF，110℃，12h；(h)CuSO 4 5H 2 O，Ascorbic acid，MeOH，H 2 O，r.t.，24h.
A preparation method of a compound with Alzheimer disease resistance shown in a general formula III comprises the following steps: to be used forIs taken as a raw material, is reacted with bromopropyne after esterification, and is hydrolyzed to obtain the +.>Reaction of methyl 6-methylnicotinate with hydrazine hydrate to give +.>And->After the reaction, the cyclization is carried out to obtainThe method comprises the steps of taking methyl anthranilate as a raw material, hydrolyzing, cyclizing, ammonolyzing by ethanolamine, acylating by p-toluenesulfonyl chloride, and reacting with TMSN3 to obtain +.>And (3) withAnd obtaining the target compound with the structure shown in the general formula III through click reaction.
(a)i：H 2 SO4，MeOH，reflux，6h；ii：3-Bromopropyne，K 2 CO 3 ，Me 2 CO，reflux，6h；iii：2NNaOH(aq)，MeOH，100℃，6h；(b)hydrazine hydrate，MeOH，reflux，6h；(c)i：CDI，DMF，110℃，5h；ii：POCl 3 ，110℃，6h；(d)NaOH，H 2 O，r.t.，12h；(e)Cyclohexanone，POCl 3 ，reflux，3h；(f)i：ethanolamine，1-pentanol，150℃，24h；ii：TsCl，TEA，DCM，60℃，18h；(g)TMSN3，DMF，110℃，12h；(h)CuSO 4 5H 2 O，Ascorbic acid，MeOH，H 2 O，r.t.，24h.
The application of the 1,2, 4-oxadiazole Nrf2 activator-tack Lin Pinge product in preparing medicines for preventing or treating Alzheimer's disease.
A pharmaceutical composition comprising a compound of the general formulae I, II, III or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 as an active ingredient or a major active ingredient and a pharmaceutically acceptable carrier.
Further, the pharmaceutical composition preparation is a tablet, a capsule, a powder, a syrup, a liquid, a suspension or an injection.
The beneficial effects are that: the invention uses acetylcholinesterase inhibition activity, nrf2 activation activity and selectivity screening and Morris water maze experiment as a carrier to evaluate the compounds shown in the general formulas I, II and III to treat Alzheimer's disease (especially moderate and severe Alzheimer's disease), and finds that the compounds have good in vitro and in vivo activity and extremely high selectivity, and can be used as a precursor substance for further developing the effect of resisting Alzheimer's disease by selectively inhibiting acetylcholinesterase and activating Nrf 2. The invention synthesizes the multi-target compound with Nrf2 activating activity and AChE inhibiting activity, can simultaneously act on a plurality of pathological processes in the AD development process, and achieves the purposes of relieving brain injury of patients and delaying disease process. In the design of multi-target cholinesterase inhibitors, tacrine is the most common cholinesterase inhibitory active fragment. As an active fragment, tacrine has the advantages of small molecular weight, high activity and the like. Therefore, firstly, a tacrine fragment is used as a cholinesterase inhibition activity fragment, and is spliced with a 1,2, 4-oxadiazole Nrf2 activator fragment, so that a novel multi-target compound is obtained through the strategy.
Drawings
FIG. 1 shows the result of determining the nerve cytotoxicity of the test compound;
FIG. 2 shows the results of an assay for the activation activity of the test compound Nrf 2;
FIG. 3 is the ROS assay result of Compound 15;
FIG. 4 shows the results of Elisa assay of compound 15 for TNF- α production;
FIG. 5 shows the results of an Elisa assay for IL-1β production by Compound 15;
FIG. 6 shows the results of Elisa experiments on the production of iNOS by Compound 15;
FIG. 7 is the average distance of mice to the platform;
FIG. 8 is the average time to reach the plateau for mice;
fig. 9 is a trace of a mouse reaching a platform.
Detailed Description
Example 1
(1) Synthesis of 3- (3- (6-methylpyridyl)) -5- (3-hydroxyphenyl) -1,2, 4-oxadiazole (intermediate 1)
3-Benzyloxybenzoic acid and N, N '-carbonyl-diimidazole are dissolved in N, N' -dimethylformamide and stirred at room temperature for 45min. 6-methyl-N-hydroxy-3-pyridine formamidine was added to the reaction flask, and then heated to 110℃and stirred for 5 hours. The reaction mixture was cooled to room temperature, poured into a saturated aqueous sodium bicarbonate solution, and a white solid was precipitated. Suction filtration is carried out, and filter cakes are washed twice. And drying to obtain white powder. The white powder was dissolved in ethanol, concentrated hydrochloric acid was added, and the mixture was refluxed overnight at 105 ℃. After cooling to room temperature, a white solid precipitated. Suction filtration, water washing twice and petroleum ether washing twice. And (5) drying to obtain white solid. 1 H NMR(300MHz，DMSO-d6)：δ10.12(s，1H)，9.11(d，J＝1.9Hz，1H)，8.31(dd，J＝8.1，2.1Hz，1H)，7.62(d，J＝7.7Hz，1H)，7.56(s，1H)，7.48(dd，J＝17.5，8.2Hz，2H)，7.12(dd，J＝8.2，2.3Hz，1H)，2.58(s，3H).
And (3) injection: the 3-benzyloxy benzoic acid is self-made, and the method comprises the following steps: 3-hydroxybenzoic acid, benzyl bromide and potassium carbonate were dissolved in acetonitrile and refluxed for 8 hours. After the reaction was completed, cooling to room temperature, adding water, extracting with dichloromethane, combining the organic layers, evaporating the solvent under reduced pressure to obtain a white solid, adding into a eggplant-shaped bottle, adding 2N aqueous sodium hydroxide solution, methanol, and heating in an oil bath to 100deg.C. The reaction solution was refluxed for about 6 hours, and after the reaction was completed, it was cooled to room temperature. The pH=3 is adjusted by using 2M hydrochloric acid aqueous solution, a large amount of white solid is precipitated, and the white solid is obtained by suction filtration and drying.
The method for preparing the 6-methyl-N-hydroxy-3-pyridine formamidine comprises the following steps: dissolving 5-cyano-2-methylpyridine in ethanol, adding hydroxylamine hydrochloride and potassium carbonate, refluxing and stirring for 10h. And (5) filtering while the reaction is hot after the reaction is finished. After the filtrate was cooled, diethyl ether was added thereto, and at this time, white crystals were precipitated. After suction filtration, the filter cake is washed twice by diethyl ether, and dried to obtain white crystals.
(2) Synthesis of N- (6-chlorohexyl) -9-amino-1, 2,3, 4-tetrahydroacridine (intermediate 2)
9-chloro-1, 2,3, 4-tetrahydroacridine and 6-amino-1-hexanol were dissolved in n-pentanol and stirred at 150℃for 24h. After the reaction, the reaction mixture was cooled to room temperature, and the reaction mixture was poured into petroleum ether. A black oil formed and the petroleum ether was decanted. The black oil was purified by column chromatography on silica gel (developer dichloromethane: methanol: triethylamine=100:2:1) to give a yellow brown oil. This was dissolved in methylene chloride with triphenylphosphine and stirred in an ice bath. Carbon tetrachloride is added into the reaction bottle in batches, and then is transferred into room temperature and stirred for 24 hours. After the reaction was completed, the reaction solution was washed twice with water, and the organic layer was dried over anhydrous sodium sulfate overnight. Anhydrous sodium sulfate was filtered off, and the solvent was evaporated under reduced pressure to give a black oil. The black oil was purified by column chromatography on silica gel (developer dichloromethane: methanol: triethylamine=200:2:1) to give a yellow brown oil. 1 H NMR(300MHz，CDCl 3 )δ7.98(dd，J＝4.1，1.0Hz，1H)，7.95(dd，J＝3.9，1.0Hz，1H)，7.57(ddd，J＝8.4，6.8，1.3Hz，1H)，7.37(ddd，J＝8.2，6.8，1.2Hz，1H)，3.53(td，J＝6.9，4.9Hz，4H)，3.09(s，2H)，2.73(s，2H)，1.94(dt，J＝6.7，3.5Hz，4H)，1.85-1.75(m，2H)，1.75-1.66(m，2H)，1.48(dd，J＝8.4，5.9Hz，4H).
And (3) injection: the method for preparing the 9-chloro-1, 2,3, 4-tetrahydroacridine comprises the following steps: adding methyl anthranilate into 2N NaOH aqueous solution, stirring for 12 hours at room temperature, dropwise adding concentrated hydrochloric acid PH to 4-5, precipitating white solid, filtering, and drying the filter cake under an infrared lamp to obtain white solid anthranilic acid.
Adding the anthranilic acid and cyclohexanone into a eggplant-shaped bottle, and adding trichlorooxyPhosphorus was added to a constant pressure dropping funnel, slowly dropped into the reaction flask under ice bath conditions, and after stirring for 5min, transferred to an oil bath to heat 115 ℃. The reaction solution was refluxed for 3h, cooled to room temperature, quenched with water in an ice bath, and saturated NaHCO 3 The pH of the solution was adjusted to 9, a yellow solid precipitated, filtered off with suction, the filter cake was dissolved in dichloromethane and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure and purified by column chromatography on silica gel (eluent petroleum ether: ethyl acetate=10:1) to give 9-chloro-1, 2,3, 4-tetrahydroacridine as a bright yellow solid.
(3) Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
3- (3- (6-methylpyridyl)) -5- (3-hydroxyphenyl) -1,2, 4-oxadiazole (intermediate 1), N- (6-chlorohexyl) -9-amino-1, 2,3, 4-tetrahydroacridine (intermediate 2) and potassium carbonate were dissolved in N, N' -dimethylformamide and stirred at 90℃for 36h. The reaction solution was cooled to room temperature, and ethyl acetate was then added. The organic layer was washed three times with saturated aqueous sodium bicarbonate and twice with brine. The solvent was evaporated under reduced pressure to give a black oil. The black oil was purified by chromatography on silica gel (developer petroleum ether: dichloromethane: methanol: triethylamine=100:100:2:1) to give a yellowish brown oil, N- (6- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methyl-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.29(d，J＝1.9Hz，1H)，8.33(dd，J＝8.1，2.2Hz，1H)，7.98(d，J＝8.5Hz，1H)，7.92(d，J＝8.1Hz，1H)，7.81(d，J＝7.7Hz，1H)，7.72(dd，J＝2.3，1.6Hz，1H)，7.57(ddd，J＝8.2，6.7，1.2Hz，1H)，7.47(t，J＝8.0Hz，1H)，7.41-7.30(m，2H)，4.08(t，J＝6.3Hz，2H)，3.53(t，J＝7.2Hz，2H)，3.08(s，2H)，2.74(s，2H)，2.67(s，3H)，1.93(t，J＝3.2Hz，4H)，1.89-1.84(m，2H)，1.77-1.71(m，2H)，1.54(dt，J＝11.1，5.9Hz，4H).HRMS(ESI)：found 534.2869，calcd for C 33 H 36 N 5 O 2 [M+H] + 534.2864.
Example 2
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-methyl-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) -5- (4-methyl-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a light brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methyl-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.29(d，J＝2.0Hz，1H)，8.33(dd，J＝8.1，2.2Hz，1H)，7.97(dd，J＝10.6，8.9Hz，2H)，7.74(dd，J＝7.7，1.0Hz，1H)，7.58(dd，J＝14.3，4.3Hz，2H)，7.38(d，J＝7.3Hz，1H)，7.31(d，J＝7.8Hz，2H)，4.12(t，J＝6.2Hz，2H)，3.55(t，J＝7.1Hz，2H)，3.09(s，2H)，2.73(s，2H)，2.67(s，3H)，2.31(s，3H)，1.93(s，6H)，1.80-1.72(m，2H)，1.62-1.52(m，4H).HRMS(ESI)：found 548.3024，calcd for C 34 H 38 N 5 O 2 [M+H] + 548.3020.
Example 3
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-methoxy-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) -5- (4-methoxy-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a light brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methoxy-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.27(s，1H)，8.32(d，J＝7.6Hz，1H)，8.15(d，J＝7.8Hz，1H)，8.06(d，J＝8.4Hz，1H)，7.84(d，J＝8.3Hz，2H)，7.68(s，1H)，7.65-7.58(m，1H)，7.40(d，J＝7.6Hz，1H)，7.31(d，J＝8.1Hz，1H)，7.01(d，J＝8.3Hz，1H)，4.16(t，J＝6.3Hz，2H)，3.95(s，3H)，3.71(t，J＝7.0Hz，2H)，3.16(s，2H)，2.66(s，5H)，1.91(s，6H)，1.86-1.77(m，2H)，1.59(s，4H).HRMS(ESI)：found 564.2967，calcd for C 34 H 38 N 5 O 3 [M+H] + 564.2969.
Example 4
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-fluoro-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) 5- (4-fluoro-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a dark brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-fluoro-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.28(s，1H)，8.38-8.28(m，1H)，7.99(d，J＝8.2Hz，1H)，7.95(d，J＝7.5Hz，1H)，7.78(dd，J＝8.0，1.7Hz，1H)，7.73(d，J＝3.5Hz，1H)，7.60-7.53(m，2H)，7.40-7.36(m，1H)，7.35-7.31(m，1H)，7.29(s，1H)，4.25-4.09(m，2H)，3.56(d，J＝6.3Hz，2H)，3.10(s，2H)，2.73(s，2H)，2.68(s，3H)，1.94(s，6H)，1.76(d，J＝6.3Hz，2H)，1.63(d，J＝5.6Hz，2H)，1.55(d，J＝5.4Hz，2H).HRMS(ESI)：found 552.2771，calcd for C 33 H 35 FN 5 O 2 [M+H] + 552.2769.
Example 5
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-chloro-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) 5- (4-chloro-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a light brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-chloro-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.27(d，J＝1.5Hz，1H)，8.32(dd，J＝8.1，2.2Hz，1H)，7.95(dd，J＝13.9，8.5Hz，2H)，7.76(dd，J＝8.2，1.6Hz，1H)，7.72(d，J＝1.5Hz，1H)，7.56(t，J＝8.2Hz，2H)，7.34(dd，J＝16.4，8.2Hz，2H)，4.18(t，J＝6.1Hz，2H)，3.54(t，J＝7.2Hz，2H)，3.08(s，2H)，2.73(s，2H)，2.67(s，3H)，1.93(s，6H)，1.75(dt，J＝14.3，7.3Hz，2H)，1.67-1.50(m，4H).HRMS(ESI)：found 568.2483，calcd for C 33 H 35 ClN 5 O 2 [M+H] + 568.2474.
Example 6
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-bromo-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) 5- (4-bromo-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a light brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-bromo-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.27(d，J＝1.5Hz，1H)，8.32(dd，J＝8.1，2.1Hz，1H)，7.95(dd，J＝15.5，8.6Hz，2H)，7.78-7.66(m，3H)，7.56(t，J＝7.1Hz，1H)，7.43-7.27(m，3H)，4.18(t，J＝6.2Hz，2H)，3.54(t，J＝7.1Hz，2H)，3.08(s，2H)，2.73(s，2H)，2.67(s，3H)，1.93(s，6H)，1.79-1.72(m，2H)，1.68-1.51(m，4H).HRMS(ESI)：found 612.1963，calcd for C 33 H 35 BrN 5 O 2 [M+H] + 612.1969.
Example 7
Synthesis of N- (6- (5- (5- (6- (2-methylpyridyl)) 1,2, 4-oxadiazolyl) -2-iodo-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic procedure of example 1, intermediate 1 in example 1 was replaced with 3- (3- (6-methylpyridinyl)) 5- (4-iodo-3-hydroxyphenyl) -1,2, 4-oxadiazole to give a light brown oil, N- (6- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-iodo-phenoxy) hexyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.27(d，J＝1.8Hz，1H)，8.32(dd，J＝8.1，2.1Hz，1H)，8.05-7.88(m，3H)，7.64-7.51(m，3H)，7.34(dd，J＝15.1，7.6Hz，2H)，4.17(t，J＝6.1Hz，2H)，3.56(t，J＝7.1Hz，2H)，3.08(s，2H)，2.74(s，2H)，2.67(s，3H)，1.93(s，6H)，1.77(dt，J＝14.4，7.3Hz，2H)，1.67(dd，J＝15.7，7.4Hz，2H)，1.61-1.52(m，2H).HRMS(ESI)：found 660.1824，calcd for C 33 H 35 IN 5 O 2 [M+H] + 660.1830.
Example 8:
(1) Synthesis of 3- (6-methylpyridin-3-yl) -5- (3- (prop-2-yl-1-yloxy) phenyl) -1,2, 4-oxadiazole (intermediate 1)
3- (prop-2-yn-1-yloxy) benzoic acid and N, N '-carbonyl-diimidazole were dissolved in N, N' -dimethylformamide and stirred at room temperature for 45min. 6-methyl-N-hydroxy-3-pyridine formamidine was added to the reaction flask, and then heated to 110℃and stirred for 5 hours. The reaction mixture was cooled to room temperature, poured into a saturated aqueous sodium bicarbonate solution, and a white solid was precipitated. Suction filtration is carried out, and filter cakes are washed twice. And drying to obtain white powder. 1 H NMR(500MHz，CDCl 3 )δ7.80(dt，J＝7.6，1.3Hz，1H)，7.74(dd，J＝2.7，1.5Hz，1H)，7.44(t，J＝8.0Hz，1H)，7.28(dd，J＝2.7，1.0Hz，1H)，4.79(d，J＝2.4Hz，2H)，2.58(d，J＝2.4Hz，1H).
Note that: 3- (prop-2-yn-1-yloxy) benzoic acid is self-made by the following method: adding 3-hydroxybenzoic acid into an eggplant-shaped bottle, adding methanol and concentrated sulfuric acid, refluxing for 6h, adding water to cool to room temperature, precipitating white, filtering, and drying to obtain white solid. The white solid was added to a eggplant-shaped bottle, bromopropyne, potassium carbonate and acetone were added, refluxed for 6 hours, cooled to room temperature, extracted three times with dichloromethane by adding water, and the organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to remove the solvent, to give a colorless oil. To the flask was added 2N aqueous sodium hydroxide solution, methanol, and heated in an oil bath at 100deg.C. The reaction solution was refluxed for about 6 hours, and after the reaction was completed, it was cooled to room temperature. Adjusting the pH to be=3 by using a 2M hydrochloric acid aqueous solution to precipitate a large amount of white solid, carrying out suction filtration and drying to obtain a white solid.
The preparation of 6-methyl-N-hydroxy-3-pyridinecarboxamidine was carried out in the same manner as in example 1 (1).
(2) Synthesis of N- (2-azidoethyl) -1,2,3, 4-tetrahydroacridin-9-amine (intermediate 2)
2- ((1, 2,3, 4-tetrahydroacridin-9-yl)) Amino) ethylmethanesulfonate and TMSN3 were dissolved in DMF, stirred at 110 ℃ for 12h and cooled to room temperature. Brine is added into a reaction bottle, dichloromethane is used for extraction three times, and a dichloromethane layer is dried over night by anhydrous sodium sulfate. Anhydrous sodium sulfate was filtered off, and the solvent was evaporated under reduced pressure to give a brown oil. The brown oil was purified by chromatography on a silica gel column (developer dichloromethane: methanol: triethylamine=100:2:1) to give a brown solid. 1 H NMR(300MHz，CDCl 3 )δ7.96(d，J＝7.6Hz，2H)，7.60(ddd，J＝8.3，6.7，1.4Hz，1H)，7.41(ddd，J＝8.1，6.8，1.2Hz，1H)，3.83(t，J＝5.8Hz，2H)，3.70(t，J＝5.4Hz，2H)，3.11(t，J＝5.3Hz，2H)，2.83(d，J＝6.1Hz，2H)，1.95(q，J＝3.4Hz，4H)。
Note that: 2- ((1, 2,3, 4-tetrahydroacridin-9-yl) amino) ethyl methanesulfonate is self-made by the method of: 9-chloro-1, 2,3, 4-tetrahydroacridine and ethanolamine were dissolved in n-pentanol and stirred at 150℃for 24h. After the completion of the reaction, the reaction mixture was cooled to room temperature, and the reaction mixture was poured into 200mL of petroleum ether. A black oil formed and the petroleum ether was decanted. The black oil was purified by column chromatography on silica gel (developer dichloromethane: methanol: triethylamine=100:2:1) to give a yellow solid. The yellow solid and 4mL of triethylamine were dissolved in dichloromethane, tsCl was added at room temperature and stirred at 60℃for 18h. The reaction solution was cooled to room temperature, aqueous hydrochloric acid was extracted twice with dichloromethane and the solvent was evaporated under reduced pressure to give a brown oil. The brown oil was purified by chromatography on a silica gel column (developer dichloromethane: methanol: triethylamine=100:2:1) to give a yellow brown oil. 1 H NMR(300MHz，CDCl 3 )δ7.96(d，J＝7.6Hz，2H)，7.60(ddd，J＝8.3，6.7，1.4Hz，1H)，7.41(ddd，J＝8.1，6.8，1.2Hz，1H)，3.83(t，J＝5.8Hz，2H)，3.70(t，J＝5.4Hz，2H)，3.13-3.09(m，3H)，2.82(d，J＝5.4Hz，2H)，1.95(q，J＝3.4Hz，4H)，1.36(t，J＝7.2Hz，2H).
The procedure of example 1 (2) was repeated except that 9-chloro-1, 2,3, 4-tetrahydroacridine was used as the starting material.
(3) Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
3- (6-methylpyridin-3-yl) -5- (3- (prop-2-yl-1-yloxy) phenyl) -1,2, 4-oxadiazole (intermediate 1) and N- (2-azidoethyl) -1,2,3, 4-tetrahydroacridin-9-amine (intermediate 2) were added to a reaction flask, water was added to the flask, catalytic amounts of copper sulphate pentahydrate and ascorbic acid were added, and stirred overnight at room temperature. The mixture was extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate for 5h. Ethyl acetate was removed by rotary evaporation under reduced pressure to give a white powder, which was purified by silica gel chromatography (developer DCM: meoh=200:1) to give a pale yellow powder, which was N- (1- (4- (2- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.24(d，J＝2.1Hz，1H)，8.30(dd，J＝8.1，2.2Hz，1H)，8.11-8.06(m，3H)，7.80(d，J＝7.7Hz，1H)，7.75(t，J＝2.0Hz，1H)，7.47(dt，J＝18.2，7.8Hz，2H)，7.32(t，J＝7.5Hz，2H)，7.21(dd，J＝8.3，2.6Hz，1H)，5.31(s，1H)，5.28(s，2H)，4.91(t，J＝5.6Hz，2H)，4.37(q，J＝5.7Hz，2H)，3.10-3.06(m，3H)，2.66(s，4H)，1.84(h，J＝6.6，5.2Hz，4H).HRMS(ESI)：found 559.2558，calcd for C 32 H 30 N 8 O 2 [M+H] + 559.2485.
Example 9
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methyl-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 5- (4-methyl-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methyl-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(500MHz，CDCl 3 )δ9.22(d，J＝2.2Hz，1H)，8.30(dd，J＝8.1，2.3Hz，1H)，7.93(d，J＝8.4Hz，1H)，7.79-7.73(m，2H)，7.69(s，1H)，7.63-7.60(m，1H)，7.54(s，1H)，7.37-7.32(m，2H)，7.32-7.29(m，1H)，5.33(s，2H)，4.62(dd，J＝6.5，4.5Hz，2H)，4.07(d，J＝5.5Hz，2H)，3.06(t，J＝6.2Hz，2H)，2.68(s，3H)，2.62(t，J＝6.1Hz，2H)，2.28(s，3H)，1.92-1.82(m，4H).HRMS(ESI)：found 573.2713，calcd for C 33 H 32 N 8 O 2 [M+H] + 573.2641.
Example 10
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methoxy-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 5- (4-methoxy-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-methoxy-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl3)δ9.22(s，1H)，8.29(dd，J＝8.2，2.2Hz，1H)，8.19(d，J＝8.5Hz，1H)，8.09(s，2H)，7.84(dd，J＝11.3，3.4Hz，2H)，7.54(t，J＝7.7Hz，1H)，7.38-7.31(m，2H)，7.00(d，J＝8.4Hz，1H)，6.85(s，1H)，5.33(s，2H)，4.94(s，2H)，4.39(d，J＝6.1Hz，2H)，3.92(s，3H)，3.06(s，2H)，2.65(s，5H)，1.84(s，4H).HRMS(ESI)：found 589.2669，calcd for C 33 H 32 N 8 O 3 [M+H] + 589.2597.
Example 11
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-fluoro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 5- (4-fluoro-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give a pale yellow powder, N- (2- (4- (2- (5- (5- (6- (2-methyl)) methyl) 5- (2-methyl) 5-1-methoxy-1-prop-2-yn-2-yl) 1-yl) oxadiazolePyridyl)) 1,2, 4-oxadiazolyl) -2-fluoro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.26(s，1H)，8.31(dd，J＝8.3，2.2Hz，1H)，8.01-7.90(m，2H)，7.88-7.80(m，1H)，7.74(d，J＝8.4Hz，1H)，7.67(s，1H)，7.56(t，J＝7.6Hz，1H)，7.38-7.28(m，3H)，5.39(s，2H)，4.58(t，J＝5.6Hz，2H)，4.04(d，J＝5.9Hz，2H)，3.05(d，J＝7.3Hz，2H)，2.64(d，J＝16.0Hz，5H)，1.93-1.83(m，4H).HRMS(ESI)：found 577.2646，calcd for C 32 H 29 FN 8 O 2 [M+H] + 577.2391.
Example 12
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-chloro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 5- (4-chloro-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-chloro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.20-9.16(s，1H)，8.23(dd，J＝8.2，2.2Hz，1H)，7.90-7.81(m，2H)，7.78-7.66(m，3H)，7.52-7.43(m，2H)，7.32-7.24(m，2H)，5.33(s，2H)，4.84(s，1H)，4.61(t，J＝5.2Hz，2H)，4.01(s，2H)，2.99(d，J＝6.2Hz，2H)，2.77(q，J＝7.3Hz，2H)，2.59(d，J＝8.3Hz，3H)，1.87-1.72(m，4H).HRMS(ESI)：found 593.2166，calcd for C 32 H 29 ClN 8 O 2 [M+H] + 593.2094.
Example 13
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-bromo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Reference to the synthetic method of example 8, example 8The intermediate 1 in (a) was replaced with 5- (4-bromo-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-bromo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.20-9.16(s，1H)，8.23(dd，J＝8.2，2.2Hz，1H)，7.90-7.81(m，2H)，7.78-7.66(m，3H)，7.52-7.43(m，2H)，7.32-7.24(m，2H)，5.33(s，2H)，4.84(s，1H)，4.61(t，J＝5.2Hz，2H)，4.01(s，2H)，2.99(d，J＝6.2Hz，2H)，2.77(q，J＝7.3Hz，2H)，2.59(d，J＝8.3Hz，3H)，1.87-1.72(m，4H).HRMS(ESI)：found 593.2166，calcd for C 32 H 29 ClN 8 O 2 [M+H] + 593.2094.
Example 14
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-iodo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 5- (4-iodo-3- (prop-2-yn-1-yloxy) phenyl) -3- (6-methylpyridin-3-yl) -1,2, 4-oxadiazole to give N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,2, 4-oxadiazolyl) -2-iodo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine as a pale yellow powder. 1 H NMR(300MHz，CDCl 3 )δ9.25(s，1H)，8.31(dd，J＝8.1，2.1Hz，1H)，8.06(d，J＝8.5Hz，1H)，8.02-7.92(m，3H)，7.74(d，J＝1.6Hz，1H)，7.60-7.48(m，2H)，7.34(t，J＝8.7Hz，2H)，5.93(s，1H)，5.39(s，2H)，4.83(s，2H)，4.28(s，2H)，3.12(s，2H)，2.66(s，5H)，1.86(s，4H).HRMS(ESI)：found 685.1526calcd for C 32 H 29 IN 8 O 2 [M+H] + 685.1454.
Example 15
(1) Synthesis of 2- (6-methylpyridin-3-yl) -5- (3- (prop-2-yn-1-yloxy) phenyl) -1,3, 4-oxadiazole (intermediate 1)
3- (prop-2-yn-1-yloxy) benzoic acid and N, N '-dicarbonyl imidazole were dissolved in N, N' -dimethylformamide and stirred at room temperature for 45min. The 6-methyl nicotinoyl hydrazide is added into the reaction flask, and then the temperature is raised to 110 ℃ and stirred for 5 hours. The reaction mixture was cooled to room temperature, poured into a saturated aqueous sodium bicarbonate solution, and a white solid was precipitated. Suction filtration is carried out, and filter cakes are washed twice. And drying to obtain white powder. The white solid was dissolved in phosphorus oxychloride at reflux for 6h. The reaction solution was cooled to room temperature, a saturated sodium carbonate solution was slowly added thereto to ph=9, gray crystals were precipitated, and after standing overnight, suction filtration was performed, and the filter cake was rinsed twice with water to obtain a gray solid. 1 H NMR(300MHz，CDCl 3 )δ9.24(s，1H)，8.33(d，J＝7.8Hz，1H)，7.76-7.62(m，2H)，7.35(t，J＝7.7Hz，2H)，4.86(q，J＝2.3Hz，2H)，2.71-2.66(m，3H)，2.60-2.55(m，1H)，2.35(d，J＝2.8Hz，3H).
And (3) injection: 3- (prop-2-yn-1-yloxy) benzoic acid was prepared in the same manner as in example 8 (1).
The method for preparing the 6-methyl nicotinate hydrazide comprises the following steps: dissolving 6-methylnicotinic acid methyl ester in methanol, adding hydrazine hydrate, refluxing and stirring for 6h. Methanol was removed by rotary evaporation under reduced pressure, petroleum ether was added thereto after cooling, and gray crystals were precipitated at this time. After suction filtration, the filter cake is washed twice by petroleum ether and dried to obtain gray crystals.
(2) Synthesis of N- (2-azidoethyl) -1,2,3, 4-tetrahydroacridin-9-amine (intermediate 2)
The procedure is as in example 8 (2).
(3) Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (6-methylpyridin-3-yl) -5- (3- (prop-2-yn-1-yloxy) phenyl) -1,3, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(500MHz，CDCl 3 )δ9.20(s，1H)，8.29(d，J＝8.1Hz，1H)，7.98(d，J＝17.4Hz，3H)，7.70(d，J＝7.2Hz，2H)，7.50(t，J＝6.9Hz，1H)，7.44(t，J＝7.6Hz，1H)，7.33(dd，J＝16.4，7.7Hz，2H)，7.16(d，J＝8.0Hz，1H)，6.01(s，1H)，5.27(s，2H)，4.82(s，2H)，4.27(s，2H)，3.07(q，J＝7.3Hz，4H)，2.67(s，3H)，1.84(s，4H).HRMS(ESI)：found 559.2567，calcd for C 32 H 30 N 8 O 2 [M+H] + 559.2492.
Example 16
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-methyl-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-methyl-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-methyl-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(500MHz，CDCl 3 )δ9.22(d，J＝2.3Hz，1H)，8.30(dd，J＝8.1，2.3Hz，1H)，7.93(d，J＝8.4Hz，1H)，7.78-7.72(m，2H)，7.69(s，1H)，7.63-7.60(m，1H)，7.54(t，J＝7.6Hz，1H)，7.36-7.29(m，3H)，5.33(s，2H)，4.62(dd，J＝6.6，4.4Hz，2H)，4.07(q，J＝6.0Hz，2H)，3.06(t，J＝6.2Hz，2H)，2.68(s，3H)，2.62(d，J＝12.2Hz，2H)，2.28(s，3H)，1.91-1.84(m，4H).HRMS(ESI)：found 573.2724，calcd for C 33 H 32 N 8 O 2 [M+H] + 573.2651.
Example 17
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-methoxy-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-methoxy-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3,4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-methoxy-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(500MHz，CDCl 3 )δ9.21(s，1H)，8.31-8.26(m，1H)，7.94(d，J＝8.3Hz，1H)，7.79(d，J＝16.7Hz，2H)，7.76-7.71(m，2H)，7.54(t，J＝7.6Hz，1H)，7.34(d，J＝8.4Hz，2H)，7.01(d，J＝8.4Hz，1H)，5.37(s，2H)，4.62(t，J＝5.5Hz，2H)，4.07(d，J＝6.4Hz，2H)，3.93(s，3H)，3.06(d，J＝6.5Hz，2H)，2.68(s，3H)，2.61(d，J＝6.0Hz，2H)，1.92-1.81(m，4H).HRMS(ESI)：found 589.2669，calcd for C 33 H 32 N 8 O 2 [M+H] + 589.2597.
Example 18
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-fluoro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-fluoro-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-fluoro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.20(s，1H)，8.30(dd，J＝8.3，2.4Hz，1H)，8.01(d，J＝7.5Hz，2H)，7.91(t，J＝6.9Hz，2H)，7.67(d，J＝5.7Hz，1H)，7.50(t，J＝7.7Hz，1H)，7.38-7.30(m，2H)，7.22(dd，J＝10.7，8.4Hz，1H)，5.87(s，1H)，5.35(s，2H)，4.81(s，2H)，4.26(s，2H)，3.09(s，2H)，2.68(s，3H)，2.61(s，2H)，1.84(s，4H).HRMS(ESI)：found 577.2472，calcd for C 32 H 29 FN 8 O 2 [M+H] + 577.24.
Example 19
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-chloro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-chloro-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-chloro-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(500MHz，CDCl 3 )δ9.21(d，J＝2.2Hz，1H)，8.30(dd，J＝8.1，2.3Hz，1H)，7.93-7.86(m，2H)，7.79(d，J＝20.2Hz，2H)，7.64(dd，J＝8.2，1.8Hz，1H)，7.51(dt，J＝7.7，3.6Hz，2H)，7.35(d，J＝8.1Hz，1H)，7.32(d，J＝7.8Hz，1H)，5.30(s，2H)，4.66(t，J＝5.6Hz，2H)，4.09(q，J＝5.8Hz，2H)，3.04(t，J＝6.0Hz，2H)，2.68(s，3H)，2.60(t，J＝6.0Hz，2H)，1.90-1.80(m，4H).HRMS(ESI)：found 593.2176，calcd for C 32 H 29 ClN 8 O 2 [M+H] + 593.2104.
Example 20
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-bromo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-bromo-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3, 4-oxadiazole to give a pale yellow powder, N- (2- (1- (4- (2- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-bromo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3, 4-tetrahydroacridine. 1 H NMR(300MHz，CDCl 3 )δ9.25(s，1H)，8.32(d，J＝8.2Hz，1H)，7.95(d，J＝8.4Hz，1H)，7.86(s，1H)，7.73(d，J＝9.4Hz，3H)，7.63-7.52(m，2H)，7.34(d，J＝12.1Hz，2H)，5.42(s，2H)，5.32(q，J＝1.2Hz，1H)，4.62(s，2H)，4.07(s，2H)，3.07(s，2H)，2.70(s，3H)，2.63(s，2H)，1.88(s，4H).HRMS(ESI)：found 637.1669，calcd for C 32 H 29 BrN 8 O 2 [M+H] + 637.1597.
Example 21
Synthesis of N- (2- (1- (4- (2- (5- (5- (6- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-iodo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3,4-tetrahydroacridine
Referring to the synthetic method of example 8, intermediate 1 in example 8 was replaced with 2- (4-iodo-3- (prop-2-yn-1-yloxy) phenyl) -5- (6-methylpyridin-3-yl) -1,3, 4-oxadiazole to give N- (2- (1- (4- (5- (5- (2-methylpyridinyl)) 1,3, 4-oxadiazolyl) -2-iodo-phenoxy) ethyl) -1,2, 3-triazolyl) ethyl) -9-amino-1, 2,3,4-tetrahydroacridine as a pale yellow powder. 1 H NMR(500MHz，CDCl 3 )δ9.21(s，1H)，8.31-8.27(m，1H)，8.21(d，J＝8.4Hz，1H)，8.17(s，1H)，8.12(d，J＝8.7Hz，1H)，7.90(d，J＝8.0Hz，1H)，7.71-7.67(m，1H)，7.52(s，1H)，7.42(d，J＝8.0Hz，1H)，7.35(d，J＝8.0Hz，2H)，7.28(s，1H)，5.33(s，2H)，5.01(t，J＝5.6Hz，2H)，4.44(d，J＝5.8Hz，2H)，3.16(d，J＝5.7Hz，2H)，2.68(d，J＝10.8Hz，5H)，1.83(d，J＝5.5Hz，4H).HRMS(ESI)：found 685.1530，calcd for C 32 H 29 IN 8 O 2 [M+H] + 685.1456.
TABLE 1 structural formulas of Compounds synthesized in examples 1-21
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The following are some of the compounds of the present invention pharmacodynamic tests and results:
1. cholinesterase inhibition activity assay:
medicine and reagent: test compounds, eeAChE (e.c. 3.1.1.7, type VI-s, selected from electric eels), equche (e.c. 3.1.1.8, selected from horse serum), huAChE (EC 3.1.1.7, selected from human sources), huBChE (EC 3.1.1.8, selected from human sources). 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB), acetylthiocholine (ATC) iodide, butyrylthiocholine (BTC) iodide were all purchased from Sigma corporation; tacrine hydrochloride (9-Amin-1, 2,3,4-tetrahydroacridine hydrochloride hydrate) was purchased from BioTrend.
Instrument: THERMO Varioskan Flash full-wavelength multifunctional enzyme-labeled instrument.
The experimental method comprises the following steps:
(1) Preparing a buffer solution: 13.6g of potassium dihydrogen phosphate was dissolved in 1L of water and the pH was adjusted to 8.0.+ -. 0.1 with potassium hydroxide. The solution was stored at 4℃for further use.
(2) Preparing a 0.01M DTNB solution: 0.396g of DTNB and 0.15g of sodium bicarbonate were dissolved in 100mL of water to prepare a 0.01M DTNB solution, which was stored at-30℃for further use.
(3) Preparing 0.075M ATC and BTC solution: dissolving 0.217g of ATC in 10mL of water to prepare 0.075M ATC and BTC solution, and preserving at-30 ℃ for later use; 0.237g of BTC was dissolved in 10mL of water to prepare a 0.075M BTC solution, which was stored at-30℃for further use.
(4) Preparing AChE and BChE solution: dissolving 5000 units of AChE in 1mL of 1% gel solution, diluting with water to 100mL to obtain AChE solution with concentration of 5 units/mL, and preserving at-30deg.C for use; 5000 units of BChE was dissolved in 1mL of a 1% gel solution, and then diluted with water to 100mL to prepare an AChE/BChE solution with a concentration of 5 units/mL, which was stored at-30℃for use.
(5) Preparing a test object solution: the test compound was dissolved in ethanol to give a concentration of 10 -3 M solution (ethanol does not affect test results) and then diluted with water to give 10 concentration respectively -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 M.
Before the experiment, the solutions were warmed to room temperature and the AChE and BuChE solutions were diluted one time with water to give a concentrateAn enzyme solution having a degree of 2.5 units/mL. Background UV absorbance was measured with blank buffer (3 mL). mu.L of test compound solution, 100. Mu.L of DTNB solution and 100. Mu.L of enzyme solution were added to 3mL of buffer solution, and after the reaction was triggered by the addition of 20. Mu.L of ATC or BTC solution, the test solution was immediately timed and simultaneously mixed rapidly and the UV absorbance was measured at 412nM wavelength after 2 min. The blank was measured using an equal volume of water instead of the test solution. All tests were run in triplicate. The absorbance of the test compound at each concentration was recorded using the UV absorbance of the blank as 100%, and the results were calculated as the corresponding IC in a nonlinear decay analysis mode (non-linear regression analysis model) using GraphPad Prism TM (GraphPad Software, san Diego, calif., USA) software 50 Values, results are shown in table 2.
Table 2 results of compound target activity assay
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The 21 compounds in Table 2 all show better inhibition activity to AChEs and BChEs, and after structural modification, the inhibition activity of the general formula II and the general formula III to ChEs is obviously enhanced, and even the nanomolar activity level can be reached. In healthy brains, the activity of ache is dominant (80%), while BChEs appear to play a supportive role only. However, in progressive AD, AChE levels in the brain gradually drop to 55-67% of normal, while BuChE levels increase to 120% of normal. And the inhibition of BChEs exhibited weak peripheral cholinergic-like side effects. This suggests that inhibition of ache and BChEs is extremely important in slowing down the pathogenesis of AD during progressive AD development. The compound has good inhibitory activity on ChEs and is expected to have good curative effect on AD.
Pc 12 neurocytotoxicity assay:
medicine and reagent: test compound, DMEM medium (01-050-1A) purchased from Biological Industries, FBS fetal bovine serum (04-001-1A) purchased from Biological Industries, MTT thiazole blue reagent (KGT 525500) purchased from Yu Kaiji organisms.
Instrument: THERMO Varioskan Flash full-wavelength multifunctional enzyme-labeled instrument.
The experimental method comprises the following steps:
(1) Inoculating cells: first, log phase cells were collected and prepared at a concentration of 1X 10 5 Per mL of cell suspension, 100. Mu.l of cell suspension per well (1X 10 per well) was added to a 96-well cell culture plate 4 Individual cells), plates were placed at 37 ℃,5% co 2 Culturing in an incubator for 24 hours.
(2) The culture was discarded, washed 1 time with PBS buffer saline, and 100. Mu.L of compound at different concentrations prepared with DMEM medium was added to each well, and the blank group was added with only DMEM medium without compound. 96-well plates were placed at 37℃with 5% CO 2 Culturing in an incubator for 24 hours,
(3) 15. Mu.L of MTT solution (5 mg/mL, i.e., 0.5% MTT) was added per well in the dark and the culture was continued for 3-4 hours.
(4) The medium was discarded, 100. Mu.L of dimethyl sulfoxide was added to each well, and the mixture was gently shaken to dissolve the crystals in dimethyl sulfoxide. The absorbance (OD) of each well was measured at 490nm in a multifunctional enzyme-mapping instrument. The normal group OD value was set to 100% and the cell viability was calculated as follows: cell viability = (dosing OD value-blank OD value)/(normal OD value-blank OD value) ×100%. The experimental results are shown in FIG. 1.
When the concentration of the compound of the general formula I with the straight hexane chain as a connecting chain is higher than 5 mu M, PC12 cells cannot survive basically, and the compound shows strong cytotoxicity to the PC12 cells. When the click chemistry reaction is used for replacing the connecting chain, the survival rate of PC12 cells can reach 90% when the concentration of the obtained compounds 8-14 in the general formula II is 15 mu M, and when the 1,2, 4-oxadiazole ring system is replaced by the 1,3, 4-oxadiazole ring system, the survival rate of PC12 cells can still be kept above 80% when the concentration of the compounds is increased to 20 mu M, and the cytotoxicity of the compounds is obviously improved.
Nrf2 activation activity assay:
the experimental method comprises the following steps:
(1) And (5) in vitro cell culture. HepG2 cells transfected with ARE luciferase reporter plasmid were incubated with 10% fetal bovine serum in RPMI-1640 medium at 37℃with 5% CO 2 Culturing under the condition.
(2) Treating HepG2-ARE-C8 cells in logarithmic growth phase with 0.25% pancreatin digest to give a concentration of 4×10 5 Is a cell suspension of (a) a cell suspension of (b). 100. Mu.L of each was added to the 96-well ELISA plate for overnight incubation.
(3) The test compounds were prepared in 2-fold test concentrations in the medium, 100. Mu.L of t-butylhydroquinone (tBHQ) was added to the corresponding wells as positive control, and DMSO was negative control. Placing the 96-well ELISA plate with the added compound at 37deg.C and 5% CO 2 Incubate under conditions for 12h.
(4) The 5 Xlysate in the luciferase assay kit was prepared as 1 Xlysate for use.
(5) The 96-well plate was removed, the culture solution was aspirated from the well, the cells were washed with 1 XPBS buffer, and PBS buffer was aspirated after the washing was completed. 25 or 30. Mu.L of 1 Xcell lysate was added to each well and lysed on ice for 15min. After the cleavage is completed, standing for 3-5min, sucking 20 mu L of supernatant and adding the supernatant into a corresponding 96-well white ELISA plate.
(6) The white enzyme-labeled plate was placed in a Thermo Scientific LuminoskanAsent chemiluminescent microwell reader, 100 μl of luciferase assay reagent (prepared by uniformly mixing 1 bottle of luciferase assay substrate in the luciferase assay kit with 1 bottle of luciferase assay buffer) was added to each well before testing, and the reading was performed within 1min after adding the assay reagent.
The results are shown in FIG. 2. The fold induction of the test compounds in the test cells at different concentrations for the ARE-luciferase is on the ordinate, and the results show that all test compounds exhibit certain ARE reporter induction activity at 5 different test concentrations. For the compounds of the general formulae II and III, unsubstituted compounds 8 and 15 are preferred, and the Nrf 2-inducing activity of the compounds decreases instead after introduction of the electron donating group. Compound 14 exhibited 6-fold Nrf 2-inducing activity, but the compound had significant cytotoxicity, and damage to cells could activate cellular defenses to some extent, up-regulating Nrf2 levels, resulting in compound superimposed cellular defensive ARE activating activity on its own Nrf2 activation.
4. Determination of anti-inflammatory Activity
ROS detection method:
experimental reagent: LPS (Sigma-Aldrich, shangghai, china), active oxygen assay kit (Shanghai Biyun Biotechnology Co., ltd.), test compound, DMEM medium (01-050-1A) purchased from Biological Industries, FBS fetal bovine serum (04-001-1A) purchased from Biological Industries, MTT thiazole blue reagent (KGT 525500) purchased from Yu Kaiji organisms.
Experimental consumables and instruments: 96-well plate (Corning company), THERMO Varioskan Flash full-wavelength multifunctional enzyme-labeled instrument.
The experimental steps are as follows:
selecting BV2 cells in logarithmic growth phase, and adjusting cell concentration to 1.0X10 5 mu.L of cell suspension per well (1X 10 per well) was added to 96-well cell culture plates per mL 4 Individual cells), placed at 37 ℃,5% co 2 Culturing in an incubator for 24 hours. The medium was discarded, washed once with PBS, DMEM medium containing different concentrations of compound was added to each well, and incubated for 2h (normal group without compound, blank group without cells, and the remaining reagents were added normally, 3 duplicate wells were set for each concentration). After 2h, LPS solution (5. Mu.g/mL) was added and incubated with the test compound for 24h. The medium was removed, and DCFH-DA 0.1mL at a concentration of 10. Mu.M in a DMEN medium formulation (1:1000 dilution) was added to each well and incubated at 37℃for 20min in a cell incubator to allow sufficient contact between the probe and the cells. The cell culture broth was removed and the cells were washed three times with serum-free DMEM medium to sufficiently remove DAFH-DA that did not enter the cells. The luminescence value is read by using a multifunctional enzyme-labeled instrument, and the whole operation is finished within 10 minutes to prevent fluorescence quenching.
ELISA detection method:
experimental reagent and instrument: lipopolysaccharides (LPS), mouse TNF-a, 1L-1 beta, iNOS Elisa assay kit (Sigma-Aldrich, shanghai, china), test compounds, DMEM medium (01-050-1A) purchased from Biological Industries, FBS fetal bovine serum (04-001-1A) purchased from Biological Industries, MTT thiazole blue reagent (KGT 525500) purchased from Yu Kaiji organisms.
Experimental consumables and instruments: THERMO Varioskan Flash full-wavelength multifunctional enzyme-labeled instrument.
Experimental procedure
BV2 cells were seeded in 96-well plates with 100. Mu.L of cell suspension per well (1X 10 4 cells well), and the test compound was added after culturing in an incubator for 24 hours. The culture medium in each well was blotted, DMEM medium containing different concentrations of compound was added to each well, and incubated for 2h (normal group without compound, blank group without cells, and the remaining reagents were added normally, 3 duplicate wells were set for each concentration). After 2h, LPS solution (5. Mu.g/mL) was added and incubated with the test compound for 24h, the test was performed according to ELISA instructions, the kit was equilibrated at room temperature for 60min, the required strips were removed from the aluminum foil bag, and the remaining strips were returned to 4℃with a self-sealing bag seal. The test comprises a standard hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are respectively added into the standard hole; the sample wells were filled with 50. Mu.L of the sample to be tested, the reaction wells were sealed with a sealing plate membrane, and incubated at 37℃for 45min after gentle mixing. The sample was removed and the plate was washed 4 times with wash solution. Except for blank holes, 50 mu L of peroxidase-labeled detection antibody is added to each of the rest holes to be detected, the reaction holes are sealed by a sealing plate film, and the reaction holes are incubated for 30min at 37 ℃. The liquid was discarded and the plate was washed 4 times with washing liquid. 50. Mu.L of horseradish peroxidase was added to each well, and after sealing the plate, the mixture was gently mixed and incubated at 37℃for 15min. The liquid was discarded and the plate was washed 4 times with washing liquid. 50. Mu.L of horseradish peroxidase substrates A and B are added into the wells, the reaction wells are sealed by a sealing plate film, and incubated for 15min at constant temperature. After 50. Mu.L of the reaction termination liquid was added and mixed slightly, the liquid in the well was observed to change from blue to yellow. The whole operation is finished within 15min after reading at 450nm by using a multifunctional enzyme label instrument so as to prevent fluorescence quenching.
As a result, as shown in FIG. 3, compound 15 exhibited a better activity in the above activity evaluation, and thus, it was examined for its anti-inflammatory activity as a preferred molecule. The level of ROS in the cells treated with LPS increased compared to the control group, and the level of ROS in the cells treated with compound 15 at different concentrations decreased to a level comparable to the control group, indicating that compound 15 has an antioxidant stress effect.
Under the stimulation of LPS, the levels of pro-inflammatory factors TNF-alpha, IL-1 beta and iNOS in BV2 cells are obviously increased, as shown in figures 4, 5 and 6, after being treated by the compound 15, the levels of the TNF-alpha, IL-1 beta and iNOS in the cells are reduced along with the increase of the concentration of the compound to be equivalent to the levels of a blank group, and the preliminary indication shows that the compound 15 has better anti-inflammatory effect.
Morris water maze study mouse behavioural study
Instrument: panlab SMART 3.0 behavioural video analyzer
Animals: adult male ICR mice (8-10 weeks, body weight 20-25 g) were purchased from the university of Yangzhou medical center.
Reagent: scopolamine hydrobromide was purchased from the company Ara Ding Shiji (NAT 107418, shanghai), tacrine (purity > 95%), compound 15.
The experimental method comprises the following steps: the 40 mice were randomly divided into 4 groups (10 mice per group): control, model, tacrine, compound 15 treatment. Tacrine, compound 15 was dissolved in CMC-Na solution (0.5 g CMC-Na,100mL distilled water) and administered by gavage (10 mg/kg body weight). After 30min, in the model group, tacrine group and compound 15 treatment group mice were intraperitoneally injected with scopolamine (1 mg/kg), and in the control group mice were intraperitoneally injected with physiological saline. Mice were tested for cognitive function and memory capacity by the water maze. An escape platform (diameter: 10 cm) is fixed in a circular water tank (diameter: 120cm, height: 60 cm), a small flag (5 cm in height) is fixed on the platform, water with a height of 40cm is filled in the water tank, and the water tank is kept at 25 ℃ to form a water maze. The mice were trained on the escape platform on days 1-2 of drug administration, the platform was placed under water 1cm on days 3-5, the mice were trained, the platform was removed on the last day (day 6), the mice were evaluated, and the time, trajectory and speed of the mice reaching the location of the platform were recorded. The experimental results are as follows.
Analysis of results: in connection with figures 7, 8, 9 and the tables, the average time and distance of arrival of the model mice at the plateau was much higher than those of the control group, indicating that scopolamine resulted in memory deficit in the mice, and that modeling was successful. The time and distance spent in the tacrine group were significantly reduced relative to the model group, indicating a significant improvement in the memory and cognitive function of the mice. While the average time and distance to the platform was slightly higher for the compound 15 mice than for the tacrine group, but significantly lower than for the model group. Compound 15 was shown to have an improving effect on mice memory and cognitive function, but slightly inferior to tacrine.

Claims (7)

1. 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product or pharmaceutically acceptable salt thereof has a structure shown in general formulas II and III:
wherein,
r represents hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxy.
2. A 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product or a pharmaceutically acceptable salt thereof according to claim 1, which is any one of the following:
3. a 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: the pharmaceutically acceptable salt is selected from hydrochloride, maleate or citrate.
4. A process for the preparation of the 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, comprising the steps of:
(1) The reaction formula for preparing the general formula II is as follows:
to be used forIs taken as a raw material, is reacted with bromopropyne after esterification, and is hydrolyzed to obtain the +.>And->The cyclization is carried out to obtain->The preparation method comprises the steps of taking methyl anthranilate as a raw material, hydrolyzing, cyclizing, aminating, then ammonolyzing by ethanolamine, carrying out acylation reaction by using p-toluenesulfonyl chloride, and then reacting with TMSN3 to obtain +.>And->Obtaining a target compound through click reaction;
(2) The reaction scheme for preparing formula III is as follows:
to be used forIs taken as a raw material, is reacted with bromopropyne after esterification, and is hydrolyzed to obtainReaction of methyl 6-methylnicotinate with hydrazine hydrate to give +.>And (3) withAfter the reaction, the cyclization is carried out to obtain +.>The method comprises the steps of taking methyl anthranilate as a raw material, hydrolyzing, cyclizing, ammonolyzing by ethanolamine, acylating by p-toluenesulfonyl chloride, and reacting with TMSN3 to obtain +.>And->Obtaining a target compound with a structure shown in a general formula III through click reaction;
the definition of R is as defined in any one of claims 1 to 3.
5. Use of the 1,2, 4-oxadiazole Nrf2 activator-tac Lin Pinge product of any one of claims 1-3 in the preparation of a medicament for preventing or treating alzheimer's disease.
6. A pharmaceutical composition characterized by: comprising the compound represented by the general formulae II and III or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 as an active ingredient or a main active ingredient and a pharmaceutically acceptable carrier.
7. The pharmaceutical composition according to claim 6, wherein: the pharmaceutical composition preparation is tablet, capsule, powder, syrup, liquid, suspension or injection.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109134350A (en) * 2017-06-13 2019-01-04 中国药科大学 Donepezil-BHT heterozygote, preparation method and its for treating Alzheimer's disease
CN110143956A (en) * 2019-06-10 2019-08-20 中国药科大学 Tacrine-pyrido thiophenes and preparation method thereof and purposes
CN110669044A (en) * 2019-09-09 2020-01-10 中国药科大学 Donepezil-oxadiazole fusion compound and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109134350A (en) * 2017-06-13 2019-01-04 中国药科大学 Donepezil-BHT heterozygote, preparation method and its for treating Alzheimer's disease
CN110143956A (en) * 2019-06-10 2019-08-20 中国药科大学 Tacrine-pyrido thiophenes and preparation method thereof and purposes
CN110669044A (en) * 2019-09-09 2020-01-10 中国药科大学 Donepezil-oxadiazole fusion compound and preparation method and application thereof

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