A mannich base compound and a new use of the compound as a drug

Abstract

The present application relates to a kind of Mannich base compound and its new use, specifically, the present application relates to the Mannich base compound as shown in general formula I, and the pharmaceutical composition of the compound or their pharmaceutical salt, as cholinesterase inhibitor for preparing the drug for treating and / or preventing, alleviate Alzheimer's disease, myasthenia gravis or its combination.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a class of Mannich base compounds and their novel uses. Specifically, this invention relates to the use of Mannich base compounds as shown in general formula (I) or pharmaceutical compositions thereof, their pharmaceutical salts, enantiomers, diastereomers and racemic compounds as cholinesterase inhibitors in the preparation of medicaments for the treatment and / or prevention and relief of diseases such as Alzheimer's disease and myasthenia gravis caused by neurological disorders. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, accounting for 50% to 75% of all dementia cases. It primarily affects people aged 65 and older, with an incidence rate of 7% to 10% in this age group. The risk of developing the disease increases significantly with age.

[0003] The main pathological features of Alzheimer's disease (AD) include senile plaques formed by the aggregation of β-amyloid protein (Aβ) in the brain, neurofibrillary tangles formed by the aggregation of hyperphosphorylated tau protein, long-term inflammatory response, and neuronal death. However, the exact pathogenesis of AD remains unclear, and effective prevention and treatment of AD remain challenges for modern medicine. Acetylcholinesterase (AChE) is a class of glycoproteins that exist in the body in various isoenzyme forms, generally classified into true cholinesterase and pseudocholinesterase. True cholinesterase, namely acetylcholinesterase (AChE), is mainly found in the synaptic cleft of cholinergic nerve endings, especially accumulating in large quantities in the folds of the postsynaptic membrane of motor nerve endplates, and is also found in cholinergic neurons and erythrocytes. AChE is an important hydrolytic enzyme present in the human synaptic cleft, and its main function is to hydrolyze acetylcholine at the nerve synapse, terminating the transmission of nerve impulses. Currently, the small molecule drugs approved for the treatment of Alzheimer's disease (AD) are mainly acetylcholinesterase inhibitors (AChEIs), such as tacrine, donepezil, galantamine, huperzine A, and rivastigmine. AChEIs increase the content of acetylcholine in the synaptic cleft by inhibiting central cholinesterase, thereby enhancing cholinergic nerve function and improving cognitive function.

[0004] Furthermore, AChEIs have wide applications in treating sensory or motor disorders, memory loss, polyneuritis, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, and cerebral palsy in children caused by neurological disorders. Therefore, researching and developing next-generation, highly effective AChEIs could be used to prepare better anti-AD drugs and to prevent or treat sensory or motor disorders, memory loss, polyneuritis, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, and cerebral palsy in children caused by neurological disorders. Summary of the Invention

[0005] The purpose of this invention is to provide a Mannich base compound and its novel uses.

[0006] Specifically, the present invention provides the use of Mannich base compounds of general formula (I) as inhibitors of cholinesterase in the preparation of medicaments for the treatment and / or prevention, relief of Alzheimer's disease, myasthenia gravis or combinations thereof.

[0007] In a first aspect of the invention, there is provided the use of a Mannich base compound of general formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, or racemic mixture thereof, characterized in that it is used as an inhibitor of cholinesterase for the preparation of a medicament for the treatment and / or prevention, or relief of Alzheimer's disease, myasthenia gravis, or combinations thereof:

[0008]

[0009] In formula (Ⅰ),

[0010] The ring Ar is an unsubstituted or substituted aromatic group consisting of 1 to 2 azirphenyl groups, benzo5 to 6-membered aromatic heterocyclic groups, each of which contains 1 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen; each of which is independently selected from the group consisting of bromine, C1 to C4 straight-chain or branched alkyl, C1 to C4 straight-chain or branched alkoxy, and amide.

[0011] R1 is hydrogen, or a C1-C4 straight-chain or branched alkyl group;

[0012] R2 is an amino group substituted with 1-2 C1-C4 straight-chain or branched alkyl groups, or a 5-7 membered nitrogen heterocyclic group that is unsubstituted or substituted with 1-2 substituents, each of which contains 0-1 heteroatom selected from oxygen, sulfur and nitrogen; the substituents are independently selected from the group consisting of halogens, C1-C4 straight-chain or branched alkyl groups, hydroxyl groups, hydroxymethyl groups, and trifluoromethyl groups.

[0013] In a second aspect of the invention, the compounds in general formula (I) are selected from the group consisting of:

[0014]

[0015]

[0016]

[0017] In a third aspect of the invention, a pharmaceutical composition comprising (a) a therapeutically effective amount of one or more Mannich base compounds of formula (I) as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, for use in the preparation of a medicament for the treatment and / or prevention and relief of Alzheimer's disease and myasthenia gravis-related diseases.

[0018] In a fourth aspect of the invention, a pharmaceutical composition comprising (a) a therapeutically effective amount of one or more Mannich base compounds as described in the second aspect of the invention, or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, is provided for use in the preparation of a medicament for the treatment and / or prevention and relief of Alzheimer's disease and myasthenia gravis-related diseases.

[0019] Within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new technical solutions.

[0020] Method of drug administration:

[0021] Because the compounds of the present invention have excellent inhibitory activity against cholinesterase, the compounds of the present invention and their pharmaceutically acceptable salts, as well as pharmaceutical compositions containing the compounds of the present invention as the main active ingredient, are all included in this patent for the treatment, prevention, and / or relief of related diseases caused by nervous system disorders.

[0022] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose.

[0023] The "pharmaceutically acceptable salt" described in this invention refers to a conventional, non-toxic salt formed by the reaction of a compound of general formula (I) with an inorganic or organic acid. The inorganic or organic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, phosphoric acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid, etc.; or a compound of general formula (I) reacting with propionic acid, oxalic acid, etc. The sodium, potassium, calcium, aluminum, or ammonium salts formed by esters of malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid, or glutamic acid with inorganic bases; or the methylamine, ethylamine, or ethanolamine salts formed by compounds of general formula (I) with organic bases; or the corresponding inorganic acid salts formed by esters of compounds of general formula (I) with lysine, arginine, or ornithine with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid, or the corresponding organic acid salts formed with formic acid, acetic acid, picric acid, methanesulfonic acid, or ethanesulfonic acid.

[0024] This invention also relates to a medicament comprising at least one compound of formula (I) of this invention, together with one or more pharmacologically acceptable excipients or carriers, and to its use for the aforementioned purposes. "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers or gel substances suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components of the composition can be mixed with and with the compounds of this invention without significantly reducing the efficacy of the compounds. The pharmaceutical carriers mentioned here include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, phosphates, glycerol, sorbic acid, potassium sorbate, vegetable oils, mixtures of partial glycerides of saturated vegetable fatty acids, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium sulfate, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin, gelatin, talc, solid lubricants, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0025] There are no particular limitations on the administration of the compound or pharmaceutical composition of formula (I) of the present invention. Representative administration methods include, but are not limited to: oral, intramuscular or intravenous injection, parenteral, pulmonary, nasal, sublingual, tongue, buccal, rectal, transdermal, conjunctival, local administration or administration in the form of an implant.

[0026] Solid dosage forms for oral administration include capsules, tablets (uncoated or coated), pills, powders, and granules. In these solid dosage forms, the active compound of formula (I) is mixed with at least one conventional inert excipient or carrier, such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol or glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0027] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes; if necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0028] Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, dimethyl sulfoxide, and cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof.

[0029] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. Besides the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0030] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous ethanol, polyol solutions, dispersions, suspensions, emulsions, lyophilized products, and sterile powders, and sterile powders for reconstitution into sterile injectable solutions or dispersions, suitable aqueous and non-aqueous carriers, and diluents.

[0031] Dosage forms suitable for other routes of administration include nasal drops / solutions, sprays; tablets or capsules, suppositories for administration to the tongue, sublingual or buccal, preparations for the ears and eyes, vaginal capsules, aqueous suspensions (lotions, shaken mixtures), lipophilic suspensions, ointments, creams, lotions, pastes, powders or implants.

[0032] The active ingredient of formula (I) can be converted into the above-described administration form using methods known per se, which can be achieved using inert, non-toxic, and suitable pharmaceutical excipients, including carriers such as microcrystalline cellulose, solvents such as liquid polyethylene glycol, emulsifiers such as sodium dodecyl sulfate, dispersants such as polyvinylpyrrolidone, synthetic and natural biopolymers, stabilizers such as antioxidants, colorants such as inorganic pigments, or flavoring agents and / or masking agents. Where suitable, the active ingredient may be present in one or more of the above-described carriers in the form of microencapsulation.

[0033] In addition to the compounds of formula (I) of this invention, the above-mentioned pharmaceutical preparations may also contain other active pharmaceutical ingredients.

[0034] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically considered effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg; of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition.

[0035] The main advantages of this invention include: This invention provides a Mannich base compound with the structure shown in formula (Ⅰ), which can effectively inhibit cholinesterase activity and has the potential to prepare drugs for treating and / or preventing and alleviating related diseases caused by nervous system disorders.

[0036] Furthermore, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0037] English abbreviations and their full Chinese names

[0038] Detailed Implementation

[0039] Through extensive and in-depth research and screening, the inventors have developed a class of active ingredients that can effectively inhibit cholinesterase, namely compounds represented by general formula (I) or their pharmaceutically acceptable salts, solvates, hydrates, enantiomers, diastereomers, or racemates. Experiments have shown that the active ingredients of this invention can efficiently inhibit hAChE activity, thus making them suitable for treating diseases related to nervous system disorders. Based on this, the present invention was completed.

[0040] The technical solution of the present invention will be clearly and completely described below. However, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] the term

[0042] The term "aromatic group" refers to an aromatic group, such as, but not limited to, the group selected from, but not limited to, phenyl and naphthyl.

[0043] The term "5- or 6-membered aromatic heterocyclic group" refers to a 5- or 6-membered aromatic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur, and can be selected from the following group: pyridinyl, pyrimidinyl, thiazolyl, isothiazolyl, furanyl, thiophene, pyrroleyl.

[0044] The term "1 to 2 azaphenyl" refers to a 6-membered aromatic group having 1 or 2 aza atoms, which can be selected from the following group: pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl.

[0045] The term "5- or 6-membered heterocyclic group" refers to a cyclic group having one or more heteroatoms selected from nitrogen, oxygen, or sulfur that are 5-, 6-, or 7-membered and can be selected from the group consisting of tetrahydrofuranyl, piperidinyl, piperazineyl, morpholinyl, pyrrolyl, tetrahydrothiazolyl, dioxaneyl, or similar groups.

[0046] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0047] The term "C1-C4 alkyl" refers to alkyl groups with one to four carbon atoms, either linear or branched, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0048] The term "C1-C4 alkoxy" refers to straight-chain or branched alkoxy groups having 1-4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups.

[0049] The term "C2-C6 ester group" refers to a ROC (=O)- group having 2-6 carbon atoms, such as -COOCH3, -COOC2H5, -COOC3H7, -COOC4H9, or similar groups.

[0050] In this document, unless otherwise specified, the term "substitution" means that one or more hydrogen atoms on a group are substituted by a substituent selected from the group consisting of: C1-C4 alkyl, C1-C4 alkoxy, halogen, hydroxyl, carboxyl, C2-C4 acyl, C2-C6 ester, amino, and phenyl; wherein the phenyl includes unsubstituted phenyl or substituted phenyl having 1-3 substituents selected from: halogen, C1-C4 alkyl, cyano, hydroxyl, nitro, C3-C6 cycloalkyl, C1-C4 alkoxy, and amino.

[0051] Example 1: Synthesis of Compound 1 (2-((diethylamino)methyl)-4-(pyridin-4-ylamino)phenol)

[0052] Synthesis route:

[0053]

[0054] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add diethylamine (1.82 g, 25 mmol) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 18 h. Concentrate the solvent under reduced pressure, add 20 mL of methyl tert-butyl ether, stir rapidly for 1 h, pour off the supernatant, concentrate under reduced pressure, and dry under vacuum to obtain a yellow oily substance, R1-3a (3.72 g, 78%). 1 H NMR (400MHz, DMSO) δ10.84(s,1H),9.63(s,1H),7.29(s,1H),7.25(d,J=8.6Hz,1H),6 .61(d,J=8.6Hz,1H),3.66(s,2H),2.76-2.50(m,4H),1.97(s,3H),1.25-0.80(m,6H). 13 C NMR (101MHz, DMSO) δ168.22,153.17,130.37,122.56,120.33,119.69,115.06,55.14,45.79,23.41,10.95.

[0055] Add R1-3a (2.36 g, 10 mmol) and 25 ml of 20% hydrochloric acid to a 100 mL round-bottom flask, heat under reflux for 2 h, allow to cool naturally, add 25 ml of ethanol, concentrate the solvent under reduced pressure, add another 25 ml of ethanol, concentrate the solvent under reduced pressure, repeat this process 2-3 times until the solvent is basically evaporated, add 25 ml of methyl tert-butyl ether, stir at room temperature for 1 h, filter, dry the filter cake under vacuum to obtain a light yellow solid, which is R1-3 (2.21 g, 83%). 1HNMR(400MHz,DMSO)δ10.91(s,1H),10.37(s,3H),10.08(s,1H),7.54(s,1H),7.33(d,J= 8.4Hz,1H),7.16(d,J=8.6Hz,1H),4.22(s,2H),3.23-2.82(m,4H),1.27(t,J=7.0Hz,6H). 13 C NMR (101MHz, DMSO) δ156.41,127.49,125.86,122.74,117.30,116.50,49.01,46.27,8.53.

[0056] R1-3 (0.80 g, 3.0 mmol) was added to a 50 mL round-bottom flask, followed by 10 mL of acetic acid and then L1N (0.34 g, 3.0 mmol). The mixture was heated under reflux for 5 h. After natural cooling, the pH was adjusted to 9–10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3 x 25 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and subjected to column chromatography (CH2Cl2:CH3OH = 30:1, v / v) to give product 1 (L1NR1-3, white solid, 0.61 g, 75%). 1 H NMR (400MHz, CDCl3) δ8.20(d,J=5.3Hz,2H),7.00(dd,J=8.5,2.4Hz,1H),6.84(d,J=2.0Hz,1H),6.80(d,J= 8.5Hz,1H),6.62(d,J=6.0Hz,2H),6.06(s,1H),3.75(s,2H),2.64(q,J=7.2Hz,4H),1.12(t,J=7.2Hz,6H). 13 CNMR(101MHz, CDCl3)δ156.12,152.40,149.97,130.20,124.71,124.48,123.07,116.89,108.52,56.83,46.39,11.22.MS(ESI + m / z (%): 272.17 [M+H] + MS (ESI+) m / z (%): 272.4 [M+H] + .

[0057] Example 2: Synthesis of compound 3(4-((4-hydroxy-3-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyridineamide)

[0058]

[0059] In a 100 mL round-bottom flask, add acetaminophen (R1, 4.2 g, 30 mmol), 50 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (4.05 g, 50 mmol). Then add N-methylpiperazine (3.1 g, 31 mmol) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 12 h. Concentrate the solvent under reduced pressure, add 50 mL of methyl tert-butyl ether, stir rapidly for 1 h, filter, and dry the filter cake under vacuum to obtain a light yellow solid, R1-4a (4.8 g, 61%). 1 HNMR (400MHz, CDCl3) δ7.31 (d, J=2.1Hz, 1H), 7.09 (dd, J=8.6, 2.4Hz, 1H), 6.7 5(d,J=8.6Hz,1H),3.68(s,2H),3.15-2.35(m,8H),2.30(s,3H),2.13(s,3H). 13 C NMR (101MHz, CDCl3) δ168.17,154.37,129.59,121.37,121.05,116.09,61.24,54.88,52.46,45.84,24.24.

[0060] Add R1-4a (2.65 g, 10 mmol) and 25 ml of 20% hydrochloric acid to a 100 mL round-bottom flask, heat under reflux for 3 h. After natural cooling, add 25 ml of ethanol and concentrate the solvent under reduced pressure. Repeat this process 2-3 times until the solvent is almost completely evaporated. Add 20 ml of ethyl acetate, stir rapidly at room temperature for 1 h, filter, and dry the filter cake under vacuum to obtain a light yellow solid R1-4 (3.1 g, 93%). 1 H NMR (400MHz, DMSO) δ7.54(d,J=2.4Hz,1H),7.37(dd,J=8.7,2.5Hz,1H),7.13(d,J=8.7Hz,1H),4.39(s,2H),3.23-3.75(m,9H),2.88(s,3H). 13 C NMR (101MHz, DMSO) δ156.54,127.91,126.37,122.03,116.54,116.18,49.05,48.43,47.33,41.72.

[0061] R1-4 (0.62 g, 2.0 mmol) was added to a 50 mL round-bottom flask, followed by 8 mL of acetic acid and then L2N (0.31 g, 2.0 mmol). The mixture was heated under reflux for 6 h. After natural cooling, the pH was adjusted to 9–10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3 x 30 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and subjected to column chromatography (CH2Cl2:CH3OH = 20:1, v / v) to give a pale yellow solid 3 (L2NR1-4, 0.40 g, 59%). 1 H NMR(400MHz,DMSO)δ8.18(d,J=6.9Hz,1H),7.59(d,J=11.0Hz,2H),7.33–7.2 5(m,2H),7.15(d,J=8.8Hz,1H),4.40(s,2H),3.66–3.28(m,8H),2.88(s,3H). 13 C NMR(101MHz,DMSO)δ160.72,157.18,156.49,155.46,128.90,128.02,127.58,126.24,12 2.06,116.88,116.51,116.02,52.68,49.12,47.37,41.74.MS(ESI+)m / z(%):342.19[M+H] + .

[0062] Example 3: Synthesis of compound 4 (4-((3-((dimethylamino)methyl)-4-hydroxyphenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0063]

[0064] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add dimethylamine (3.75 g, 25 mmol, 30% ethanol solution) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 24 h. Concentrate the solvent under reduced pressure, add 20 mL of methyl tert-butyl ether, stir rapidly for 1 h, filter, and dry the filter cake under vacuum to obtain a light yellow solid, R1-1a (2.8 g, 67%). 1 H NMR (400MHz, CDCl3) δ7.32(s,1H),7.21(s,1H),7.06(d,J=8.5Hz,1H),6.76(d,J=8.6Hz,1H),3.61(s,2H),2.40–2.26(m,6H),2.13(s,3H). 13C NMR (101MHz, CDCl3) δ168.14,155.01,129.41,122.09,121.11,121.00,116.10,62.74,44.48,24.33.

[0065] Add R1-1a (2.08 g, 10 mmol) and 25 ml of 20% hydrochloric acid to a 100 mL round-bottom flask, heat under reflux for 3 h, allow to cool naturally, add 25 ml of ethanol, concentrate the solvent under reduced pressure, add another 25 ml of ethanol, concentrate the solvent under reduced pressure, repeat this process 2-3 times until the solvent is basically evaporated, add 25 ml of methyl tert-butyl ether, stir at room temperature for 1 h, filter, dry the filter cake to obtain a white solid, R1-1 (2.20 g, 92%). 1 H NMR (400MHz, DMSO) δ10.92(s,1H),10.66-9.91(s,4H),7.48(s,1H),7.35(d,J=8.2Hz,1H),7.17(d,J=8.4Hz,1H),4.24(s,2H),2.71(s,6H). 13 C NMR (101MHz, DMSO) δ156.19,127.24,126.12,121.91,117.44,116.37,54.57,42.06.

[0066] Add R1-1 (0.72 g, 3.0 mmol) to a 50 mL round-bottom flask, add 10 mL of acetic acid, then add L3N (0.71 g, 3.0 mmol), and heat under reflux for 4 h. After natural cooling, adjust the pH to 9–10 with 1 M NaOH aqueous solution, extract with CH2Cl2 (3 x 25 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 30:1, v / v) to give white solid 4 (L3NR1-1, 0.77 g, 70%). 1 H NMR (400MHz, DMSO) δ14.55(d,J=4.9Hz,1H),10.94(s,1H),10.86(s,1H),10.68(s,1H),9.05(s,1H),8.38(t,J=6.3Hz,1H),7.90(s,1H),7.84(s,1 H),7.60(s,2H),7.35(dd,J=8.6,2.3Hz,1H),7.21(d,J=8.6Hz,1H),6.82 (d,J=7.1Hz,1H),4.26(d,J=4.9Hz,2H),4.02(s,3H),2.86-2.65(m,6H). 13C NMR (101MHz, DMSO) δ165.40,160.10,155.89,154.93,142.54,141.00,129.86,128.41,128.16,126.3 7,125.98,117.57,116.85,110.49,99.84,99.73,56.47,53.94,41.79.MS(ESI+)m / z(%):367.17[M+H] + .

[0067] Example 4: Synthesis of compound 5 (4-(4-hydroxy-3-(pyrrolidone-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0068]

[0069] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add tetrahydropyrrole (1.78 g, 25 mmol, 30% ethanol solution) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 16 h. Concentrate the solvent under reduced pressure, add 20 mL of methyl tert-butyl ether, stir rapidly for 1 h, and dry the filter cake under vacuum to obtain a pale yellow solid, R1-2a (3.8 g, 81%). 1 H NMR (400MHz, CDCl3) δ7.33(s,1H),7.06(d,J=8.5Hz,1H),6.74(d,J=8.6Hz,1H),3.79(s,2H),2.78-2.41(m,4H),2.12(s,3H),1.91-1.61(m,4H). 13 CNMR (101MHz, CDCl3) δ168.17,154.98,129.33,122.64,120.83,120.70,115.96,58.75,53.48,24.31,23.66.

[0070] R1-2a (2.34 g, 10 mmol) and 25 ml of 20% hydrochloric acid were added to a 100 mL round-bottom flask and heated under reflux for 3 h. After natural cooling, 25 ml of ethanol was added, and the solvent was concentrated under reduced pressure. This process was repeated 2-3 times until the solvent was almost completely evaporated. 25 ml of methyl tert-butyl ether was added, and the mixture was stirred at room temperature for 1 h. The mixture was then filtered, and the filter cake was dried under vacuum to obtain a white solid, R1-2 (2.35 g, 89%). 1H NMR (400MHz, DMSO) δ11.19–9.83(m,5H),7.51(s,1H),7.32(d,J=8.3Hz,1H),7. 15(d,J=8.6Hz,1H),4.29(s,2H),3.37(s,2H),3.10(s,2H),2.15-1.52(m,4H).

[0071] Add R2-1 (0.53 g, 2.0 mmol) to a 50 mL round-bottom flask, then add 8 mL of acetic acid, followed by L3N (0.47 g, 2.0 mmol). Heat under reflux for 5 h. Allow to cool naturally, adjust the pH to 9–10 with 1 M NaOH aqueous solution, and extract with CH2Cl2 (3 x 20 mL). Combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 20:1, v / v) to give a white solid 5 (L3NR1-2, 0.62 g, 79%). 1 H NMR (400MHz, DMSO) δ9.02 (s, 1H), 8.81 (s, 1H), 8.31 (d, J = 5.4Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.29 (s, 1H), 7.1 5-6.95(m,2H),6.79(d,J=8.3Hz,1H),6.51(d,J=5.4Hz,1H),3.98(s,3H),3.74(s,2H),2.56(s,4H),1.75(s,4H). 13 CNMR(101MHz,DMSO)δ166.52,157.13,153.74,152.06,151.17,149.94,130.64,125.71,124.8 1,124.37,124.00,122.68,115.68,112.99,107.59,99.45,55.97,55.83,53.07,23.14.MS(ESI + m / z (%): 393.19 [M+H] + .

[0072] Example 5: Synthesis of compound 8 (4-(4-hydroxy-3-(morpholinomethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0073]

[0074] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add morpholine (1.83 g, 21 mmol) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 16 h. Concentrate the solvent under reduced pressure, add 20 mL of ethyl acetate, stir rapidly for 1 h, pour off the supernatant, evaporate the residue to dryness under reduced pressure, and dry under vacuum to obtain a light yellow oily substance, R1-5a (3.8 g, 81%). 1 H NMR (400MHz, CDCl3) δ7.35 (s, 1H), 7.08 (dd, J = 8.5, 2.5Hz, 1H), 6.77 (dd, J = 7. 4,5.0Hz,1H),4.80(s,2H),3.89-3.67(m,4H),2.78-2.33(m,4H),2.13(s,3H). 13 C NMR (101MHz, CDCl3) δ168.22,154.38,129.79,121.55,121.22,120.89,116.21,66.76,61.77,52.90,24.30.

[0075] Add R1-5a (2.51 g, 10 mmol) and 25 ml of 20% hydrochloric acid to a 100 mL round-bottom flask, heat under reflux for 3 h, allow to cool naturally, add 25 ml of ethanol, concentrate the solvent under reduced pressure, add another 25 ml of ethanol, concentrate the solvent under reduced pressure, repeat this process 2-3 times until the solvent is basically evaporated, add 25 ml of methyl tert-butyl ether, stir at room temperature for 1 h, filter, dry the filter cake under vacuum to obtain a white solid, R1-5 (2.45 g, 87%). 1 H NMR (400MHz, DMSO) δ7.54 (d, J=2.1Hz, 1H), 7.36 (dd, J=8.6, 2.2Hz, 1H), 7.12 (d, J= 8.7Hz,1H),4.32(s,2H),4.09-3.91(m,2H),3.85-3.60(m,4H),3.40-3.26(m,3H).

[0076] Add R1-5 (0.28 g, 1.0 mmol) to a 50 mL round-bottom flask, add 8 mL of acetic acid, then add L3N (0.24 g, 1.0 mmol), and heat under reflux for 5 h. After natural cooling, adjust the pH to 9–10 with 1 M NaOH aqueous solution, extract with CH2Cl2 (3 x 15 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 20:1, v / v) to give a white solid 8 (L3NR1-5, 0.31 g, 75%). 1H NMR (400MHz, DMSO) δ14.57(s,1H),11.73–11.36(m,1H),10.93(s,1H),10.83(s,1H),9.05(s,1H),8.38(s,1H),7.91(s,1H),7.84(s,1H),7.6 9(d,J=1.8Hz,1H),7.59(s,1H),7.41–7.30(m,1H),7.21(d,J=8.7Hz,1 H),6.88(d,J=7.0Hz,1H),4.31(s,2H),4.02(s,3H),4.00-3.75(m,8H). 13 C NMR(101MHz,DMSO)δ165.41,160.06,156.12,154.78,142.51,140.98,130.36,128.42,12 8.13,126.35,125.96,116.87,116.39,110.47,99.83,62.93,56.46,55.97,50.66.MS(ESI + m / z (%): 409.18 [M+H] + .

[0077] Example 6: Synthesis of compound 9 (4-(4-hydroxy-3-(piperidin-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0078]

[0079] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add piperidine (1.87 g, 22 mmol) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 14 h. Concentrate the solvent under reduced pressure, add 20 mL of methyl tert-butyl ether, stir rapidly for 1 h, pour off the supernatant, evaporate the residue to dryness under reduced pressure, and dry under vacuum to obtain a light yellow oily substance, R1-6a (3.8 g, 77%). 1 H NMR (400MHz, CDCl3) δ7.30 (d, J=2.1Hz, 1H), 7.06 (dd, J=8.6, 2.3Hz, 1H), 6.73 (d, J=8.6Hz ,1H),3.62(s,2H),3.13–2.20(m,4H),2.12(s,3H),1.77–1.56(m,4H),1.55-1.29(m,2H). 13C NMR (101MHz, CDCl3) δ168.18,154.74,129.40,121.90,121.22,120.74,115.96,62.06,53.90,25.85,24.24,23.95.

[0080] Add R1-6a (2.49 g, 10 mmol) and 25 ml of 20% hydrochloric acid to a 100 mL round-bottom flask, heat under reflux for 3 h, allow to cool naturally, add 30 ml of ethanol, concentrate the solvent under reduced pressure, add another 30 ml of ethanol, concentrate the solvent under reduced pressure, repeat this process 2-3 times until the solvent is basically evaporated, add 25 ml of ethyl acetate, stir at room temperature for 1 h, filter, dry the filter cake under vacuum to obtain a white solid, R1-6 (2.51 g, 90%). 1 H NMR (400MHz, DMSO) δ7.53(d,J=2.4Hz,1H),7.35(dd,J=8.7,2.5Hz,1H),7.11(d,J=8.7Hz,1H),4. 23(s,2H),3.33(d,J=11.9Hz,2H),2.94(t,J=10.9Hz,2H),2.04–1.60(m,5H),1.51–1.31(m,1H). 13 C NMR (101MHz, DMSO) δ156.64,127.95,125.96,122.53,116.77,116.56,52.88,51.50,22.15,21.18.

[0081] Add R1-5 (0.28 g, 1.0 mmol) to a 50 mL round-bottom flask, add 6 mL of acetic acid, then add L3N (0.24 g, 1.0 mmol), and heat under reflux for 4 h. After natural cooling, adjust the pH to 9–10 with 1 M NaOH aqueous solution, extract with CH2Cl2 (3 x 20 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 20:1, v / v) to give white solid 9 (L3NR1-6, 0.28 g, 69%). 1H NMR (400MHz, DMSO) δ14.66 (d, J = 4.3Hz, 1H), 10.94 (s, 1H), 10.93-10.73 (m, 2H), 9.06 (s, 1H), 8.38 (t ,J=6.0Hz,1H),7.92(s,1H),7.84(s,1H),7.70(d,J=2.3Hz,1H),7.61(s,1H),7.53(s,1H),7.40(s,1H ),7.34(dd,J=8.6,2.3Hz,1H),7.28(s,1H),7.23(d,J=8.7Hz,1H),6.83(d,J=7.1Hz,1H),4.22(d,J= 4.4Hz,2H),4.02(s,3H),3.39-3.24(m,2H),3.03–2.84(m,2H),1.95–1.59(m,5H),1.47–1.32(m,1H). 13 CNMR(101MHz,DMSO)δ165.44,160.04,156.12,154.82,142.46,140.98,130.31,128.26,128.0 9,126.32,125.98,116.96,116.85,110.46,99.79,56.45,52.98,51.55,22.06,21.26.MS(ESI + m / z (%): 407.20 [M+H] + .

[0082] Example 7: Synthesis of Compound 11 (4-(ethyl(4-hydroxy-3-(pyrrolidone-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0083]

[0084] Add L3NR1-2 (0.20 g, 0.5 mmol) and 10 mL of acetonitrile to a 50 mL round-bottom flask, then add iodoethane (0.10 g, 0.6 mmol), heat under reflux for 1 h, allow to cool naturally, and perform direct column chromatography (CH2Cl2:CH3OH = 30:1, v / v) to give a pale yellow solid 11 (L3NR1-8, 0.15 g, 68%). 1HNMR (400MHz, DMSO) δ8.71(s,1H),8.32(d,J=5.4Hz,1H),7.30(s,1H),7.09(d,J=5.9Hz,2H),6.84(d,J=9.2Hz,1H),6.52(d, J=5.5Hz,1H),4.34(q,J=7.1Hz,2H),3.93(s,3H),3.75(s,2H),2.61-2.43(m,4H),1.83-1.61(m,4H),1.35(t,J=7.1Hz,3H). 13 C NMR (101MHz, DMSO) δ165.69,157.55,154.03,152.46,151.38,150.09,130.26,125.82,125.19,124.42,120. 17,115.80,112.43,107.86,99.59,60.90,55.89,55.77,53.03,23.10,14.10.MS(ESI+)m / z(%):421.22[M+H] + .

[0085] Example 8: Synthesis of compound 16(4-((6,7-dimethoxyquinoline-4-yl)amino)-2-((4-(hydroxymethyl)piperidin-1-yl)methyl)phenol)

[0086]

[0087] In a 100 mL round-bottom flask, add acetaminophen (R1, 3.1 g, 20 mmol), 30 mL of ethanol, and formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol). Then add 4-piperidinemethanol (2.53 g, 22 mmol) and 0.2 mL of concentrated hydrochloric acid. Heat under reflux for 16 h. Concentrate the solvent under reduced pressure, add 20 mL of methyl tert-butyl ether, stir rapidly for 1 h, filter, and perform column chromatography on the filter cake (CH2Cl2:CH3OH = 20:1, v / v) to give a pale yellow solid, R1-7a (3.4 g, 61%). 1 H NMR (400MHz, DMSO) δ9.64(s,1H),7.37–7.20(m,2H),6.62(d,J=8.3Hz,1H),3.57(s,2H),3.25(d,J=6.2Hz,2H),2.8 7(d,J=11.3Hz,2H),2.08-1.98(m,2H),1.97(s,3H),1.67(d,J=12.7Hz,2H),1.46-1.26(m,1H),1.23-0.98(m,2H). 13C NMR (101MHz, DMSO) δ167.45,152.83,130.85,122.14,120.22,119.38,115.04,65.64,59.65,52.49,28.61,23.71.

[0088] R1-7a (2.79 g, 10 mmol) and 25 ml of 20% hydrochloric acid were added to a 100 mL round-bottom flask and heated under reflux for 4 h. After natural cooling, 30 ml of ethanol was added and the solvent was concentrated under reduced pressure. This process was repeated 2-3 times until the solvent was almost completely evaporated. 25 ml of ethyl acetate was added and the mixture was stirred at room temperature for 1 h. The mixture was then filtered, and the filter cake was dried under vacuum to obtain a white solid, R1-7 (2.93 g, 95%). 1 H NMR (400MHz, DMSO) δ10.82(s,1H),10.58-10.12(m,4H),7.55(d,J=1.7Hz,1H),7.34(dd,J=8.6,2.2Hz,1H),7.18(t,J=11.8Hz,1H),4.29(s,1H ),4.19(s,2H),3.36(d,J=11.4Hz,2H),3.23(d,J=5.4Hz,2H),3.04-2.8 1(m,2H),1.79(d,J=12.7Hz,2H),1.71-1.56(m,1H),1.55-1.33(m,2H). 13 C NMR (101MHz, DMSO) δ156.43,127.83,125.99,122.00,116.44,64.64,53.28,51.45,35.48,25.59.

[0089] Add R1-7 (0.31 g, 1.0 mmol) to a 50 mL round-bottom flask, add 6 mL of acetic acid, then add L4N (0.22 g, 1.0 mmol), and heat under reflux for 3 h. After natural cooling, adjust the pH to 9–10 with 1 M NaOH aqueous solution, extract with CH2Cl2 (3 x 20 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 20:1, v / v) to give a white solid 16 (L4NR1-7, 0.29 g, 68%). 1H NMR (400MHz, DMSO) δ14.37 (s, 1H), 10.72 (s, 2H), 10.6-10.25 (m, 1H), 8.29 (d, J = 6.7Hz, 1H),8.21(s,1H),7.68(d,J=1.9Hz,1H),7.48(s,1H),7.36(dd,J=8.6,2.2Hz,1H),7.20 (d,J=8.6Hz,1H),6.74(d,J=7.0Hz,1H),4.22(s,2H),4.00(s,3H),3.97(s,3H),3.50-3 .35(m,2H),3.31-3.18(m,2H),3.12-2.86(m,2H),1.96-1.73(m,2H),1.76–1.43(m,3H). 13 C NMR (101MHz, DMSO) δ155.61,154.61,153.38,149.40,139.52,134.81,130.47,128.77,128.32,116. 98,116.65,111.06,102.33,99.72,99.08,64.67,56.63,56.21,51.54,48.43,35.54,25.61.MS(ESI + m / z (%): 424.22 [M+H] + .

[0090] Example 9: Synthesis of compound 21 (4-((1H-pyrazolo[3,4-b]pyridin-4-yl)amino)-2-((diethylamino)methyl)phenol)

[0091]

[0092] Add R1-3 (0.27 g, 1.0 mmol) to a 50 mL round-bottom flask, add 6 mL of acetic acid, then add L7N (0.18 g, 1.2 mmol), and heat under reflux for 5 h. After natural cooling, adjust the pH to 9–10 with 1 M NaOH aqueous solution, extract with CH2Cl2 (3 x 20 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution, dry with anhydrous Na2SO4, and perform column chromatography (CH2Cl2:CH3OH = 15:1, v / v) to give white solid 21 (L7NR1-3, 0.20 g, 63%). 1H NMR (400MHz, DMSO) δ13.11(s,1H),8.93(s,1H),7.99(d,J=5.5Hz,1H),7.94(s,1H),7.06(d,J=7.1Hz,2H) ,6.76(d,J=8.8Hz,1H),6.31(d,J=5.5Hz,1H),3.74(s,2H),2.57(q,J=7.1Hz,4H),1.04(t,J=7.1Hz,6H). 13 C NMR (101MHz, DMSO) δ154.55,153.28,149.61,146.88,131.49,130.46,124.61,123.88,123.81,115.85,104.26,96.93,54.76,45.87,11.07.MS(ESI + m / z (%): 312.18 [M+H] + .

[0093] Following a similar method to Examples 1-9, the following compounds have been synthesized:

[0094]

[0095]

[0096]

[0097]

[0098] Example 10: Test of the compound's ability to inhibit AChE:

[0099] 1. Experimental Objective

[0100] The present invention relates to the testing and calculation of the IC50 of Mannich bases as represented by general formula (I) for AChE. 50 value.

[0101] 2. Experimental Materials

[0102] Acetylthiocholine iodine (ATCh), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and hAChE (derived from human erythrocytes) were used. Equipment included: centrifuge (SIGMA 3-18K), microplate reader (Bio Rad Microplate Reader Model 550), balance (METTLERTOLEDO), incubator (Blue-part, Shanghai Hengke), multi-channel pipette (BIOHIT), and 96-well plates.

[0103] 3. Preparation methods for experimental reagents

[0104] 3.1 Preparation methods for buffer solutions with different pH values:

[0105] Solution A: 31.202 g of 0.2 M NaH2PO4, NaH2PO4·2H2O (MW 156.01) plus 1000 mL of distilled water;

[0106] Solution B: 71.628 g of 0.2 M Na₂HPO₄·12H₂O (MW 358.14) plus 1000 mL of distilled water;

[0107] pH=7.0 PBS buffer: 39 mL of solution A, 61 mL of solution B, and 100 mL of distilled water.

[0108] pH=7.4 PBS buffer: 19 mL of solution A, 81 mL of solution B, and 100 mL of distilled water.

[0109] 3.2 DTNB Preparation Method

[0110] Dissolve 29.7 mg / 100 mL pH 7.0 PBS buffer to a concentration of 0.75 mM, store at 0-4℃, and use immediately on the same day.

[0111] 3.3 ATCh Preparation Method

[0112] Dissolve 8.7 mg / 10 mL pH 7.4 PBS buffer to a concentration of 3 mM, store at 0-4℃, and use immediately on the same day.

[0113] 3.4 Enzyme Pretreatment

[0114] hAChE(sigma) was diluted to the appropriate concentration with PBS (pH=7.4, 0.1% BSA) and stored at 0-4℃ for immediate use.

[0115] 3.5 Drug Dissolution and Dilution

[0116] Weigh out the appropriate mass of the drug and dissolve it in DMSO to prepare a concentration of 1*10. -2 Drug stock solution M (as tested in control, the solvent had no effect on the experimental results). Drug dilution: Dilute the above drug stock solution with PBS (pH=7.4) in 96-well plates to 48μM, 24μM, 12μM, 3μM, 0.3μM, and 0.03μM, respectively, and use immediately.

[0117] 4. Test of the enzyme inhibition rate of the compound

[0118] 4.1 Principle of Activity Testing

[0119] The activity assay employed the micro-DTNB method (Formula II). ATCh is an analogue of ACh and can be catalytically hydrolyzed by AChE to generate acetic acid and thiocholine iodide (TCh) (see reaction A). TCh can react rapidly and quantitatively with DTNB to generate the yellow anion 5-thio-2-nitrobenzoic acid (RS-) (reaction B). The latter has maximum absorption at a wavelength of 412 nm. The activity of AChE can be calculated by measuring the absorbance (OD value) of the generated RS- and thus quantifying the generated TCh.

[0120]

[0121] For a typical enzyme-catalyzed reaction (Formula III), E represents the enzyme, S represents the substrate, ES is the enzyme-substrate complex, P is the product, and k represents the relevant positive and negative reaction rate constants.

[0122]

[0123] Since the concentration of the enzyme used in the experiment is much smaller than the concentration of the substrate, i.e., [ES] << [S], [ES] can be approximated as constant, thus formula 3-1 applies:

[0124]

[0125] Let [E]0 represent the initial concentration of the enzyme, then [E]0 = [E] + [ES], that is, [E] = [E]0 - [ES]. Substituting this into formula 3-1, we can obtain the formula...

[0126] Equation 3-2 is shown below:

[0127]

[0128] Because the concentration of the intermediate ES is very small, the substrate consumption concentration can be approximated as equal to the product formation concentration, i.e., -d[S] / dt = d[P] / dt. Therefore, the product formation rate can be expressed as Equation 3-3:

[0129]

[0130] Substituting 3-2 into 3-3, for reaction A in equation II, [H2O] can be considered constant, and its k -2 =0, thus we get formula 3-4:

[0131]

[0132] Let K M =(k -1 +k2) / k1, K M Since Michaelis is a constant, we get Formula 3-5:

[0133]

[0134] As can be seen from Equation 3-5, the reaction rate is only related to the substrate concentration. If the substrate concentration is high, it can be assumed that the substrate concentration remains constant for a period of time at the beginning of the reaction, and therefore the reaction rate is constant. If the product concentration at a fixed reaction time point is measured, the reaction rate, i.e., the enzyme's catalytic activity, can be calculated proportionally from this. In reaction B of Equation II, the colorimetric reaction of the products TCh and DTNB obtained by AChE catalysis is completed instantaneously and quantitatively, and the final measured OD value is proportional to RS. - The concentration, which is directly proportional to the concentration of the catalytic hydrolysis product TCh, can be determined by measuring the colorimetric substance RS. - The OD value is used to reflect enzyme activity. The substrate concentration (ATCh, 3.0 mM) used in this experiment has been proven by preliminary experiments to fully meet the above conditions. Within 50 min after the start of the enzymatic reaction, the OD value was found to be directly proportional to the reaction time. Therefore, the OD value measured at a fixed time point (e.g., 30 min) after the start of the enzymatic reaction can be used to measure AChE activity.

[0135] 4.2 Test of enzyme inhibition rate

[0136] This experiment first incubated different drugs with hAChE, then added ATCh for reaction, and finally added DTNB for color development. The enzyme activity rate was calculated by comparing the results with those of normal hAChE testing, and then the enzyme inhibition rate was calculated. The IC50 was calculated by measuring the enzyme inhibition rate at multiple concentrations (16, 8, 4, 1, 0.1, 0.01 μM). 50 value.

[0137] a) Enzyme dilution: Dilute the AChE stock solution (20 U / mL) 2000 times with PBS (0.1M, pH=7.4, 0.1% BSA) and store at 0-4℃.

[0138] b) Enzyme inhibition: Add 20 μL of the diluted enzyme solution to 20 μL of the corresponding drug concentration (operation in a 96-well plate, final drug concentration 16, 8, 4, 1, 0.1, 0.01 μM), and inhibit the enzyme at 25℃ for 15 min. The normal enzyme control is 20 μL of PBS buffer instead of the corresponding drug concentration.

[0139] c) Reaction: Add 30 μL LATCH (3.0 mM, pH = 7.4 PBS) to the above enzyme at room temperature and react for 30 min.

[0140] d) Color development: Add 150 μL DTNB (0.75 mM, 0.1 M PBS, pH = 7.0) and measure the OD value at 412 nM using a microplate reader.

[0141] Enzyme inhibition rate calculation method: %Activity = (SB) * 100 / (PB). S = OD value of the inhibited group. P = OD value of the normal group, B = OD value of the PBS blank group (solvent background value). Enzyme inhibition rate: %Inhibition = 1 - %Activity.

[0142] IC 50 Calculation method (The following formula is used to perform nonlinear fitting on the enzyme inhibition rate at different concentrations to calculate IC50) 50 Value): %Activity = 100 * IC 50 / (IC 50 +[S]), where [S] represents the drug concentration.

[0143] Table 1: IC50 of the synthesized compounds against hAChE according to the examples 50 (μM)

[0144]

[0145] It should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a Mannich base compound represented by general formula (I), characterized in that, As a cholinesterase inhibitor, it is used in the preparation of drugs for the treatment and / or prevention, or relief of Alzheimer's disease, myasthenia gravis, or combinations thereof: In formula (Ⅰ), The ring Ar is an unsubstituted or substituted aromatic group consisting of 1 to 2 azirphenyl groups, benzo5 to 6-membered aromatic heterocyclic groups, each of which contains 1 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen; each of which is independently selected from the group consisting of bromine, C1 to C4 straight-chain or branched alkyl, C1 to C4 straight-chain or branched alkoxy, and amide. R1 is hydrogen, or a C1-C4 straight-chain or branched alkyl group; R2 is an amino group substituted with 1-2 C1-C4 straight-chain or branched alkyl groups, or a 5-7 membered nitrogen heterocyclic group that is unsubstituted or substituted with 1-2 substituents, each of which contains 0-1 heteroatom selected from oxygen, sulfur and nitrogen; the substituents are independently selected from the group consisting of halogens, C1-C4 straight-chain or branched alkyl groups, hydroxyl groups, hydroxymethyl groups, and trifluoromethyl groups.

2. The use as described in claim 1, characterized in that, The compounds in general formula (I) shown are selected from the following group: 。 3. The use of a pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more Mannich base compounds of general formula (I) of claim 1 or pharmaceutically acceptable salts thereof, as cholinesterase inhibitors, for the preparation of medicaments for the treatment and / or prevention, relief of Alzheimer's disease, myasthenia gravis or combinations thereof.

4. The use of a pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more Mannich base compounds as described in claim 2 or pharmaceutically acceptable salts thereof, as cholinesterase inhibitors, for use in the preparation of medicaments for the treatment and / or prevention, relief of Alzheimer's disease, myasthenia gravis, or combinations thereof.

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