Mannich base compound and novel pharmaceutical application thereof

Abstract

The invention relates to a Mannich base compound and a new application thereof, in particular to a Mannich base compound shown in a general formula I, a medicinal composition of the compound, and medicinal salts, solvates, hydrates, enantiomer forms, diastereoisomers and racemic compounds of the Mannich base compound, the medicinal composition and the medicinal salts, the solvates, the hydrates, the enantiomer forms, the diastereoisomers and the racemic compounds of the Mannich base compound and the medicinal composition. The compound can be used as a cholinesterase inhibitor for preparing medicines for treating and / or preventing and relieving related diseases caused by nervous system diseases. The related diseases are selected from the group consisting of Alzheimer's disease, sensory or dyskinesia, hypomnesia, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, polyneuritis and complications thereof, or a combination thereof. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a class of Mannich base compounds and new uses thereof. Specifically, the present invention relates to the use of Mannich base compounds as shown in general formula (I) or pharmaceutical compositions thereof, their pharmaceutical salts, enantiomeric forms, diastereoisomers and racemic compounds as cholinesterase inhibitors in the preparation of drugs for treating and / or preventing and alleviating Alzheimer's disease, sensory or motor disorders, memory loss, polyneuritis, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis and cerebral palsy in children and other related diseases caused by nervous system diseases. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, accounting for 50% to 75% of all dementia cases. The disease mainly affects the elderly population aged 65 and above, 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 AD include senile plaques formed by the aggregation of beta-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 is still unclear, and effective prevention and treatment of AD remains a difficult problem in modern medicine. Acetylcholinesterase (AChE) is a class of glycoproteins that exists in the body in the form of multiple isozymes and can generally be divided into true cholinesterase and pseudocholinesterase. True cholinesterase, namely acetylcholinesterase (AChE), is mainly present in the synaptic cleft of cholinergic nerve endings, especially in the wrinkles of the postsynaptic membrane of the motor nerve end plate. It is also present in cholinergic neurons and red blood cells. AChE is an important hydrolase existing in the synaptic cleft of the human body. Its main function is to hydrolyze acetylcholine at the synaptic site and terminate the conduction of nerve impulses. At present, the small molecule drugs approved for the treatment of AD are mainly acetylcholinesterase inhibitors (AChE inhibitor, AChEI), such as tacrine, donepezil, galantamine, Huperzine A and rivastigmine, etc. AChEI increases the content of acetylcholine in the synaptic cleft by inhibiting central cholinesterase, enhances cholinergic nerve function, and improves cognitive function.

[0004] In addition, AChEI has a wide range of uses in related diseases such as sensory or motor disorders, memory loss, multiple neuritis, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, and cerebral palsy in children caused by diseases of the nervous system. Therefore, the research and development of a new generation of highly effective AChEI can be used to prepare better anti-AD drugs, and can also be used to prevent or treat sensory or motor disorders, memory loss, multiple neuritis, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, and cerebral palsy in children caused by diseases of the nervous system. Summary of the invention

[0005] The purpose of the present invention is to provide a Mannich base compound and a new use thereof.

[0006] Specifically, the present invention provides a Mannich base compound represented by the general formula (I) as a cholinesterase inhibitor, for use in the preparation of a drug for treating and / or preventing or alleviating related diseases caused by nervous system disorders; the related diseases are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof.

[0007] In the first aspect of the present invention, there is provided a use of a Mannich base compound represented by general formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate thereof, characterized in that it is used as a reversible inhibitor of cholinesterase for preparing a drug for treating and / or preventing or alleviating related diseases caused by nervous system disorders:

[0008]

[0009] In formula (I),

[0010] Ring Ar is the following aromatic group which is unsubstituted or substituted by 1-2 substituents: 1-2 azaphenyl groups, benzo 5-6 membered aromatic heterocyclic groups, 1-2 azaphenyl 5-6 membered aromatic heterocyclic groups, each of which contains 1-3 heteroatoms selected from oxygen, sulfur and nitrogen; each of which is independently selected from the following group: bromine, C1-C4 straight chain or branched alkyl, C1-C4 straight chain or branched alkoxy, C2-C6 straight chain or branched ester, cyano, nitro, hydroxyl, amino, aminomethyl, dimethylamino, hydroxymethyl, trifluoromethyl, carboxyl, mercapto, C2-C4 acyl, amide, or two adjacent substituents together with the carbon atoms connected thereto form a 5-7 membered ring;

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

[0012] R2 is an amino group substituted by 1-2 C1~C4 straight or branched alkyl groups, a 5-7 membered nitrogen heterocyclic group which is unsubstituted or substituted by 1-2 substituents, each of which further contains 0-1 heteroatoms selected from oxygen, sulfur and nitrogen; and the substituents are independently selected from the following group: halogen, C1~C4 straight or branched alkyl groups, hydroxyl, hydroxymethyl, trifluoromethyl, carboxyl, C2~C4 acyl, and amide.

[0013] The related diseases caused by nervous system disorders are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof.

[0014] The compound in the general formula (I) is selected from the following group:

[0015]

[0016]

[0017] In a second aspect of the present invention, there is provided a Mannich base compound as shown in formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate thereof:

[0018] In formula (I),

[0019] Ring Ar is a pyrido 5- to 6-membered aromatic heterocyclic group, a pyridyl group or a benzopyridyl group substituted by 1-2 substituents, each of the aromatic heterocyclic groups contains 1-2 nitrogen heteroatoms, and the substituents are each independently selected from the following group: bromine, amide, hydroxyl, hydroxymethyl, cyano, nitro, dimethylamino, C1-C4 straight-chain or branched alkoxy;

[0020] R1 is hydrogen, C1-C4 straight or branched alkyl;

[0021] R2 is an amino group substituted by 1-2 C1, C3, C4 straight or branched alkyl groups, a 5-7 membered nitrogen heterocyclic group which is unsubstituted or substituted by 1 substituent, each of which further contains 0-1 heteroatoms selected from oxygen, sulfur and nitrogen; and the substituents are independently selected from the following group: C1-C4 straight or branched alkyl groups, hydroxyl, hydroxymethyl, trifluoromethyl, carboxyl, and amide.

[0022] The compound in the general formula (I) is selected from compounds 2-5, 7-13, 15-18, 20-22 in the above group.

[0023] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising: a therapeutically effective amount of one or more Mannich base compounds represented by the general formula (I) described in the second aspect of the present invention, or a pharmaceutically acceptable salt thereof, and an optional pharmaceutically acceptable carrier.

[0024] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising (a) a therapeutically effective amount of one or more Mannich base compounds of the general formula (I) according to the first aspect of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate or prodrug thereof, and (b) a pharmaceutically acceptable carrier, for use in the preparation of a drug for treating and / or preventing or alleviating related diseases caused by nervous system disorders.

[0025] The related diseases caused by nervous system disorders are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof.

[0026] In the fifth aspect of the present invention, a method for treating, preventing, and / or alleviating related diseases caused by nervous system disorders is provided, comprising the steps of: administering a safe and effective amount of a Mannich base compound represented by the general formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate thereof to a subject in need, wherein the Mannich base compound represented by the general formula (I) is as described in the first aspect of the present invention.

[0027] The object is a primate mammal, such as a human.

[0028] Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new technical solution.

[0029] Method of drug administration:

[0030] Since the compounds of the present invention have excellent inhibitory activity on cholinesterase, the compounds of the present invention and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions containing the compounds of the present invention as the main active ingredient, which can be used to treat, prevent, and / or alleviate related diseases caused by nervous system diseases are all included in this patent.

[0031] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose.

[0032] The "pharmaceutically acceptable salt" of the present invention is a conventional non-toxic salt formed by the reaction of the compound of formula (I) with an inorganic acid or an organic acid. The inorganic acid 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, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or the compound of formula (I) reacts with propionic acid, oxalic acid, The sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by ester formation with malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid and then reacting with an inorganic base; or the methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of the general formula (I) and an organic base; or the corresponding inorganic acid salt formed by ester formation with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid or the corresponding organic acid salt formed with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

[0033] The present invention also relates to a medicament comprising at least one compound of formula (I) of the present invention, together with one or more pharmacologically acceptable excipients or carriers, and also to its use for the above-mentioned purpose. "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. The pharmaceutical carriers here include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, phosphates, glycerol, sorbic acid, potassium sorbate, vegetable oils, partial glyceride mixtures of saturated vegetable fatty acids, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium sulfate, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, gelatin, talc, solid lubricants, emulsifiers, wetting agents, colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0034] There is no particular limitation on the mode of administration of the compound of formula (I) or the pharmaceutical composition of the present invention. Representative modes of administration include, but are not limited to, oral administration, intramuscular or intravenous injection, parenteral administration, pulmonary administration, nasal administration, sublingual administration, lingual administration, buccal administration, rectal administration, transdermal administration, conjunctival administration, topical administration or administration in the form of an implant.

[0035] 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 ingredients: (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrators, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, sodium carbonate; (e) solubilizers, such as paraffin; (f) absorption accelerators, such as quaternary ammonium salt 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 lauryl sulfate, or mixtures thereof. Capsules, tablets and pills may also contain buffers.

[0036] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes; if necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0037] Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent 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-butylene glycol, dimethylformamide, dimethyl sulfoxide, and cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0038] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances. In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances.

[0039] Compositions for parenteral injection may include 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.

[0040] Dosage forms suitable for other routes of administration include nasal drops / solutions, sprays; tablets or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the ears and eyes, vaginal capsules, aqueous suspensions (lotions, shaken mixtures), lipophilic suspensions, ointments, creams, emulsions, pastes, dusting powders or implants.

[0041] The active ingredient of formula (I) can be converted into the above-mentioned administration form by methods known per se, which can be achieved with suitable inert non-toxic pharmaceutical excipients, including carriers such as microcrystalline cellulose, solvents such as liquid polyethylene glycol, emulsifiers such as sodium lauryl sulfate, dispersants such as polyvinyl pyrrolidone, synthetic and natural biopolymers, stabilizers such as antioxidants, colorants such as inorganic pigments or flavoring agents and / or taste masking agents. In appropriate cases, the active ingredient can be present in one or more of the above-mentioned carriers in the form of microencapsulation.

[0042] In addition to the compound of formula (I) of the present invention, the above-mentioned pharmaceutical preparation may also contain other pharmaceutically active ingredients.

[0043] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage applied is a pharmaceutically effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg; of course, the specific dosage should also refer to factors such as the route of administration and the patient's health status.

[0044] The advantages of the present invention mainly include: the present invention provides a Mannich base compound with a structure shown in formula (I), which can effectively inhibit the activity of cholinesterase and has the potential to prepare drugs for treating and / or preventing and alleviating related diseases caused by nervous system diseases.

[0045] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0046] English abbreviations and their full Chinese names

[0047] DETAILED DESCRIPTION

[0048] After extensive and in-depth research and a large number of screenings, the inventors have developed a class of active ingredients that can effectively inhibit cholinesterase, namely, the compound represented by the general formula (I) or its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate. Experiments have shown that the active ingredients of the present invention can effectively inhibit the activity of hAChE, and thus can be used for related diseases caused by nervous system diseases, and the present invention has been completed on this basis.

[0049] The technical solution of the present invention will be described clearly and completely below, but the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] the term

[0051] The term "aromatic ring group" refers to a group having aromaticity, such as selected from the following group but not limited to: phenyl, naphthyl.

[0052] The term "5- to 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, which can be selected from the following group: pyridyl, pyrimidinyl, thiazolyl, isothiazolyl, furanyl, thienyl, pyrrolyl.

[0053] The term "1 to 2 azaphenyl groups" refers to a 6-membered aromatic group having 1 or 2 nitrogen heteroatoms, which can be selected from the following group: pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl.

[0054] The term "5- to 6-membered heterocyclic group" refers to a 5-, 6- or 7-membered cyclic group having one or more heteroatoms selected from nitrogen, oxygen or sulfur, which can be selected from the following group: tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, tetrahydrothiazolyl, dioxane, or similar groups.

[0055] The term "halogen" refers to fluorine, chlorine, bromine, iodine.

[0056] The term "C1-C4 alkyl" refers to a straight or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0057] The term "C1-C4 alkoxy" refers to a straight or branched alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

[0058] 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.

[0059] In this document, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C4 alkyl, C1-C4 alkoxy, halogen, hydroxyl, carboxyl, C2-C4 acyl, C2-C6 ester, amino, phenyl; the phenyl group includes unsubstituted phenyl or substituted phenyl with 1-3 substituents selected from the group consisting of halogen, C1-C4 alkyl, cyano, hydroxyl, nitro, C3-C6 cycloalkyl, C1-C4 alkoxy, amino.

[0060] Example 1: Synthesis of Compound 1 (2-((diethylamino)methyl)-4-(pyridin-4-ylamino)phenol)

[0061] Synthesis route:

[0062]

[0063] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) into a 100 mL round-bottom flask, then add diethylamine (1.82 g, 25 mmol), 0.2 ml of concentrated hydrochloric acid, and heat to reflux for 18 h. Concentrate the solvent under reduced pressure, add 20 ml of methyl tert-butyl ether, stir rapidly for 1 h, pour out the clear liquid, concentrate under reduced pressure, and dry in vacuo to obtain a yellow oil, which is 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.

[0064] R1-3a (2.36 g, 10 mmol) and 25 ml of 20% hydrochloric acid were added to a 100 mL round-bottom flask and heated under reflux for 2 h. After cooling naturally, 25 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of methyl tert-butyl ether was added and stirred at room temperature for 1 h. The filter cake was vacuum dried to obtain a light yellow solid, which was R1-3 (2.21 g, 83%). 1 HNMR(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.

[0065] R1-3 (0.80 g, 3.0 mmol) was added to a 50 mL round-bottom flask, 10 ml of acetic acid was added, and then L1N (0.34 g, 3.0 mmol) was added, and the mixture was heated to reflux for 5 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with a 1 M NaOH aqueous solution, and the mixture was extracted with CH2Cl2 (3*25 mL). The organic phases were combined, washed with a saturated sodium chloride aqueous solution, dried with anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=30:1, v / v) to obtain 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] + .

[0066] Example 2: Synthesis of Compound 3 (4-((4-hydroxy-3-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyridineamide)

[0067]

[0068] Add acetaminophen (R1, 4.2 g, 30 mmol), 50 ml of ethanol, formaldehyde aqueous solution (aq 37%) (4.05 g, 50 mmol) into a 100 mL round-bottom flask, then add N-methylpiperazine (3.1 g, 31 mmol), 0.2 ml of concentrated hydrochloric acid, and heat to 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 vacuum dry the filter cake 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.

[0069] R1-4a (2.65 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 cooling naturally, 25 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 20 ml of ethyl acetate was added and the mixture was rapidly stirred at room temperature for 1 h. The mixture was filtered and the filter cake was dried 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). 13C NMR (101MHz, DMSO) δ156.54,127.91,126.37,122.03,116.54,116.18,49.05,48.43,47.33,41.72.

[0070] R1-4 (0.62 g, 2.0 mmol) and 8 ml of acetic acid were added to a 50 mL round-bottom flask, and then L2N (0.31 g, 2.0 mmol) was added and heated to reflux for 6 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*30 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried with anhydrous Na2SO4, and subjected to column chromatography (CH2Cl2:CH3OH=20:1, v / v) to obtain a light 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] + .

[0071] Example 3: Synthesis of Compound 4 (4-((3-((dimethylamino)methyl)-4-hydroxyphenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0072]

[0073] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) to a 100 mL round-bottom flask, then add dimethylamine (3.75 g, 25 mmol, 30% ethanol solution), 0.2 ml of concentrated hydrochloric acid, and heat to 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 vacuum dry the filter cake to obtain a light yellow solid, R1-1a (2.8 g, 67%). 1H 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). 13 C NMR (101MHz, CDCl3) δ168.14,155.01,129.41,122.09,121.11,121.00,116.10,62.74,44.48,24.33.

[0074] R1-1a (2.08 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 cooling naturally, 25 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of methyl tert-butyl ether was added and stirred at room temperature for 1 h. The mixture was filtered and the filter cake was dried to obtain a white solid, which was 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.

[0075] R1-1 (0.72 g, 3.0 mmol) and 10 ml of acetic acid were added to a 50 mL round-bottom flask, and then L3N (0.71 g, 3.0 mmol) was added and heated to reflux for 4 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*25 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=30:1, v / v) to obtain a white solid 4 (L3NR1-1, 0.77 g, 70%). 1H 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). 13 C NMR (101 MHz, DMSO)δ

[0076] 165.40,160.10,155.89,154.93,142.54,141.00,129.86,128.41,128.16,126.37,125.98, 117.57,116.85,110.49,99.84,99.73,56.47,53.94,41.79.MS(ESI+)m / z(%):367.17[M+H] + .

[0077] Example 4: Synthesis of Compound 5 (4-(4-hydroxy-3-(pyrrolidin-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0078]

[0079] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) to a 100 mL round-bottom flask, then add tetrahydropyrrole (1.78 g, 25 mmol, 30% ethanol solution), 0.2 ml of concentrated hydrochloric acid, and heat to 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 in vacuum to obtain a light 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). 13C NMR (101MHz, CDCl3) δ168.17,154.98,129.33,122.64,120.83,120.70,115.96,58.75,53.48,24.31,23.66.

[0080] 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 cooling naturally, 25 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of methyl tert-butyl ether was added and stirred at room temperature for 1 h. The filter cake was vacuum dried to obtain a white solid, which was R1-2 (2.35 g, 89%). 1 H 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).

[0081] R2-1 (0.53 g, 2.0 mmol) and 8 ml of acetic acid were added to a 50 mL round-bottom flask, and then L3N (0.47 g, 2.0 mmol) was added and heated to reflux for 5 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*20 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=20:1, v / v) to obtain 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). 13CNMR(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] + .

[0082] Example 5: Synthesis of Compound 8 (4-(4-hydroxy-3-(morpholinomethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0083]

[0084] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) into a 100 mL round-bottom flask, then add morpholine (1.83 g, 21 mmol), 0.2 ml of concentrated hydrochloric acid, and heat to reflux for 16 h. Concentrate the solvent under reduced pressure, add 20 ml of ethyl acetate, stir rapidly for 1 h, pour out the clear liquid, evaporate the residue under reduced pressure, and dry in vacuo to obtain a light yellow oil, 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.

[0085] R1-5a (2.51 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 cooling naturally, 25 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of methyl tert-butyl ether was added and stirred at room temperature for 1 h. The filter cake was vacuum dried to obtain a white solid, which was R1-5 (2.45 g, 87%). 1H 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).

[0086] R1-5 (0.28 g, 1.0 mmol) and 8 ml of acetic acid were added to a 50 mL round-bottom flask, and then L3N (0.24 g, 1.0 mmol) was added and heated to reflux for 5 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*15 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=20:1, v / v) to obtain a white solid 8 (L3NR1-5, 0.31 g, 75%). 1 H 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] + .

[0087] Example 6: Synthesis of Compound 9 (4-(4-hydroxy-3-(piperidin-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0088]

[0089] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) into a 100 mL round-bottom flask, then add piperidine (1.87 g, 22 mmol) and 0.2 ml of concentrated hydrochloric acid, and heat to reflux for 14 h. Concentrate the solvent under reduced pressure, add 20 ml of methyl tert-butyl ether, stir rapidly for 1 h, pour out the clear liquid, evaporate the residue under reduced pressure, and dry under vacuum to obtain a light yellow oil, which is 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). 13 C 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.

[0090] R1-6a (2.49 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 cooling naturally, 30 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of ethyl acetate was added and stirred at room temperature for 1 h. The filter cake was vacuum dried to obtain a white solid, which was 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.

[0091] R1-5 (0.28 g, 1.0 mmol) and 6 ml of acetic acid were added to a 50 mL round-bottom flask, and then L3N (0.24 g, 1.0 mmol) was added and heated to reflux for 4 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*20 mL), and 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 obtain a white solid 9 (L3NR1-6, 0.28 g, 69%). 1 H 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] + .

[0092] Example 7: Synthesis of Compound 11 (4-(ethyl(4-hydroxy-3-(pyrrolidin-1-ylmethyl)phenyl)amino)-7-methoxyquinoline-6-carboxamide)

[0093]

[0094] L3NR1-2 (0.20 g, 0.5 mmol), 10 ml of acetonitrile, and iodoethane (0.10 g, 0.6 mmol) were added to a 50 mL round-bottom flask and heated under reflux for 1 h. After cooling naturally, the reaction solution was directly subjected to column chromatography (CH2Cl2:CH3OH=30:1, v / v) to obtain a light yellow solid 11 (L3NR1-8, 0.15 g, 68%). 1 HNMR (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] + .

[0095] Example 8: Synthesis of Compound 16 (4-((6,7-dimethoxyquinolin-4-yl)amino)-2-((4-(hydroxymethyl)piperidin-1-yl)methyl)phenol)

[0096]

[0097] Add acetaminophen (R1, 3.1 g, 20 mmol), 30 ml of ethanol, formaldehyde aqueous solution (aq 37%) (2.45 g, 30 mmol) to a 100 mL round-bottom flask, then add 4-piperidinol (2.53 g, 22 mmol), 0.2 ml of concentrated hydrochloric acid, and heat to 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 filter the cake by column chromatography (CH2Cl2:CH3OH=20:1, v / v) to obtain a light yellow solid, R1-7a (3.4 g, 61%). 1H 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). 13 C 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.

[0098] 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 cooling naturally, 30 ml of ethanol was added and the solvent was concentrated under reduced pressure. Then 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 basically evaporated to dryness. 25 ml of ethyl acetate was added and stirred at room temperature for 1 h. The filter cake was vacuum dried to obtain a white solid, which was 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.

[0099] R1-7 (0.31 g, 1.0 mmol) and 6 ml of acetic acid were added to a 50 mL round-bottom flask, and then L4N (0.22 g, 1.0 mmol) was added and heated to reflux for 3 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*20 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=20:1, v / v) to obtain a white solid 16 (L4NR1-7, 0.29 g, 68%). 1 H 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] + .

[0100] Example 9: Synthesis of Compound 21 (4-((1H-pyrazolo[3,4-b]pyridin-4-yl)amino)-2-((diethylamino)methyl)phenol)

[0101]

[0102] R1-3 (0.27 g, 1.0 mmol) and 6 ml of acetic acid were added to a 50 mL round-bottom flask, and then L7N (0.18 g, 1.2 mmol) was added and heated to reflux for 5 h. The mixture was cooled naturally, and the pH was adjusted to 9-10 with 1 M NaOH aqueous solution. The mixture was extracted with CH2Cl2 (3*20 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous Na2SO4, and purified by column chromatography (CH2Cl2:CH3OH=15:1, v / v) to obtain a white solid 21 (L7NR1-3, 0.20 g, 63%). 1 H 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] + .

[0103] According to the similar method of Example 1-Example 9, the following compounds have been synthesized:

[0104]

[0105]

[0106]

[0107]

[0108] Example 10: Test of the inhibitory ability of compounds on AChE:

[0109] 1. Purpose of the experiment

[0110] The present invention relates to the Mannich base compounds represented by the general formula (I) and the IC of AChE. 50 value.

[0111] 2. Experimental Materials

[0112] Acetylthiocholine iodine (ATCh), 5,5'-Dithiobis(2-nitrobenzonic acid) (DTNB) and hAChE (from human erythrocytes). Centrifuge (SIGMA 3-18K), microplate reader (Bio Rad Microplate Reader Model 550), balance (METTLERTOLEDO), incubator (Blue-part, Shanghai Hengke), sample gun (BIOHIT), 96-well plate.

[0113] 3. Preparation of experimental reagents

[0114] 3.1 Preparation method of buffer solutions with different pH values:

[0115] Solution A: 0.2M NaH2PO4, NaH2PO4·2H2O (MW 156.01) 31.202g plus 1000mL distilled water;

[0116] Solution B: 0.2M Na2HPO4, Na2HPO4·12H2O (MW358.14) 71.628g plus 1000mL distilled water;

[0117] pH = 7.0 PBS buffer: 39 mL of solution A, 61 mL of solution B, and 100 mL of distilled water.

[0118] pH = 7.4 PBS buffer: 19 mL of solution A, 81 mL of solution B, and 100 mL of distilled water.

[0119] 3.2 DTNB preparation method

[0120] Dissolve in 29.7mg / 100mL pH=7.0 PBS buffer, concentration 0.75mM, store at 0-4℃, use on the same day.

[0121] 3.3ATCh preparation method

[0122] 8.7mg / 10mL pH=7.4 PBS buffer solution, concentration 3mM, stored at 0-4℃, use on the same day.

[0123] 3.4 Enzyme pretreatment

[0124] hAChE (Sigma) was diluted to the corresponding concentration with PBS (pH=7.4, 0.1% BSA), stored at 0-4°C, and used on the same day.

[0125] 3.5 Dissolution and dilution of drugs

[0126] Weigh the corresponding amount of drug and dissolve it in DMSO to prepare a concentration of 1*10 -2 Dilution of drug: dilute the above drug solutions to 48 μM, 24 μM, 12 μM, 3 μM, 0.3 μM, and 0.03 μM respectively with PBS (pH=7.4) in a 96-well plate and use immediately.

[0127] 4. Test of compound enzyme inhibition rate

[0128] 4.1. Activity Testing Principle

[0129] The activity test uses the micro-DTNB method (Formula II). ATCh is an analog of ACh, which can be catalyzed by AChE to hydrolyze into acetic acid and thiocholine iodide (TCh) (see reaction A). TCh can react with DTNB to quickly and quantitatively generate yellow anion 5-sulfanyl-2-nitrobenzoic acid (RS-) (reaction B), which has a maximum absorption at a wavelength of 412nm. The generated TCh can be quantified by measuring the absorbance (OD value) of the generated RS-, thereby calculating the activity of AChE.

[0130]

[0131] For a general enzymatic 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 related positive and negative reaction rate constants.

[0132]

[0133] Since the concentration of the enzyme used in the experiment is much smaller than the substrate concentration, that is, [ES]<<[S], it can be approximately considered that [ES] is constant, so there is formula 3-1:

[0134]

[0135] [E]0 represents the initial concentration of the enzyme, then [E]0 = [E] + [ES], that is, [E] = [E]0 - [ES]. Substituting it into formula 3-1, we get

[0136] Formula 3-2 is as follows:

[0137]

[0138] Because the concentration of the intermediate ES is very small, it can be approximately considered that the consumption concentration of the substrate is equal to the generation concentration of the product, that is, -d[S] / dt=d[P] / dt. Therefore, the generation rate of the product can be expressed as formula 3-3:

[0139]

[0140] Substituting 3-2 into 3-3, for reaction A of formula II, [H2O] can be considered constant, and its k -2 =0, so we get formula 3-4:

[0141]

[0142] Let K M =(k -1 +k2) / k1,K M is the Michaelis constant, so we get formula 3-5:

[0143]

[0144] It can be seen from formula 3-5 that the reaction rate is only related to the substrate concentration. If the substrate concentration is very high, it can be considered that the substrate concentration remains unchanged during the initial period of the reaction, so the reaction rate is also constant. If the product concentration at a fixed reaction time point is measured, the reaction rate can be calculated in direct proportion to the catalytic activity of the enzyme. In reaction B of formula II, the color reaction of the product TCh and DTNB catalyzed by AChE is completed instantly and quantitatively, and the final OD value measured is proportional to RS - The concentration is proportional to the concentration of the catalytic hydrolysis product TCh, so it can be measured by measuring the color substance RS - The OD value is used to reflect the enzyme activity. The substrate concentration (ATCh, 3.0mM) selected in this experiment has been proven by preliminary experiments to fully meet the above conditions. Within 50 minutes after the start of the enzymatic reaction, the OD value tested is directly proportional to the reaction time. Therefore, the OD value measured by color development at a fixed time point (such as 30 minutes) after the start of the enzymatic reaction can be used to measure AChE activity.

[0145] 4.2. Test of enzyme inhibition rate

[0146] In this experiment, different drugs were first incubated with hAChE, then ATCh was added for reaction, and finally DTNB was added for color development. By comparing with the normal hAChE test results, the enzyme activity rate was calculated, and then the enzyme inhibition rate was calculated. By measuring the enzyme inhibition rate at multiple concentrations (16, 8, 4, 1, 0.1, 0.01 μM), the IC 50 value.

[0147] a) Dilution of enzyme: AChE stock solution (20 U / mL) was diluted 2000 times with PBS (0.1 M, pH=7.4, 0.1% BSA) and stored at 0-4°C.

[0148] b) Inhibition of enzyme: Take 20 μL of the above enzyme dilution and add it to 20 μL of the corresponding concentration of drug (operated in 96-well plate, the final drug concentration is 16, 8, 4, 1, 0.1, 0.01 μM), and inhibit the enzyme for 15 minutes at 25℃. The normal enzyme control is 20 μL PBS buffer instead of the corresponding concentration of drug.

[0149] c) Reaction: Add 30 μL ATCh (3.0 mM, pH=7.4 PBS) to the above enzyme at room temperature and react for 30 min.

[0150] 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 an enzyme marker.

[0151] Calculation method of enzyme inhibition rate: % Activity = (SB) * 100 / (PB). S = OD value of enzyme inhibition group. P = OD value of normal group, B = OD value of PBS blank group (solvent background value). Enzyme inhibition rate: % Inhibition = 1-% Activity.

[0152] IC 50 Calculation method (the following formula is used to perform nonlinear fitting on the enzyme inhibition rate at different concentrations to calculate IC 50 Value): %Activity = 100*IC 50 / (IC 50 + [S]), where [S] represents the drug concentration.

[0153] Table 1: IC values ​​of the compounds synthesized according to the examples for hAChE 50 (μM)

[0154]

[0155] It should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a Mannich base compound represented by general formula (I), characterized in that: As a cholinesterase inhibitor, it is used to prepare a drug for treating and / or preventing and alleviating related diseases caused by nervous system diseases, and the related diseases are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof: In formula (I), Ring Ar is the following aromatic group which is unsubstituted or substituted by 1-2 substituents: 1-2 azaphenyl groups, benzo 5-6 membered aromatic heterocyclic groups, 1-2 azaphenyl 5-6 membered aromatic heterocyclic groups, each of which contains 1-3 heteroatoms selected from oxygen, sulfur and nitrogen; each of which is independently selected from the following group: bromine, C1-C4 straight chain or branched alkyl, C1-C4 straight chain or branched alkoxy, C2-C6 straight chain or branched ester, cyano, nitro, hydroxyl, amino, aminomethyl, dimethylamino, hydroxymethyl, trifluoromethyl, carboxyl, mercapto, C2-C4 acyl, amide, or two adjacent substituents together with the carbon atoms connected thereto form a 5-7 membered ring; R1 is hydrogen, C1-C4 straight or branched alkyl; R2 is an amino group substituted by 1-2 C1~C4 straight or branched alkyl groups, a 5-7 membered nitrogen heterocyclic group which is unsubstituted or substituted by 1-2 substituents, each of which further contains 0-1 heteroatoms selected from oxygen, sulfur and nitrogen; and the substituents are independently selected from the following group: halogen, C1~C4 straight or branched alkyl groups, hydroxyl, hydroxymethyl, trifluoromethyl, carboxyl, C2~C4 acyl, and amide.

2. A compound represented by general formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate thereof: In formula (I), Ring Ar is a pyrido 5- to 6-membered aromatic heterocyclic group, a pyridyl group or a benzopyridyl group substituted by 1-2 substituents, each of the aromatic heterocyclic groups contains 1-2 nitrogen heteroatoms, and the substituents are each independently selected from the following group: bromine, amide, hydroxyl, hydroxymethyl, cyano, nitro, dimethylamino, C1-C4 straight-chain or branched alkoxy; R1 is hydrogen, C1-C4 straight or branched alkyl; R2 is an amino group substituted by 1-2 C1, C3, C4 straight or branched alkyl groups, a 5-7 membered nitrogen heterocyclic group which is unsubstituted or substituted by 1 substituent, each of which further contains 0-1 heteroatoms selected from oxygen, sulfur and nitrogen; and the substituents are independently selected from the following group: C1-C4 straight or branched alkyl groups, hydroxyl, hydroxymethyl, trifluoromethyl, carboxyl, and amide.

3. The use according to claim 1, characterized in that The compound in the general formula (I) is selected from the following group:

4. A Mannich base compound according to claim 2, or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer or racemate thereof, characterized in that: The compound in the general formula (I) is selected from the compounds 2-5, 7-13, 15-18, 20-22 in the above group.

5. A pharmaceutical composition, comprising: A therapeutically effective amount of one or more Mannich base compounds of general formula (I) as claimed in claim 2, a pharmaceutically acceptable salt thereof, and an optional pharmaceutically acceptable carrier.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition contains one or more Mannich base compounds of the general formula (I) according to claim 1 or pharmaceutically acceptable salts, solvates, hydrates, enantiomers, diastereomers or racemates thereof as cholinesterase inhibitors, and is used to prepare drugs for treating and / or preventing and alleviating related diseases caused by nervous system disorders, wherein the related diseases are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof.

7. A method for treating, preventing, and / or alleviating diseases related to nervous system disorders, characterized in that: The method comprises the steps of administering a safe and effective amount of a Mannich base compound represented by the general formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate thereof to a subject in need thereof, wherein the Mannich base compound represented by the general formula (I) is as described in claim 1.

8. The method according to claim 7, characterized in that The related diseases caused by nervous system disorders are selected from the following group: Alzheimer's disease, sensory or motor disorders, memory loss, myasthenia gravis, progressive muscular dystrophy, sequelae of poliomyelitis, cerebral palsy in children, multiple neuritis and its complications, or a combination thereof.

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