Deuterated huperzine A compound or pharmaceutically acceptable salt thereof as well as preparation method and application of deuterated huperzine A compound or pharmaceutically acceptable salt thereof

Abstract

The invention relates to the technical field of medicines, in particular to a deuterated huperzine A compound or pharmaceutically acceptable salt thereof as well as a preparation method and application of the deuterated huperzine A compound. The structure of the deuterated huperzine A compound is shown in the specification, and R1-R9 are respectively and independently H or D and are not H at the same time. The compound provided by the invention has the advantages of high chemical stability, long metabolic half-life period, low toxicity, obvious synergistic effect with the Bramer's olantin in the aspect of treating the Alzheimer's disease, and capability of reducing the toxicity of the Bramer's olantin. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. Background Art

[0002] Huperzine A is an organic compound with the chemical formula C 15 H 18 N2O is mainly used as a cholinesterase inhibitor for the treatment of benign memory impairment and various types of dementia, memory cognitive function and emotional and behavioral disorders in the middle-aged and elderly, and can also be used to treat myasthenia gravis. Huperzine A can effectively improve patients' cognitive function and living ability in the treatment of Alzheimer's disease, but the efficacy needs to be further improved.

[0003] Deuterated compounds refer to new compounds obtained by replacing hydrogen atoms in one or some carbon-hydrogen bonds in a compound molecule with deuterium atoms. Since carbon-deuterium bonds are more stable than carbon-hydrogen bonds, deuteration reactions can be used to improve the pharmacokinetic characteristics of drugs in drug development. Based on its unique properties, deuterium modification is one of the methods to improve drug properties. By replacing the active sites of drug molecules with deuterium, drug metabolism time can be significantly prolonged and drug dosage can be reduced.

[0004] However, it is extremely difficult to deuterate huperzine A. So far, no deuterated compound of huperzine A has been successfully synthesized, making it impossible to conduct related research on deuterated compounds.

[0005] Alzheimer's disease (AD) is a complex neurodegenerative disease whose pathogenesis has not yet been fully elucidated, but it is generally believed to be closely related to β-amyloid protein deposition, abnormal tau protein phosphorylation, and neuronal damage. At present, the treatment of AD mainly relies on drug intervention, aiming to improve cognitive function and delay the progression of the disease. However, most existing drugs can only partially improve cognitive function, and have shortcomings such as limited efficacy and obvious side effects.

[0006] Bremelanotide (PT141), as a melanocortin receptor agonist, can activate MC1R and MC4R receptors, thus showing potential in neuroprotection, anti-neuroinflammation and improving cognitive function. It is considered to be a promising drug for the treatment of Alzheimer's disease.

[0007] However, in practical applications, bremelanotide has significant disadvantages and shortcomings when used alone, as follows: (1) Low drug activity: In AD model cell experiments, bremelanotide showed a high EC50 value (i.e., the concentration required to achieve half the maximum effect concentration). A high EC50 value means that a higher drug concentration is required to achieve the desired therapeutic effect, which not only increases the cost of medication, but also may bring potential safety risks.

[0008] (2) Poor cell survival and recovery rates: Although bremelanotide has a certain neuroprotective effect, when used alone, it does not significantly improve the survival rate and functional recovery rate of AD model cells. This limits the efficacy of bremelanotide in clinical applications and the degree of benefit to patients.

[0009] (3) Potential side effects: Increasing the drug dosage to achieve therapeutic effects may increase the patient's risk of adverse reactions, such as nausea, vomiting, headache, etc., affecting the patient's treatment experience and compliance.

[0010] Therefore, the poor drug activity of bremelanotide when used alone limits its clinical application effect in the treatment of Alzheimer's disease. Developing new strategies that can enhance the drug activity of bremelanotide to improve its therapeutic effect and reduce the risk of side effects has important clinical significance and practical application value.

[0011] No relevant research has been found on the combination of bremelanotide and huperzine A or its deuterated derivatives for the treatment of Alzheimer's disease. Summary of the invention

[0012] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, which has high chemical stability, long metabolic half-life, low toxicity, obvious pharmacodynamic synergy with bremelanotide in the treatment of Alzheimer's disease and can reduce the toxicity of bremelanotide, and a preparation method and application thereof, a pharmaceutical composition and application thereof.

[0013] The present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, wherein the deuterated huperzine A compound has the following structure: Figure 1 ): ; wherein R1-R9 are each independently H or D, and are not H at the same time.

[0014] As an embodiment of the present invention, at least one of R1-R6 is D.

[0015] As a preferred embodiment of the present invention, at least one of R1-R3 is D, and at least one of R4-R6 is D.

[0016] As an embodiment of the present invention, R7 is D.

[0017] As an embodiment of the present invention, at least one of R8-R9 is D.

[0018] As an embodiment of the present invention, the structure of the deuterated huperzine A compound includes any one of the following: , , , , .

[0019] The second aspect of the present invention provides a method for preparing the above-mentioned deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, comprising the following steps: S1 uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as the starting material to prepare compound 5; S2 reacts compound 5 with methanesulfonyl chloride to obtain compound 6; S3 reacts compound 6 with sodium acetate to obtain compound 7; S4 reacts compound 7 with compound Y to obtain compound 8; S5: reacting compound 8 with thiophenol to obtain compound 9; S6: reacting compound 9 under an inert atmosphere and alkaline conditions to obtain compound 10; S7: Compound 10 is reacted with triethylamine and diphenylphosphoryl azide to obtain compound 11; S8: Compound 11 is reacted with iodotrimethylsilane to obtain compound 12; S9 separating compound 12 by chiral preparative high performance liquid chromatography to obtain the target product; The structure of compound Y is as follows: ; Among them, R 10 -R 14 are independently H or D.

[0020] As an implementation mode of the present invention, step S1 includes the following steps: S11 uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as a starting material and reacts with compound X to obtain compound 2; S12: reducing compound 2 under hydrogen gas to obtain compound 3; S13: Compound 3 is oxidized with an oxidant to obtain compound 4; S14: Compound 4 is reacted with tetramethylguanidine to obtain compound 5; The structure of compound X is as follows:

[0021] Among them, R 15 -R 19 are each independently H or D, and are not H at the same time.

[0022] As a preferred embodiment of the present invention, step S1 includes the following steps: S11: Compound X and potassium carbonate are added to a dimethylformamide solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate, and the mixture is stirred at 20-80° C. for 10-24 h in an inert atmosphere to obtain compound 2; S12 Pd / C and Pd(OH)2 / C were added to the ethyl acetate solution of compound 2, and the mixture was stirred at 20°C-reflux temperature for 10-24 hours under hydrogen conditions, and the mixture was filtered and concentrated to obtain compound 3; S13: Add Dess-Martin periodinane to a mixture of compound 3 and sodium bicarbonate in dichloromethane at -10-20°C, and stir at -10-20°C under nitrogen for 1-5 hours to obtain compound 4; S14: Tetramethylguanidine is added to a dichloromethane solution of compound 4, and the resulting mixture is stirred at 0-40°C for 10-24 h under an inert atmosphere to obtain compound 5.

[0023] As a more preferred embodiment of the present invention, after the stirring in S11 is completed, the mixture is quenched with water, extracted with ethyl acetate, washed and purified to obtain compound 2.

[0024] As a more preferred embodiment of the present invention, after the stirring in S13 is completed, the mixture is quenched with an aqueous sodium thiosulfate solution and a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, washed, filtered, and concentrated to obtain Compound 4.

[0025] As a more preferred embodiment of the present invention, after the stirring in S14 is completed, the mixture is concentrated under reduced pressure, and the residue is chromatographed on a silica gel column using a mixed solvent of dichloromethane and ethyl acetate as eluent to obtain Compound 5.

[0026] As an implementation mode of the present invention, step S1 includes the following steps: S11′ uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as a starting material and reacts it with tetramethylguanidine and methacrolein to obtain compound 5.

[0027] As a preferred embodiment of the present invention, step S1 includes the following steps: S11′ To a dichloromethane solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate was added a dichloromethane solution of tetramethylguanidine and methacrolein, and the mixture was stirred at 0-40° C. for 10-24 h under an inert atmosphere to obtain compound 5.

[0028] As an embodiment of the present invention, S2 adds triethylamine and methanesulfonyl chloride to a dichloromethane solution of compound 5, and the resulting mixture is stirred at 0-40° C. for 2-8 hours under an inert atmosphere to obtain compound 6.

[0029] As an embodiment of the present invention, S3 adds sodium acetate to the acetic acid solution of compound 6, and the resulting mixture is stirred at 90-120° C. for 40-72 h under an inert atmosphere to obtain compound 7.

[0030] As an embodiment of the present invention, S4 cools the tetrahydrofuran solution of compound 7 to -78-0°C, adds n-butyl lithium, stirs at 0-40°C for 20-30 minutes, and adds a solution of compound Y in tetrahydrofuran to react to obtain compound 8.

[0031] As an embodiment of the present invention, S5, azobisisobutyronitrile and thiophenol are added to a toluene solution of compound 8, and the mixture is stirred at 70-110° C. for 16-36 hours to obtain compound 9.

[0032] As an embodiment of the present invention, S6 adds potassium hydroxide to a solution of compound 9 in dimethyl sulfoxide and tetrahydrofuran, and stirs at 60-120° C. for 36-48 h under an inert atmosphere to obtain compound 10.

[0033] As an embodiment of the present invention, S7 adds triethylamine and a solution of diphenylphosphoryl azide in toluene to a toluene solution of compound 10, and the resulting mixture is stirred at 60-110° C. for 2-6 h under an inert atmosphere to obtain compound 11.

[0034] As an embodiment of the present invention, S8: to a solution of compound 11 in chloroform, add iodotrimethylsilane at 0-40°C, and stir at 40-75°C for 10-24h under an inert atmosphere to obtain compound 12.

[0035] As an embodiment of the present invention, when compound X is not used in the preparation process of compound 5, R in compound Y 10 -R 14 Not H at the same time.

[0036] As an embodiment of the present invention, the compound Y includes any one of the following structures: , , , .

[0037] As a preferred embodiment of the present invention, the preparation method of the compound E comprises the following steps: adding ethyltriphenylphosphonium bromide to deuterated water, then adding triethylamine, heating under reflux reaction under inert gas conditions, and removing the solvent under reduced pressure to obtain the compound E.

[0038] As a more preferred embodiment of the present invention, the preparation method of compound E comprises the following steps: adding ethyltriphenylphosphonium bromide to deuterated water, then adding triethylamine, heating under reflux at 100-120°C under inert gas conditions for 12-24h, and removing the solvent under reduced pressure to obtain compound E.

[0039] As a further preferred embodiment of the present invention, the preparation method of the compound E comprises the following steps: adding ethyltriphenylphosphonium bromide to deuterated water, then adding triethylamine, heating under reflux at 110° C. for 16 hours under inert gas conditions, and removing the solvent under reduced pressure to obtain the compound E.

[0040] As a preferred embodiment of the present invention, the preparation method of compound F comprises the following steps: stirring a mixture of 1-bromoethane-2,2,2-d3 and triphenylphosphine bromide in toluene at 100-120°C for 12-24 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain compound F.

[0041] As a preferred embodiment of the present invention, the preparation method of compound G comprises the following steps: stirring a mixture of 1-bromoethane-1,1,2,2,2-d5 and triphenylphosphine bromide in toluene at 100-120°C for 12-24 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain compound G.

[0042] As an embodiment of the present invention, the compound X includes any one of the following structures: , , .

[0043] As a preferred embodiment of the present invention, the preparation method of compound A comprises the following steps: (1) adding potassium carbonate and deuterated iodomethane CD3I to a mixture of diethyl malonate (A-1) and DMF to react, pouring the resulting reactant into water, and post-treating to obtain compound A-2; (2) Add n-butyl lithium to a mixture of compound A-2 and anhydrous tetrahydrofuran, stir under inert gas conditions, add ((chloromethoxy)methyl)benzene (A-3) to the above solution, quench the reaction with water, and post-treat to obtain compound A-4; (3) adding potassium hydroxide solution to the ethanol solution of compound A-4, removing ethanol from the obtained reactant under reduced pressure, adding water to dilute, adjusting the pH to 1-4, and post-treating to obtain compound A-5; (4) Compound A-5 is added to xylene and stirred. The obtained reactant is concentrated and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound A-6; (5) Under inert gas conditions, borane-tetrahydrofuran is added to a tetrahydrofuran solution of compound A-6 for reaction. After the reaction is completed, methanol is used to quench the reaction, and post-treatment is performed to obtain compound A-7; (6) adding iodine to a dichloromethane solution containing triphenylphosphine and imidazole, stirring, adding compound A-7 to react, quenching the reaction with an aqueous sodium sulfite solution after the reaction is completed, and post-treating to obtain compound A; The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

[0044] As a more preferred embodiment of the present invention, the preparation method of compound A comprises the following steps: (1) Add potassium carbonate and deuterated iodomethane CD3I to a mixture of diethyl malonate (A-1) and DMF for reaction at 30-80°C for 48-72h, pour the obtained reactant into water, and post-treat to obtain compound A-2; (2) Add n-butyl lithium to a mixture of compound A-2 in anhydrous tetrahydrofuran at -78-0°C, stir under inert gas for 1-3 hours, then add ((chloromethoxy)methyl)benzene at -78-0°C, stir at -78°C to room temperature for 2 hours, quench the reaction with water, and post-treat to obtain compound A-4; (3) Add potassium hydroxide solution to the ethanol solution of compound A-4, stir at 60-100°C for 1-4h, remove ethanol from the obtained reactant under reduced pressure, dilute with water, wash with dichloromethane, adjust the pH to 1-4, and post-treat to obtain compound A-5; (4) Compound A-5 was added to xylene and stirred at 120-150°C for 12-24h. The obtained reactant was concentrated and purified by column chromatography using dichloromethane / methanol = 1 / 99 to obtain compound A-6; (5) Under inert gas conditions, add borane-tetrahydrofuran to a tetrahydrofuran solution of compound A-6 at -5-0°C, react at 50-80°C for 12-24h, cool to 0°C, quench the reaction with methanol, and post-treat to obtain compound A-7; (6) Add iodine to a dichloromethane solution containing triphenylphosphine and imidazole, stir at room temperature for 1-3 h, add compound A-7 and react for 1-4 h. After the reaction is completed, quench the reaction with 10% sodium sulfite aqueous solution, and post-treat to obtain compound A.

[0045] Further preferably, the preparation method of compound A comprises the following steps: (1) Add potassium carbonate and deuterated iodomethane CD3I to a mixture of diethyl malonate (A-1) and DMF for reaction at 50°C for 72h. Pour the obtained reactant into water and post-treat to obtain compound A-2; (2) Add n-butyl lithium to a mixture of compound A-2 in anhydrous tetrahydrofuran at -78°C, stir under inert gas for 1 hour, then add ((chloromethoxy)methyl)benzene (A-3) at -78°C, stir at -78°C to room temperature for 2 hours, quench the reaction with water, and post-treat to obtain compound A-4; (3) Add potassium hydroxide solution to the ethanol solution of compound A-4, stir at 90°C for 2h, remove ethanol from the obtained reactant under reduced pressure, dilute with water, wash with dichloromethane, adjust the pH to 1-4 (preferably 2), and post-treat to obtain compound A-5; (4) Compound A-5 was added to xylene and stirred at 149°C for 16 hours. The obtained reactant was concentrated and purified by column chromatography using dichloromethane / methanol = 1 / 99 to obtain compound A-6; (5) Under inert gas conditions, borane-tetrahydrofuran was added to a tetrahydrofuran solution of compound A-6 at 0°C, reacted at 70°C for 16 h, cooled to 0°C, quenched with methanol, and post-treated to obtain compound A-7; (6) Add iodine to a dichloromethane solution containing triphenylphosphine and imidazole, stir at room temperature for 1 h, add compound A-7 and react for 2 h. After the reaction is completed, quench the reaction with 10% sodium sulfite aqueous solution, and post-treat to obtain compound A.

[0046] As a preferred embodiment of the present invention, the preparation method of compound B comprises the following steps: 1) Add n-butyl lithium to a mixture of diethyl 2-methylmalonate (B-1) and anhydrous tetrahydrofuran, stir under inert gas conditions, add ((chloromethoxy)methyl)benzene (A-3) to the above solution, quench the reaction with water, and post-treat to obtain compound B-2; 2) Add potassium hydroxide solution to the ethanol solution of compound B-2, stir under inert gas conditions, remove ethanol from the obtained reactant under reduced pressure, dilute with water, adjust the pH to 2-6 (preferably 4), and post-treat to obtain compound B-3; 3) Under inert gas conditions, compound B-3 is added to xylene and stirred, the obtained reactant is diluted with water, and post-treated to obtain compound B-4; 4) Add concentrated sulfuric acid to the methanol solution of compound B-4, stir under inert gas conditions, quench the reaction with saturated sodium bicarbonate, and post-treat to obtain compound B-5; 5) Add lithium aluminum deuteride to a tetrahydrofuran solution of compound B-5, stir under inert gas conditions, quench the reaction with sodium sulfate decahydrate, and post-treat to obtain compound B-6; 6) adding imidazole and iodine to a dichloromethane solution containing triphenylphosphine, stirring under inert gas conditions, adding compound B-6 to react, quenching the reaction with sodium thiosulfate after the reaction is completed, extracting with dichloromethane, and drying to obtain compound B; The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

[0047] As a more preferred embodiment of the present invention, the preparation method of compound B comprises the following steps: 1) Add n-butyl lithium to a mixture of diethyl 2-methylmalonate (B-1) and anhydrous tetrahydrofuran at -78-0°C, stir for 1-3 hours under inert gas conditions, add ((chloromethoxy)methyl)benzene to the above solution, stir at room temperature for 3 hours, quench the reaction with water, and post-treat to obtain compound B-2; 2) Add potassium hydroxide solution to the ethanol solution of compound B-2 under inert gas conditions at -5-5°C, stir at 70-100°C for 1-3h, remove ethanol from the obtained reactant under reduced pressure, dilute with water, adjust the pH to 2-6, and post-treat to obtain compound B-3; 3) Under inert gas conditions, compound B-3 is added to xylene, stirred at 145-155°C for 12-18h, the obtained reactant is diluted with water, and post-treated to obtain compound B-4; 4) Add concentrated sulfuric acid to the methanol solution of compound B-4, stir at 60-90°C for 12-18h under inert gas conditions, quench the reaction with saturated sodium bicarbonate, and post-treat to obtain compound B-5; 5) Add lithium aluminum deuteride to a tetrahydrofuran solution of compound B-5 at -5-5°C, stir at -5-5°C for 2-4h under inert gas conditions, quench the reaction with sodium sulfate decahydrate, and post-treat to obtain compound B-6; 6) Add imidazole and iodine to a dichloromethane solution containing triphenylphosphine at -5-5°C, stir at room temperature for 1-2 hours under inert gas conditions, add compound B-6 and continue stirring for 1-3 hours. After the reaction is completed, quench the reaction with sodium thiosulfate, extract with dichloromethane, and dry to obtain compound B.

[0048] As a further preferred embodiment of the present invention, the preparation method of compound B comprises the following steps: 1) At -78°C, n-butyl lithium was added to a mixture of diethyl 2-methylmalonate (B-1) and anhydrous tetrahydrofuran, and the mixture was stirred for 1 h under inert gas conditions. ((chloromethoxy)methyl)benzene (A-3) was added to the above solution, and the mixture was stirred at room temperature for 3 h. The reaction was quenched with water, and post-treated to obtain compound B-2; 2) Under inert gas conditions at 0°C, potassium hydroxide solution was added to the ethanol solution of compound B-2, and the mixture was stirred at 90°C for 2h. The ethanol was removed from the obtained reactant under reduced pressure, and the mixture was diluted with water, and the pH was adjusted to 4. After post-treatment, compound B-3 was obtained. 3) Under inert gas conditions, compound B-3 was added to xylene and stirred at 149°C for 16 hours. The obtained reactant was diluted with water and post-treated to obtain compound B-4; 4) Add concentrated sulfuric acid to the methanol solution of compound B-4, stir at 80°C for 16h under inert gas conditions, quench the reaction with saturated sodium bicarbonate, and post-treat to obtain compound B-5; 5) Add lithium aluminum deuteride to a tetrahydrofuran solution of compound B-5 at 0°C, stir at 0°C for 3h under inert gas conditions, quench the reaction with sodium sulfate decahydrate, and post-treat to obtain compound B-6; 6) At 0°C, imidazole and iodine were added to a dichloromethane solution containing triphenylphosphine. The mixture was stirred at room temperature for 1.5 h under inert gas conditions. Compound B-6 was added and stirring was continued for 2 h. After the reaction was completed, the reaction was quenched with sodium thiosulfate, extracted with dichloromethane, and dried to obtain compound B.

[0049] As a preferred embodiment of the present invention, the preparation method of compound C comprises the following steps: (i) adding concentrated sulfuric acid to a methanol solution of compound A-6, stirring under inert gas conditions, concentrating the obtained reactant, diluting it with water, and post-treating it to obtain compound C-1; (ii) adding lithium aluminum deuteride to a tetrahydrofuran solution of compound C-1, stirring under inert gas conditions, quenching the reaction with sodium sulfate decahydrate, and post-treating to obtain compound C-2; (iii) adding imidazole and iodine to a dichloromethane solution containing triphenylphosphine, stirring under inert gas conditions, adding compound C-2 for reaction, quenching the reaction with sodium thiosulfate after the reaction is completed, extracting with dichloromethane, and drying to obtain compound C.

[0050] As a more preferred embodiment of the present invention, the preparation method of compound C comprises the following steps: (i) under inert gas conditions, concentrated sulfuric acid is added to a methanol solution of compound A-6, and the mixture is stirred at 80-100° C. for 1-3 h. The obtained reactant is concentrated and then diluted with water, and post-treated to obtain compound C-1; (ii) adding lithium aluminum deuteride to a tetrahydrofuran solution of compound C-1 at 0°C, stirring for 2-4 hours under inert gas conditions, quenching the reaction with sodium sulfate decahydrate, and post-treating to obtain compound C-2; (iii) Add imidazole and iodine to a dichloromethane solution containing triphenylphosphine at 0°C, stir at room temperature for 1-3 hours under inert gas conditions, add compound C-2 and continue stirring for 1-3 hours. After the reaction is completed, quench the reaction with sodium thiosulfate, extract with dichloromethane, and dry to obtain compound C.

[0051] As a further preferred embodiment of the present invention, the preparation method of compound C comprises the following steps: (i) under inert gas conditions, concentrated sulfuric acid was added to a methanol solution of compound A-6, and the mixture was stirred at 90°C for 2h. The obtained reactant was concentrated and diluted with water, and post-treated to obtain compound C-1; (ii) adding lithium aluminum deuteride to a tetrahydrofuran solution of compound C-1 at 0°C, stirring for 3 h under inert gas conditions, quenching the reaction with sodium sulfate decahydrate, and post-treating to obtain compound C-2; (iii) Add imidazole and iodine to a dichloromethane solution containing triphenylphosphine at 0°C, stir at room temperature for 1.5 h under inert gas conditions, add compound C-2 and continue stirring for 2 h. After the reaction is completed, quench the reaction with sodium thiosulfate, extract with dichloromethane, and dry to obtain compound C.

[0052] The third aspect of the present invention provides the use of the above-mentioned deuterated huperzine A compound or a pharmaceutically acceptable salt thereof in the preparation of a cholinesterase inhibitor.

[0053] As an embodiment of the present invention, the cholinesterase inhibitor includes an acetylcholinesterase inhibitor.

[0054] The fourth aspect of the present invention provides use of the above-mentioned deuterated huperzine A compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease.

[0055] The fifth aspect of the present invention provides use of the above-mentioned deuterated huperzine A compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in combination with bremelanotide.

[0056] The sixth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned deuterated huperzine A compound or a pharmaceutically acceptable salt thereof and bremelanotide.

[0057] As an embodiment of the present invention, the mass ratio of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof to bremelanotide is in the range of 5:30-5:60.

[0058] As a preferred embodiment of the present invention, the mass ratio of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof to bremelanotide is in the range of 5:43.75.

[0059] A seventh aspect of the present invention provides use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating Alzheimer's disease.

[0060] The beneficial effects of the present invention are: 1. The present invention utilizes deuteration technology to partially or completely replace the hydrogen atoms in the huperzine A molecule, and successfully synthesizes deuterated huperzine A with a purity of more than 95%.

[0061] 2. The deuterated huperzine A of the present invention has the same effect as huperzine A in inhibiting the activities of acetylcholinesterase and butyrylcholinesterase, but the deuterated huperzine A of the present invention has higher chemical stability, lower toxicity, and significantly prolonged metabolic half-life.

[0062] 3. The present invention uses deuterated huperzine A and bremelanotide in combination, and finds that there is a synergistic effect between deuterated huperzine A and bremelanotide. When the two are used in combination, the effect in treating Alzheimer's disease can be significantly enhanced, while the toxic effects of bremelanotide can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is the general structural formula of the deuterated huperzine A compound of the present invention.

[0064] Figure 2 This is the dose-response curve of tacrine on acetylcholinesterase (ACHE).

[0065] Figure 3 This is the dose-response curve of huperzine A and deuterated huperzine A on acetylcholinesterase (ACHE).

[0066] Figure 4 This is the EC50 curve of bremelanotide in the Aβ model.

[0067] Figure 5 This is the EC50 curve of deuterated huperzine A on the Aβ model.

[0068] Figure 6 This is the EC50 curve of bremelanotide and deuterated huperzine A combined in the Aβ model. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0070] Example 1 Synthesis of Compound (WP102-3) - Deuterated Huperzine A General steps for the preparation of compound E:

[0071] Triethylamine TEA (0.5 ml) was added to a solution of ethyltriphenylphosphine bromide (Compound E-1, 6.0 g, 16.2 mmol, 1.0 eq.) in deuterated water D2O (16 ml). The reaction mixture was heated under reflux at 110°C for 16 h under nitrogen. The solvent was removed under reduced pressure to obtain Compound E (5.8 g, 96%) as a white solid.

[0072] Liquid chromatography-mass spectrometry (LCMS): 293 [M-79]+; 1H NMR (400 MHz, CDCl3) 7.95 - 7.65 (m, 15H), 1.39 (d, J = 20.0 Hz, 3H).

[0073] General steps for the preparation of compound 2:

[0074] Under stirring conditions, tetramethylguanidine (compound 1a, 979 mg, 8.5 mmol, 0.2 eq) and methacrolein (compound D, 7.4 g, 106.3 mmol, 2.5 eq) in dichloromethane (10 ml) were added to a solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (compound 1, 10.0 g, 42.5 mmol, 1.0 eq) in dichloromethane (150 ml). The resulting mixture was stirred at 25 ° C for 16 h under a nitrogen atmosphere. LCMS showed a new peak and the starting material disappeared. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: ethyl acetate = 7: 3) to give racemic compound (+) 2 (11.0 g, 85%) as a white solid.

[0075] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf (compound 1) = 0.6; Rf (compound 2) = 0.2; Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0076] General steps for the preparation of compound 3:

[0077] To a solution of racemic methyl (5S,9R)-8-hydroxy-2-methoxy-7-methyl-11-oxo-7,8,9,10-tetrahydro-5,9-carboxylic octyl[b]pyridine-5(6H)-carboxylate (compound (+) 2, 11.0 g, 36.0 mmol, 1.0 eq) in dichloromethane (150 ml) were added triethylamine TEA (40 ml, 288.2 mmol, 8.0 eq), 4-dimethylaminopyridine DMAP (439 mg, 3.6 mmol, 0.1 eq) and methanesulfonyl chloride MsCl (16.4 g, 144.1 mmol, 4.0 eq) dropwise. The resulting mixture was stirred at room temperature (rt) for 4 h under nitrogen atmosphere. The mixture was washed with saturated aqueous ammonium chloride solution (50 ml) and brine (50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:2) to give racemic compound (+) 3 (10.0 g, 72%) as a white solid.

[0078] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2; Rf value of compound 2 = 0.2; Rf value of compound 3 = 0.6; Liquid chromatography-mass spectrometry (LCMS): 384.2 [M+H]+.

[0079] General steps for the preparation of compound 4:

[0080] To a solution of racemic (5S,9R)-2-methoxy-7-methyl-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (compound (+) 3, 9.8 g, 25.5 mmol, 1.0 eq) in acetic acid HOAc (300 ml) was added sodium acetate NaOAc (4.2 g, 51.0 mmol, 2.0 eq). The resulting mixture was stirred at reflux temperature for 48 h under N2 atmosphere. The mixture was concentrated to remove the solvent HOAc, diluted with ethyl acetate EA (200 ml), washed with saturated NaHCO3 aqueous solution (50 ml), washed with brine (50 ml), dried over anhydrous Na2SO4, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate EA / petroleum ether PE = 2 / 3) to give racemic compound (+) 4 (2.7 g, 37%) as a white solid.

[0081] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf value of compound 3 = 0.3; Rf value of compound 4 = 0.5; Liquid chromatography-mass spectrometry (LCMS): 288.1 [M+H]+.

[0082] General steps for the preparation of compound 5:

[0083] A solution of (ethyl-1,1-d2)triphenylphosphine bromide (Compound E, 1.63 g, 4.35 mmol, 2.5 eq) in tetrahydrofuran (THF) (15 ml) was cooled to -78°C, a hexane solution (3.65 mmol, 2.1 eq) of n-butyllithium nBuLi (1.46 ml, 2.5 M) was added dropwise over 10 minutes, and the mixture was stirred at room temperature (rt) for 20 minutes. After the solution was cooled to -78°C, a solution of racemic (5S,9R)-2-methoxy-7-methyl-11-oxo-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound (+) 4, 500 mg, 1.74 mmol, 1.0 eq) in THF (5 ml) was added dropwise over 10 minutes. The resulting solution was stirred at room temperature for 1.5 hours. LC-MS showed that the reaction was complete. The reaction was quenched with brine (20 ml), extracted with ethyl acetate (100 ml × 3), the organic layers were combined and dried over anhydrous Na2SO4, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate EA: petroleum ether PE = 1:4) to give racemic compound (+) 5 (Z / E = 4 / 1, 376 mg, 72%) as a colorless oil.

[0084] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf value of compound 4 = 0.3; Rf value of compound 5 = 0.6; Liquid chromatography-mass spectrometry (LCMS): 301.1 [M+H]+.

[0085] General steps for the preparation of compound 6:

[0086] Azobisisobutyronitrile AIBN (598 mg, 3.65 mmol, 1.1 eq) and thiophenol PhSH (1.3 g, 11.63 mmol, 3.5 eq) were added to a toluene (30 ml) solution of racemic methyl (5R,9R)-11-(ethylidene-1-d)-2-methoxy-7-methyl-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylate (compound (+) 5, 1.0 g, 3.32 mmol, 1.0 eq). The resulting mixture was stirred at 95°C for 24 h, and AIBN (298 mg, 1.82 mmol, 0.55 eq) and thiophenol (730 mg, 6.64 mmol, 2.0 eq) were added, followed by stirring at 95°C for 24 h. The reaction solution was concentrated and then subjected to NMR detection. 1H NMR showed Z configuration / E configuration = 1 / 9. The solution was diluted with ethyl acetate (150 mL), washed with NaOH (1M, 20 mL × 2) and brine (20 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (EA:PE = 1:4) to give racemic compound (+) 6 (950 mg, 95%) as a yellow solid.

[0087] TLC: ethyl acetate / petroleum ether = 1 / 10; Rf (compound 5) = 0.5; Rf (compound 6) = 0.55; LCMS: 301.1 [M+H]+.

[0088] General steps for the preparation of compound 7:

[0089] To a solution of racemic methyl (5R,9R,E)-11-(ethylidene-1-d)-2-methoxy-7-methyl-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylate (compound (+) 6, 950 mg, 3.16 mmol, 1.0 eq.) in dimethyl sulfoxide (DMSO) (10 mL) was added potassium hydroxide (12%, 50 mL). The resulting mixture was stirred at 100°C for 40 h under nitrogen atmosphere. LCMS showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0°C, the pH was adjusted to 4-5 with 4M aqueous hydrochloric acid solution, extracted with dichloromethane / methanol (9 / 1, 200 mL × 5), i.e., extracted 5 times, 200 mL each time, the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give racemic compound (+) 7 (900 mg, 99%) as a white solid. The product was used directly in the next step without further purification.

[0090] TLC: methanol / dichloromethane = 1 / 10; Rf (compound 6) = 0.8; Rf (compound 7) = 0.4; LCMS: 287.3 [M+H]+.

[0091] General steps for the preparation of compound 8:

[0092] To a mixture of racemic (5R,9R,E)-11-(ethylidene-1-d)-2-methoxy-7-methyl-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid (compound (+) 7, 750 mg, 2.61 mmol, 1.0 eq) in toluene (40 mL) was added dropwise TEA (782 mg, 7.84 mmol, 3.0 eq) and diphenylphosphoryl azide DPPA (1.4 g, 5.22 mmol, 2.0 eq) in toluene PhMe (3 mL). The resulting mixture was stirred at 85 °C for 3 h under nitrogen atmosphere. LCMS showed that compound 7 was consumed. Methanol MeOH (40 mL) was added to the reaction and refluxed for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EA:PE=3:7) to give racemic compound (+) 8 (1.1 g, crude) as a colorless oil.

[0093] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1; Rf (compound 7) = 0.8; Rf (compound 8) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 316.3 [M+H]+.

[0094] General steps for the preparation of compound 9:

[0095] Under nitrogen protection, trimethylsilyl iodide TMSI (3.4 ml, 25.32 mmol, 10 equivalents) was added to a solution of the racemic compound (compound (+) 8, 800 mg, 2.53 mmol) in chloroform CHCl3 (50 ml) at room temperature, and the mixture was heated at 65°C for 16 h. After cooling, methanol (30 ml) was added, and the resulting mixture was stirred at 65°C for 6 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was diluted with dichloromethane / methanol (10 / 1, 100 ml), washed with sodium thiosulfate (10%, 3 ml), saturated sodium bicarbonate (3 ml) and brine (3 ml) in sequence. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) and then by preparative thin layer chromatography (dichloromethane / methanol = 9 / 1) to give racemic compound (+) 9 (350 mg, two-step yield 55%) as a white solid.

[0096] Thin layer chromatography (TLC): dichloromethane / methanol = 9 / 1; Rf (compound 8) = 0.6; Rf (compound 9) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 244.3 [M+H]+.

[0097] General steps for the preparation of deuterated huperzine A (WP102-3):

[0098] The racemic compound (+) 9 (500 mg, 2.05 mmol) was separated by chiral preparative HPLC SFC to give deuterated huperzine A WP102-3 (125.5 mg, 25%, purity 99.44%) as a white solid.

[0099] Chiral preparative HPLC conditions (same conditions as in Examples 1 to 5): Instrument: Supercritical fluid chromatograph SFC002; Chromatographic column: CHIRALPAK AD-H 10 mm × 250 mm × 5 μm; Detection wavelength: 220nm; Column oven temperature: 40°C; Injection volume: 1000 μl (20 mg); Flow rate: 10.0ml / min; Diluent: Isopropyl alcohol IPA; Mobile phase: Phase A: CO2; Phase B: isopropanol containing 0.1% diethylamine.

[0100] The elution conditions are shown in Table 1.

[0101] Table 1

[0102] Thin layer chromatography (TLC): dichloromethane / methanol = 9 / 1; Rf (compound 9) = 0.6; Rf(WP102-3)=0.4; Liquid chromatography-mass spectrometry (LCMS): 244.3 [M+H]+.

[0103] 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ 11.26 (s, 1H), 7.84 (d, J = 9.6 Hz, 1H), 6.17 (d, J = 9.6 Hz, 1H), 5.45 (d, J = 5.2 Hz, 1H), 3.62-3.55 (m, 1H), 2.66 (dd, J = 17.2, 5.0 Hz, 1H), 2.53 (d, J = 13.2 Hz, 1H), 2.15-1.99 (m, 2H), 1.83 (brs, 2H), 1.67 (s, 3H), 1.55 (s, 3H).

[0104] Example 2: Synthesis of Compound (WP102-1) - Deuterated Huperzine A General preparation steps of compound A-2:

[0105] Potassium carbonate (K2CO3, 207 g, 1.5 mol, 1.5 equiv) and deuterated iodomethane CD3I (145 g, 1.0 mol, 1.0 equiv) were added to a mixture of diethyl malonate (compound A-1, 160 g, 1.0 mol, 1.0 equiv) in dimethylformamide DMF (1.6 liters). The mixture was stirred at 50 ° C for 72 h. TLC showed the formation of new spots. The mixture was poured into water (1.6 liters) and extracted with ethyl acetate (EtOAc, 1.0 liter × 3). The combined organic phase was washed with brine (500 ml × 6), dried over sodium sulfate, filtered and concentrated by rotary evaporation. The resulting residue was purified by column chromatography with ethyl acetate / petroleum ether = 1 / 99 to obtain compound A-2 (110 g, 62%) as a yellow oil.

[0106] TLC: petroleum ether / ethyl acetate = 20 / 1 Rf (Compound A-1) = 0.4 Rf (Compound A-2) = 0.5 1H NMR (400 MHz, deuterated chloroform CDCl3): δ 4.09 (m, 4H), 3.30 (s, 1H), 1.17 (dd, J = 7.1, 1.6 Hz, 6H).

[0107] General preparation steps of compound A-4:

[0108] To a mixture of diethyl 2-(methyl-d3)malonate (Compound A-2, 110 g, 0.62 mol, 1.0 eq) in anhydrous tetrahydrofuran (THF, 1.1 L), n-butyl lithium (nBuLi, 2.5 M, 248 mL, 0.62 mol, 1.0 eq) was added at -78 °C and stirred for 1 h under N2 atmosphere. ((Chloromethoxy)methyl)benzene (Compound A-3, 97.2 g, 0.62 mol, 1.0 eq) was added to the above solution at -78 °C and stirred for 2 h at -78 °C. The mixture was quenched with H2O (1 L) and extracted with ethyl acetate (EtOAc, 500 mL x 3). The combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated by rotary evaporation. The obtained residue was purified by column chromatography with ethyl acetate / petroleum ether = 1 / 99 to give compound A-4 (134 g, 72%) as a yellow oil.

[0109] TLC: petroleum ether / ethyl acetate = 20 / 1; Rf (Compound A-2) = 0.5; Rf (Compound A-4) = 0.45; 1H NMR (400 MHz, CDCl3): δ 7.34 – 7.20 (m, 5H), 4.53 (s, 2H), 4.17 (q, J = 7.1Hz, 4H), 3.80 (s, 2H), 1.22 (t, J = 7.1 Hz, 6H).

[0110] General preparation steps of compound A-5:

[0111] To a solution of compound diethyl 2-((benzyloxy)methyl)-2-(methyl-d3)malonate (compound A-4, 134 g, 0.45 mol, 1.0 eq) in ethanol (1.3 L) was added a solution of potassium hydroxide (126 g, 2.25 mol, 5.0 eq) in water (1 L) at room temperature under stirring. The reaction mixture was stirred at 90 ° C for 2 h. Ethanol was evaporated under reduced pressure and the solution was diluted with H2O (1 L). The aqueous phase was washed with dichloromethane (DCM, 600 ml) and the pH was adjusted to 2 using 6N HCl. The product was extracted with ethyl acetate (EtOAc, 1 L × 3). The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated by rotary evaporation to give compound A-5 (108 g, 99%) as a yellow solid.

[0112] Thin layer chromatography: petroleum ether / ethyl acetate = 20 / 1; Rf (Compound A-4) = 0.45; Rf (Compound A-5) = 0.01; 1H NMR (400 MHz, deuterated dimethyl sulfoxide (DMSO-d6)): δ 7.28 (m, 5H), 4.46 (s, 2H), 3.67 (s, 2H).

[0113] General preparation steps of compound A-6:

[0114] A mixture of 2-((benzyloxy)methyl)-2-(methyl-d3)malonic acid (compound A-5, 108 g, 0.448 mol) in xylene (1 liter) was stirred at 149 ° C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The resulting residue was purified by column chromatography with dichloromethane / methanol = 1 / 99 to give A-6 (88 g, 99.7%) as a yellow oil.

[0115] Thin layer chromatography: dichloromethane / methanol = 20 / 1; Rf (Compound A-5) = 0.2; Rf (Compound A-6) = 0.5; 1H NMR (400 MHz, deuterated chloroform (CHCl3-d)): δ 7.37 (m, 5H), 4.58 (s, 2H), 3.72 (m, 1H), 3.57 (dd, J = 9.2, 5.7 Hz, 1H), 2.83 (t, J = 6.3 Hz, 1H).

[0116] General preparation steps of compound A-7:

[0117] Under nitrogen protection, borane-tetrahydrofuran (BH3-THF, 733 ml, 0.733 mol, 3.0 equiv) was added dropwise to a mixture of 2-((benzyloxy)methyl)propanoic acid-3,3,3-d3 acid (compound A-6, 48.2 g, 244 mmol, 1.0 equiv) in tetrahydrofuran (500 ml) at 0°C. The mixture was stirred at 70°C for 16h. The mixture was cooled to 0°C and quenched with methanol (100 ml). The mixture was extracted with ethyl acetate (500 ml × 3). The combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography with ethyl acetate / petroleum ether = 1 / 1 to give A-7 (40 g, 89.3%) as a yellow oil.

[0118] Thin layer chromatography: dichloromethane / methanol = 20 / 1; Rf (Compound A-6) = 0.5; Rf (Compound A-7) = 0.8; 1H NMR (400 MHz, deuterated chloroform (CHCl3-d)): δ 7.33 (m, 5H), 4.52 (m, 2H), 3.59 (m, 3H), 3.42 (m, 1H), 2.06 (m, 1H).

[0119] General preparation steps of compound A:

[0120] To a mixture of triphenylphosphine (PPh3, 85.9 g, 0.328 mol, 1.5 eq) and imidazole (22.3 g, 0.328 mmol, 1.5 eq) in dichloromethane (DCM, 1 L) was added iodine (I2, 83.2 g, 0.328 mol, 1.5 eq) at room temperature. The mixture was stirred at room temperature (rt) for 1 h. 2-((benzyloxy)methyl)propan-3,3,3-d3-1-ol (compound A-7, 40 g, 218 mmol, 1.0 eq) was added to the above solution at room temperature and stirred for 2 h. The reaction was quenched with 10% aqueous sodium sulfite solution (100 ml). The mixture was extracted with ethyl acetate (200 ml × 3). The combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated by rotary evaporation. The obtained residue was purified by column chromatography with ethyl acetate / petroleum ether = 5 / 95 to give compound A (54 g, yield: 84.3%) as a colorless oil.

[0121] Thin layer chromatography (TLC): petroleum ether (PE) / ethyl acetate (EtOAc) = 20 / 1; Rf (Compound A-7) = 0.3; Rf (Compound A) = 0.9; 1H NMR (400 MHz, deuterated chloroform (CHCl3-d)): δ 7.33 (d, J = 19.8 Hz, 5H), 4.53 (s, 2H), 3.39 (s, 1H), 3.32 (s, 2H), 1.78 (s, 1H).

[0122] General steps for the preparation of compound 2-2:

[0123] To a stirred solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 1, 14.0 g, 42.5 mmol, 1.0 eq) in dimethylformamide (DMF) (150 ml) were added ((2-(iodomethyl)propoxy-3,3,3-d3)methyl)benzene (Compound A, 18.6 g, 63.8 mmol, 1.5 eq) and potassium carbonate (K2CO3) (7.4 g, 106.3 mmol, 3.0 eq). The resulting mixture was stirred at 50°C for 16 h under a nitrogen atmosphere. The mixture was quenched with H2O (100 ml), extracted with ethyl acetate (EtOAc) (100 ml x 3), washed with brine (50 ml x 5), and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1) to give compound 2-2 (14.6 g, 61%) as a yellow oil.

[0124] Thin layer chromatography (TLC): ethyl acetate (EtOAc) / petroleum ether (PE) = 1 / 5; Rf (compound 1) = 0.2; Rf (compound 2-2) = 0.6; Liquid chromatography-mass spectrometry (LCMS): 401.1 [M+H]+.

[0125] General steps for the preparation of compound 2-3:

[0126] Pd / C (1.7 g) and Pd(OH)2 / C (1.7 g) were added to a solution of 5-(2-((benzyloxy)methyl)propyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 2-2, 7.5 g, 18.8 mmol, 1.0 equivalent) in ethyl acetate (EtOAc) (150 ml), and stirred at 60° C. for 16 h under hydrogen (H2). The mixture was filtered and concentrated to give compound 2-3 (4.8 g crude product) as a colorless oil.

[0127] Thin layer chromatography (TLC): ethyl acetate (EtOAc) / petroleum ether (PE) = 1 / 2; Rf (compound 2-2) = 0.6; Rf (compound 2-3) = 0.2; Liquid chromatography-mass spectrometry (LCMS): 311.1 [M+H]+.

[0128] General steps for the preparation of compound 2-4:

[0129] To a mixture of 5-(2-(hydroxymethyl)propyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (Compound 2-3, 5.0 g, 16.1 mmol, 1.0 eq) and sodium bicarbonate (NaHCO3) (6.8 g, 129 mmol, 8.0 eq) in dichloromethane (DCM) (300 ml) was added Dess-Martin periodinane (8.2 g, 19.4 mmol, 1.2 eq) at 0°C. The reaction mixture was stirred at 0°C under nitrogen for 1.5 h. The mixture was quenched with 10% sodium thiosulfate (Na2S2O3) aqueous solution (30 ml) and saturated sodium bicarbonate (NaHCO3) aqueous solution (30 ml), and extracted with dichloromethane (DCM) (100 ml×3). The combined organic layers were washed with saturated aqueous sodium bicarbonate (NaHCO3) solution (50 ml x 2), dried over anhydrous sodium sulfate, filtered and concentrated to give crude compound 2-4 (4.3 g crude) as a yellow oil.

[0130] Liquid chromatography-mass spectrometry (LCMS): 309.1 [M+H]+.

[0131] General steps for the preparation of compound 2-5:

[0132] Tetramethylguanidine (TMG, 2.87 g, 24.3 mmol, 0.5 eq.) was added to a stirred solution of 5-(2-formylpropyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 2-4, 15.0 g, 48.7 mmol, 1.0 eq.) in dichloromethane (150 ml). The resulting mixture was stirred at 30 ° C for 16 h under a nitrogen atmosphere. Liquid chromatography-mass spectrometry (LCMS) showed the appearance of a new peak and the disappearance of the starting material. The solution was concentrated under reduced pressure. The residue was chromatographed on a silica gel column, eluted with dichloromethane / ethyl acetate = 7 / 3, to give the racemic compound 2-5 (8.0 g, 53% total yield in three steps) as a yellow solid.

[0133] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf (compound 2-4) = 0.6; Rf (compound 2-5) = 0.2; Liquid chromatography-mass spectrometry (LCMS): 309.1 [M+H]+.

[0134] General steps for the preparation of compound 2-6:

[0135] To a solution of racemic (5S,9R)-8-hydroxy-2-methoxy-7-(methyl-d3)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 2-5, 8.0 g, 25.9 mmol, 1.0 eq) in dichloromethane (150 ml) were added triethylamine (29 ml, 207.8 mmol, 8.0 eq) and methanesulfonyl chloride (MsCl, 11.8 g, 103.9 mmol, 4.0 eq) dropwise. The resulting mixture was stirred at room temperature (rt) for 4 h under nitrogen atmosphere. The mixture was washed with saturated aqueous ammonium chloride solution (50 ml) and brine (50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2 / 3) to give racemic compound 2-6 (7.2 g, 70%) as a yellow solid.

[0136] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2; Rf (compound 2-5) = 0.2; Rf (compound 2-6) = 0.6; Liquid chromatography-mass spectrometry (LCMS): 387.2 [M+H]+.

[0137] General steps for the preparation of compound 2-7:

[0138] To a solution of racemic (5S,9R)-2-methoxy-7-(methyl-d3)-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 2-6, 10 g, 25.8 mmol, 1.0 eq) in acetic acid (300 ml) was added sodium acetate (4.23 g, 51.6 mmol, 2.0 eq). The resulting mixture was stirred at 120°C for 48 h under a nitrogen atmosphere. The mixture was concentrated to remove the solvent acetic acid, diluted with ethyl acetate (200 ml), washed with saturated sodium bicarbonate aqueous solution (50 ml), brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 1:4) to give racemic compound 2-7 (2.57 g, 34%) as a white solid.

[0139] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf (compound 2-6) = 0.3; Rf (compound 2-7) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 291.1 [M+H]+.

[0140] General steps for the preparation of compound 2-8:

[0141] The preparation method of compound F comprises the following steps: stirring a mixture of 1-bromoethane-2,2,2-d3 and triphenylphosphine bromide in toluene at 110° C. for 20 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain compound F.

[0142] A solution of (ethyl-2,2,2-d3)triphenylphosphine bromide (Compound F, 2.57 g, 6.89 mmol, 2.5 eq) in tetrahydrofuran (24 ml) was cooled to -78 °C, n-butyl lithium (2.31 ml, 2.5 M in hexane, 5.79 mmol, 2.1 eq) was added dropwise over 10 min, and stirred at room temperature for 20 min. After the solution was cooled to -78 °C again, a solution of racemic methyl (5S, 9R)-2-methoxy-7-(methyl-d3)-11-oxo-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylate (Compound 2-7, 800 mg, 2.75 mmol, 1.0 eq) in tetrahydrofuran (8 ml) was added dropwise over 10 min. The resulting solution was stirred at room temperature for 1.5 h. LC-MS showed that the reaction was complete. The reaction was quenched with brine (20 ml), extracted with ethyl acetate (50 ml x 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to give racemic compound 2-8 (Z / E = 4 / 1, 710 mg, 84%) as a white solid.

[0143] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf (compound 2-7) = 0.3; Rf (compound 2-8) = 0.6; Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0144] General steps for the preparation of compound 2-9:

[0145] To a solution of racemic methyl (5R,9R)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylate (compound 2-8, 1.5 g, 4.90 mmol, 1.0 eq) in toluene (PhMe, 60 ml) were added azobisisobutyronitrile (AIBN, 884 mg, 5.39 mmol, 1.1 eq) and thiophenol (PhSH, 1.89 g, 17.15 mmol, 3.5 eq). The resulting mixture was stirred at 105 ° C for 24 h, and then azobisisobutyronitrile (884 mg, 5.39 mmol, 1.1 eq) and thiophenol (1.89 g, 17.15 mmol, 3.5 eq) were added and stirred at 105 ° C for 24 h. 1H NMR showed Z configuration / E configuration = 1 / 9. The solution was diluted with ethyl acetate (150 ml), washed with sodium hydroxide (1M, 30 ml × 2) and brine (30 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give racemic compound 2-9 (1.3 g, 86%) as a yellow oil.

[0146] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf (compound 2-8) = 0.5; Rf (compound 2-9) = 0.55; Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0147] General steps for the preparation of compound 2-10:

[0148] To a mixed solution of racemic (5R,9R,E)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 2-9, 1.3 g, 4.26 mmol, 1.0 eq.) in dimethyl sulfoxide (DMSO, 12 ml) and tetrahydrofuran (THF, 12 ml) was added potassium hydroxide solution (12%, 48 ml). The resulting mixture was stirred at 100°C for 40 h under nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0°C, the pH was adjusted to 4-5 with 4M aqueous hydrochloric acid solution, extracted with methanol / dichloromethane = 1 / 9 (200 ml × 5), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give racemic compound 2-10 (1.1 g, 88%) as a white solid. The product was used directly in the next step without further purification.

[0149] Thin layer chromatography (TLC): methanol / dichloromethane = 1 / 10; Rf (compound 2-9) = 0.9; Rf (compound 2-10) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 292.3 [M+H]+.

[0150] General steps for the preparation of compound 2-11:

[0151] To a toluene (20 ml) solution of racemic (5R,9R,E)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid (compound 2-10, 320 mg, mmol, 1.0 eq) was added dropwise triethylamine (TEA, 782 mg, 7.84 mmol, 3.0 eq) and diphenylphosphoryl azide (DPPA) (1.4 g, 5.22 mmol, 2.0 eq) in toluene (3 ml). The resulting mixture was stirred at 85°C for 3 h under nitrogen atmosphere. LC-MS showed that compound 10 had been consumed. Methanol (MeOH, 40 ml) was added to the reaction and refluxed for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 7) to give racemic compound 2-11 (150 mg, crude product) as a colorless oil.

[0152] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1; Rf (compound 2-10) = 0.8; Rf (compound 2-11) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 316.3 [M+H]+.

[0153] General steps for the preparation of compound 2-12:

[0154] To a solution of racemic methyl ((5R,9R,E)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridin-5(6H)-yl)carbamate (Compound 2-11, 150 mg, 0.46 mmol, 1.0 eq) in chloroform (CHCl3, 10 ml) was added trimethylsilyl iodide (TMSI, 0.66 ml, 4.68 mmol, 10 eq) at room temperature and stirred at 65°C for 16 h under nitrogen. After cooling, methanol (6 ml) was added and the resulting mixture was stirred at 65°C for 6 h. After completion of the reaction, the mixture was concentrated under vacuum. The residue was diluted with dichloromethane / methanol (10 / 1, 100 ml), washed with sodium thiosulfate (10%, 3 ml), saturated sodium bicarbonate (3 ml) and brine (3 ml) in sequence. The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 15 / 85) to give racemic compound 2-12 (70 mg, 25% for two steps) as a white solid.

[0155] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1; Rf (compound 2-11) = 0.6; Rf (compound 2-12) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 249.3 [M+H]+.

[0156] General steps for the preparation of deuterated huperzine A (WP102-1):

[0157] Racemic (5R,9R,E)-5-amino-11-(ethylidene-2,2,2-d3)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanocyclooctyl[b]pyridin-2(1H)-one (compound 2-12, 70 mg, 0.28 mmol) was separated by chiral preparative HPLC to give deuterated huperzine A WP102-1 (29.5 mg, 42%, purity 98.03%) as a white solid.

[0158] Liquid chromatography-mass spectrometry (LCMS): 249.0 [M+H]+; 1H NMR (400 MHz, deuterated methanol (methanol-d4)): δ 7.91 (d, J = 9.5 Hz, 1H), 6.36 (d, J = 9.4 Hz, 1H), 5.54 (s, 1H), 5.43 (dd, J = 5.3, 2.1 Hz, 1H), 3.64 (t, J = 5.1 Hz, 1H), 2.78 (dd, J = 17.0, 5.1 Hz, 1H), 2.59 (dd, J = 17.0, 1.8 Hz, 1H), 2.25 – 2.09 (m, 2H).

[0159] Example 3: Synthesis of Compound (WP102-2) - Deuterated Huperzine A General preparation steps of compound B-2:

[0160] Under nitrogen protection, a solution of diethyl 2-methylmalonate (compound B-1, 80 g, 0.46 mmol, 1.0 eq.) in tetrahydrofuran (THF, 800 ml) was added with n-butyl lithium (nBuLi, 202 ml, 0.506 mol, 1.1 eq., 2.5 M in THF) at -78 °C. The reaction mixture was stirred at -78 °C for 1 h. Then ((chloromethoxy)methyl)benzene (A-3, 72 g, 0.46 mmol, 1.0 eq.) was added to the solution and stirred at room temperature for 3 h under nitrogen protection. The reaction was quenched with H2O (100 ml), extracted with ethyl acetate (EtOAc, 200 ml × 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to give compound B-2 (120 g, 88%) as a colorless oil.

[0161] TLC: petroleum ether / ethyl acetate (PE / EtOAc) = 20 / 1; Rf (Compound B-1) = 0.4; Rf (Compound B-2) = 0.5; LCMS: 295.1 [M+H]+.

[0162] General preparation steps of compound B-3:

[0163] Under nitrogen protection, a solution of 2-((benzyloxy)methyl)-2-methylmalonic acid diethyl ester (compound B-2, 150 g, 0.508 mol, 1.0 equiv) in ethanol (EtOH, 1.5 L) was added to a solution of potassium hydroxide (KOH, 142.6 g, 2.54 mol, 5.0 equiv) in water (1.0 L) at 0 ° C. The reaction mixture was stirred at 90 ° C for 2 h under nitrogen protection. The reaction was concentrated and diluted with H2O (500 ml). The aqueous phase was washed with dichloromethane (DCM, 200 ml × 3) and the pH was adjusted to 4 with 6N HCl. The mixture was extracted with ethyl acetate (EtOAc, 300 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated to give compound B-3 (120 g, 98%) as a colorless oil.

[0164] TLC: PE / EtOAc=20 / 1; Rf (Compound B-2) = 0.45; Rf (Compound B-3) = 0.01; LCMS: 239.1 [M+H]+.

[0165] General preparation steps of compound B-4:

[0166] Under nitrogen protection, a solution of 2-((benzyloxy)methyl)-2-methylmalonic acid (compound B-3, 120 g, 0.52 mol, 1.0 eq.) in xylene (1.5 L) was stirred at 149 ° C for 16 h. The reaction was concentrated and diluted with H2O (500 ml), extracted with ethyl acetate (EtOAc, 300 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 99) to give compound B-4 (80 g, 81%) as a colorless oil.

[0167] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 20 / 1; Rf (Compound B-3) = 0.01; Rf (Compound B-4) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 195.1 [M+H]+.

[0168] General steps for the preparation of compound B-5:

[0169] At room temperature, concentrated sulfuric acid (10 ml) was added to a solution of 3-(benzyloxy)-2-methylpropionic acid (compound B-4, 80 g, 0.41 mol, 1.0 equivalent) in methanol (1.5 liters). The reaction mixture was stirred at 80 ° C for 16 h under nitrogen protection. The reaction mixture was concentrated and quenched with saturated sodium bicarbonate (200 ml), extracted with ethyl acetate (200 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 99) to give compound B-5 (75 g, 87%) as a colorless oil.

[0170] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 20 / 1; Rf (Compound B-4) = 0.5; Rf (Compound B-5) = 0.8; Liquid chromatography-mass spectrometry (LCMS): 209.1 [M+H]+.

[0171] General steps for the preparation of compound B-6:

[0172] At 0°C, lithium aluminum deuteride (LiAlD4, 21 g, 0.5 mol, 2.0 equiv) was added to a solution of methyl 3-(benzyloxy)-2-methylpropanoate (compound B-5, 52 g, 249.8 mmol, 1.0 equiv) in tetrahydrofuran (870 ml). The reaction mixture was stirred at 0°C for 3 h under nitrogen protection. The reaction was quenched by adding sodium sulfate decahydrate (50 g), extracted with ethyl acetate (200 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 99) to give compound B-7 (40 g, 76%) as a colorless oil.

[0173] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 3; Rf (Compound B-5) = 0.5; Rf (Compound B-6) = 0.3; Liquid chromatography-mass spectrometry (LCMS): 183.1 [M+H]+.

[0174] General steps for the preparation of compound B:

[0175] At 0°C, imidazole (21.8 g, 0.32 mmol, 1.5 eq) and iodine (I2, 81.3 g, 0.32 mmol, 1.5 eq) were added to a solution of triphenylphosphine (PPh3, 83.9 g, 0.32 mol, 1.5 eq) in dichloromethane (550 ml). The reaction mixture was stirred at room temperature for 1.5 h under nitrogen protection. Compound B-6 (38.9 g, 213.6 mmol, 1.0 eq) was then added to the above solution and stirred for 2 h at room temperature. The reaction was quenched by adding sodium thiosulfate (100 ml), extracted with dichloromethane (100 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 99) to obtain compound B (40 g, 64%) as a colorless oil.

[0176] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 20; Rf value of compound B-6 = 0.01; Rf value of compound B = 0.8; Liquid chromatography-mass spectrometry (LCMS): 293.1 [M+H]+.

[0177] General steps for the preparation of compound 3-2:

[0178] To a stirred solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 1, 14.0 g, 42.5 mmol, 1.0 eq) in dimethylformamide (DMF, 150 ml) were added ((2-(iodomethyl)propoxy-3,3,3-d3)methyl)benzene (Compound B, 18.6 g, 63.8 mmol, 1.5 eq) and potassium carbonate (K2CO3, 14.7 g, 106.3 mmol, 3.0 eq). The resulting mixture was stirred at 50°C for 16 h under a nitrogen atmosphere. The mixture was quenched with H2O (100 ml) and extracted with ethyl acetate (100 ml x 3). The combined organic layers were washed with brine (50 ml x 5) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 9 / 1) to give racemic compound 3-2 (14.6 g, 61%) as a yellow oil.

[0179] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf value of compound 1 = 0.2; Rf value of compound 3-2 = 0.6; Liquid chromatography-mass spectrometry (LCMS): 400.1 [M+H]+.

[0180] General steps for the preparation of compound 3-3:

[0181] Pd / C (2.5 g) and Pd(OH)2 / C (2.5 g) were added to a stirred solution of 5-(3-(benzyloxy)-2-methylpropyl-1,1-d2)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 3-2, 12.5 g, 31.2 mmol, 1.0 eq.) in ethyl acetate (300 ml). The resulting mixture was stirred at 60 °C for 16 h under a hydrogen atmosphere. Liquid chromatography-mass spectrometry (LCMS) showed a new peak and the starting material disappeared. The mixture was filtered and concentrated to give compound 3-3 (10.1 g crude product) as a colorless oil.

[0182] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2; Rf value of compound 3-2 = 0.6; Rf value of compound 3-3 = 0.2; Liquid chromatography-mass spectrometry (LCMS): 310.2 [M+H]+.

[0183] General steps for the preparation of compound 3-4:

[0184] To a stirred solution of methyl 5-(3-hydroxy-2-methylpropyl-1,1-d2)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (compound 3-3, 6.2 g, 20.1 mmol, 1.0 eq) in dichloromethane (DCM, 500 ml) was added sodium bicarbonate (NaHCO3, 8.43 g, 100.3 mmol, 5 eq) and Dess-Martin periodinane DMP (Dess-Martin, 10.2 g, 24.1 mmol, 1.2 eq) at 0°C. The reaction was stirred at 0°C under nitrogen for 1.5 h. Liquid chromatography-mass spectrometry (LCMS) showed a new peak and the starting material disappeared. The mixture was quenched with sodium thiosulfate (Na2S2O3, 50 ml) and extracted with dichloromethane (50 ml×3). The mixture was washed with sodium bicarbonate (100 ml x 2) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to give racemic compound 3-4 (9.0 g crude product) as a yellow oil.

[0185] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 3; Rf (compound 3-3) = 0.4; Rf (compound 3-4) = 0.5; LC-MS: 308.1[M+H]+.

[0186] General steps for the preparation of compound 3-5:

[0187] Tetramethylguanidine (TMG, 2.7 g, 23.4 mmol, 0.5 eq.) was added to a stirred solution of 2-methoxy-5-(2-methyl-3-oxopropyl-1,1-d2)-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 3-4, 14.4 g, 46.8 mmol, 1.0 eq.) in dichloromethane (700 ml). The resulting mixture was stirred at 30 ° C for 16 h under a nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 7 / 3) to give racemic compound 3-5 (8.2 g, 53%) as a yellow solid.

[0188] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 5; Rf (compound 3-4) = 0.6; Rf (compound 3-5) = 0.2; LC-MS: 308.1[M+H]+.

[0189] General steps for the preparation of compound 3-6:

[0190] To a solution of racemic (5S,9R)-8-hydroxy-2-methoxy-7-methyl-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (compound 3-5, 8.2 g, 26.7 mmol, 1.0 eq) in dichloromethane (240 ml) was added triethylamine (21.6 g, 213.6 mmol, 8.0 eq) and methanesulfonyl chloride (22.0 g, 106.8 mmol, 4.0 eq) dropwise. The resulting mixture was stirred at room temperature for 4 h under a nitrogen atmosphere. The mixture was washed with saturated aqueous ammonium chloride solution (50 ml) and brine (50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 2) to give racemic compound 3-6 (9.0 g, 70%) as a yellow solid.

[0191] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 2; Rf (compound 3-5) = 0.2; Rf (compound 3-6) = 0.6; LC-MS: 386.2[M+H]+.

[0192] General steps for the preparation of compound 3-7:

[0193] To a solution of racemic (5S,9R)-2-methoxy-7-methyl-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (Compound 3-6, 9.0 g, 23.3 mmol, 1.0 eq) in acetic acid (300 ml) was added sodium acetate (3.83 g, 46.6 mmol, 2.0 eq). The resulting mixture was stirred at 120°C for 48 h under a nitrogen atmosphere. The mixture was concentrated to remove the solvent acetic acid, diluted with ethyl acetate (200 ml), washed with saturated sodium bicarbonate aqueous solution (50 ml), brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give racemic compound 3-7 (2.0 g, 34%) as a white solid.

[0194] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 5; Rf (compound 3-6) = 0.3; Rf (compound 3-7) = 0.5; LC-MS: 290.1[M+H]+.

[0195] General steps for the preparation of compound 3-8:

[0196] A solution of ethyltriphenylphosphine bromide (compound H, 3.5 g, 9.5 mmol, 2.5 eq) in tetrahydrofuran (24 ml) was cooled to -78°C, n-butyllithium (3.2 ml, 2.5 M in hexane, 7.99 mmol, 2.1 eq) was added dropwise over 10 minutes, and the mixture was stirred at room temperature for 20 minutes. After the solution was cooled to -78°C again, a solution of racemic (5S, 9R)-2-methoxy-7-methyl-11-oxo-9,10-dihydro-5,9-methanecyclooctane [b] pyridine-5 (6H)-carboxylic acid methyl ester-6,6-d2 (compound 3-7, 1.1 g, 3.8 mmol, 1.0 eq) in tetrahydrofuran (8 ml) was added dropwise over 10 minutes. The resulting solution was stirred at room temperature for 1.5 h. LC-MS showed that the reaction was complete. The reaction was quenched with brine (20 ml), extracted with ethyl acetate (100 ml x 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to give racemic compound 3-8 (Z / E = 4 / 1, 950 mg, 82%) as a white solid.

[0197] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 10; Rf (compound 3-7) = 0.3; Rf (compound 3-8) = 0.6; LC-MS: 302.1[M+H]+.

[0198] General steps for the preparation of compound 3-9:

[0199] Azobisisobutyronitrile (568 mg, 3.46 mmol, 1.1 eq) and thiophenol (PhSH, 1.22 g, 11.05 mmol, 3.5 eq) were added to a toluene (50 ml) solution of racemic (5R, 6S, 9R)-11-ethylidene-2-methoxy-6,7-dimethyl-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6-d (compound 3-8, 950 mg, 3.15 mmol, 1.0 eq). The resulting mixture was stirred at 105° C. for 24 h, and azobisisobutyronitrile (568 mg, 3.46 mmol, 1.1 eq) and thiophenol (1.22 g, 11.05 mmol, 3.5 eq) were added and stirred at 105° C. for 24 h. 1HNMR showed Z configuration / E configuration = 1 / 9. The solution was diluted with ethyl acetate (150 ml), washed with sodium hydroxide (1M, 30 ml × 2) and brine (30 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give racemic compound 3-9 (1.0 g, crude product) as a yellow oil.

[0200] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 10; Rf (compound 3-8) = 0.5; Rf (compound 3-9) = 0.55; LC-MS: 302.1[M+H]+.

[0201] General steps for the preparation of compound 3-10:

[0202] To a mixed solution of racemic (5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (compound 3-9, 1.0 g, 3.3 mmol, 1.0 eq.) in dimethyl sulfoxide (12 ml) and tetrahydrofuran (4 ml) was added potassium hydroxide solution (12%, 48 ml). The resulting mixture was stirred at 100 °C for 40 h under a nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0 °C, the pH was adjusted to 4-5 with 4M aqueous hydrochloric acid solution, extracted with methanol / dichloromethane = 1 / 9 (200 ml × 5), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give racemic compound 3-10 (750 mg, 78%) as a white solid. The product was used directly in the next step without further purification.

[0203] Thin layer chromatography: methanol / dichloromethane = 1 / 10; Rf (compound 3-9) = 0.9; Rf (compound 3-10) = 0.4; LC-MS: 288.3[M+H]+.

[0204] General steps for the preparation of compound 3-11:

[0205] To a solution of racemic (5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid-6,6-d2 acid (compound 3-10, 750 mg, 2.6 mmol, 1.0 eq) in toluene (20 ml) was added dropwise triethylamine (789 mg, 7.8 mmol, 3.0 eq) and diphenylphosphoryl azide (DPPA, 1.4 g, 5.22 mmol, 2.0 eq) in toluene (3 ml). The resulting mixture was stirred at 85 °C for 3 h under nitrogen atmosphere. LC-MS showed that compound 3-10 had been consumed. Methanol (40 ml) was added to the reaction and refluxed for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 3) to give racemic compound 3-11 (450 mg, crude product) as a colorless oil.

[0206] Thin layer chromatography: petroleum ether / ethyl acetate = 3 / 1; Rf (compound 3-10) = 0.8; Rf (compound 3-11) = 0.5; LC-MS: 317.3[M+H]+.

[0207] General steps for the preparation of compound 3-12:

[0208] To a solution of racemic methyl ((5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methancyclooctane[b]pyridin-5(6H)-yl-6,6-d2)carbamate (Compound 3-11, 450 mg, 1.42 mmol, 1.0 eq) in chloroform (30 ml) was added trimethylsilyl iodide (2.84 g, 14.2 mmol, 10 eq) at room temperature under nitrogen atmosphere, and the mixture was stirred at 65°C for 16 h. After cooling, methanol (30 ml) was added, and the resulting mixture was stirred at 65°C for 6 h. After completion of the reaction, the mixture was concentrated under vacuum. The residue was diluted with dichloromethane / methanol (10 / 1, 100 ml) and washed successively with sodium thiosulfate (10%, 5 ml), saturated sodium bicarbonate (6 ml) and brine (6 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to give racemic compound 3-12 (190 mg, 54% for two steps) as a white solid.

[0209] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1; Rf (compound 3-11) = 0.6; Rf (compound 3-12) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 245.3 [M+H]+.

[0210] General steps for the preparation of deuterated huperzine A (WP102-2):

[0211] Racemic (5R,9R,E)-5-amino-11-ethylidene-7-methyl-5,6,9,10-tetrahydro-5,9-carboxycyclooctyl[b]pyridin-2(1H)-one-6,6-d2 (compound 3-12, 190 mg, 0.77 mmol) was separated by chiral preparative HPLC SFC to give deuterated huperzine A WP102-2 (64.4 mg, 33%, purity 99.61%) as a white solid.

[0212] Liquid chromatography-mass spectrometry (LCMS): 245.3 [M+H]+; 1H NMR (400 MHz, CD3OD-d4): δ 7.91 (d, J = 9.4 Hz, 1H), 6.37 (d, J = 9.6, 0.6 Hz, 1H), 5.56 (d, J = 6.8 Hz, 1H), 5.45 (dd, J = 5.2, 1.5 Hz, 1H), 3.65 (s, 1H), 2.77 (dd, J = 5.2, 1.0 Hz, 1H), 2.59 (dd, J = 17.0, 1.8 Hz, 1H), 1.70 (d, J = 6.8 Hz, 3H), 1.55 (t, J = 1.2 Hz, 3H).

[0213] Example 4: Synthesis of Compound (WP102-4) - Deuterated Huperzine A General steps for the preparation of compound 4-2:

[0214] The preparation method of compound G comprises the following steps: stirring a mixture of 1-bromoethane-1,1,2,2,2-d5 and triphenylphosphine bromide in toluene at 110° C. for 20 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain compound G.

[0215] A solution of (ethyl-d5)triphenylphosphine bromide (compound G, 3.87 g, 10.34 mmol, 2.5 eq) in tetrahydrofuran (THF, 36 ml) was cooled to -78 °C, a hexane solution (2.5 M) of n-butyl lithium (3.47 ml, 8.69 mmol, 2.1 eq) was added dropwise over 10 minutes, and stirred at room temperature for 20 minutes. After the solution was cooled to -78 °C again, a solution of racemic methyl (5S, 9R)-2-methoxy-7-(methyl-d3)-11-oxo-9,10-dihydro-5,9-methanecyclooctane [b] pyridine-5 (6H) -carboxylate (compound 2-7, 1.2 g, 4.13 mmol, 1.0 eq) in tetrahydrofuran (15 ml) was added dropwise over 10 minutes. The resulting solution was stirred at room temperature for 1.5 h. LCMS showed that the reaction was complete. The reaction was quenched with brine (40 ml), extracted with ethyl acetate (100 ml x 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to give racemic compound 4-2 (Z / E = 4 / 1, 1.03 g, 81%) as a white solid.

[0216] TLC: ethyl acetate / petroleum ether = 1 / 10; Rf (compound 2-7) = 0.3; Rf (compound 4-2) = 0.6; LCMS: 307.1 [M+H]+.

[0217] General steps for the preparation of compound 4-3:

[0218] Azobisisobutyronitrile (AIBN, 884 mg, 5.39 mmol, 1.1 eq) and thiophenol (PhSH, 1.89 g, 17.15 mmol, 3.5 eq) were added to a toluene (60 ml) solution of racemic methyl (5R, 9R)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylate (compound 4-2, 1.5 g, 4.90 mmol, 1.0 eq). The resulting mixture was stirred at 105°C for 24 h, and azobisisobutyronitrile (884 mg, 5.39 mmol, 1.1 eq) and thiophenol (1.89 g, 17.15 mmol, 3.5 eq) were added and stirred at 105°C for 24 h. The reaction solution was detected by 1H NMR, and the Z configuration / E configuration = 1 / 9 was achieved. The solution was diluted with ethyl acetate (150 ml), washed with NaOH (1M, 30 ml × 2) and brine (30 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4) to obtain racemic compound 4-3 (1.31 g, 87%) as a yellow oil.

[0219] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf (compound 4-2) = 0.5; Rf (compound 4-3) = 0.55; Liquid chromatography-mass spectrometry (LCMS): 307.1 [M+H]+.

[0220] General steps for the preparation of compound 4-4:

[0221] To a mixed solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (compound 4-3, 1.3 g, 4.26 mmol, 1.0 equiv) in dimethyl sulfoxide (12 ml) and tetrahydrofuran (12 ml) was added potassium hydroxide solution (12%, 48 ml). The resulting mixture was stirred at 100°C for 40 h under a nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0°C, the pH was adjusted to 4-5 with 4M aqueous hydrochloric acid solution, extracted with methanol / dichloromethane = 1 / 9 solution (200 ml × 5), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give racemic compound 4-4 (1.12 g, 90%) as a white solid. The product was used directly in the next step without further purification.

[0222] Thin layer chromatography (TLC): methanol / dichloromethane = 1 / 10; Rf (compound 4-3) = 0.9; Rf (compound 4-4) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 293.3 [M+H]+.

[0223] General steps for the preparation of compound 4-5:

[0224] To a toluene (30 ml) solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid (compound 4-4, 500 mg, 1.71 mmol, 1.0 eq) was added dropwise triethylamine (519 mg, 5.13 mmol, 3.0 eq) and diphenylphosphoryl azide (942 mg, 3.42 mmol, 2.0 eq) in toluene (3 ml). The resulting mixture was stirred at 85°C for 3 h under nitrogen atmosphere. LC-MS showed that compound 4 had been completely consumed. Methanol (30 ml) was added to the reaction and refluxed for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give racemic compound 4-5 (345 mg, crude product) as a colorless oil.

[0225] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1; Rf (compound 4-4) = 0.8; Rf (compound 4-5) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 322.3 [M+H]+.

[0226] General steps for the preparation of compound 4-6:

[0227] To a solution of racemic methyl ((5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridin-5(6H)-yl)carbamate (Compound 4-5, 345 mg, 1.07 mmol, 1.0 eq) in chloroform (8 ml) was added trimethylsilyl iodide (1.5 ml, 10.7 mmol, 10 eq) at room temperature under nitrogen atmosphere, and the mixture was heated at 65°C for 16 h. After cooling, methanol (30 ml) was added, and the resulting mixture was stirred at 65°C for 6 h. After completion of the reaction, the mixture was concentrated under vacuum. The residue was diluted with dichloromethane / methanol (10 / 1, 100 ml), washed successively with 10% sodium thiosulfate (3 ml), saturated sodium bicarbonate (3 ml) and brine (3 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) and then by preparative thin layer chromatography (dichloromethane / methanol, 9 / 1) to give racemic compound 4-6 (50 mg, 23% for two steps) as a white solid.

[0228] Thin layer chromatography (TLC): dichloromethane / methanol = 9 / 1; Rf (compound 4-5) = 0.6; Rf (compound 4-6) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 250.3 [M+H]+.

[0229] General steps for the preparation of deuterated huperzine A WP102-4 compound:

[0230] Racemic (5R,9R,E)-5-amino-11-(ethylidene-d4)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanocyclooctyl[b]pyridin-2(1H)-one (compound 4-6, 50 mg, 0.28 mmol) was separated by chiral preparative HPLC SFC to give deuterated huperzine A WP102-4 (6.5 mg, 13%, purity 98.95%) as a white solid.

[0231] Liquid chromatography-mass spectrometry (LCMS): 250.3 [M+H]+; 1H NMR (400 MHz, CD3OD-d4): δ 7.89 (d, J = 9.5 Hz, 1H), 6.37 (d, J = 9.4 Hz, 1H), 5.44 (dd, J = 5.1, 2.0 Hz, 1H), 3.65 (t, J = 4.9 Hz, 1H), 2.79 (dd, J = 17.0, 5.2 Hz, 1H), 2.62 – 2.47 (m, 1H), 2.27-2.13 (m, 2H).

[0232] Example 5: Synthesis of Compound (WP102-5) - Deuterated Huperzine A General steps for the preparation of compound C-1:

[0233] Under nitrogen protection, concentrated H2SO4 (9.1 g, 51.3 mmol, 0.1 eq) was added to a stirred solution of 2-((benzyloxy)methyl)propanoic acid-3,3,3-d3 acid (compound A-6, 101 g, 0.513 mol, 1.0 eq) in methanol (1 L), and stirred at 90 ° C for 2 h. The mixture was concentrated, diluted with water (400 ml), and extracted with ethyl acetate (300 ml × 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography with petroleum ether / ethyl acetate = 99 / 1 to obtain compound C-1 (72 g, 66.6%) as a colorless oil.

[0234] TLC: petroleum ether / ethyl acetate = 20 / 1; Rf (Compound A-6) = 0.9; Rf (Compound C-1) = 0.8; 1H NMR (400 MHz, CDCl3-d4): δ 7.31 (m, 6H), 4.51 (s, 2H), 3.66 (m, 4H), 3.49 (m, 1H), 2.76 (t, J = 6.5 Hz, 1H).

[0235] General steps for the preparation of compound C-2:

[0236] At 0°C, lithium aluminum deuteride LiAlD4 (17.9 g, 0.426 mol, 2.0 eq) was added to a solution of methyl 2-((benzyloxy)methyl)propanoate-3,3,3-d3 (compound C-1, 45 g, 213.2 mmol, 1.0 eq) in tetrahydrofuran (900 ml). The reaction mixture was stirred at 0°C for 3 h under nitrogen. LCMS showed that the reaction was complete. The reaction was concentrated and quenched with sodium sulfate decahydrate (50 g) and extracted with ethyl acetate (200 ml x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 3) to give compound C-2 (30 g, 75%) as a colorless oil.

[0237] TLC: ethyl acetate / petroleum ether = 1 / 3; Rf (Compound C-1) = 0.5; Rf (Compound C-2) = 0.3; LCMS: 186.2 [M+H]+.

[0238] General steps for the preparation of compound C:

[0239] To a solution of triphenylphosphine (PPh3, 63.6 g, 243.0 mmol, 1.5 eq) in dichloromethane (500 ml) were added imidazole (16.5 g, 243.0 mmol, 1.5 eq) and iodine (I2, 61.6 g, 243.0 mmol, 1.5 eq) at 0°C. The reaction mixture was stirred at room temperature (rt) for 1.5 h under nitrogen. Compound C-2 (30 g, 162.0 mmol, 1.0 eq) was then added to the above solution at room temperature and stirred for 2 h. The reaction was quenched with 10% Na2S2O3 (100 ml), extracted with dichloromethane (100 ml×3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (eluent: a mixed solution of ethyl acetate and petroleum ether, ethyl acetate / petroleum ether = 1 / 99) to give compound C (41.6 g, 86%) as a colorless oil.

[0240] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 20; Rf (Compound C-2) = 0.01; Rf (Compound C) = 0.8; Liquid chromatography-mass spectrometry (LCMS): 296.1 [M+H]+.

[0241] General steps for the preparation of compound 5-2:

[0242] To a stirred solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (compound 1, 20 g, 85.0 mmol, 1.0 eq) in dimethylformamide (DMF) (300 ml) were added ((3-iodo-2-(methyl-d3)propoxy-3,3-d2)methyl)benzene (compound C, 37.6 g, 127.5 mmol, 1.5 eq) and potassium carbonate (K2CO3) (35.3 g, 255 mmol, 3.0 eq). The resulting mixture was stirred at 50°C for 16 h under nitrogen atmosphere. The mixture was quenched with H2O (100 ml) and extracted with ethyl acetate (300 ml x 3). The mixture was washed with brine (50 ml x 5) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 9 / 1) to give racemic compound 5-2 (27 g, 78%) as a yellow oil.

[0243] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf (compound 1) = 0.2; Rf (compound 5-2) = 0.6; Liquid chromatography-mass spectrometry (LCMS): 403.1 [M+H]+.

[0244] General steps for the preparation of compound 5-3:

[0245] Pd / C (4 g) and Pd(OH)2 / C (4 g) were added to a stirred solution of 5-(2-((benzyloxy)methyl)propyl-1,1,3,3,3-d5)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 5-2, 20 g, 49.6 mmol, 1.0 eq.) in ethyl acetate (300 ml). The resulting mixture was stirred at 60 °C for 16 h under a hydrogen atmosphere. Liquid chromatography-mass spectrometry (LCMS) showed a new peak and the disappearance of the starting material. The mixture was filtered and concentrated to give compound 5-3 (15.2 g crude product) as a colorless oil.

[0246] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2; Rf (compound 5-2) = 0.6; Rf (compound 5-3) = 0.2; Liquid chromatography-mass spectrometry (LCMS): 313.1 [M+H]+.

[0247] General steps for the preparation of compound 5-4:

[0248] To a stirred solution of methyl 5-(2-(hydroxymethyl)propyl-1,1,3,3,3-d5)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (compound 5-3, 15.2 g, 48.5 mmol, 1.0 eq) in dichloromethane (DCM) (1.0 L) was added sodium bicarbonate (NaHCO3) (20.4 g, 242.7 mmol, 5.0 eq) and Dess-Martin periodinane DMP (24.6 g, 58.2 mmol, 1.2 eq) at 0°C. The reaction was stirred at 0°C under nitrogen for 1.5 h. Liquid chromatography-mass spectrometry (LCMS) showed a new peak and the disappearance of the starting material. The mixture was quenched with sodium thiosulfate (Na2S2O3) (100 ml) and extracted with dichloromethane (100 ml x 3). The mixture was washed with sodium bicarbonate (100 ml x 2) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to give racemic compound 5-4 (17 g crude product) as a yellow oil.

[0249] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 3; Rf (compound 5-3) = 0.3; Rf (compound 5-4) = 0.6; LC-MS: 311.1[M+H]+.

[0250] General steps for the preparation of compound 5-5:

[0251] Tetramethylguanidine TMG (3.1 g, 27.3 mmol, 0.5 eq) was added to a stirred solution of 5-(2-formylpropyl-1,1,3,3,3-d5)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester (compound 5-4, 17 g, 54.6 mmol, 1.0 eq) in dichloromethane (1 liter). The resulting mixture was stirred at 30 ° C for 16 h under a nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 7 / 3) to give racemic compound 5-5 (10.0 g, 58%) as a yellow solid.

[0252] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 5; Rf (compound 5-4) = 0.4; Rf (compound 5-5) = 0.2; LC-MS: 311.2[M+H]+.

[0253] General steps for the preparation of compound 5-6:

[0254] To a solution of racemic (5S,9R)-8-hydroxy-2-methoxy-7-(methyl-d3)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (compound 5-5, 10.0 g, 32.1 mmol, 1.0 eq) in dichloromethane (400 ml) were added triethylamine (26.0 g, 257.1 mmol, 8.0 eq) and methanesulfonyl chloride (25 g, 128.4 mmol, 4.0 eq) dropwise. The resulting mixture was stirred at room temperature for 4 h under nitrogen atmosphere. The mixture was quenched with saturated aqueous ammonium chloride solution (50 ml) and washed with brine (50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 2) to give racemic compound 5-6 (11 g, 88%) as a yellow solid.

[0255] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 2; Rf (compound 5-5) = 0.2; Rf (compound 5-6) = 0.6; LC-MS: 389.2[M+H]+.

[0256] General steps for the preparation of compound 5-7:

[0257] To a solution of racemic (5S,9R)-2-methoxy-7-(methyl-d3)-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (Compound 5-6, 11 g, 28.2 mmol, 1.0 eq) in acetic acid (300 ml) was added sodium acetate (4.64 g, 56.5 mmol, 2.0 eq). The resulting mixture was stirred at 120°C for 48 h under nitrogen atmosphere. The mixture was concentrated to remove the solvent acetic acid, diluted with ethyl acetate (200 ml), washed with saturated sodium bicarbonate aqueous solution (50 ml), brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give racemic compound 5-7 (2.5 g, 30%) as a white solid.

[0258] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5; Rf value of compound 5-6 = 0.3; Rf value of compound 5-7 = 0.5; Liquid chromatography-mass spectrometry (LCMS): 293.1 [M+H]+.

[0259] General steps for the preparation of compounds 5-8:

[0260] A solution of (ethyl-d5)triphenylphosphine bromide (compound G, 3.85 g, 10 mmol, 2.5 eq) in tetrahydrofuran (24 ml) was cooled to -78°C, n-butyl lithium (3.4 ml, 2.5 M in hexane, 8.6 mmol, 2.1 eq) was added dropwise over 10 minutes, and the mixture was stirred at room temperature for 20 minutes. After the solution was cooled to -78°C again, a solution of racemic methyl (5S, 9R)-2-methoxy-7-(methyl-d3)-11-oxo-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylate-6,6-d2 (compound 5-7, 1.0 g, 4.0 mmol, 1.0 eq) in tetrahydrofuran (8 ml) was added dropwise over 10 minutes. The resulting solution was stirred at room temperature for 1.5 h. LC-MS showed that the reaction was complete. The reaction was quenched with brine (20 ml), extracted with ethyl acetate (100 ml x 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to give racemic compound 5-8 (Z / E = 4 / 1, 780 mg, 63%) as a white solid.

[0261] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf value of compound 5-7 = 0.3; Rf value of compound 5-8 = 0.6; Liquid chromatography-mass spectrometry (LCMS): 309.1 [M+H]+.

[0262] General steps for the preparation of compound 5-9:

[0263] Azobisisobutyronitrile (AIBN, 454 mg, 2.77 mmol, 1.1 eq) and thiophenol (PhSH, 974 mg, 8.82 mmol, 3.5 eq) were added to a toluene (40 ml) solution of racemic methyl (5R, 9R)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylate-6,6-d2 (Compound 5-8, 780 mg, 2.52 mmol, 1.0 eq). The resulting mixture was stirred at 105°C for 24 h, and azobisisobutyronitrile (454 mg, 2.77 mmol, 1.1 eq) and thiophenol (974 mg, 8.82 mmol, 3.5 eq) were added, followed by stirring at 105°C for 24 h. 1H NMR showed Z configuration / E configuration = 1 / 9. The solution was diluted with ethyl acetate (150 ml), washed with sodium hydroxide (1M, 30 ml × 2) and brine (30 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give racemic compound 5-9 (750 mg, 96%) as a yellow oil.

[0264] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10; Rf (compound 5-8) = 0.5; Rf (compound 5-9) = 0.55; Liquid chromatography-mass spectrometry (LCMS): 302.1 [M+H]+.

[0265] General steps for the preparation of compounds 5-10:

[0266] To a mixed solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (Compound 5-9, 700 mg, 2.3 mmol, 1.0 eq.) in dimethyl sulfoxide (24 ml) and tetrahydrofuran (6 ml) was added potassium hydroxide solution (12%, 24 ml). The resulting mixture was stirred at 100 °C for 40 h under nitrogen atmosphere. LC-MS showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0°C, the pH was adjusted to 4-5 with 4M aqueous hydrochloric acid solution, extracted with methanol / dichloromethane = 1 / 9 solution (200 ml x 5), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain racemic compound 5-10 (630 mg, 92%) as a white solid. The product was used directly in the next step without further purification.

[0267] Thin layer chromatography (TLC): methanol / dichloromethane = 1 / 10; Rf (compound 5-9) = 0.9; Rf (compound 5-10) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 295.3 [M+H]+.

[0268] General steps for the preparation of compound 5-11:

[0269] To a toluene (20 ml) solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanecyclooctane[b]pyridine-5(6H)-carboxylic acid-6,6-d2 acid (compound 5-10, 630 mg, 2.1 mmol, 1.0 eq) was added dropwise triethylamine (647 mg, 6.4 mmol, 3.0 eq) and diphenylphosphoryl azide DPPA (1.1 g, 4.2 mmol, 2.0 eq) in toluene (3 ml). The resulting mixture was stirred at 85°C for 3 h under nitrogen atmosphere. LC-MS showed that compound 10 was completely consumed. Methanol (40 ml) was added to the reaction and refluxed for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 3) to give racemic compound 5-11 (400 mg, crude) as a colorless oil.

[0270] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1; Rf (compound 5-10) = 0.8; Rf (compound 5-11) = 0.5; Liquid chromatography-mass spectrometry (LCMS): 324.3 [M+H]+.

[0271] General steps for the preparation of compound 5-12:

[0272] To a solution of racemic methyl ((5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methancyclooctane[b]pyridin-5(6H)-yl-6,6-d2)carbamate (Compound 5-11, 400 mg, 1.23 mmol, 1.0 eq) in chloroform (30 ml) was added trimethylsilyl iodide (2.46 g, 12.3 mmol, 10 eq) at room temperature under nitrogen, and the mixture was heated at 65°C for 16 h. After cooling, methanol (30 ml) was added, and the resulting mixture was stirred at 65°C for 6 h. After completion of the reaction, the mixture was concentrated under vacuum. The residue was diluted with dichloromethane / methanol (10 / 1, 100 ml) and washed successively with sodium thiosulfate (10%, 5 ml), saturated sodium bicarbonate (6 ml) and brine (6 ml). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to give racemic compound 5-12 (187 mg, 35% for two steps) as a white solid.

[0273] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1; Rf (compound 5-11) = 0.6; Rf (compound 5-12) = 0.4; Liquid chromatography-mass spectrometry (LCMS): 252.1 [M+H]+.

[0274] General steps for the preparation of deuterated huperzine A WP102-5 compound:

[0275] Racemic (5R,9R,E)-5-amino-11-(ethylidene-d4)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanocyclooctane[b]pyridin-2(1H)-one-6,6-d2 (compound 5-12, 187 mg, 0.74 mmol) was separated by chiral preparative HPLC to give deuterated huperzine A WP102-5 (82.3 mg, 44%, purity 97.53%) as a white solid.

[0276] Liquid chromatography-mass spectrometry (LCMS): 252.1 [M+H]+; 1H NMR (400 MHz, CD3OD-d4) δ 7.91 (d, J = 9.4 Hz, 1H), 6.37 (d, J = 9.4 Hz, 1H), 5.44 (d, J = 5.2 Hz, 1H), 3.64 (t, J = 5.2 Hz, 1H), 2.79 (dd, J = 17.0, 5.2 Hz, 1H), 2.59 (dd, J = 17.0, 1.8 Hz, 1H).

[0277] Test Example 1 In vitro inhibition study of recombinant human acetylcholinesterase by huperzine A and deuterated huperzine A The present invention determines the inhibitory activity of huperzine A and deuterated huperzine A (WP102-3) on acetylcholinesterase (ACHE), and compares the in vitro activity test of ACHE with tacrine. The ACHE colorimetric reaction is based on the improved Ellman method, using alternative substrates thioacetylcholine (ATC) and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to quantify the amount of thiocholine produced by ACHE hydrolysis of ATC, and the absorption intensity of the generated DTNB adduct is used to measure the amount of thiocholine product produced, which is proportional to the ACHE activity.

[0278] The reagents, consumables and equipment information used in this test example are shown in Table 2.

[0279] Table 2

[0280] The samples to be tested were prepared using the concentrations listed in Table 3.

[0281] Table 3

[0282] The ACHE enzyme inhibition assay steps are as follows: 1. No Compound Control (ZPE) Wells - Add 10 μL of solvent and 20 μL of ACHE to both wells.

[0283] 2. Sample Wells - Add 10 μL of 5X solution and 20 μL of ACHE to two wells.

[0284] 3. Blank Wells - Add 10 μL of solvent and 20 μL of buffer to two wells.

[0285] 4. Incubate the plate at 25°C for 30 minutes.

[0286] 5. Add 20 μL ATC / DNTB and mix to start the reaction.

[0287] 6. Incubate the plate at 25°C for 5 minutes.

[0288] 7. Read the plate at absorbance of 405 nm.

[0289] Each well: 0.1 ng ACHE, 100 μM DTNB, 200 μM ATC.

[0290] Buffer: 0.1 M sodium phosphate, 0.05% (w / v) non-ionic detergent Brij-35, pH = 7.5.

[0291] The inhibition rate was calculated using the following formula: Inhibition rate % = (sample well signal value - ZPE well average signal value) / (blank well average signal value - ZPE well average signal value) × 100%. The results are as follows Figure 2-Figure 3 shown.

[0292] IC50 values ​​were calculated based on sigmoidal curve fitting using GraphPad Prism software, and the results are shown in Table 4.

[0293] Table 4 IC50 values ​​of compounds

[0294] The results showed that the IC50 values ​​of huperzine A and deuterated huperzine A (WP102-3) on ACHE were 13.28 nM and 14.73 nM, respectively.

[0295] Test Example 2 Study on the Chemical Stability of Huperzine A and Deuterated Huperzine A In order to compare the stability of huperzine A and deuterated huperzine A, the strong degradation stability and stability influencing factors of huperzine A (codenamed WP102) and deuterated huperzine A series compounds (WP102-1~WP102-5) were studied.

[0296] 1. Strong degradation destroys stability WP102, WP102-1, WP102-2, WP102-3, WP102-4 and WP102-5 were subjected to oxidative destruction, and the growth of impurities in each compound before and after destruction was compared. The solvent of the stock solution of each compound was 0.01 mol / L hydrochloric acid solution.

[0297] Oxidative destruction conditions: Measure 1 ml of 0.5 mg / ml stock solution of each compound, place it in a 5 ml volumetric flask, add 1 ml of 3% hydrogen peroxide, bathe at 60°C for 1 hour, and dilute to the mark with solvent.

[0298] The growth of impurities in each compound is shown in Table 5.

[0299] Table 5 Impurity growth of each compound

[0300] From the results, it can be seen that under oxidative conditions, the impurity growth of WP102 is the most obvious, the impurity growth of WP102-1 to WP102-4 is relatively slow, and WP102-3 is the most stable.

[0301] 2. Stability of influencing factors High temperature and light tests were conducted on WP102, WP102-1, WP102-2, WP102-3, WP102-4, and WP102-5, and the growth of impurities before and after the test was compared for each compound.

[0302] High temperature conditions: 60℃ for 10 days.

[0303] Lighting conditions: 5000 lux for 10 days.

[0304] The growth of impurities and changes in properties of each compound are shown in Tables 6 and 7.

[0305] Table 6 Changes in properties of each compound

[0306] Table 7 Impurity growth of each compound

[0307] The results show that under high temperature conditions, the colors of all compounds tend to turn yellow, and the impurity growth of WP102-1 is the most stable and better than WP102.

[0308] Under light conditions, the colors of all compounds tend to turn yellow, and the impurity growth of WP102-1 and WP102-3 is the most stable, and is better than WP102.

[0309] Test Example 3 Study on the Metabolic Stability of Huperzine A and Deuterated Huperzine A in Monkey Liver Microsomes The cynomolgus monkey liver microsomes used in this test example were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., batch number: LMCY-2403V077; protein content: 23.2 mg / mL; gender: male.

[0310] Preparation of NADPH solution: NADPH was prepared into a 50 mM solution using PBS buffer (0.1 M sodium phosphate, pH 7.4).

[0311] Preparation of MgCl2 solution: Prepare MgCl2 into a 200 mM solution using water.

[0312] Preparation of stock solutions: Testosterone, huperzine A, and deuterated huperzine A compounds WP102-1 to WP102-5 were prepared into 10 mM stock solutions using DMSO.

[0313] Preparation of working solution: Take the above stock solutions and add 50% acetonitrile solution to prepare a 10µM working solution.

[0314] Preparation of internal standard working solution: Tolbutamide, diclofenac, and metolazone were added with acetonitrile to prepare 100 ng / mL internal standard working solution.

[0315] Test method: 1. Add the following order: 467 µL PBS buffer (0.1 M sodium phosphate, pH 7.4), 7.5 µL MgCl2 solution (200 mM), 10.78 µL liver microsome solution (23.2 mg / mL), 5 µL working solution (10 µM) to a 96-well plate in triplicate and vortex gently.

[0316] 2. Incubate the 96-well plate in a water bath at 37°C and 75 rpm for 5 minutes.

[0317] 3. Add 10 µL of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) solution to start the reaction and vortex.

[0318] 4. Incubate the 96-well plate in a water bath at 37°C and 75 rpm for 60 minutes.

[0319] 5. At designated times (0, 5, 10, 15, 30, and 60 min), transfer 60 µL aliquots to a 96-well plate, add 3 volumes of the internal standard working solution, and vortex for 10 min.

[0320] 6. Centrifuge at 6000 g for 10 minutes at 4°C.

[0321] 7. After centrifugation, transfer 100 µL aliquots of the supernatant to a 96-well plate containing 100 µL of water per well.

[0322] 8. Sample analysis.

[0323] The metabolic half-life data are shown in Table 8.

[0324] Table 8 Metabolic half-life results of each compound

[0325] Test Example 4 Toxicity Effect Test of Deuterated Huperzine A 4.1 Toxicity effect test in rats Animal type: Male SD rats, body weight 275±15g; Injection method: intravenous injection.

[0326] Test articles: bremelanotide (codenamed WP203), huperzine A and deuterated huperzine A (WP102-3).

[0327] The mice were randomly divided into three groups, 5 in each group, and the specific dosages were as follows: Group 1: 1.75 mg / kg / d WP203; Group 2: 1.75 mg / kg / d WP203 + 0.2 mg / kg / d deuterated huperzine A (WP102-3); Group 3: 1.75 mg / kg / d WP203 + 0.2 mg / kg / d huperzine A; The mice were fasted overnight and resumed eating 4 hours after administration. The injections were continued for 4 consecutive days.

[0328] The results of animal mortality after administration are shown in Table 9.

[0329] Table 9 Animal death results

[0330] According to the experimental results in Table 9, Group 1 was injected with bremelanotide alone, and the final mortality rate was 60%. Group 3 was injected with bremelanotide and huperzine A at the same time, and the final mortality rate was 60%. Group 2 was injected with bremelanotide and deuterated huperzine A at the same time, and the final mortality rate was only 20%.

[0331] According to the above experimental results, the toxicity of bremelanotide alone was relatively high, with a mortality rate of 60% in rats. The mortality rate of rats injected with bremelanotide and huperzine A at the same time remained unchanged at 60%, indicating that huperzine A had no positive effect on reducing the toxicity of bremelanotide. However, the simultaneous injection of bremelanotide and deuterated huperzine A reduced the mortality rate of rats to 20%, indicating that deuterated huperzine A can significantly reduce the toxicity of bremelanotide.

[0332] 4.2 HT-22 cytotoxicity test The cytotoxicity of the samples was evaluated by measuring their effects on the activity of HT-22 mouse hippocampal neurons.

[0333] Preparation of test samples: Huperzine A and deuterated huperzine A (WP102-4) were prepared into a stock solution with a concentration of 100 mg / mL using DMSO; in the experiment, they were diluted with DMEM culture medium to a solution with a concentration of 200 mg / L and 1000 mg / L.

[0334] The revived HT-22 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2. When the cells grew to 80% confluence, they were digested with trypsin and inoculated in a 96-well plate, and the cells adhered for 24 h. Then different concentrations of the samples to be tested were added as the test group, and a blank control group was set up. After incubation for 24 h, the cell activity was measured (CCK-8 method), and the cell survival rate was calculated according to the following formula: Cell survival rate % = absorbance value of experimental group / absorbance value of blank control group × 100%.

[0335] If the cell viability of the test sample is less than 80% and is significantly different from that of the blank control group, it is considered to be cytotoxic.

[0336] The results are shown in Table 10.

[0337] Table 10 HT-22 cytotoxicity effect data

[0338] Note: Compared with the blank control group, ***p<0.001.

[0339] Test Example 5 Deuterated Huperzine A in Alzheimer's Disease Cell Model Test This test case explored the protective effects of bremelanotide, deuterated huperzine A, and the combination group on the Aβ cell injury model. HT-22 neuronal cells were damaged by Aβ 1-42, and then the test substances were administered to evaluate the neuroprotective effect of the samples in restoring cell activity.

[0340] 5.1 Experimental materials and reagents HT-22 mouse hippocampal neuronal cells, DMEM high glucose serum-free medium, DMEM high glucose complete medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0341] 5.2 Main Equipment Clean workbench, carbon dioxide incubator, analytical balance, pipette, multifunctional microplate reader.

[0342] 5.3 Aβ drug treatment and modeling methods Modeling agent information: Name or code: β-amyloid (1-42), Aβ 1-42; Source: Provided by Pony Biopharmaceutical Technology (Shanghai) Co., Ltd. CAS: 166090-74-0; Description of action: Aβ 1-42 is a 42-amino acid peptide that is toxic to hippocampal slices and is used in the study of Alzheimer's disease; Physical properties: powder; Storage conditions: -20℃; Preparation method: This product is unstable in solution state, it is recommended to prepare it fresh and use it immediately.

[0343] Specific experimental methods: 1. Take 1 mg of bottled amyloid-β protein, add 45 μL DMSO, mix well and transfer all to a centrifuge tube to obtain a 5 mM Aβ protein DMSO solution. Then add 950 μL DMEM high-glucose serum-free medium to the solution to obtain a 250 μM Aβ working solution.

[0344] 2. Before use, transfer 1 mL of 250 μM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin to make a 12.5 μM Aβ solution, and preheat it in a 37°C incubator.

[0345] 3. Complete medium replacement: add medium containing 12.5 μM Aβ solution to a 96-well plate and treat with drugs for 24 h. Then discard the medium (day 2) and add medium containing the test drug for 24 h. On day 3, use CCK-8 to detect cell viability.

[0346] 5.4 Sample preparation (1) Sample A is bremelanotide. 8 mg of the sample was placed in a glass reagent bottle and 1.94 mL of ddH2O was added to prepare a stock solution with a concentration of 3500 mg / L. In the experiment, the sample was diluted with DMEM high-glucose serum-free medium at a ratio of 1:200 to a maximum concentration of 17.5 mg / L, and then diluted 3 times in stages. (2) Sample B is deuterated huperzine A (WP102-3). 1.092 mg of the sample was placed in a reagent bottle and 2.65 mL of DMSO was added to prepare a stock solution with a concentration of 400 mg / L. In the experiment, it was first diluted to a maximum concentration of 2 mg / L using DMEM high-glucose serum-free medium at a ratio of 1:200, and then diluted 3 times in stages. (3) Sample C is a combination of sample A and sample B. The stock solution of sample A and sample B were mixed at a volume ratio of 1:1 to prepare a stock solution containing 1750 mg / L sample A + 200 mg / L sample B. In the experiment, DMEM high-glucose serum-free medium was used to dilute the solution at a ratio of 1:100 to the highest concentration, and then diluted 3 times in stages to ensure that the ratio of sample A:sample B in the solution was 1.75 / 0.2, and the fixed ratio remained unchanged.

[0347] 5.5 Test steps Cell viability test: The revived HT-22 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2. When the cells grew to 80% confluence, they were digested with trypsin and inoculated into 96-well plates.

[0348] The medium containing 12.5 μM Aβ was added to a 96-well plate and treated with drugs for 24 h; the medium containing the modeling agent was then discarded.

[0349] Follow these steps: ① Add sample A bremelanotide and test 44.0, 8.8, 1.76, 0.35, 0.07, and 0.014 mg / L, for a total of 6 concentrations; ② Add sample B deuterated huperzine A (WP102-3) and test 6 concentrations: 5.0, 1.0, 0.2, 0.04, 0.008, and 0.0016 mg / L; ③ Add sample C (combination group), which is prepared by bremelanotide + deuterated huperzine A, to ensure that the ratio of bremelanotide: deuterated huperzine A = 1.75:0.2 remains unchanged, wherein the concentration range of bremelanotide is 44.0 ~ 0.014 mg / L, and the concentration range of deuterated huperzine A is 5.0 ~ 0.0016 mg / L.

[0350] A blank control group and a model group were set up; the blank control group did not use the modeling agent Aβ, but added the same DMSO solvent as the highest drug dose (5.0 mg / L deuterated huperzine A) as a solvent control; the model group used the modeling agent Aβ to damage cells, and no drug treatment was given.

[0351] Determine cell activity (CCK-8 method) and calculate the relative cell activity according to the following formula: Cell survival rate % = absorbance value of experimental group / absorbance value of blank control group × 100%; Cell recovery rate % = (absorbance value of the experimental group – absorbance value of the model group) / (absorbance value of the blank control – absorbance value of the model group) × 100%.

[0352] The absorbance value of the blank well in the experimental platform (96-well plate) was subtracted from the absorbance value of each group to reduce the system error.

[0353] EC50 curve fitting was used, and the data point on the curve where the cell recovery rate reached 50% was the EC50 drug concentration.

[0354] 5.6 Statistical methods The experimental data were statistically analyzed using Graphpad Prism 10.1.2. The EC50 value was calculated by using the logistic curve in the nonlinear fitting mode “[Agonist] vs. normalized response – Variable slope” for regression analysis.

[0355] 5.7 Cell viability test results 5.7.1 Protective effect of bremelanotide on Aβ model After treatment with Aβ (12.5 μM, 24 hr), the cell survival rate in the model group was 63.92%, and that in the blank control group was 100%, indicating that Aβ caused significant cell damage.

[0356] After treatment with bremelanotide (44 mg / L), the cell survival rate was 71.43%, and the HT-22 cell damage was partially restored, with a cell recovery rate of 20.82%, as shown in Table 11 for details.

[0357] Through calculation at 6 concentrations, the EC50 value of bremelanotide in the Aβ model was 119.2 mg / L.

[0358] Table 11 Cell recovery rate of different concentrations of bremelanotide in Aβ model

[0359] Based on the cell activity data, the EC50 of the sample was calculated using GraphPad software, as follows: Figure 4 shown.

[0360] 5.7.2 Protective effect of deuterated huperzine A on Aβ model After treatment with Aβ (12.5 μM, 24 hr), the cell survival rate in the model group was 63.92%, and that in the blank control group was 100%, indicating that Aβ caused significant cell damage.

[0361] After treatment with deuterated huperzine A (5 mg / L), the cell survival rate was 87.56%, and the HT-22 cell damage was largely restored, with a cell recovery rate of 65.53%, as shown in Table 12 for details.

[0362] Through calculation at 6 concentrations, the EC50 value of deuterated huperzine A in the Aβ model was 2.49 mg / L.

[0363] Table 12 Cell recovery rate of different concentrations of deuterated huperzine A on Aβ model

[0364] Based on the cell activity data, the EC50 of the sample was calculated using GraphPad software, as follows: Figure 5 shown.

[0365] 5.7.3 Protective effect of the combination group on Aβ model After treatment with Aβ (12.5 μM, 24 hr), the cell survival rate in the model group was 63.92%, and that in the blank control group was 100%, indicating that Aβ caused significant cell damage.

[0366] After drug treatment in the combination group (44 mg / L bremelanotide + 5 mg / L deuterated huperzine A), the cell survival rate was 108.37%, and the HT-22 cell damage was completely restored. The cell recovery rate was 123.21%, as shown in Table 13 for details.

[0367] Through calculation at 6 concentrations, the EC50 of the combination group in the Aβ model was equivalent to the combined preparation containing 11.65 mg / L bremelanotide + 1.33 mg / L deuterated huperzine A.

[0368] Table 13 Cell recovery rate of different concentration combination groups on Aβ model

[0369] Based on the cell activity data, the EC50 of the sample was calculated using GraphPad software, as follows: Figure 6 As shown, the abscissa represents the concentration of bremelanotide in the combination group.

[0370] The synergistic effect between bremelanotide and deuterated huperzine A was evaluated using the combined effect evaluation formula, as follows:

[0371] In the formula, EA and EB represent the effects of sample A and sample B alone, respectively; E(A+B) represents the effect of the combined use of sample A and sample B. If the Q value is greater than 1, it means that sample A and sample B have a synergistic effect.

[0372] The cell survival rate after treatment with bremelanotide (44 mg / L) was 71.43%, the cell survival rate after treatment with deuterated huperzine A (5 mg / L) was 87.56%, and the cell survival rate after treatment with the combination group (44 mg / L bremelanotide + 5 mg / L deuterated huperzine A) was 108.37%. Substituting into the combined effect evaluation formula for calculation, Q value = 1.12> 1, indicating that the combined use of bremelanotide and deuterated huperzine A has a significant synergistic effect in improving cell survival rate.

[0373] The cell recovery rate after treatment with bremelanotide (44 mg / L) was 20.82%, the cell recovery rate after treatment with deuterated huperzine A (5 mg / L) was 65.53%, and the cell recovery rate after treatment with the combination group (44 mg / L bremelanotide + 5 mg / L deuterated huperzine A) was 123.21%. Substituting into the combined effect evaluation formula for calculation, the Q value = 1.69> 1, indicating that the combined use of bremelanotide and deuterated huperzine A has a significant synergistic effect in improving the cell recovery rate.

[0374] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the deuterated huperzine A compound is as follows: ； wherein R1-R9 are each independently H or D, and are not H at the same time.

2. The deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: At least one of R1-R6 is D.

3. The deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: At least one of R1-R3 is D, and at least one of R4-R6 is D.

4. The deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R7 is D.

5. The deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: At least one of R8-R9 is D.

6. The deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The structure of the deuterated huperzine A compound includes any one of the following: 、 、 、 、 。 7. A method for preparing the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, characterized in that: The steps include: S1 uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as the starting material to prepare compound 5; S2 reacts compound 5 with methanesulfonyl chloride to obtain compound 6; S3 reacts compound 6 with sodium acetate to obtain compound 7; S4 reacts compound 7 with compound Y to obtain compound 8; S5: reacting compound 8 with thiophenol to obtain compound 9; S6: reacting compound 9 under an inert atmosphere and alkaline conditions to obtain compound 10; S7: Compound 10 is reacted with triethylamine and diphenylphosphoryl azide to obtain compound 11; S8: Compound 11 is reacted with iodotrimethylsilane to obtain compound 12; S9: separating compound 12 by chiral preparative high performance liquid chromatography to obtain the target product; The structure of compound Y is as follows: ； Among them, R 10 -R 14 are independently H or D.

8. The preparation method according to claim 7, characterized in that: Step S1 includes the following steps: S11 uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as a starting material and reacts with compound X to obtain compound 2; S12: Compound 2 is reduced under hydrogen to obtain compound 3; S13: Compound 3 is oxidized with an oxidant to obtain compound 4; S14: Compound 4 is reacted with tetramethylguanidine to obtain compound 5; The structure of compound X is as follows: ； Among them, R 15 -R 19 are independently H or D, and are not H at the same time; Or S11′ uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as a starting material and reacts it with tetramethylguanidine and methacrolein to obtain compound 5.

9. The preparation method according to claim 8, characterized in that: Step S1 includes the following steps: S11: Compound X and potassium carbonate are added to a dimethylformamide solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate, and the mixture is stirred at 20-80° C. for 10-24 h in an inert atmosphere to obtain compound 2; S12 Pd / C and Pd(OH)2 / C were added to the ethyl acetate solution of compound 2, and the mixture was stirred at 20°C-reflux temperature for 10-24 hours under hydrogen conditions, and the mixture was filtered and concentrated to obtain compound 3; S13: Add Dess-Martin periodinane to a mixture of compound 3 and sodium bicarbonate in dichloromethane at -10-20°C, and stir at -10-20°C under nitrogen for 1-5 hours to obtain compound 4; S14: tetramethylguanidine is added to a dichloromethane solution of compound 4, and the resulting mixture is stirred at 0-40° C. for 10-24 h under an inert atmosphere to obtain compound 5; Or S11′: to a dichloromethane solution of methyl 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate, add a dichloromethane solution of tetramethylguanidine and methacrolein, and stir at 0-40° C. for 10-24 h under an inert atmosphere to obtain compound 5.

10. The preparation method according to claim 9, characterized in that: After the stirring in S11 is completed, the mixture is quenched with water, extracted with ethyl acetate, and washed and purified to obtain compound 2; After the stirring in S13 is completed, the mixture is quenched with an aqueous sodium thiosulfate solution and a saturated sodium bicarbonate solution, extracted with dichloromethane, washed, filtered, and concentrated to obtain compound 4; After the stirring in S14 is completed, the mixture is concentrated under reduced pressure, and the residue is chromatographed on a silica gel column using a mixed solvent of dichloromethane and ethyl acetate as eluent to obtain Compound 5.

11. The preparation method according to claim 7, characterized in that: S2: triethylamine and methanesulfonyl chloride are added to a dichloromethane solution of compound 5, and the resulting mixture is stirred at 0-40° C. for 2-8 hours under an inert atmosphere to obtain compound 6; S3: adding sodium acetate to the acetic acid solution of compound 6, and stirring the resulting mixture at 90-120° C. for 40-72 h under an inert atmosphere to obtain compound 7; S4: Cool the tetrahydrofuran solution of compound 7 to -78-0°C, add n-butyl lithium, stir at 0-40°C for 20-30 minutes, add a solution of compound Y in tetrahydrofuran to react, and obtain compound 8; S5. Add azobisisobutyronitrile and thiophenol to the toluene solution of compound 8, and stir at 70-110° C. for 16-36 h to obtain compound 9; S6: adding potassium hydroxide to a solution of compound 9 in dimethyl sulfoxide and tetrahydrofuran, and stirring at 60-120° C. for 36-48 h under an inert atmosphere to obtain compound 10; S7: adding triethylamine and a solution of diphenylphosphoryl azide in toluene to a toluene solution of compound 10, and stirring the resulting mixture at 60-110° C. for 2-6 h under an inert atmosphere to obtain compound 11; S8 To a solution of compound 11 in chloroform, add iodotrimethylsilane at 0-40°C, and stir at 40-75°C for 10-24 h under an inert atmosphere to obtain compound 12.

12. The preparation method according to claim 8, characterized in that: When compound X is not used in the preparation process of compound 5, R in compound Y 10 -R 14 Not H at the same time.

13. The preparation method according to claim 7, characterized in that: The compound Y includes any one of the following structures: 、 、 、 。 14. The preparation method according to claim 13, characterized in that: The preparation method of the compound E comprises the following steps: Ethyl triphenylphosphonium bromide is added to deuterated water, and then triethylamine is added, and the mixture is heated to reflux at 100-120° C. under inert gas conditions for 12-24 hours, and the solvent is removed under reduced pressure to obtain compound E; The preparation method of the compound G comprises the following steps: stirring a mixture of 1-bromoethane-1,1,2,2,2-d5 and triphenylphosphine bromide in toluene at 100-120° C. for 12-24 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain the compound G; The preparation method of the compound F comprises the following steps: stirring a mixture of 1-bromoethane-2,2,2-d3 and triphenylphosphine bromide in toluene at 100-120° C. for 12-24 hours, cooling the reaction mixture to room temperature to obtain a white solid precipitate, filtering and collecting the solid, and drying to obtain compound F.

15. The preparation method according to claim 8, characterized in that: The compound X includes any one of the following structures: 、 、 。 16. The preparation method according to claim 15, characterized in that: The preparation method of compound A comprises the following steps: (1) adding potassium carbonate and deuterated iodomethane CD3I to a mixture of diethyl malonate and DMF for reaction, pouring the obtained reactant into water, and post-treating to obtain compound A-2; (2) adding n-butyl lithium to a mixture of compound A-2 and anhydrous tetrahydrofuran, stirring under inert gas conditions, adding ((chloromethoxy)methyl)benzene, quenching the reaction with water, and post-treating to obtain compound A-4; (3) adding potassium hydroxide solution to the ethanol solution of compound A-4, removing ethanol from the obtained reactant under reduced pressure, adding water to dilute, adjusting the pH to 1-4, and post-treating to obtain compound A-5; (4) Compound A-5 is added to xylene and stirred. The obtained reactant is concentrated and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound A-6; (5) Under inert gas conditions, a tetrahydrofuran solution of borane is added to a tetrahydrofuran solution of compound A-6 for reaction. After the reaction is completed, the reaction is quenched with methanol and post-treated to obtain compound A-7; (6) adding iodine to a dichloromethane solution containing triphenylphosphine and imidazole, stirring, adding compound A-7 to react, quenching the reaction with an aqueous sodium sulfite solution after the reaction is completed, and post-treating to obtain compound A; The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

17. The preparation method according to claim 16, characterized in that: The preparation method of compound A comprises the following steps: (1) adding potassium carbonate and deuterated iodomethane CD3I to a mixture of diethyl malonate and DMF for reaction at 30-80°C for 48-72h, pouring the obtained reactant into water, and post-treating to obtain compound A-2; (2) Add n-butyl lithium to a mixture of compound A-2 in anhydrous tetrahydrofuran at -78-0°C, stir under inert gas for 1-3 hours, then add ((chloromethoxy)methyl)benzene at -78-0°C, stir at -78°C to room temperature for 2 hours, quench the reaction with water, and post-treat to obtain compound A-4; (3) Add potassium hydroxide solution to the ethanol solution of compound A-4, stir at 60-100°C for 1-4h, remove ethanol from the obtained reactant under reduced pressure, dilute with water, wash with dichloromethane, adjust the pH to 1-4, and post-treat to obtain compound A-5; (4) Compound A-5 was added to xylene and stirred at 120-150°C for 12-24h. The obtained reactant was concentrated and purified by column chromatography using dichloromethane / methanol = 1 / 99 to obtain compound A-6; (5) Under inert gas conditions, add a tetrahydrofuran solution of borane to a tetrahydrofuran solution of compound A-6 at -5-0°C, react at 50-80°C for 12-24h, cool to 0°C, quench the reaction with methanol, and post-treat to obtain compound A-7; (6) Add iodine to a dichloromethane solution containing triphenylphosphine and imidazole, stir at room temperature for 1-3 h, add compound A-7 and react for 1-4 h. After the reaction is completed, quench the reaction with 10% sodium sulfite aqueous solution, and post-treat to obtain compound A.

18. The preparation method according to claim 15, characterized in that: The preparation method of the compound B comprises the following steps: 1) Add n-butyl lithium to a mixture of diethyl 2-methylmalonate in anhydrous tetrahydrofuran, stir under inert gas conditions, add ((chloromethoxy)methyl)benzene, quench the reaction with water, and post-treat to obtain compound B-2; 2) Add potassium hydroxide solution to the ethanol solution of compound B-2, stir under inert gas conditions, remove ethanol from the obtained reactant under reduced pressure, dilute with water, adjust the pH to 2-6, and post-treat to obtain compound B-3; 3) Under inert gas conditions, compound B-3 is added to xylene and stirred, the obtained reactant is diluted with water, and post-treated to obtain compound B-4; 4) Add concentrated sulfuric acid to the methanol solution of compound B-4, stir under inert gas conditions, quench the reaction with saturated sodium bicarbonate, and post-treat to obtain compound B-5; 5) Add lithium aluminum deuteride to a tetrahydrofuran solution of compound B-5, stir under inert gas conditions, quench the reaction with sodium sulfate decahydrate, and post-treat to obtain compound B-6; 6) adding imidazole and iodine to a dichloromethane solution containing triphenylphosphine, stirring under inert gas conditions, adding compound B-6 to react, quenching the reaction with sodium thiosulfate after the reaction is completed, extracting with dichloromethane, and drying to obtain compound B; The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

19. The preparation method according to claim 18, characterized in that: The preparation method of the compound B comprises the following steps: 1) Add n-butyl lithium to a mixture of diethyl 2-methylmalonate in anhydrous tetrahydrofuran at -78-0°C, stir for 1-3 hours under inert gas conditions, add ((chloromethoxy)methyl)benzene, stir at room temperature for 3 hours, quench the reaction with water, and post-treat to obtain compound B-2; 2) Add potassium hydroxide solution to the ethanol solution of compound B-2 under inert gas conditions at -5-5°C, stir at 70-100°C for 1-3h, remove ethanol from the obtained reactant under reduced pressure, dilute with water, adjust the pH to 2-6, and post-treat to obtain compound B-3; 3) Under inert gas conditions, compound B-3 is added to xylene, stirred at 145-155°C for 12-18h, the obtained reactant is diluted with water, and post-treated to obtain compound B-4; 4) Add concentrated sulfuric acid to the methanol solution of compound B-4, stir at 60-90°C for 12-18h under inert gas conditions, quench the reaction with saturated sodium bicarbonate, and post-treat to obtain compound B-5; 5) Add lithium aluminum deuteride to a tetrahydrofuran solution of compound B-5 at -5-5°C, stir at -5-5°C for 2-4h under inert gas conditions, quench the reaction with sodium sulfate decahydrate, and post-treat to obtain compound B-6; 6) Add imidazole and iodine to a dichloromethane solution containing triphenylphosphine at -5-5°C, stir at room temperature for 1-2 hours under inert gas conditions, add compound B-6 and continue stirring for 1-3 hours. After the reaction is completed, quench the reaction with sodium thiosulfate, extract with dichloromethane, and dry to obtain compound B.

20. The preparation method according to claim 17, characterized in that: The preparation method of the compound C comprises the following steps: (i) adding concentrated sulfuric acid to a methanol solution of compound A-6, stirring under inert gas conditions, concentrating the obtained reactant, diluting it with water, and post-treating it to obtain compound C-1; (ii) adding lithium aluminum deuteride to a tetrahydrofuran solution of compound C-1, stirring under inert gas conditions, quenching the reaction with sodium sulfate decahydrate, and post-treating to obtain compound C-2; (iii) adding imidazole and iodine to a dichloromethane solution containing triphenylphosphine, stirring under inert gas conditions, adding compound C-2 for reaction, quenching the reaction with sodium thiosulfate after the reaction is completed, extracting with dichloromethane, and drying to obtain compound C.

21. The preparation method according to claim 20, characterized in that: The preparation method of the compound C comprises the following steps: (i) under inert gas conditions, concentrated sulfuric acid is added to a methanol solution of compound A-6, and the mixture is stirred at 80-100° C. for 1-3 h. The obtained reactant is concentrated and then diluted with water, and post-treated to obtain compound C-1; (ii) adding lithium aluminum deuteride to a tetrahydrofuran solution of compound C-1 at 0°C, stirring for 2-4 hours under inert gas conditions, quenching the reaction with sodium sulfate decahydrate, and post-treating to obtain compound C-2; (iii) Add imidazole and iodine to a dichloromethane solution containing triphenylphosphine at 0°C, stir at room temperature for 1-3 hours under inert gas conditions, add compound C-2 and continue stirring for 1-3 hours. After the reaction is completed, quench the reaction with sodium thiosulfate, extract with dichloromethane, and dry to obtain compound C.

22. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a cholinesterase inhibitor.

23. The use according to claim 22, characterized in that The cholinesterase inhibitors include acetylcholinesterase inhibitors.

24. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a drug for treating Alzheimer's disease.

25. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a medicament for use in combination with bremelanotide.

26. A pharmaceutical composition, characterized in that The invention comprises the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 and bremelanotide.

27. The pharmaceutical composition according to claim 26, characterized in that The mass ratio of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof to bremelanotide is in the range of 5:30-5:

60.

28. The pharmaceutical composition according to claim 27, characterized in that The mass ratio of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof to bremelanotide is 5:43.

75.

29. Use of the pharmaceutical composition according to any one of claims 26 to 28 in the preparation of a drug for treating Alzheimer's disease.

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