A deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof

Abstract

The present invention relates to the field of pharmaceutical technology, and particularly relates to a deuterated huperzine A compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. The structure of the deuterated huperzine A compound is as follows, wherein R1-R9 are each independently H or D, and are not simultaneously H. The present invention has the advantages of high chemical stability, long metabolic half-life, low toxicity, obvious synergistic effect with bremelanotide in the treatment of Alzheimer's disease, and can reduce the toxicity of bremelanotide. #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 N 2 Huperzine A is mainly used as a cholinesterase inhibitor for the treatment of benign memory impairment and various types of dementia, memory cognitive function and emotional behavior 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:

[0008] (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.

[0009] (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.

[0010] (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.

[0011] 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.

[0012] 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

[0013] 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.

[0014] The present invention is achieved through the following technical solutions:

[0015] The first aspect of 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 ):

[0016] ;

[0017] Among them, R 1 -R 9 are each independently H or D, and are not H at the same time.

[0018] As an embodiment of the present invention, R 1 -R 6 At least one of them is D.

[0019] As a preferred embodiment of the present invention, R 1 -R 3 At least one of them is D, and R 4 -R 6 At least one of them is D.

[0020] As an embodiment of the present invention, R 7 is D.

[0021] As an embodiment of the present invention, R 8 -R 9 At least one of them is D.

[0022] As an embodiment of the present invention, the structure of the deuterated huperzine A compound includes any one of the following:

[0023] , , , , .

[0024] 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:

[0025] S1 uses 2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylic acid methyl ester as the starting material to prepare compound 5;

[0026] S2 reacts compound 5 with methanesulfonyl chloride to obtain compound 6;

[0027] S3 reacts compound 6 with sodium acetate to obtain compound 7;

[0028] S4 reacts compound 7 with compound Y to obtain compound 8;

[0029] S5: reacting compound 8 with thiophenol to obtain compound 9;

[0030] S6: reacting compound 9 under an inert atmosphere and alkaline conditions to obtain compound 10;

[0031] S7: Compound 10 is reacted with triethylamine and diphenylphosphoryl azide to obtain compound 11;

[0032] S8: Compound 11 is reacted with iodotrimethylsilane to obtain compound 12;

[0033] S9 separating compound 12 by chiral preparative high performance liquid chromatography to obtain the target product;

[0034] The structure of compound Y is as follows:

[0035] ;

[0036] Among them, R 10 -R 14 are independently H or D.

[0037] As an implementation mode of the present invention, step S1 includes the following steps:

[0038] 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;

[0039] S12: reducing compound 2 under hydrogen gas to obtain compound 3;

[0040] S13: Compound 3 is oxidized with an oxidant to obtain compound 4;

[0041] S14: Compound 4 is reacted with tetramethylguanidine to obtain compound 5;

[0042] The structure of compound X is as follows:

[0043]

[0044] Among them, R 15 -R 19 are each independently H or D, and are not H at the same time.

[0045] As a preferred embodiment of the present invention, step S1 includes the following steps:

[0046] 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;

[0047] S12 Add Pd / C and Pd(OH) to the ethyl acetate solution of compound 2 2 / C, stirred at 20°C-reflux temperature under hydrogen conditions for 10-24 hours, the mixture was filtered and concentrated to obtain compound 3;

[0048] 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;

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 and eluted with a mixed solvent of dichloromethane and ethyl acetate to obtain Compound 5.

[0053] As an implementation mode of the present invention, step S1 includes the following steps:

[0054] 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.

[0055] As a preferred embodiment of the present invention, step S1 includes the following steps:

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] As an embodiment of the present invention, the compound Y includes any one of the following structures:

[0066] , , , .

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] As an embodiment of the present invention, the compound X includes any one of the following structures:

[0073] , , .

[0074] As a preferred embodiment of the present invention, the preparation method of compound A comprises the following steps:

[0075] (1) Add potassium carbonate and deuterated iodomethane (CD) to a mixture of diethyl malonate (A-1) and DMF. 3 I reaction, the obtained reactant is poured into water, and post-treated to obtain compound A-2;

[0076] (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;

[0077] (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;

[0078] (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;

[0079] (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;

[0080] (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;

[0081] The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

[0082] As a more preferred embodiment of the present invention, the preparation method of compound A comprises the following steps:

[0083] (1) Add potassium carbonate and deuterated iodomethane (CD) to a mixture of diethyl malonate (A-1) and DMF. 3 I reaction, 30-80°C for 48-72h, the obtained reactant is poured into water, and post-treated to obtain compound A-2;

[0084] (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;

[0085] (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;

[0086] (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;

[0087] (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;

[0088] (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.

[0089] Further preferably, the preparation method of compound A comprises the following steps:

[0090] (1) Add potassium carbonate and deuterated iodomethane (CD) to a mixture of diethyl malonate (A-1) and DMF. 3I reaction, 50 ° C for 72h, the obtained reactant was poured into water, and post-treated to obtain compound A-2;

[0091] (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;

[0092] (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;

[0093] (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;

[0094] (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;

[0095] (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.

[0096] As a preferred embodiment of the present invention, the preparation method of compound B comprises the following steps:

[0097] 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;

[0098] 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;

[0099] 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;

[0100] 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;

[0101] 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;

[0102] 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;

[0103] The work-up includes extraction with ethyl acetate and the resulting organic phase is washed with brine and dried.

[0104] As a more preferred embodiment of the present invention, the preparation method of compound B comprises the following steps:

[0105] 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;

[0106] 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;

[0107] 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;

[0108] 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;

[0109] 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;

[0110] 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.

[0111] As a further preferred embodiment of the present invention, the preparation method of compound B comprises the following steps:

[0112] 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;

[0113] 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.

[0114] 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;

[0115] 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;

[0116] 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;

[0117] 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.

[0118] As a preferred embodiment of the present invention, the preparation method of compound C comprises the following steps:

[0119] (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;

[0120] (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;

[0121] (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.

[0122] As a more preferred embodiment of the present invention, the preparation method of compound C comprises the following steps:

[0123] (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;

[0124] (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;

[0125] (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.

[0126] As a further preferred embodiment of the present invention, the preparation method of compound C comprises the following steps:

[0127] (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;

[0128] (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;

[0129] (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.

[0130] 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.

[0131] As an embodiment of the present invention, the cholinesterase inhibitor includes an acetylcholinesterase inhibitor.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] The beneficial effects of the present invention are:

[0139] 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%.

[0140] 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.

[0141] 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

[0142] Figure 1 is the general structural formula of the deuterated huperzine A compound of the present invention.

[0143] Figure 2 This is the dose-response curve of tacrine on acetylcholinesterase (ACHE).

[0144] Figure 3 This is the dose-response curve of huperzine A and deuterated huperzine A on acetylcholinesterase (ACHE).

[0145] Figure 4 This is the EC50 curve of bremelanotide in the Aβ model.

[0146] Figure 5 This is the EC50 curve of deuterated huperzine A on the Aβ model.

[0147] Figure 6 This is the EC50 curve of bremelanotide and deuterated huperzine A combined in the Aβ model. DETAILED DESCRIPTION

[0148] 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.

[0149] Example 1 Synthesis of Compound (WP102-3) - Deuterated Huperzine A

[0150] General steps for the preparation of compound E:

[0151]

[0152] To deuterated water D containing ethyltriphenylphosphonium bromide (Compound E-1, 6.0 g, 16.2 mmol, 1.0 eq.) 2 Triethylamine TEA (0.5 ml) was added to the 2-nitrogen (16 ml) solution. The reaction mixture was heated under reflux at 110°C for 16 h under nitrogen. The solvent was removed under reduced pressure to give compound E (5.8 g, 96%) as a white solid.

[0153] Liquid chromatography-mass spectrometry (LCMS): 293 [M-79]+;

[0154] 1H NMR (400 MHz, CDCl 3 ) 7.95 - 7.65 (m, 15H), 1.39 (d, J = 20.0 Hz, 3H).

[0155] General steps for the preparation of compound 2:

[0156]

[0157] 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.

[0158] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0159] Rf (compound 1) = 0.6;

[0160] Rf (compound 2) = 0.2;

[0161] Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0162] General steps for the preparation of compound 3:

[0163]

[0164] 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.

[0165] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2;

[0166] Rf value of compound 2 = 0.2;

[0167] Rf value of compound 3 = 0.6;

[0168] Liquid chromatography-mass spectrometry (LCMS): 384.2 [M+H]+.

[0169] General steps for the preparation of compound 4:

[0170]

[0171] To a solution of racemic (5S,9R)-2-methoxy-7-methyl-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methancyclooctane[b]pyridine-5(6H)-carboxylic acid methyl ester (compound (+) 3, 9.8 g, 25.5 mmol, 1.0 eq) in HOAc (300 ml) was added sodium acetate (NaOAc) (4.2 g, 51.0 mmol, 2.0 eq). The resulting mixture was stirred at N 2 The mixture was stirred at reflux temperature for 48 h under a 4% atmosphere. The mixture was concentrated to remove the HOAc solvent, diluted with ethyl acetate (200 ml), and washed with saturated NaHCO 3 The mixture was washed with water (50 ml), brine (50 ml), and anhydrous Na 2 SO 4 Dry, filter and concentrate to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate EA / petroleum ether PE = 2 / 3) to obtain racemic compound (+) 4 (2.7 g, 37%) as a white solid.

[0172] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0173] Rf value of compound 3 = 0.3;

[0174] Rf value of compound 4 = 0.5;

[0175] Liquid chromatography-mass spectrometry (LCMS): 288.1 [M+H]+.

[0176] General steps for the preparation of compound 5:

[0177]

[0178] 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 x 3), the organic layers were combined and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate to obtain a crude product. The residue was purified by silica gel column chromatography (ethyl acetate EA: petroleum ether PE = 1:4) to obtain racemic compound (+) 5 (Z / E = 4 / 1, 376 mg, 72%) as a colorless oil.

[0179] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0180] Rf value of compound 4 = 0.3;

[0181] Rf value of compound 5 = 0.6;

[0182] Liquid chromatography-mass spectrometry (LCMS): 301.1 [M+H]+.

[0183] General steps for the preparation of compound 6:

[0184]

[0185] 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.

[0186] TLC: ethyl acetate / petroleum ether = 1 / 10;

[0187] Rf (compound 5) = 0.5;

[0188] Rf (compound 6) = 0.55;

[0189] LCMS: 301.1 [M+H]+.

[0190] General steps for the preparation of compound 7:

[0191]

[0192] 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.

[0193] TLC: methanol / dichloromethane = 1 / 10;

[0194] Rf (compound 6) = 0.8;

[0195] Rf (compound 7) = 0.4;

[0196] LCMS: 287.3 [M+H]+.

[0197] General steps for the preparation of compound 8:

[0198]

[0199] 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.

[0200] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1;

[0201] Rf (compound 7) = 0.8;

[0202] Rf (compound 8) = 0.5;

[0203] Liquid chromatography-mass spectrometry (LCMS): 316.3 [M+H]+.

[0204] General steps for the preparation of compound 9:

[0205]

[0206] Under nitrogen protection, the racemic compound (compound (+) 8, 800 mg, 2.53 mmol) was added to chloroform (CHCl) at room temperature. 3To the (50 ml) solution was added iodotrimethylsilane TMSI (3.4 ml, 25.32 mmol, 10 eq.), 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 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 (+) 9 (350 mg, two-step yield 55%) as a white solid.

[0207] Thin layer chromatography (TLC): dichloromethane / methanol = 9 / 1;

[0208] Rf (compound 8) = 0.6;

[0209] Rf (compound 9) = 0.4;

[0210] Liquid chromatography-mass spectrometry (LCMS): 244.3 [M+H]+.

[0211] General steps for the preparation of deuterated huperzine A (WP102-3):

[0212]

[0213] 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.

[0214] Chiral preparative HPLC conditions (same conditions as in Examples 1 to 5):

[0215] Instrument: Supercritical fluid chromatograph SFC002;

[0216] Chromatographic column: CHIRALPAK AD-H 10 mm × 250 mm × 5 μm;

[0217] Detection wavelength: 220nm;

[0218] Column oven temperature: 40°C;

[0219] Injection volume: 1000 μl (20 mg);

[0220] Flow rate: 10.0ml / min;

[0221] Diluent: Isopropyl alcohol IPA;

[0222] Mobile phase: Phase A: CO 2 ; Phase B contains 0.1% diethylamine in isopropanol.

[0223] The elution conditions are shown in Table 1.

[0224] Table 1

[0225]

[0226] Thin layer chromatography (TLC): dichloromethane / methanol = 9 / 1;

[0227] Rf (compound 9) = 0.6;

[0228] Rf(WP102-3)=0.4;

[0229] Liquid chromatography-mass spectrometry (LCMS): 244.3 [M+H]+.

[0230] 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).

[0231] Example 2: Synthesis of Compound (WP102-1) - Deuterated Huperzine A

[0232] General preparation steps of compound A-2:

[0233]

[0234] To a mixture of diethyl malonate (Compound A-1, 160 g, 1.0 mol, 1.0 eq.) in dimethylformamide (DMF) (1.6 L) was added potassium carbonate (K 2 CO 3 , 207 g, 1.5 mol, 1.5 eq.) and deuterated iodomethane CD 3I (145 g, 1.0 mol, 1.0 eq). 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 L) and extracted with ethyl acetate (EtOAc, 1.0 L × 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 give compound A-2 (110 g, 62%) as a yellow oil.

[0235] TLC: petroleum ether / ethyl acetate = 20 / 1

[0236] Rf (Compound A-1) = 0.4

[0237] Rf (Compound A-2) = 0.5

[0238] 1H NMR (400 MHz, deuterated chloroform CDCl 3 ): δ4.09 (m, 4H), 3.30 (s, 1H), 1.17 (dd, J=7.1, 1.6Hz, 6H).

[0239] General preparation steps of compound A-4:

[0240]

[0241] 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) was added n-butyl lithium (nBuLi, 2.5 M, 248 mL, 0.62 mol, 1.0 eq.) at -78 °C and stirred at N 2 Stir for 1 h under 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. 2 The mixture was quenched with 1% 4% dHO (1 L) and extracted with ethyl acetate (EtOAc, 500 mL × 3). The combined organic phase was washed with brine, 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 give compound A-4 (134 g, 72%) as a yellow oil.

[0242] TLC: petroleum ether / ethyl acetate = 20 / 1;

[0243] Rf (Compound A-2) = 0.5;

[0244] Rf (Compound A-4) = 0.45;

[0245] 1H NMR (400 MHz, CDCl 3 ): 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).

[0246] General preparation steps of compound A-5:

[0247]

[0248] 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 washed with H 2 The solution was diluted with 4% paraformaldehyde (4% paraformaldehyde) (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 phases were 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.

[0249] Thin layer chromatography: petroleum ether / ethyl acetate = 20 / 1;

[0250] Rf (Compound A-4) = 0.45;

[0251] Rf (Compound A-5) = 0.01;

[0252] 1H NMR (400 MHz, deuterated dimethyl sulfoxide (DMSO-d6)): δ 7.28 (m, 5H), 4.46 (s, 2H), 3.67 (s, 2H).

[0253] General preparation steps of compound A-6:

[0254]

[0255] 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.

[0256] Thin-layer chromatography: dichloromethane / methanol = 20 / 1;

[0257] Rf (Compound A-5) = 0.2;

[0258] Rf (Compound A-6) = 0.5;

[0259] 1H Nuclear Magnetic Resonance (400 MHz, chloroform-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).

[0260] General procedure for the preparation of Compound A-7:

[0261]

[0262] Under nitrogen protection, at 0 °C, borane-tetrahydrofuran (BH 3 -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). The mixture was stirred at 70 °C for 16 h. 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.

[0263] Thin-layer chromatography: dichloromethane / methanol = 20 / 1;

[0264] Rf (Compound A-6) = 0.5;

[0265] Rf (Compound A-7) = 0.8;

[0266] 1H Nuclear Magnetic Resonance (400 MHz, chloroform-d): δ 7.33 (m, 5H), 4.52 (m, 2H), 3.59 (m, 3H), 3.42 (m, 1H), 2.06 (m, 1H).

[0267] General procedure for the preparation of Compound A:

[0268]

[0269] At room temperature, triphenylphosphine (PPh 3, 85.9 g, 0.328 mol, 1.5 eq) and imidazole (22.3 g, 0.328 mmol, 1.5 eq) were added all at once to a mixture of iodine (I 2 , 83.2 g, 0.328 mol, 1.5 eq) in dichloromethane (DCM, 1 L). The mixture was stirred at room temperature (rt) for 1 h. 2-((Benzyloxy)methyl)propane-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 resulting 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.

[0270] Thin-layer chromatography (TLC): Petroleum ether (PE) / ethyl acetate (EtOAc) = 20 / 1;

[0271] Rf (Compound A-7) = 0.3;

[0272] Rf (Compound A) = 0.9;

[0273] 1H nuclear magnetic resonance (400 MHz, chloroform-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).

[0274] General procedure for the preparation of Compound 2-2:

[0275]

[0276] 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) was added ((2-(iodomethyl)propoxy-3,3,3-d3)methyl)benzene (Compound A, 18.6 g, 63.8 mmol, 1.5 eq) and potassium carbonate (K 2 CO 3 )(7.4 g, 106.3 mmol, 3.0 eq). The resulting mixture was stirred at 50 °C under a nitrogen atmosphere for 16 h. With H 2A quenching mixture of O (100 mL) was extracted with ethyl acetate (EtOAc) (100 mL × 3), washed with brine (50 mL × 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.

[0277] Thin-layer chromatography (TLC): ethyl acetate (EtOAc) / petroleum ether (PE) = 1 / 5;

[0278] Rf (compound 1) = 0.2;

[0279] Rf (compound 2-2) = 0.6;

[0280] Liquid chromatography-mass spectrometry (LCMS): 401.1 [M+H]+.

[0281] General procedure for the preparation of compound 2-3:

[0282]

[0283] To a solution of methyl 5-(2-((benzyloxy)methyl)propyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (compound 2-2, 7.5 g, 18.8 mmol, 1.0 equiv) in ethyl acetate (EtOAc) (150 mL) was added Pd / C (1.7 g) and Pd(OH) 2 / C (1.7 g), and the mixture was stirred at 60 °C under hydrogen (H 2 ) for 16 h. The mixture was filtered and concentrated to give compound 2-3 (4.8 g crude) as a colorless oil.

[0284] Thin-layer chromatography (TLC): ethyl acetate (EtOAc) / petroleum ether (PE) = 1 / 2;

[0285] Rf (compound 2-2) = 0.6;

[0286] Rf (compound 2-3) = 0.2;

[0287] Liquid chromatography-mass spectrometry (LCMS): 311.1 [M+H]+.

[0288] General procedure for the preparation of compound 2-4:

[0289]

[0290] To a mixture of methyl 5-(2-(hydroxymethyl)propyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 2-3, 5.0 g, 16.1 mmol, 1.0 eq) and sodium bicarbonate (NaHCO 3 )(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% aqueous sodium thiosulfate (Na 2 S 2 O 3 ) (30 mL) and saturated aqueous sodium bicarbonate (NaHCO 3 ) (30 mL), and extracted with dichloromethane (DCM) (100 mL × 3). The combined organic layers were washed with saturated aqueous sodium bicarbonate (NaHCO 3 )(50 mL × 2), dried over anhydrous sodium sulfate, filtered and concentrated to give crude Compound 2-4 (4.3 g crude), as a yellow oil.

[0291] LCMS: 309.1 [M+H]+.

[0292] General procedure for the preparation of Compound 2-5:

[0293]

[0294] To a stirred solution of methyl 5-(2-formylpropyl-3,3,3-d3)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 2-4, 15.0 g, 48.7 mmol, 1.0 eq) in dichloromethane (150 mL) was added tetramethylguanidine (TMG, 2.87 g, 24.3 mmol, 0.5 eq). The resulting mixture was stirred at 30 °C under nitrogen for 16 h. 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 purified by silica gel column chromatography, eluting with dichloromethane / ethyl acetate = 7 / 3, to give racemic Compound 2-5 (8.0 g, 53% overall yield over three steps), as a yellow solid.

[0295] TLC: ethyl acetate / petroleum ether = 1 / 5;

[0296] Rf (Compound 2-4) = 0.6;

[0297] Rf (Compound 2-5) = 0.2;

[0298] Liquid chromatography - mass spectrometry (LCMS): 309.1 [M+H]+.

[0299] General procedure for the preparation of compounds 2 - 6:

[0300]

[0301] To a solution of racemic (5S,9R)-8-hydroxy-2-methoxy-7-(methyl-d3)-11-oxo-7,8,9,10-tetrahydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 2 - 5, 8.0 g, 25.9 mmol, 1.0 equiv) in dichloromethane (150 mL) was added dropwise triethylamine (29 mL, 207.8 mmol, 8.0 equiv) and methanesulfonyl chloride (MsCl, 11.8 g, 103.9 mmol, 4.0 equiv). The resulting mixture was stirred at room temperature (rt) for 4 h under a nitrogen atmosphere. The mixture was washed with saturated aqueous ammonium chloride solution (50 mL) and brine (50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2 / 3) to afford racemic Compound 2 - 6 (7.2 g, 70%) as a yellow solid.

[0302] Thin-layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2;

[0303] Rf (Compound 2 - 5) = 0.2;

[0304] Rf (Compound 2 - 6) = 0.6;

[0305] Liquid chromatography - mass spectrometry (LCMS): 387.2 [M+H]+.

[0306] General procedure for the preparation of compounds 2 - 7:

[0307]

[0308] To a solution of racemic (5S,9R)-methyl 2-methoxy-7-(methyl-d3)-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate (Compound 2-6, 10 g, 25.8 mmol, 1.0 equiv) in acetic acid (300 mL) was added sodium acetate (4.23 g, 51.6 mmol, 2.0 equiv). 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 aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the 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.

[0309] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0310] Rf (Compound 2-6) = 0.3;

[0311] Rf (Compound 2-7) = 0.5;

[0312] Liquid chromatography-mass spectrometry (LCMS): 291.1 [M+H]+.

[0313] General procedure for the preparation of Compound 2-8:

[0314]

[0315] The preparation method of Compound F comprises the following steps: A mixture of 1-bromoethane-2,2,2-d3 and triphenylphosphonium bromide in toluene was stirred at 110 °C for 20 h. The reaction mixture was cooled to room temperature to obtain a white solid precipitate. The solid was collected by filtration, dried to obtain Compound F.

[0316] A solution of (ethyl-2,2,2-d3) triphenylphosphonium bromide (Compound F, 2.57 g, 6.89 mmol, 2.5 eq) in tetrahydrofuran (24 mL) was cooled to -78 °C, and n-butyllithium (2.31 mL, 2.5 M in hexane, 5.79 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-methanocyclooct[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 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 (50 mL × 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the 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.

[0317] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0318] Rf (Compound 2-7) = 0.3;

[0319] Rf (Compound 2-8) = 0.6;

[0320] Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0321] General procedure for the preparation of Compound 2-9:

[0322]

[0323] To a solution of racemic methyl (5R,9R)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate (Compound 2-8, 1.5 g, 4.90 mmol, 1.0 equiv) in toluene (PhMe, 60 mL) was added azobisisobutyronitrile (AIBN, 884 mg, 5.39 mmol, 1.1 equiv) and benzenethiol (PhSH, 1.89 g, 17.15 mmol, 3.5 equiv). The resulting mixture was stirred at 105 °C for 24 h, then azobisisobutyronitrile (884 mg, 5.39 mmol, 1.1 equiv) and benzenethiol (1.89 g, 17.15 mmol, 3.5 equiv) were added, and the mixture was 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) and washed with sodium hydroxide (1 M, 30 mL × 2) and brine (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the 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.

[0324] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0325] Rf (Compound 2-8) = 0.5;

[0326] Rf (Compound 2-9) = 0.55;

[0327] Liquid chromatography-mass spectrometry (LCMS): 306.1 [M+H]+.

[0328] General procedure for the preparation of Compound 2-10:

[0329]

[0330] Racemic (5R,9R,E)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 2-9, 1.3 g, 4.26 mmol, 1.0 equiv) was added to a mixed solution of dimethyl sulfoxide (DMSO, 12 mL) and tetrahydrofuran (THF, 12 mL), followed by the addition of potassium hydroxide solution (12%, 48 mL). The resulting mixture was stirred at 100 °C for 40 h under a nitrogen atmosphere. LC-MS indicated the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0 °C, and the pH was adjusted to 4-5 with 4 M aqueous hydrochloric acid. The mixture was extracted with methanol / dichloromethane = 1 / 9 (200 mL × 5), and the combined organic layers were 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.

[0331] Thin-layer chromatography (TLC): methanol / dichloromethane = 1 / 10;

[0332] Rf (Compound 2-9) = 0.9;

[0333] Rf (Compound 2-10) = 0.4;

[0334] LC-MS: 292.3 [M+H]+.

[0335] General procedure for the preparation of Compound 2-11:

[0336]

[0337] A solution of triethylamine (TEA, 782 mg, 7.84 mmol, 3.0 equiv) and diphenylphosphoryl azide (DPPA) (1.4 g, 5.22 mmol, 2.0 equiv) in toluene (3 mL) was added dropwise to a solution of racemic (5R,9R,E)-11-(ethylidene-2,2,2-d3)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5-carboxylic acid (Compound 2-10, 320 mg, mmol, 1.0 equiv) in toluene (20 mL). The resulting mixture was stirred at 85 °C for 3 h under a 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) as a colorless oil.

[0338] Thin-layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1;

[0339] Rf (Compound 2-10) = 0.8;

[0340] Rf (Compound 2-11) = 0.5;

[0341] Liquid chromatography-mass spectrometry (LCMS): 316.3 [M+H]+.

[0342] General procedure for the preparation of Compound 2-12:

[0343]

[0344] 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-methanocyclooct[b]pyridin-5(6H)-yl)carbamate (Compound 2-11, 150 mg, 0.46 mmol, 1.0 eq) in chloroform (CHCl 3 , 10 mL) at room temperature was added trimethylsilyl iodide (TMSI, 0.66 mL, 4.68 mmol, 10 eq), and the mixture was stirred at 65 °C under nitrogen for 16 h. 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 in vacuo. The residue was diluted with dichloromethane / methanol (10 / 1, 100 mL) and washed successively with sodium thiosulfate (10%, 3 mL), saturated sodium bicarbonate (3 mL), and brine (3 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 15 / 85) to give racemic Compound 2-12 (70 mg, 25% over two steps) as a white solid.

[0345] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1;

[0346] Rf (Compound 2-11) = 0.6;

[0347] Rf (Compound 2-12) = 0.4;

[0348] Liquid chromatography-mass spectrometry (LCMS): 249.3 [M+H]+.

[0349] General procedure for the preparation of deuterated huperzine A (WP102-1):

[0350]

[0351] The racemic (5R,9R,E)-5-amino-11-(ethylidene-2,2,2-d3)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanoazocino[b]pyridin-2(1H)-one (Compound 2-12, 70 mg, 0.28 mmol) was separated by chiral preparative high performance liquid chromatography to give huperzine A WP102-1 (29.5 mg, 42%, purity 98.03%) as a white solid.

[0352] Liquid chromatography-mass spectrometry (LCMS): 249.0 [M+H]+;

[0353] 1H nuclear magnetic resonance (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).

[0354] Example 3 Compound (WP102-2) - Synthesis of Huperzine A

[0355] General procedure for the preparation of Compound B-2:

[0356]

[0357] Under nitrogen protection, a solution of diethyl 2-methylmalonate (Compound B-1, 80 g, 0.46 mmol, 1.0 equiv) in tetrahydrofuran (THF, 800 mL) was added dropwise with n-butyllithium (nBuLi, 202 mL, 0.506 mol, 1.1 equiv, 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 equiv) was added to the solution, and the mixture was stirred at room temperature for 3 h under nitrogen protection. The reaction was quenched with H 2 2O (100 mL), extracted with ethyl acetate (EtOAc, 200 mL × 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the 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.

[0358] TLC: Petroleum ether / ethyl acetate (PE / EtOAc) = 20 / 1;

[0359] Rf (Compound B-1) = 0.4;

[0360] Rf (Compound B-2) = 0.5;

[0361] LCMS: 295.1 [M+H]+.

[0362] General preparation procedure for Compound B-3:

[0363]

[0364] Under nitrogen protection, a solution of diethyl 2-((benzyloxy)methyl)-2-methylmalonate (Compound B-2, 150 g, 0.508 mol, 1.0 eq) in ethanol (EtOH, 1.5 L) was added to a solution of potassium hydroxide (KOH, 142.6 g, 2.54 mol, 5.0 eq) 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 H 2 O (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.

[0365] TLC: PE / EtOAc = 20 / 1;

[0366] Rf (Compound B-2) = 0.45;

[0367] Rf (Compound B-3) = 0.01;

[0368] LCMS: 239.1 [M+H]+.

[0369] General preparation procedure for Compound B-4:

[0370]

[0371] 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 H 2 O (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.

[0372] Thin Layer Chromatography (TLC): Dichloromethane (DCM) / Methanol (MeOH) = 20 / 1;

[0373] Rf (Compound B-3) = 0.01;

[0374] Rf (Compound B-4) = 0.5;

[0375] Liquid Chromatography-Mass Spectrometry (LCMS): 195.1 [M+H]+.

[0376] General Procedure for the Preparation of Compound B-5:

[0377]

[0378] At room temperature, concentrated sulfuric acid (10 mL) was added to a solution of 3-(benzyloxy)-2-methylpropanoic acid (Compound B-4, 80 g, 0.41 mol, 1.0 equiv) in methanol (1.5 L). 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 obtain the 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.

[0379] Thin Layer Chromatography (TLC): Dichloromethane (DCM) / Methanol (MeOH) = 20 / 1;

[0380] Rf (Compound B-4) = 0.5;

[0381] Rf (Compound B-5) = 0.8;

[0382] Liquid Chromatography-Mass Spectrometry (LCMS): 209.1 [M+H]+.

[0383] General Procedure for the Preparation of Compound B-6:

[0384]

[0385] At 0 °C, lithium aluminum deuteride (LiAlD 4, 21 g, 0.5 mol, 2.0 eq). 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 obtain the 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.

[0386] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 3;

[0387] Rf (compound B-5) = 0.5;

[0388] Rf (compound B-6) = 0.3;

[0389] Liquid chromatography-mass spectrometry (LCMS): 183.1 [M+H]+.

[0390] General procedure for the preparation of compound B:

[0391]

[0392] At 0 °C, imidazole (21.8 g, 0.32 mmol, 1.5 eq) and iodine (I 3 , 81.3 g, 0.32 mmol, 1.5 eq) were added to a solution of triphenylphosphine (PPh 2 , 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. Then compound B-6 (38.9 g, 213.6 mmol, 1.0 eq) was added to the above solution and stirred at room temperature for 2 h. 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 the crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 99) to give compound B (40 g, 64%) as a colorless oil.

[0393] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 20;

[0394] Rf value of compound B-6 = 0.01;

[0395] Rf value of compound B = 0.8;

[0396] Liquid chromatography-mass spectrometry (LCMS): 293.1 [M+H]+.

[0397] General procedure for the preparation of compound 3-2:

[0398]

[0399] 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), add ((2-(iodomethyl)propoxy-3,3,3-d3)methyl)benzene (Compound B, 18.6 g, 63.8 mmol, 1.5 eq) and potassium carbonate (K 2 CO 3 , 14.7 g, 106.3 mmol, 3.0 eq). The resulting mixture was stirred at 50 °C for 16 h under a nitrogen atmosphere. Quench the mixture with H 2 O (100 mL), and extract with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (50 mL × 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.

[0400] Thin-layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0401] Rf value of Compound 1 = 0.2;

[0402] Rf value of Compound 3-2 = 0.6;

[0403] Liquid chromatography-mass spectrometry (LCMS): 400.1 [M+H]+.

[0404] General procedure for the preparation of Compound 3-3:

[0405]

[0406] To a stirred solution of methyl 5-(3-(benzyloxy)-2-methylpropyl-1,1-d2)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 3-2, 12.5 g, 31.2 mmol, 1.0 eq) in ethyl acetate (300 mL), add Pd / C (2.5 g) and Pd(OH) 2 / C (2.5 g). The resulting mixture was stirred at 60 °C for 16 h under a hydrogen atmosphere. LCMS showed a new peak and the starting material disappeared. Filter the mixture and concentrate to give Compound 3-3 (10.1 g crude) as a colorless oil.

[0407] Thin-layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 2;

[0408] Rf value of Compound 3-2 = 0.6;

[0409] The Rf value of Compound 3-3 = 0.2;

[0410] Liquid chromatography-mass spectrometry (LCMS): 310.2 [M+H]+.

[0411] General procedure for the preparation of Compound 3-4:

[0412]

[0413] 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 (NaHCO 3 , 8.43 g, 100.3 mmol, 5 eq) and Dess-Martin periodinane (DMP, 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. LCMS showed the appearance of a new peak and the disappearance of the starting material. The mixture was quenched with sodium thiosulfate (Na 2 S 2 O 3 , 50 mL) and extracted with dichloromethane (50 mL × 3). The extract was washed with sodium bicarbonate (100 mL × 2) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to give racemic Compound 3-4 (9.0 g crude) as a yellow oil.

[0414] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 3;

[0415] Rf (Compound 3-3) = 0.4;

[0416] Rf (Compound 3-4) = 0.5;

[0417] LCMS: 308.1 [M+H]+.

[0418] General procedure for the preparation of Compound 3-5:

[0419]

[0420] To a solution of methyl 2-methoxy-5-(2-methyl-3-oxopropyl-1,1-d2)-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 3-4, 14.4 g, 46.8 mmol, 1.0 eq) in dichloromethane (700 mL) under stirring was added tetramethylguanidine (TMG, 2.7 g, 23.4 mmol, 0.5 eq). The resulting mixture was stirred at 30 °C for 16 h under a nitrogen atmosphere. LC-MS indicated 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.

[0421] TLC: ethyl acetate / petroleum ether = 1 / 5;

[0422] Rf (Compound 3-4) = 0.6;

[0423] Rf (Compound 3-5) = 0.2;

[0424] LC-MS: 308.1 [M+H]+.

[0425] General procedure for the preparation of Compound 3-6:

[0426]

[0427] To a solution of racemic methyl (5S,9R)-8-hydroxy-2-methoxy-7-methyl-11-oxo-7,8,9,10-tetrahydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate-6,6-d2 (Compound 3-5, 8.2 g, 26.7 mmol, 1.0 eq) in dichloromethane (240 mL) was added dropwise triethylamine (21.6 g, 213.6 mmol, 8.0 eq) and methanesulfonyl chloride (22.0 g, 106.8 mmol, 4.0 eq). 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 combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the 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.

[0428] TLC: ethyl acetate / petroleum ether = 1 / 2;

[0429] Rf (Compound 3-5) = 0.2;

[0430] Rf (Compound 3-6) = 0.6;

[0431] LC-MS: 386.2 [M+H]+.

[0432] General procedure for the preparation of Compounds 3-7:

[0433]

[0434] To a solution of racemic methyl (5S,9R)-2-methoxy-7-methyl-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanocycloocta[b]pyridine-5(6H)-carboxylate-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 aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to afford racemic Compound 3-7 (2.0 g, 34%) as a white solid.

[0435] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 5;

[0436] Rf (Compound 3-6) = 0.3;

[0437] Rf (Compound 3-7) = 0.5;

[0438] Liquid chromatography-mass spectrometry: 290.1 [M+H]+.

[0439] General procedure for the preparation of Compound 3-8:

[0440]

[0441] A solution of ethyltriphenylphosphonium bromide (Compound H, 3.5 g, 9.5 mmol, 2.5 eq) in tetrahydrofuran (24 mL) was cooled to -78 °C, and n-butyllithium (3.2 mL, 2.5 M in hexane, 7.99 mmol, 2.1 eq) was added dropwise over 10 minutes. 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)-methyl 2-methoxy-7-methyl-11-oxo-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate-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 × 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the 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.

[0442] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 10;

[0443] Rf (Compound 3-7) = 0.3;

[0444] Rf (Compound 3-8) = 0.6;

[0445] LC-MS: 302.1 [M+H]+.

[0446] General procedure for the preparation of Compound 3-9:

[0447]

[0448] To a solution of racemic (5R,6S,9R)-11-ethylidene-2-methoxy-6,7-dimethyl-9,10-dihydro-5,9-methanocycloocta[b]pyridine-5(6H)-carboxylic acid methyl ester-6-d (Compound 3-8, 950 mg, 3.15 mmol, 1.0 equiv) in toluene (50 mL) was added azobisisobutyronitrile (568 mg, 3.46 mmol, 1.1 equiv) and benzenethiol (PhSH, 1.22 g, 11.05 mmol, 3.5 equiv). The resulting mixture was stirred at 105 °C for 24 h, then azobisisobutyronitrile (568 mg, 3.46 mmol, 1.1 equiv) and benzenethiol (1.22 g, 11.05 mmol, 3.5 equiv) were added, and the mixture was 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) and washed with sodium hydroxide (1 M, 30 mL × 2) and brine (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the 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) as a yellow oil.

[0449] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 10;

[0450] Rf (Compound 3-8) = 0.5;

[0451] Rf (Compound 3-9) = 0.55;

[0452] Liquid chromatography-mass spectrometry: 302.1 [M+H]+.

[0453] General procedure for the preparation of Compound 3-10:

[0454]

[0455] To a solution of racemic (5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methanocycloocta[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (Compound 3-9, 1.0 g, 3.3 mmol, 1.0 equiv) in a mixture of 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. Liquid chromatography-mass spectrometry indicated the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0 °C, and the pH was adjusted to 4-5 with 4 M aqueous hydrochloric acid. It was extracted with methanol / dichloromethane = 1 / 9 (200 mL × 5). The combined organic layers were 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.

[0456] Thin layer chromatography: methanol / dichloromethane = 1 / 10;

[0457] Rf (Compound 3-9) = 0.9;

[0458] Rf (Compound 3-10) = 0.4;

[0459] Liquid chromatography-mass spectrometry: 288.3 [M+H]+.

[0460] General procedure for the preparation of Compound 3-11:

[0461]

[0462] To a solution of racemic (5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylic acid-6,6-d2 acid (Compound 3-10, 750 mg, 2.6 mmol, 1.0 equiv) in toluene (20 mL) was added dropwise a solution of triethylamine (789 mg, 7.8 mmol, 3.0 equiv) and diphenylphosphoryl azide (DPPA, 1.4 g, 5.22 mmol, 2.0 equiv) in toluene (3 mL). The resulting mixture was stirred at 85 °C for 3 h under a nitrogen atmosphere. Liquid chromatography-mass spectrometry 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) as a colorless oil.

[0463] Thin layer chromatography: petroleum ether / ethyl acetate = 3 / 1;

[0464] Rf (Compound 3-10) = 0.8;

[0465] Rf (Compound 3-11) = 0.5;

[0466] Liquid chromatography-mass spectrometry: 317.3 [M+H]+.

[0467] General procedure for the preparation of Compound 3-12:

[0468]

[0469] To a solution of racemic methyl ((5R,9R,E)-11-ethylidene-2-methoxy-7-methyl-9,10-dihydro-5,9-methanocyclooct[b]pyridin-5(6H)-yl-6,6-d2)carbamate (Compound 3-11, 450 mg, 1.42 mmol, 1.0 equiv) in chloroform (30 mL) was added trimethylsilyl iodide (2.84 g, 14.2 mmol, 10 equiv) under a nitrogen atmosphere at room temperature. 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 in vacuo. 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 combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to afford racemic Compound 3-12 (190 mg, 54% over two steps) as a white solid.

[0470] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1;

[0471] Rf (Compound 3-11) = 0.6;

[0472] Rf (Compound 3-12) = 0.4;

[0473] Liquid chromatography-mass spectrometry (LCMS): 245.3 [M+H]+.

[0474] General procedure for the preparation of deuterated huperzine A (WP102-2):

[0475]

[0476] Racemic (5R,9R,E)-5-amino-11-ethylidene-7-methyl-5,6,9,10-tetrahydro-5,9-methanocyclooct[b]pyridin-2(1H)-one-6,6-d2 (Compound 3-12, 190 mg, 0.77 mmol) was separated by chiral preparative high performance liquid chromatography SFC to give deuterated huperzine A WP102-2 (64.4 mg, 33%, purity 99.61%) as a white solid.

[0477] Liquid chromatography-mass spectrometry (LCMS): 245.3 [M+H]+;

[0478] 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).

[0479] Example 4 Compound (WP102-4) - Synthesis of Deuterated Huperzine A

[0480] General procedure for the preparation of Compound 4-2:

[0481]

[0482] The preparation method of Compound G comprises the following steps: A mixture of 1-bromoethane-1,1,2,2,2-d5 and triphenylphosphonium bromide in toluene was stirred at 110 °C for 20 hours. The reaction mixture was cooled to room temperature to obtain a white solid precipitate. The solid was collected by filtration, dried, to obtain Compound G.

[0483] A solution of (ethyl-d5)triphenylphosphonium bromide (Compound G, 3.87 g, 10.34 mmol, 2.5 eq) in tetrahydrofuran (THF, 36 mL) was cooled to -78 °C. A solution of n-butyllithium (3.47 mL, 8.69 mmol, 2.1 eq) in hexane (2.5 M) was added dropwise thereto 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-methanocyclooct[b]pyridine-5(6H)-carboxylate (Compound 2-7, 1.2 g, 4.13 mmol, 1.0 eq) in tetrahydrofuran (15 mL) was added dropwise thereto over 10 minutes. The resulting solution was stirred at room temperature for 1.5 h. LCMS showed the completion of the reaction. The reaction was quenched with brine (40 mL), extracted with ethyl acetate (100 mL × 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to obtain racemic Compound 4-2 (Z / E = 4 / 1, 1.03 g, 81%) as a white solid.

[0484] TLC: ethyl acetate / petroleum ether = 1 / 10;

[0485] Rf (Compound 2-7) = 0.3;

[0486] Rf (Compound 4-2) = 0.6;

[0487] LCMS: 307.1 [M+H]+.

[0488] General procedure for the preparation of Compound 4-3:

[0489]

[0490] To a solution of racemic methyl (5R,9R)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate (Compound 4-2, 1.5 g, 4.90 mmol, 1.0 equiv) in toluene (60 mL) was added azobisisobutyronitrile (AIBN, 884 mg, 5.39 mmol, 1.1 equiv) and benzenethiol (PhSH, 1.89 g, 17.15 mmol, 3.5 equiv). The resulting mixture was stirred at 105 °C for 24 h, then azobisisobutyronitrile (884 mg, 5.39 mmol, 1.1 equiv) and benzenethiol (1.89 g, 17.15 mmol, 3.5 equiv) were added, and the mixture was stirred at 105 °C for 24 h. The reaction solution was detected by 1H NMR, and the ratio of Z configuration / E configuration reached 1 / 9. The solution was diluted with ethyl acetate (150 mL) and washed with NaOH (1 M, 30 mL × 2) and brine (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the 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.

[0491] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0492] Rf (Compound 4-2) = 0.5;

[0493] Rf (Compound 4-3) = 0.55;

[0494] Liquid chromatography-mass spectrometry (LCMS): 307.1 [M+H]+.

[0495] General procedure for the preparation of Compound 4-4:

[0496]

[0497] Racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylic acid methyl ester (Compound 4-3, 1.3 g, 4.26 mmol, 1.0 eq) was added to a mixed solution of dimethyl sulfoxide (12 mL) and tetrahydrofuran (12 mL), and potassium hydroxide solution (12%, 48 mL) was added. 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, and the pH was adjusted to 4-5 with 4 M aqueous hydrochloric acid. The mixture was extracted with a methanol / dichloromethane = 1 / 9 solution (200 mL × 5), and the combined organic layers were 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.

[0498] Thin layer chromatography (TLC): methanol / dichloromethane = 1 / 10;

[0499] Rf (Compound 4-3) = 0.9;

[0500] Rf (Compound 4-4) = 0.4;

[0501] Liquid chromatography-mass spectrometry (LCMS): 293.3 [M+H]+.

[0502] General procedure for the preparation of Compound 4-5:

[0503]

[0504] To a solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5-carboxylic acid (Compound 4-4, 500 mg, 1.71 mmol, 1.0 eq) in toluene (30 mL) was added dropwise a solution of 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 a nitrogen atmosphere. LC-MS showed that Compound 4 had been consumed completely. 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) as a colorless oil.

[0505] Thin layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1;

[0506] Rf (Compound 4-4) = 0.8;

[0507] Rf (Compound 4-5) = 0.5;

[0508] Liquid chromatography-mass spectrometry (LCMS): 322.3 [M+H]+.

[0509] General procedure for the preparation of Compound 4-6:

[0510]

[0511] To a solution of racemic methyl ((5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridin-5(6H)-yl)carbamate (Compound 4-5, 345 mg, 1.07 mmol, 1.0 equiv) in chloroform (8 mL) was added trimethylsilyl iodide (1.5 mL, 10.7 mmol, 10 equiv) at room temperature under a nitrogen atmosphere. 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 in vacuo. The residue was diluted with dichloromethane / methanol (10 / 1, 100 mL) and washed successively with 10% sodium thiosulfate (3 mL), saturated sodium bicarbonate (3 mL), and brine (3 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the 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% over two steps) as a white solid.

[0512] Thin-layer chromatography (TLC): dichloromethane / methanol = 9 / 1;

[0513] Rf (Compound 4-5) = 0.6;

[0514] Rf (Compound 4-6) = 0.4;

[0515] Liquid chromatography-mass spectrometry (LCMS): 250.3 [M+H]+.

[0516] General procedure for the preparation of deuterated huperzine A WP102-4 compound:

[0517]

[0518] The racemic (5R,9R,E)-5-amino-11-(ethylidene-d4)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanoazocino[b]pyridin-2(1H)-one (Compound 4-6, 50 mg, 0.28 mmol) was separated by chiral preparative high performance liquid chromatography SFC to obtain deuterated huperzine A WP102-4 (6.5 mg, 13%, purity 98.95%), which was a white solid.

[0519] Liquid chromatography-mass spectrometry (LCMS): 250.3 [M+H]+;

[0520] 1H nuclear magnetic resonance (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).

[0521] Example 5 Compound (WP102-5) - Synthesis of deuterated huperzine A

[0522] General procedure for the preparation of Compound C-1:

[0523]

[0524] Under nitrogen protection, concentrated H 2 SO 4 (9.1 g, 51.3 mmol, 0.1 equivalent) was added to a solution of 2-((benzyloxy)methyl)propanoic acid-3,3,3-d3 (Compound A-6, 101 g, 0.513 mol, 1.0 equivalent) in methanol (1 L) with stirring, and the mixture was 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 layers were washed with brine, dried over anhydrous Na 2 SO 4 , 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%), which was a colorless oil.

[0525] TLC: petroleum ether / ethyl acetate = 20 / 1;

[0526] Rf (Compound A-6) = 0.9;

[0527] Rf (Compound C-1) = 0.8;

[0528] 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).

[0529] General procedure for the preparation of compound C-2:

[0530]

[0531] At 0 °C, to a solution of methyl 2-((benzyloxy)methyl)propionate-3,3,3-d3 (compound C-1, 45 g, 213.2 mmol, 1.0 equiv) in tetrahydrofuran (900 mL) was added lithium aluminum deuteride LiAlD 4 (17.9 g, 0.426 mol, 2.0 equiv). The reaction mixture was stirred at 0 °C under nitrogen for 3 h. LCMS showed the reaction was complete. The reaction was concentrated and quenched with sodium sulfate decahydrate (50 g), and extracted with ethyl acetate (200 mL × 3). The combined organic layers were 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 = 7 / 3) to give compound C-2 (30 g, 75%) as a colorless oil.

[0532] TLC: ethyl acetate / petroleum ether = 1 / 3;

[0533] Rf (compound C-1) = 0.5;

[0534] Rf (compound C-2) = 0.3;

[0535] LCMS: 186.2 [M+H]+.

[0536] General procedure for the preparation of compound C:

[0537]

[0538] At 0 °C, to a solution of triphenylphosphine (PPh 3 , 63.6 g, 243.0 mmol, 1.5 equiv) in dichloromethane (500 mL) was added imidazole (16.5 g, 243.0 mmol, 1.5 equiv) and iodine (I 2 , 61.6 g, 243.0 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature (rt) under nitrogen for 1.5 h. Then at room temperature, compound C-2 (30 g, 162.0 mmol, 1.0 equiv) was added to the above solution and stirred for 2 h. The reaction was quenched with 10% Na 2 S 2 O 3Quench with (100 mL), extract with dichloromethane (100 mL × 3), combine the organic phases, dry over anhydrous Na 2 SO 4 , filter and concentrate to obtain the 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.

[0539] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 20;

[0540] Rf (compound C-2) = 0.01;

[0541] Rf (compound C) = 0.8;

[0542] Liquid chromatography - mass spectrometry (LCMS): 296.1 [M+H]+.

[0543] General procedure for the preparation of compound 5-2:

[0544]

[0545] 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 equiv) in dimethylformamide (DMF) (300 mL) was added ((3-iodo-2-(methyl-d3)propoxy-3,3-d2)methyl)benzene (compound C, 37.6 g, 127.5 mmol, 1.5 equiv) and potassium carbonate (K 2 CO 3 )(35.3 g, 255 mmol, 3.0 equiv). The resulting mixture was stirred at 50 °C for 16 h under a nitrogen atmosphere. Quench the mixture with H 2 O (100 mL), extract with ethyl acetate (300 mL × 3). Wash with brine (50 mL × 5), dry 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.

[0546] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0547] Rf (compound 1) = 0.2;

[0548] Rf (compound 5-2) = 0.6;

[0549] Liquid chromatography - mass spectrometry (LCMS): 403.1 [M+H]+.

[0550] General procedure for the preparation of Compound 5-3:

[0551]

[0552] To a stirred solution of methyl 5-(2-((benzyloxy)methyl)propyl-1,1,3,3,3-d5)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 5-2, 20 g, 49.6 mmol, 1.0 eq) in ethyl acetate (300 mL) was added Pd / C (4 g) and Pd(OH) 2 / C (4 g). The resulting mixture was stirred at 60 °C for 16 h under a hydrogen atmosphere. 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) as a colorless oil.

[0553] TLC: ethyl acetate / petroleum ether = 1 / 2;

[0554] Rf (Compound 5-2) = 0.6;

[0555] Rf (Compound 5-3) = 0.2;

[0556] LCMS: 313.1 [M+H]+.

[0557] General procedure for the preparation of Compound 5-4:

[0558]

[0559] 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) at 0 °C was added sodium bicarbonate (NaHCO 3 )(20.4 g, 242.7 mmol, 5.0 eq) and Dess-Martin periodinane (DMP) (24.6 g, 58.2 mmol, 1.2 eq). The reaction was stirred at 0 °C under nitrogen for 1.5 h. LCMS showed a new peak and the disappearance of the starting material. The mixture was quenched with sodium thiosulfate (Na 2 S 2 O 3 )(100 mL), and extracted with dichloromethane (100 mL × 3). Washed with sodium bicarbonate (100 mL × 2) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to give racemic Compound 5-4 (17 g crude) as a yellow oil.

[0560] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 3;

[0561] Rf (Compound 5-3) = 0.3;

[0562] Rf (Compound 5-4) = 0.6;

[0563] Liquid chromatography - mass spectrometry: 311.1 [M+H]+.

[0564] General procedure for the preparation of Compound 5-5:

[0565]

[0566] To a stirred solution of methyl 5-(2-formylpropyl-1,1,3,3,3-d5)-2-methoxy-6-oxo-5,6,7,8-tetrahydroquinoline-5-carboxylate (Compound 5-4, 17 g, 54.6 mmol, 1.0 equiv) in dichloromethane (1 L) was added tetramethylguanidine TMG (3.1 g, 27.3 mmol, 0.5 equiv). The resulting mixture was stirred at 30 °C for 16 h under a nitrogen atmosphere. Liquid chromatography - mass spectrometry indicated 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.

[0567] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 5;

[0568] Rf (Compound 5-4) = 0.4;

[0569] Rf (Compound 5-5) = 0.2;

[0570] Liquid chromatography - mass spectrometry: 311.2 [M+H]+.

[0571] General procedure for the preparation of Compound 5-6:

[0572]

[0573] To a solution of racemic (5S,9R)-8-hydroxy-2-methoxy-7-(methyl-d3)-11-oxo-7,8,9,10-tetrahydro-5,9-methanocycloocta[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) was added dropwise triethylamine (26.0 g, 257.1 mmol, 8.0 eq) and methanesulfonyl chloride (25 g, 128.4 mmol, 4.0 eq). The resulting mixture was stirred at room temperature for 4 h under a nitrogen atmosphere. The mixture was quenched with saturated aqueous ammonium chloride solution (50 mL) and washed with brine (50 mL). The combined organic layers were 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.

[0574] Thin layer chromatography: ethyl acetate / petroleum ether = 1 / 2;

[0575] Rf (Compound 5-5) = 0.2;

[0576] Rf (Compound 5-6) = 0.6;

[0577] Liquid chromatography-mass spectrometry: 389.2 [M+H]+.

[0578] General procedure for the preparation of Compound 5-7:

[0579]

[0580] To a solution of racemic (5S,9R)-2-methoxy-7-(methyl-d3)-8-((methylsulfonyl)oxy)-11-oxo-7,8,9,10-tetrahydro-5,9-methanocycloocta[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 a nitrogen atmosphere. The mixture was concentrated to remove the solvent acetic acid, diluted with ethyl acetate (200 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), brine (50 mL), 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 = 4 / 1) to give racemic Compound 5-7 (2.5 g, 30%) as a white solid.

[0581] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 5;

[0582] Rf value of Compound 5-6 = 0.3;

[0583] The Rf value of Compound 5-7 = 0.5;

[0584] Liquid chromatography-mass spectrometry (LCMS): 293.1 [M+H]+.

[0585] General procedure for the preparation of Compound 5-8:

[0586]

[0587] A solution of (ethyl-d5)triphenylphosphonium bromide (Compound G, 3.85 g, 10 mmol, 2.5 eq) in tetrahydrofuran (24 mL) was cooled to -78 °C, and n-butyllithium (3.4 mL, dissolved in hexane at a concentration of 2.5 M, 8.6 mmol, 2.1 eq) was added dropwise over 10 minutes. 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-methanocyclooct[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. LCMS showed that the reaction was complete. The reaction was quenched with brine (20 mL), extracted with ethyl acetate (100 mL × 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9) to obtain racemic Compound 5-8 (Z / E = 4 / 1, 780 mg, 63%), as a white solid.

[0588] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0589] The Rf value of Compound 5-7 = 0.3;

[0590] The Rf value of Compound 5-8 = 0.6;

[0591] Liquid chromatography-mass spectrometry (LCMS): 309.1 [M+H]+.

[0592] General procedure for the preparation of Compound 5-9:

[0593]

[0594] A solution of racemic methyl (5R,9R)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylate-6,6-d2 (Compound 5-8, 780 mg, 2.52 mmol, 1.0 equiv) in toluene (40 mL) was added azobisisobutyronitrile (AIBN, 454 mg, 2.77 mmol, 1.1 equiv) and benzenethiol (PhSH, 974 mg, 8.82 mmol, 3.5 equiv). The resulting mixture was stirred at 105 °C for 24 h, then azobisisobutyronitrile (454 mg, 2.77 mmol, 1.1 equiv) and benzenethiol (974 mg, 8.82 mmol, 3.5 equiv) were added, and then the mixture was 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) and washed with sodium hydroxide (1 M, 30 mL × 2) and brine (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the 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.

[0595] Thin layer chromatography (TLC): ethyl acetate / petroleum ether = 1 / 10;

[0596] Rf (Compound 5-8) = 0.5;

[0597] Rf (Compound 5-9) = 0.55;

[0598] Liquid chromatography-mass spectrometry (LCMS): 302.1 [M+H]+.

[0599] General procedure for the preparation of Compound 5-10:

[0600]

[0601] Racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5(6H)-carboxylic acid methyl ester-6,6-d2 (Compound 5-9, 700 mg, 2.3 mmol, 1.0 eq) was added to a mixed solution of dimethyl sulfoxide (24 mL) and tetrahydrofuran (6 mL), followed by the addition of potassium hydroxide solution (12%, 24 mL). The resulting mixture was stirred at 100 °C for 40 h under a nitrogen atmosphere. LC-MS analysis showed the appearance of a new peak and the disappearance of the starting material. The mixture was cooled to 0 °C, and the pH was adjusted to 4-5 with 4 M aqueous hydrochloric acid. The mixture was extracted with a methanol / dichloromethane = 1 / 9 solution (200 mL × 5), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give racemic Compound 5-10 (630 mg, 92%) as a white solid. The product was used directly in the next step without further purification.

[0602] Thin-layer chromatography (TLC): methanol / dichloromethane = 1 / 10;

[0603] Rf (Compound 5-9) = 0.9;

[0604] Rf (Compound 5-10) = 0.4;

[0605] LC-MS: 295.3 [M+H]+.

[0606] General procedure for the preparation of Compound 5-11:

[0607]

[0608] To a solution of racemic (5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[b]pyridine-5-carboxylic acid-6,6-d2 (Compound 5-10, 630 mg, 2.1 mmol, 1.0 eq) in toluene (20 mL) was added dropwise a solution of 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 a nitrogen atmosphere. LC-MS analysis showed the complete consumption of Compound 10. 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.

[0609] Thin-layer chromatography (TLC): petroleum ether / ethyl acetate = 3 / 1;

[0610] Rf (Compound 5-10) = 0.8;

[0611] Rf (Compound 5-11) = 0.5;

[0612] Liquid chromatography-mass spectrometry (LCMS): 324.3 [M+H]+.

[0613] General procedure for the preparation of Compound 5-12:

[0614]

[0615] To a solution of racemic methyl ((5R,9R,E)-11-(ethylidene-d4)-2-methoxy-7-(methyl-d3)-9,10-dihydro-5,9-methanocyclooct[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. 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 in vacuo. 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 combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to give racemic Compound 5-12 (187 mg, 35% over two steps) as a white solid.

[0616] Thin layer chromatography (TLC): dichloromethane (DCM) / methanol (MeOH) = 9 / 1;

[0617] Rf (Compound 5-11) = 0.6;

[0618] Rf (Compound 5-12) = 0.4;

[0619] Liquid chromatography-mass spectrometry (LCMS): 252.1 [M+H]+.

[0620] General procedure for the preparation of deuterated huperzine A WP102-5 compound:

[0621]

[0622] The racemic (5R,9R,E)-5-amino-11-(ethylidene-d4)-7-(methyl-d3)-5,6,9,10-tetrahydro-5,9-methanoazocino[b]pyridin-2(1H)-one-6,6-d2 (Compound 5-12, 187 mg, 0.74 mmol) was separated by chiral preparative high performance liquid chromatography to obtain deuterated huperzine A WP102-5 (82.3 mg, 44%, purity 97.53%) as a white solid.

[0623] Liquid chromatography-mass spectrometry (LCMS): 252.1 [M+H]+;

[0624] 1H nuclear magnetic resonance (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).

[0625] Test Example 1 In vitro inhibition study of huperzine A and deuterated huperzine A on recombinant human acetylcholinesterase

[0626] In the present invention, the inhibitory activities of huperzine A and deuterated huperzine A (WP102-3) on acetylcholinesterase (ACHE) were determined, and an in vitro activity test comparison with tacrine for ACHE was carried out. The ACHE colorimetric reaction was based on the improved Ellman method, using the alternative substrate acetylthiocholine (ATC) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to quantify the amount of thiocholine produced by ACHE hydrolysis of ATC. The absorption intensity of the generated DTNB adduct was used to measure the amount of thiocholine product produced, and this amount was proportional to the ACHE activity.

[0627] The information on the reagents, consumables and equipment used in this test example is shown in Table 2.

[0628] Table 2

[0629]

[0630] The samples to be tested were prepared at the concentrations recorded in Table 3.

[0631] Table 3

[0632]

[0633] The steps of the ACHE enzyme inhibition experiment are as follows:

[0634] 1. No-compound control (ZPE) wells - Add 10 μL of solvent and 20 μL of AChE to two wells.

[0635] 2. Sample wells - Add 10 μL of 5X solution and 20 μL of AChE to two wells.

[0636] 3. Blank wells - Add 10 μL of solvent and 20 μL of buffer to two wells.

[0637] 4. Incubate the plate at 25 °C for 30 minutes.

[0638] 5. Add 20 μL of ATC / DNTB mixture to start the reaction.

[0639] 6. Incubate the culture plate at 25 °C for 5 minutes.

[0640] 7. Read the plate at an absorbance of 405 nm.

[0641] Per well: 0.1 ng AChE, 100 μM DTNB, 200 μM ATC.

[0642] Buffer: 0.1 M sodium phosphate, 0.05% (w / v) nonionic detergent Brij-35, pH = 7.5.

[0643] Calculate the inhibition rate using the following formula:

[0644] Inhibition rate % = (Sample well signal value - Average ZPE well signal value) / (Average blank well signal value - Average ZPE well signal value) × 100%. The results are as Figure 2-Figure 3 shown.

[0645] Calculate the IC50 value using GraphPad Prism software based on sigmoidal curve fitting. The results are shown in Table 4.

[0646] Table 4 IC50 values of the compounds

[0647]

[0648] The results show that the IC50 values of huperzine A and deuterated huperzine A (WP102-3) on AChE are 13.28 nM and 14.73 nM, respectively.

[0649] Test Example 2 Study on the chemical stability of huperzine A and deuterated huperzine A

[0650] To compare the compound stabilities of huperzine A and deuterated huperzine A, studies on the strong degradation stability and stress stability of huperzine A (code name WP102) and a series of deuterated huperzine A compounds (WP102-1 to WP102-5) were conducted.

[0651] 1. Forced degradation to evaluate stability

[0652] Oxidative degradation was performed on WP102, WP102-1, WP102-2, WP102-3, WP102-4, and WP102-5. The increase in impurities before and after degradation of each compound was compared. The solvent for the stock solution of each compound was 0.01 mol / L hydrochloric acid solution.

[0653] Oxidative degradation conditions: Measure 1 ml of the stock solution of each compound at 0.5 mg / ml, place it in a 5-ml volumetric flask, add 1 ml of 3% hydrogen peroxide, heat in a water bath at 60 °C for 1 hour, and dilute to the mark with the solvent.

[0654] The increase in impurities of each compound is shown in Table 5.

[0655] Table 5 Increase in impurities of each compound

[0656]

[0657] It can be seen from the results that under oxidative conditions, the increase in impurities of WP102 is the most obvious, the increase in impurities of WP102-1 to WP102-4 is relatively slow, and WP102-3 is the most stable.

[0658] 2. Stress stability

[0659] High temperature and light tests were performed on WP102, WP102-1, WP102-2, WP102-3, WP102-4, and WP102-5. The increase in impurities before and after the test of each compound was compared.

[0660] High temperature conditions: Place at 60 °C for 10 days.

[0661] Light conditions: Place at an illuminance of 5000 lux for 10 days.

[0662] The increase in impurities and changes in properties of each compound are shown in Tables 6 - 7.

[0663] Table 6 Changes in properties of each compound

[0664]

[0665] Table 7 Increase in impurities of each compound

[0666]

[0667] The results show that under high temperature conditions, the color of each compound has a tendency to turn yellow, and the increase in impurities of WP102-1 is the most stable, and is better than that of WP102.

[0668] Under light conditions, the colors of all compounds tend to turn yellow, and the growth of impurities is most stable in WP102-1 and WP102-3, and is better than that of WP102.

[0669] Test Example 3 Study on the Metabolic Stability of Huperzine A and Deuterated Huperzine A in Monkey Liver Microsomes

[0670] 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.

[0671] Preparation of NADPH solution: NADPH was prepared into a 50 mM solution using PBS buffer (0.1 M sodium phosphate, pH 7.4).

[0672] MgCl 2 Preparation of solution: MgCl 2 was prepared into a 200 mM solution using water.

[0673] Preparation of stock solution: Testosterone, huperzine A, deuterated huperzine A compounds WP102-1 to WP102-5 were respectively prepared into 10 mM stock solutions using DMSO.

[0674] Preparation of working solution: The above stock solutions were respectively taken and added with 50% acetonitrile solution to prepare 10 μM working solutions.

[0675] Preparation of internal standard working solution: Tolbutamide, diclofenac, and metolazone were respectively added with acetonitrile to prepare 100 ng / mL internal standard working solutions.

[0676] Test method:

[0677] 1. Add in the following order: 467 μL PBS buffer (0.1 M sodium phosphate, pH 7.4), 7.5 μL MgCl 2 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 gently vortex.

[0678] 2. Incubate the 96-well plate in a water bath at 37˚C and 75 revolutions per minute for 5 minutes.

[0679] 3. Add 10 μL of nicotinamide adenine dinucleotide phosphate (NADPH) solution to initiate the reaction and vortex.

[0680] 4. Incubate the 96-well plate in a water bath at 37˚C and 75 revolutions per minute for 60 minutes.

[0681] 5. Transfer 60 µL aliquots to a 96-well plate at specified times (0, 5, 10, 15, 30, and 60 minutes), add three volumes of the internal standard working solution, and vortex for 10 minutes.

[0682] 6. Centrifuge at 6000 g for 10 minutes at 4 °C.

[0683] 7. After centrifugation, transfer 100 µL aliquots of the supernatant to a 96-well plate containing 100 µL of water in each well.

[0684] 8. Sample analysis.

[0685] The metabolic half-life data are shown in Table 8.

[0686] Table 8 Metabolic half-life results of each compound

[0687]

[0688] Test Example 4 Toxicity effect test of deuterated huperzine A

[0689] 4.1 Rat toxicity effect test

[0690] Animal type: Male SD rats, weighing 275 ± 15 g; injection method: intravenous injection.

[0691] Test articles: Bremelanotide (code name WP203), huperzine A, and deuterated huperzine A (WP102-3).

[0692] Randomly divide into three groups, with 5 rats in each group. The specific dosing doses are as follows:

[0693] Group 1: 1.75 mg / kg / d WP203;

[0694] Group 2: 1.75 mg / kg / d WP203 + 0.2 mg / kg / d deuterated huperzine A (WP102-3);

[0695] Group 3: 1.75 mg / kg / d WP203 + 0.2 mg / kg / d huperzine A;

[0696] Overnight fasting, resume feeding 4 hours after dosing, and inject continuously for 4 days.

[0697] The animal death results after dosing are shown in Table 9.

[0698] Table 9 Animal death results

[0699]

[0700] Combined with the experimental results in Table 9, Bremelanotide was injected alone in Group 1, and the final mortality rate was 60%. In Group 3, Bremelanotide and Huperzine A were injected simultaneously, and the final mortality rate was 60%. In Group 2, Bremelanotide and deuterated Huperzine A were injected simultaneously, and the final mortality rate was only 20%.

[0701] Based on the above experimental results, it can be seen that injecting Bremelanotide alone has greater toxicity, and the mortality rate of rats is 60%. When Bremelanotide and Huperzine A were injected simultaneously, the mortality rate of rats remained unchanged at 60%, indicating that Huperzine A has no positive effect on reducing the toxicity of Bremelanotide. However, when Bremelanotide and deuterated Huperzine A were injected simultaneously, the mortality rate of rats decreased to 20%, indicating that deuterated Huperzine A can significantly reduce the toxicity of Bremelanotide.

[0702] 4.2 HT-22 Cell Toxicity Effect Test

[0703] The cytotoxicity of the samples was evaluated by measuring the effect of the samples on the activity of HT-22 mouse hippocampal neuronal cells.

[0704] Preparation of the samples to be tested: Huperzine A and deuterated Huperzine A (WP102-4) were respectively prepared into stock solutions with a concentration of 100 mg / mL using DMSO; in the experiment, they were diluted into solutions with concentrations of 200 mg / L and 1000 mg / L using DMEM medium.

[0705] Resuscitate HT-22 cells and culture them in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The culture conditions are 37°C and 5% CO 2 . When the cells grow to 80% confluence, digest them with trypsin and inoculate them into a 96-well plate. The cells adhere to the wall for 24 h. Subsequently, add different concentrations of the samples to be tested as the experimental groups, and set a blank control group. After incubating for 24 h, measure the cell activity (CCK-8 method), and calculate the cell survival rate according to the following formula:

[0706] Cell survival rate % = absorbance value of the experimental group / absorbance value of the blank control group × 100%.

[0707] If the cell survival rate of the test sample is less than 80% and there is a significant difference compared with the blank control group, it is considered to have cytotoxicity.

[0708] The results are shown in Table 10.

[0709] Table 10 HT-22 Cell Toxicity Effect Data

[0710]

[0711] Note: Compared with the blank control group, ***p < 0.001.

[0712] Test Example 5 Alzheimer's Disease Cell Model Test of Deuterated Huperzine A

[0713] This test example explores the protective effects of Bremelanotide, deuterated huperzine A, and the combination group on the Aβ cell damage model. HT-22 neurons were damaged by Aβ 1-42, and then the test substances were administered to evaluate the neuroprotective effects of the samples in restoring cell viability.

[0714] 5.1 Test Materials and Reagents

[0715] HT-22 mouse hippocampal neuron cells, high-glucose DMEM serum-free medium, high-glucose DMEM complete medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0716] 5.2 Main Equipment

[0717] Clean bench, carbon dioxide incubator, analytical balance, pipette, multifunctional microplate reader.

[0718] 5.3 Aβ Drug Treatment and Modeling Method

[0719] Modeling agent information:

[0720] Name or code: β-amyloid (1-42), Aβ 1-42;

[0721] Source: Provided by PONY Testing International Group Biopharmaceutical Technology (Shanghai) Co., Ltd.;

[0722] CAS: 166090-74-0;

[0723] Function description: Aβ 1-42 is a polypeptide composed of 42 amino acids, which is toxic to hippocampal slices and is used in the study of Alzheimer's disease;

[0724] Physical properties: Powder;

[0725] Storage conditions: -20°C;

[0726] Preparation method: This product is unstable in solution state. It is recommended to prepare and use it immediately.

[0727] Specific experimental method:

[0728] 1. Take 1 mg of bottled β-amyloid protein, add 45 μL of DMSO, mix well and transfer all to a centrifuge tube to obtain a 5 mM Aβ protein DMSO solution. Then add 950 μL of high-glucose DMEM serum-free medium to the solution to obtain a 250 μM Aβ working solution.

[0729] 2. Immediately before use, transfer 1 mL of 250 μM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin to prepare a 12.5 μM Aβ solution, and preheat it in an incubator at 37°C.

[0730] 3. Replace the medium completely. Add the medium containing 12.5 μM Aβ solution to a 96-well plate, treat with the drug for 24 h, then discard the medium (on the second day), add the medium containing the test drug, treat for 24 h, and detect the cell viability using CCK-8 on the third day.

[0731] 5.4 Sample preparation

[0732] (1) Sample A is Bremelanotide. 8 mg of the sample is placed in a glass reagent bottle, and 1.94 mL of ddH 2 O is added to prepare a stock solution with a concentration of 3500 mg / L. In the experiment, it is first diluted to the highest concentration of 17.5 mg / L with high-glucose DMEM serum-free medium at a ratio of 1:200, and then serially diluted 3-fold.

[0733] (2) Sample B is deuterated huperzine A (WP102-3). 1.092 mg of the sample is placed in a reagent bottle, and 2.65 mL of DMSO is added to prepare a stock solution with a concentration of 400 mg / L. In the experiment, it is first diluted to the highest concentration of 2 mg / L with high-glucose DMEM serum-free medium at a ratio of 1:200, and then serially diluted 3-fold.

[0734] (3) Sample C is the combination of Sample A and Sample B. The stock solutions of Sample A and Sample B are mixed according to a volume ratio of 1:1 to prepare a stock solution containing 1750 mg / L of Sample A + 200 mg / L of Sample B. In the experiment, it is first diluted to the highest concentration with high-glucose DMEM serum-free medium at a ratio of 1:100, and then serially diluted 3-fold to ensure that the ratio of Sample A:Sample B in the solution is 1.75 / 0.2, and the fixed ratio remains unchanged.

[0735] 5.5 Test procedure

[0736] Cell viability test:

[0737] Resuscitate HT-22 cells and culture them in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the culture conditions are 37°C, 5% CO 2 . When the cells grow to 80% confluence, digest them with trypsin and inoculate them in a 96-well plate.

[0738] Add the medium containing 12.5 μM Aβ to a 96-well plate and treat with the drug for 24 h; then discard the medium containing the modeling agent.

[0739] According to the following method:

[0740] ① Add sample A Bremelanotide, and test at 44.0, 8.8, 1.76, 0.35, 0.07, 0.014 mg / L, a total of 6 concentrations;

[0741] ② Add sample B deuterated Huperzine A (WP102-3), and test at 5.0, 1.0, 0.2, 0.04, 0.008, 0.0016 mg / L, a total of 6 concentrations;

[0742] ③ Add sample C (combination group), which is prepared from Bremelanotide + deuterated Huperzine A, ensuring that the ratio of Bremelanotide to deuterated Huperzine A remains 1.75:0.2 unchanged. Among them, 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.

[0743] In addition, set a blank control group and a model group; the blank control group does not use the modeling agent Aβ, and adds the same DMSO solvent as the highest drug dose (5.0 mg / L deuterated Huperzine A) as the solvent control; the model group uses the modeling agent Aβ to damage cells and does not give drug treatment.

[0744] Measure cell viability (CCK-8 method), and calculate the relative cell viability according to the following formula:

[0745] Cell survival rate % = absorbance value of the experimental group / absorbance value of the blank control group × 100%;

[0746] 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%.

[0747] The absorbance values of each group are subtracted by the absorbance value of the blank well of the experimental platform (96-well plate) to reduce the systematic error.

[0748] Use EC50 curve fitting. The data point on the curve where the cell recovery rate reaches 50% is the EC50 drug concentration.

[0749] 5.6 Statistical methods

[0750] The experimental data were statistically analyzed using Graphpad Prism 10.1.2. Regression analysis was performed using the logistic curve in the "agonist vs. normalized response - variable slope" mode of non-linear fitting to calculate the EC50 value.

[0751] 5.7 Results of cell viability assay

[0752] 5.7.1 Protective effect of Bremelanotide on Aβ model

[0753] After treatment with Aβ (12.5 μM, 24 h), the cell viability of the model group was 63.92%, and that of the blank control group was 100%, indicating that Aβ caused obvious cell damage.

[0754] After treatment with Bremelanotide (44 mg / L), the cell viability was 71.43%, partially restoring the damage of HT-22 cells, and the cell recovery rate was 20.82%. See Table 11 for details.

[0755] Calculated by six concentrations, the EC50 value of Bremelanotide on the Aβ model was 119.2 mg / L.

[0756] Table 11 Cell recovery rate of different concentrations of Bremelanotide on Aβ model

[0757]

[0758] According to the cell activity data, the EC50 of the sample was calculated using GraphPad software, as Figure 4 shown.

[0759] 5.7.2 Protective effect of deuterated Huperzine A on Aβ model

[0760] After treatment with Aβ (12.5 μM, 24 h), the cell viability of the model group was 63.92%, and that of the blank control group was 100%, indicating that Aβ caused obvious cell damage.

[0761] After treatment with deuterated Huperzine A (5 mg / L), the cell viability was 87.56%, mostly restoring the damage of HT-22 cells, and the cell recovery rate was 65.53%. See Table 12 for details.

[0762] Calculated by six concentrations, the EC50 value of deuterated Huperzine A on the Aβ model was 2.49 mg / L.

[0763] Table 12 Cell recovery rate of different concentrations of deuterated Huperzine A on Aβ model

[0764]

[0765] According to the cell activity data, the EC50 of the sample was calculated using GraphPad software, as Figure 5 shown.

[0766] 5.7.3 Protective effect of combination group on Aβ model

[0767] After treatment with Aβ (12.5 μM, 24 hr), the cell viability of the model group was 63.92%, and that of the blank control group was 100%, indicating that Aβ caused obvious cell damage.

[0768] After treatment with the combined group (44 mg / L Bremelanotide + 5 mg / L deuterated Huperzine A), the cell viability was 108.37%, completely restoring the damage of HT-22 cells, and the cell recovery rate was 123.21%. See Table 13 for details.

[0769] Calculated through 6 concentrations, on the Aβ model, the EC50 of the combined group was equivalent to a combined preparation containing 11.65 mg / L Bremelanotide + 1.33 mg / L deuterated Huperzine A.

[0770] Table 13 Cell recovery rate of the combined group at different concentrations on the Aβ model

[0771]

[0772] Based on the cell activity data, the EC50 of the sample was calculated using GraphPad software. As Figure 6 shown, the abscissa represents the concentration of Bremelanotide in the combined group.

[0773] The combined action evaluation formula was used to evaluate the synergistic effect between Bremelanotide and deuterated Huperzine A, as follows:

[0774] In the formula, EA and EB represent the effects of the two drugs, sample A and sample B, when used alone; E(A+B) represents the effect of sample A and sample B when used in combination. If the Q value > 1, it indicates that sample A and sample B have a synergistic effect.

[0775] After treatment with Bremelanotide (44 mg / L), the cell viability was 71.43%. After treatment with deuterated Huperzine A (5 mg / L), the cell viability was 87.56%. After treatment with the combined group (44 mg / L Bremelanotide + 5 mg / L deuterated Huperzine A), the cell viability was 108.37%. Substituting into the combined action evaluation formula for calculation, the Q value = 1.12 > 1, indicating that the combined use of Bremelanotide and deuterated Huperzine A has a significant synergistic effect in improving cell viability.

[0776] After treatment with Bremelanotide (44 mg / L), the cell recovery rate was 20.82%. After treatment with deuterated Huperzine A (5 mg / L), the cell recovery rate was 65.53%. After treatment with the combination group (44 mg / L Bremelanotide + 5 mg / L deuterated Huperzine A), the cell recovery rate 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.

[0777] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change without departing from the present invention should be included within 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 includes any one of the following: 、 、 。 2. A method for preparing the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1, 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; Compound Y includes any of the following structures: 、 。 3. The preparation method according to claim 2, 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: 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; Compound X includes any of the following structures: 、 ； 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.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 4, 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.

6. The preparation method according to claim 2, 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.

7. The preparation method according to claim 2, 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 compound G.

8. The preparation method according to claim 3, 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.

9. The preparation method according to claim 8, 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.

10. The preparation method according to claim 9, 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.

11. The preparation method according to claim 10, 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.

12. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a cholinesterase inhibitor.

13. The use according to claim 12, characterized in that: The cholinesterase inhibitor is an acetylcholinesterase inhibitor.

14. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating Alzheimer's disease.

15. Use of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for use in combination with bremelanotide.

16. A pharmaceutical composition, characterized in that It comprises the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof as described in claim 1 and bremelanotide.

17. The pharmaceutical composition according to claim 16, 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.

18. The pharmaceutical composition according to claim 17, characterized in that The mass ratio of the deuterated huperzine A compound or a pharmaceutically acceptable salt thereof to bremelanotide is 5:43.

75.

19. Use of the pharmaceutical composition according to any one of claims 16 to 18 in the preparation of a drug for treating Alzheimer's disease.

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