N-(3,5,6-trimethylpyrazin-2-ylmethyl)-huperzine A and its synthetic method and application

Abstract

The application provides N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A and a synthetic method and application thereof. A structural formula is as follows: a preparation method thereof comprises the following steps: (1) carrying out bromination reaction on tetramethylpyrazine to obtain 2-bromomethyl-3,5,6-trimethylpyrazine; (2) carrying out condensation reaction on huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A. The compound not only has acetylcholinesterase inhibition effect, but also can prevent brain nerve tissue aging and cell apoptosis by eliminating ROS and free radicals and resisting oxidative damage, so as to intervene in the development of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, and relates to a new compound N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A, its synthesis method, and its application. Background Technology

[0002] Alzheimer's disease is a progressive neurodegenerative disease characterized by gradual memory loss and progressive damage to neurons in the hippocampus and cortical areas of the brain. It primarily affects middle-aged and elderly individuals. Clinically, Alzheimer's disease manifests as a gradual decline or even complete loss of language, memory, and motor abilities, ultimately leading to death within 3 to 10 years. The causes of Alzheimer's disease are complex, involving multiple mechanisms, primarily: abnormal levels of acetylcholine in the brain; and free radical-induced aging of brain tissue and cell apoptosis. Currently, no single drug can simultaneously target both of these pathogenic mechanisms. Many studies employ the combined administration of two or more drugs, each targeting different aspects of Alzheimer's disease, demonstrating a significant advantage over single-drug therapy.

[0003] Huperzine A has strong blood-brain barrier permeability and is an effective, competitive, and reversible acetylcholinesterase inhibitor. It is commonly used to prevent the degradation of endogenous acetylcholine, thereby increasing acetylcholine levels and enhancing memory function in Alzheimer's patients. Compared to other commonly used cholinesterase inhibitors (tacrine, donepezil, levanstigmine), huperzine A has high affinity and selectivity, preferentially inhibiting AChE tetramer (G4) in the brain. Therefore, compared to these drugs, huperzine A has higher or comparable efficacy in enhancing memory function in Alzheimer's patients.

[0004] However, huperzine A primarily targets abnormal acetylcholine levels in the brain, improving benign memory impairment by increasing acetylcholine levels. It has a very weak effect on preventing and treating brain neural tissue aging, damage, and apoptosis caused by free radicals, thus proving ineffective in improving aphasia, apraxia, agnosia, visuospatial skill impairment, and executive dysfunction. Summary of the Invention

[0005] To address the problems of the prior art, the present invention provides N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A, its synthesis method, and its applications.

[0006] This invention is achieved through the following technical solution:

[0007] An N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A has the following structural formula:

[0008]

[0009] Preferably, it includes the following steps:

[0010] (1) Tetramethylpyrazine was subjected to bromination to give 2-bromomethyl-3,5,6-trimethylpyrazine;

[0011] (2) Huperzine A was condensed with 2-bromomethyl-3,5,6-trimethylpyrazine to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A.

[0012] Furthermore, step (1) specifically includes the following steps:

[0013] 1) Using tetramethylpyrazine as a raw material, N-bromosuccinimide was reacted with it to produce crude 2-bromomethyl-3,5,6-trimethylpyrazine.

[0014] 2) The crude product of 2-bromomethyl-3,5,6-trimethylpyrazine was purified by silica gel column chromatography and eluted with petroleum ether-ethyl acetate buffer to give 2-bromomethyl-3,5,6-trimethylpyrazine.

[0015] Further, step 1) specifically involves adding tetramethylpyrazine and N-bromosuccinimide to a CCl4 solvent, adding a catalytic amount of benzoyl peroxide, and refluxing at 40–70°C under light to obtain the crude product 2-bromomethyl-3,5,6-trimethylpyrazine.

[0016] Furthermore, in step 1), the molar ratio of the tetramethylpyrazine to N-bromosuccinimide is 1.0:(0.5-3.0).

[0017] Furthermore, in step 2), the volume ratio of petroleum ether to ethyl acetate is 1.0:(0.1-3.0).

[0018] Further, step (2) specifically involves dissolving huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine in DMF, reacting at high temperature, crystallizing at low temperature, filtering, washing the filter cake with water, and drying under vacuum to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A.

[0019] Furthermore, in step (2), the reaction temperature is 100–150 °C.

[0020] Furthermore, in step (2), the molar ratio of huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine is 1.0:(0.5-3.0).

[0021] The application of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A in the preparation of drugs for treating Alzheimer's disease.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a novel compound, N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A, with the molecular formula C0. 23 H 28 N4O, with a relative molecular mass of 376.51, is a pale yellow to white crystalline powder. It is a novel compound, and there are currently no reports of its existence in domestic or international literature. The compound of this invention is a huperzine A derivative containing a pyrazine ring. It not only exhibits excellent acetylcholinesterase inhibition, preventing the degradation of endogenous acetylcholine and increasing acetylcholine levels, thus enhancing memory function in Alzheimer's patients; it also intervenes in the development of Alzheimer's disease by scavenging ROS and free radicals, resisting oxidative damage, and preventing aging of brain nerve tissue and apoptosis. Therefore, it can serve as a potential drug for intervening in Alzheimer's disease.

[0024] The compound of this invention is obtained by using huperzine A as a raw material and undergoing multiple reaction steps to obtain the final product. The synthesis yield reaches more than 70%. Experiments have confirmed that this is a reasonable, economical and efficient preparation method. Attached Figure Description

[0025] Figure 1 ESI-MS chromatogram of the product 2-bromomethyl-3,5,6-trimethylpyrazine from Example 2;

[0026] Figure 2 ESI-MS image of the final product N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A from Example 3. Detailed Implementation

[0027] The present invention will now be described in detail. Before proceeding with the description, it should be understood that the terminology used in this specification and the appended claims should not be construed as limited to its general or dictionary meaning, but rather should be interpreted according to the meaning and concept corresponding to the technical aspects of the invention, based on the principle that the inventors are allowed to appropriately define the terms for the best interpretation. Therefore, the description presented herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention. It should be understood that other equivalents or modifications can be obtained from it without departing from the spirit and scope of the invention.

[0028] The following embodiments are merely examples illustrating implementations of the present invention and do not constitute any limitation on the present invention. Those skilled in the art will understand that modifications made without departing from the spirit and concept of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0029] The structural formula of the compound N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A described in this invention is as follows:

[0030]

[0031] The above-mentioned chemical synthesis method of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A includes the following steps:

[0032] (1) Bromination of tetramethylpyrazine yields 2-bromomethyl-3,5,6-trimethylpyrazine, the bromination product of tetramethylpyrazine.

[0033] (2) The amide bond of huperzine A undergoes a condensation reaction with the brominated product of tetramethylpyrazine, and the final product N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A is obtained by purification.

[0034] In this invention, step (1) specifically includes the following steps:

[0035] Step 1), Synthesis of the brominated product of tetramethylpyrazine: Using tetramethylpyrazine as a raw material, it is brominated with N-bromosuccinimide to obtain the crude product of 2-bromomethyl-3,5,6-trimethylpyrazine.

[0036] Step 2), purification of 2-bromomethyl-3,5,6-trimethylpyrazine: The crude product was purified by silica gel column chromatography to obtain a product with a purity ≥95%.

[0037] Step 1) specifically involves: using tetramethylpyrazine and N-bromosuccinimide as raw materials, adding them to a CCl4 solvent, adding a catalytic amount of benzoyl peroxide, and refluxing at 40–70°C under incandescent light. After the reaction is completed, post-treatment is performed to obtain the crude product 2-bromomethyl-3,5,6-trimethylpyrazine; wherein the molar ratio of tetramethylpyrazine to N-bromosuccinimide is 1.0:(0.5–3.0).

[0038] Step 2) specifically involves purifying the crude product 2-bromomethyl-3,5,6-trimethylpyrazine from step 1) using a silica gel column, eluting with petroleum ether-ethyl acetate eluent, wherein the volume ratio of petroleum ether to ethyl acetate is 1.0:(0.1-3.0).

[0039] In this invention, step (2) specifically includes the following steps: dissolving huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine in DMF, reacting at high temperature (100-150°C), crystallizing at low temperature, filtering, washing the filter cake with water, and drying under vacuum to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A final product; wherein, the molar ratio of huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine is 1.0:(0.5-3.0).

[0040] The following specific embodiments illustrate the synthesis method of the present invention in detail:

[0041] Example 1:

[0042] Synthesis of 2-bromomethyl-3,5,6-trimethylpyrazine: 13.6 g of tetramethylpyrazine and 17.8 g of N-bromosuccinimide were dissolved in 0.2 L of carbon tetrachloride solution, and then 0.12 g of benzoyl peroxide was added. The mixture was stirred and refluxed under incandescent light for 12 h, and the solvent was removed under reduced pressure to obtain the crude product.

[0043] The synthesis route is shown below:

[0044]

[0045] Purification of 2-bromomethyl-3,5,6-trimethylpyrazine: The crude product was loaded onto a silica gel column and purified with petroleum ether:ethyl acetate = 9:1 to obtain a product with a purity ≥ 95%; its ESI-MS chromatogram is shown below. Figure 1 ESI-MS, m / z: 217.0 [M+H] + .

[0046] Synthesis of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A: 9.0 g of huperzine A was dissolved in 200 mL of DMF, and then 8.0 g of the purified product 2-bromomethyl-3,5,6-trimethylpyrazine was added. The reaction mixture was heated to 120 °C and stirred for 5 h. After cooling to room temperature, the solution was poured into 500 mL of ice water, resulting in a pale yellow precipitate. The precipitate was filtered, washed with water (3 × 50 mL), and dried under vacuum overnight to give 9.6 g of a pale yellow solid, with a yield of 69.8%. ESI-MS, m / z: 377.3 [M+H] + .

[0047] The synthesis route is shown below:

[0048]

[0049] The ESI-MS chromatogram of the final product N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A is shown in Figure 1. Figure 2 .

[0050] Example 2:

[0051] Synthesis of 2-bromomethyl-3,5,6-trimethylpyrazine: 13.6 g of tetramethylpyrazine and 53.4 g of N-bromosuccinimide were dissolved in 0.5 L of carbon tetrachloride solution, and then 0.12 g of benzoyl peroxide was added. The mixture was stirred and refluxed under incandescent light for 12 h, and the solvent was removed under reduced pressure to obtain the crude product.

[0052] Purification of 2-bromomethyl-3,5,6-trimethylpyrazine: The crude product was loaded onto a silica gel column and purified with petroleum ether:ethyl acetate = 9:1 to obtain a product with a purity ≥95%;

[0053] Synthesis of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A: 9.0 g of huperzine A was dissolved in 300 mL of DMF, and then 24.0 g of the purified product 2-bromomethyl-3,5,6-trimethylpyrazine was added. The reaction mixture was heated to 120 °C and stirred for 5 h. After cooling to room temperature, the solution was poured into 500 mL of ice water, resulting in a pale yellow precipitate. The precipitate was filtered, washed with water (3 × 50 mL), and dried under vacuum overnight to obtain 9.9 g of a pale yellow solid.

[0054] Example 3

[0055] Synthesis of 2-bromomethyl-3,5,6-trimethylpyrazine: 13.6 g of tetramethylpyrazine and 15.0 g of N-bromosuccinimide were dissolved in 0.2 L of carbon tetrachloride solution, and then 0.12 g of benzoyl peroxide was added. The mixture was stirred and refluxed under incandescent light for 12 h, and the solvent was removed under reduced pressure to obtain the crude product.

[0056] Purification of 2-bromomethyl-3,5,6-trimethylpyrazine: The crude product was loaded onto a silica gel column and purified with petroleum ether:ethyl acetate = 9:1 to obtain a product with a purity ≥95%;

[0057] Synthesis of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A: 9.0 g of huperzine A was dissolved in 200 mL of DMF, and then 7.0 g of the purified product 2-bromomethyl-3,5,6-trimethylpyrazine was added. The reaction mixture was heated to 120 °C and stirred for 5 h. After cooling to room temperature, the solution was poured into 500 mL of ice water, resulting in a pale yellow precipitate. The precipitate was filtered, washed with water (3 × 50 mL), and dried under vacuum overnight to obtain 8.4 g of a pale yellow solid.

[0058] Example 4: Effect of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A on an animal model induced by acetylcholine receptor blockers.

[0059] A scopolamine-induced memory loss animal model was established and administered: Rats were housed for 5 days to acclimatize. Then, the rats were randomly divided into a control group, a scopolamine group, a huperzine A group, and a drug group. The control group and the scopolamine group received intraperitoneal injections of physiological saline (1 mg / kg) daily, the huperzine A group received huperzine A via gavage (2 μg / kg) daily, and the drug group received N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A via gavage (2 μg / kg) daily. After 14 days of continuous administration, a water maze test was performed, with continued administration during the water maze test. Half an hour before the water maze test, the control group received intraperitoneal injections of physiological saline, while the scopolamine group, the huperzine A group, and the drug group received intraperitoneal injections of scopolamine (1 mg / kg).

[0060] Water Maze Experiment: The water maze consisted of a cylindrical pool with a diameter of 150 cm and a height of 50 cm, with black inner walls and bottom. The interior was divided into four quadrants. A 30 cm transparent glass platform was placed in the center of the third quadrant. The positions of the pool and platform remained constant throughout the experiment. The water temperature was maintained at 25℃. In each experiment, rats were placed into the water from the midpoint of each quadrant at the edge of the pool. The time it took for the rat to find the platform within 60 seconds was recorded; this was the latency period. Regardless of whether the rat found the platform, it was placed on the platform for 30 seconds. This training was repeated for 7 days. On the 9th day, the latency period and the number of times the rat crossed the platform were measured.

[0061] The results showed that huperzine A and N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A significantly improved the learning and memory abilities of dementia rats. In the control group, rats could quickly find the plateau starting from day 4, and could find it from day 5 to day 7. On day 9, when memory was tested, the scopolamine group rats failed to find the plateau, with a latency of (60.0 ± 0.0) s. In the drug group and the huperzine A group, rats began to find the plateau from day 5, and the latency gradually decreased. On day 9, when memory was tested, the latency in the drug group was longer than that in the control group but shorter than that in the huperzine A group.

[0062] Example 5: Effects of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A on an aging animal model

[0063] A mouse model of aging induced by D-galactose combined with aluminum trichloride was established: Mice were housed for 5 days to acclimatize. Then, the mice were randomly divided into a control group and a model group. The model group was treated with D-galactose (60 mg / kg, subcutaneously) combined with aluminum trichloride (5 mg / kg, intragastric gavage) to establish the mouse aging model, with a modeling period of 42 days. The learning and memory abilities of the mice were then assessed using the water maze test, and Alzheimer's disease model mice were selected. The Alzheimer's disease model mice were randomly divided into a model group, a huperzine A group, and a drug group. The control group and model group were administered physiological saline by gavage daily, the huperzine A group was administered huperzine A by gavage daily (2 μg / kg), and the drug group was administered N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A by gavage daily (2 μg / kg). All groups continued to receive the modeling agent concurrently with the drug administration.

[0064] Open field test: The open field test was conducted on days 7 and 18 after drug administration. Mice were placed in the central area of ​​a 3x3 grid, and the operator immediately moved away from the open field. The entire experiment was conducted in a well-ventilated, quiet, and dimly lit indoor space. The distance the mice traveled and the time they remained in the central area were recorded over 5 minutes to reflect anxiety-like behavior. After each mouse's experiment, the experimental equipment was cleaned with 75% ethanol and mouse excrement was removed. The results showed that mice with lower anxiety levels tended to travel more distance and remain in the central, open area of ​​the open field for longer periods. Both the huperzine A group and the drug group increased the movement distance and dwell time in the central region of an open field in aging mice. However, the drug group was far more effective than the huperzine A group. The drug group mice moved a shorter distance and stayed longer in the central region than the huperzine A group, indicating that the anxiety level of the mice in the drug group was lower than that in the huperzine A group. In other words, the improved N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A of this invention greatly enhances the effect of huperzine A in preventing and treating aging, damage and apoptosis of brain nerve tissue caused by free radicals.

Claims

1. An N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A, characterized in that, Its structural formula is:

2. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 1, characterized in that, Includes the following steps: (1) Tetramethylpyrazine was subjected to bromination to give 2-bromomethyl-3,5,6-trimethylpyrazine; (2) Huperzine A was condensed with 2-bromomethyl-3,5,6-trimethylpyrazine to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A.

3. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 2, characterized in that, Step (1) specifically includes the following steps: 1) Using tetramethylpyrazine as a raw material, N-bromosuccinimide was reacted with it to produce crude 2-bromomethyl-3,5,6-trimethylpyrazine. 2) The crude product of 2-bromomethyl-3,5,6-trimethylpyrazine was purified by silica gel column chromatography and eluted with petroleum ether-ethyl acetate buffer to give 2-bromomethyl-3,5,6-trimethylpyrazine.

4. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, Step 1) specifically involves adding tetramethylpyrazine and N-bromosuccinimide to a CCl4 solvent, adding a catalytic amount of benzoyl peroxide, and refluxing at 40–70°C under light to obtain the crude product 2-bromomethyl-3,5,6-trimethylpyrazine.

5. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, In step 1), the molar ratio of tetramethylpyrazine to N-bromosuccinimide is 1.0:(0.5-3.0).

6. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, In step 2), the volume ratio of petroleum ether to ethyl acetate is 1.0:(0.1-3.0).

7. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, Step (2) specifically involves dissolving huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine in DMF, reacting at high temperature, crystallizing at low temperature, filtering, washing the filter cake with water, and drying under vacuum to obtain N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A.

8. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, In step (2), the reaction temperature is 100-150℃.

9. The method for synthesizing N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A according to claim 3, characterized in that, In step (2), the molar ratio of huperzine A and 2-bromomethyl-3,5,6-trimethylpyrazine is 1.0:(0.5-3.0).

10. The use of N-(3,5,6-trimethylpyrazine-2-methylene)-huperzine A as described in claim 1 in the preparation of a drug for treating Alzheimer's disease.

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