A tacrine-pyrazine derivative and its use

Abstract

The present invention discloses a tacrine-pyrazine derivative and its use. The structure of the tacrine-pyrazine derivative is represented by the following general formula (I), general formula (II), and general formula (III): The tacrine-pyrazine derivative of the present invention uses the acetylcholinesterase inhibitor tacrine as a parent nucleus and is combined with a functional group of ligustrazine (tetramethylpyrazine) having an anti-platelet aggregation effect. Other tacrine-pyrazine derivatives have activities such as inhibiting acetylcholinesterase, resisting platelet aggregation, and inhibiting tau protein hyperphosphorylation, thereby being able to better protect neurons from damage and improve impaired learning and memory in rats. The tacrine-pyrazine derivative has high blood-brain barrier permeability and relatively low toxicity, and can be used for the prevention and treatment of vascular dementia.

Description

Technical Field

[0001] The present invention relates to a drug for vascular dementia, in particular to a tacrine-pyrazine derivative designed based on the dual effects of acetylcholinesterase (AChE) inhibition (tacrine) and platelet aggregation resistance (ligustrazine) and its use. Background Art

[0002] Vascular dementia (VaD) is a syndrome of brain dysfunction caused by cerebrovascular disease, characterized by cognitive impairment, psychiatric and behavioral symptoms, and decreased functional ability. Its incidence is second only to Alzheimer's disease (AD), and its risk and mortality rates are higher than those of AD. This disease severely impacts the quality of life of patients and their families, placing a significant psychological and financial burden on patients. Currently, there is no specific treatment for VaD. The 2019 Guidelines for the Diagnosis and Treatment of Vascular Cognitive Impairment in China (Full Version) recommend the use of acetylcholinesterase inhibitors (AChEIs), excitatory amino acid receptor (NDMA) antagonists, and calcium ion antagonists (such as nimodipine). In addition, antioxidants and traditional Chinese medicines (such as ginkgo biloba and Chuanxiong) are also recommended to improve cognitive function, increase cerebral blood flow, and protect neurons. In short, this approach involves dual treatment targeting both dementia-like behaviors and cerebrovascular symptoms. In the Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke (2020) and its related guidelines for the prevention and treatment of cerebrovascular diseases, the therapeutic status of anti-platelet aggregation has been widely recognized.

[0003] Tacrine is the first drug approved by the FDA for AD. It has good acetylcholinesterase inhibitory activity. However, it was withdrawn from the market due to hepatotoxicity caused by long-term use. Many studies have focused on reducing its hepatotoxicity and enhancing or maintaining its acetylcholinesterase activity. Tacrine derivatives such as thiol-tacrine hybrids, tacrine-caffeic acid mixtures, coumarin-tacrine, and tacrine-diphenyl diesters have been designed by modifying groups or connecting active groups. Novel tacrine-related drugs are potential candidates for the treatment of AD. Alzheimer's disease. Bioorg. Med. Chem. Lett., 2013, 23: 1916-1922; Research progress on the design of multi-target drugs based on tacrine and donepezil and their anti-Alzheimer's disease activity [Chinese Journal of Pharmaceutical Sciences, 2019, 54(5): 352-359]. Many derivatives have been shown to have low hepatotoxicity and synergistic effects, which enables tacrine as an acetylcholinesterase inhibitory pharmacophore to participate in the design of multi-target and multi-action mechanism drugs, providing ideas and basis for the design of new anti-VaD drugs.

[0004] Ligustrazine is a traditional Chinese medicine commonly used in the clinical treatment of cerebrovascular diseases. It belongs to the pyrazine alkaloid class and has the effects of dilating blood vessels, increasing arterial blood flow, and inhibiting platelet aggregation [Experimental study on the antiplatelet aggregation effect of ligustrazine. Laboratory Medicine, 2014, 9(9):976-977]. In addition, ligustrazine injection can improve learning and memory in rats with vascular dementia model [Study on the effect of ligustrazine injection on learning and memory in rats with vascular dementia model and its mechanism of action, Master's thesis of University of South China] and is a promising neuroprotective agent for the treatment of VaD [Protective effect and mechanism of ligustrazine on rats with vascular dementia (VaD) and PC12 cells with oxygen and glucose deprivation (OGD) Journal of Medical Research, 2020, 49(9):144-149]. Summary of the Invention

[0005] The present invention aims to provide a tacrine-pyrazine derivative and its use. The technical problem to be solved is to select a suitable molecular structure. The most important thing is to screen the connecting arm between the two parent structures of tacrine and pyrazine so that it has better AChE and platelet aggregation inhibitory activity, and can also add an inhibitory effect on Tau protein hyperphosphorylation, thereby better protecting neurons and improving impaired learning and memory. It also has the characteristics of low toxicity, high blood-brain barrier penetration rate, and easy preparation.

[0006] The tacrine-pyrazine derivative of the present invention has multiple functions such as inhibiting AChE, resisting platelet aggregation, inhibiting Tau protein hyperphosphorylation, improving learning and memory impairment, and protecting neurons, and can be used for the prevention and treatment of VaD.

[0007] The tacrine-pyrazine derivative of the present invention is a composition of tacrine and bromomethylpyrazine connected by an alkyldiamine, and its structure is represented by the general formula (I):

[0008]

[0009] In the general formula (I), R1, R2, R3, and R4 each independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO2; R5, R6, and R7 each independently represent -H, -Me, and -MeO, and n=1 to 7.

[0010] The preparation method of the tacrine-pyrazine derivative represented by general formula (I) comprises amination reaction, bromination reaction, substitution reaction and post-treatment unit processes:

[0011] The amination reaction comprises mixing benzene ring-substituted 9-chloro-1,2,3,4-tetrahydroacridine, sodium iodide, and phenol in a molar ratio of 1:0.2:(10-15), reacting at 90° C. for 2 hours; then adding 7 molar equivalents of alkyl diamine (based on 9-chloro-1,2,3,4-tetrahydroacridine), raising the temperature to 120° C., and continuing the reaction for 2 hours to obtain intermediate A;

[0012] The general structural formula of the benzene ring substituted 9-chloro-1,2,3,4-tetrahydroacridine is:

[0013]

[0014] wherein R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO2.

[0015] The general structural formula of the alkyl diamine is:

[0016]

[0017] Where n=1~7.

[0018] The general structural formula of the intermediate A is:

[0019]

[0020] The bromination reaction is to add substituted 2-methylpyrazine and N-bromosuccinimide (NBS) in a molar ratio of 1:0.9 to CCl4, reflux for 1-3 hours under 100W light, and vacuum decompression fractionation to obtain brominated intermediate B;

[0021] The substituted 2-methylpyrazine has the general structural formula:

[0022]

[0023] wherein R5, R6, and R7 independently represent -H, -Me, and -MeO.

[0024] The general structural formula of the intermediate B is:

[0025]

[0026] wherein R5, R6, and R7 independently represent -H, -Me, and -MeO.

[0027] The substitution reaction comprises adding intermediate A, intermediate B and a base in a molar ratio of 1:(1.5-2.0):(1.5-3.0) to an organic solvent, reacting under reflux under stirring for 8-10 hours, and separating and purifying by column chromatography (dichloromethane / methanol = 20:1-15:1, V / V) to obtain the target product (I).

[0028] The base in the substitution reaction is selected from alkali metal carbonate, alkali metal bicarbonate, triethylamine or pyridine.

[0029] The organic solvent in the substitution reaction is selected from one or more of acetonitrile, ethanol, tetrahydrofuran, acetone and dichloromethane.

[0030] The reaction route is as follows:

[0031]

[0032]

[0033] The tacrine-pyrazine derivative of the present invention is a composition of tacrine and bromomethylpyrazine connected by piperazine-alkyl alcohol, and its structure is represented by the general formula (II):

[0034]

[0035] In the general formula (II), R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO2; R5, R6, and R7 independently represent -H, -Me, and -MeO, and m=1 to 3.

[0036] The preparation method of the tacrine-pyrazine derivative represented by general formula (II) comprises the following unit processes: piperazine amination reaction, bromohydrin substitution reaction, bromomethylpyrazine substitution reaction, and post-treatment:

[0037] The piperazine amination reaction comprises mixing benzene ring-substituted 9-chloro-1,2,3,4-tetrahydroacridine, sodium iodide, and phenol in a molar ratio of 1:0.2:(10-15), reacting at 90° C. for 2 hours; then adding 7 molar equivalents of piperazine (based on 9-chloro-1,2,3,4-tetrahydroacridine), raising the temperature to 120° C., and continuing the reaction for 2 hours to obtain intermediate C;

[0038] The general structural formula of the benzene ring substituted 9-chloro-1,2,3,4-tetrahydroacridine is:

[0039]

[0040] wherein R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO2.

[0041] The general structural formula of the intermediate C is:

[0042]

[0043] wherein R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, or -NO2.

[0044] The bromohydrin substitution reaction is to add intermediate C, bromohydrin, and potassium carbonate in a molar ratio of 1:1:1.5 to dichloromethane, heat at room temperature to 50°C for 5-8 hours, and separate and purify by column chromatography (dichloromethane / methanol = 15:1-10:1, V / V) to obtain intermediate D;

[0045] The general structural formula of the bromohydrin is:

[0046] Wherein, m=1-3.

[0047] The general structural formula of the intermediate D is:

[0048]

[0049] wherein R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, or -NO2; and m=1-3.

[0050] The bromomethylpyrazine substitution reaction is to add intermediate D, substituted bromomethylpyrazine (intermediate B) and a base in a molar ratio of 1: (1-1.2): (1.5-3.0) to an organic solvent, heat and stir at room temperature to 85° C. for 8-10 hours, and separate and purify by column chromatography to obtain the target product (II).

[0051] The general structural formula of the substituted bromomethylpyrazine is:

[0052]

[0053] wherein R5, R6, and R7 independently represent -H, -Me, and -MeO.

[0054] The base in the substitution reaction is selected from alkali metal carbonates, alkali metal bicarbonates, and triethylamine.

[0055] The organic solvent in the substitution reaction is selected from one or more of acetonitrile, tetrahydrofuran, acetone and dichloromethane.

[0056] The reaction route is as follows:

[0057]

[0058] The tacrine-pyrazine derivative of the present invention is an amide composition in which tacrine and pyrazine carboxylic acid are connected via an alkyl diamine, and its structure is represented by the general formula (III):

[0059]

[0060] In the general formula (III), R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO2.

[0061] R5 and R6 each independently represent -H, -Br, -Cl, -Me, or -MeO, and n=1 to 7.

[0062] The preparation method of the tacrine-pyrazinamide compound represented by general formula (III) comprises amination reaction, amidation reaction and post-treatment unit processes, and is characterized in that:

[0063] The amination reaction comprises mixing benzene ring-substituted 9-chloro-1,2,3,4-tetrahydroacridine, sodium iodide and phenol in a molar ratio of 1:0.2:10, reacting at 90° C. for 2 hours, then adding 7 molar equivalents of alkyl diamine (calculated as 9-chloro-1,2,3,4-tetrahydroacridine), raising the temperature to 120° C., and continuing the reaction for 2 hours to obtain intermediate A.

[0064] The general structural formula of the intermediate A is:

[0065]

[0066] wherein R1, R2, R3, and R4 independently represent -H, -F, -Cl, -Br, -Me, -MeO, or -NO2, and n=1 to 7.

[0067] The amidation reaction comprises adding intermediate A, substituted pyrazine carboxylic acid, a dehydrating agent and a catalyst in a molar ratio of 1:(1-1.5):(0.6-0.8):0.4 to an organic solvent, reacting at room temperature for 15-20 hours, and separating and purifying by column chromatography (dichloromethane / methanol=15:1-10:1, V / V) to obtain the target product (III).

[0068] The general structural formula of the substituted pyrazine carboxylic acid is:

[0069]

[0070] R5 and R6 each independently represent -H, -Br, -Cl, -Me, or -MeO.

[0071] The dehydrating agent is dicyclohexylcarbodiimide, and the catalyst is 4-dimethylaminopyridine (i.e., a DCC / DMAP combination); alternatively, the dehydrating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the catalyst is 4-dimethylaminopyridine (EDC / DMAP combination); alternatively, the dehydrating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the catalyst is 1-hydroxybenzotriazole (EDC / HOBt combination).

[0072] The organic solvent is selected from one or more of acetonitrile, ethanol, tetrahydrofuran, acetone, N,N'-dimethylformamide, and dichloromethane.

[0073] The reaction route is as follows:

[0074]

[0075] The use of the tacrine-pyrazine derivatives represented by general formula (I), general formula (II) and general formula (III) of the present invention is to prepare pharmaceutical preparations using the tacrine-pyrazine derivatives. The pharmaceutical preparations have multiple effects such as inhibiting AChE, resisting platelet aggregation, inhibiting Tau protein hyperphosphorylation, protecting neurons, and improving impaired learning and memory. They can be used as improved AChE inhibitors or antiplatelet aggregation drugs for the prevention and / or treatment of VaD.

[0076] The tacrine-pyrazine derivatives designed in the present invention exhibit excellent AChE inhibition and platelet aggregation resistance, as well as Tau protein hyperphosphorylation inhibition. The derivatives of tacrine and pyrazine linked via an alkyldiamine or piperazine (target products (I) and (II)) are particularly active, demonstrating AChE inhibitory activity nearly equivalent to that of tacrine while enhancing or retaining the antiplatelet aggregation activity of ligustrazine. Furthermore, their Tau protein hyperphosphorylation inhibition activity is nearly equivalent to that of the positive control drug, Rember (Tau protein aggregation inhibitor, Phase III clinical trial). More importantly, they exhibit minimal hepatotoxicity and high blood-brain barrier permeability.

[0077] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0078] 1. Compared to existing drugs such as tacrine or tetramethylpyrazine (tetramethylpyrazine), the tacrine-pyrazine derivatives of the present invention retain or enhance AChE inhibitory and antiplatelet aggregation activities, while significantly reducing the hepatotoxicity inherent to tacrine. More importantly, these compounds also exhibit potent inhibitory activity against Tau protein hyperphosphorylation, ultimately potentially intervening in multiple pathways to address the neuronal dysfunction and learning and memory impairment caused by Vascular Degeneration (VD).

[0079] 2. The tacrine-pyrazine derivatives of the present invention have high blood-brain barrier permeability, the drug concentration in the brain can be well maintained and the degradation rate is slowed down, which makes it easy to reduce the frequency and dosage of medication, thereby avoiding the metabolic load and safety risks of long-term VaD medication. DETAILED DESCRIPTION

[0080] Example 1: Synthesis of Intermediate A-1—N-(1,2,3,4-tetrahydroacridine-9-)-hexane-1,6-diamine

[0081]

[0082] 5 g of 9-chloro-1,2,3,4-tetrahydroacridine, 0.75 g of sodium iodide, and 20 g of phenol were weighed, mixed, and reacted at 90° C. for 2 hours. 18.3 g of 1.6-hexanediamine was then added, and the temperature was raised to 120° C. for 2 hours. After the reaction, the mixture was cooled to room temperature, and a 10 wt % NaOH solution was added to adjust the pH to 9-10. The mixture was extracted twice with dichloromethane. The two extracts were combined, washed with water, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by silica gel column chromatography (eluent, by volume: dichloromethane / methanol / triethylamine = 15:1:0.1). After concentration, a brown oily product, namely, intermediate A-1—N-(1,2,3,4-tetrahydroacridine-9-)-hexane-1,6-diamine 4.8 g, was obtained with a yield of 70%.

[0083] 1 H NMR(500MHz, CDCl3)7.90-7.97(m,2H),7.52-7.58(m,1H),7.32-7.37(m,1H),4.01(br,1H),3.45-3.52(m,2 H),3.07(m,2H),2.66-2.72(m,4H),2.01(br,2H),1.91-1.95(m,4H),1.62-1.72(m,4H),1.32-1.49(m,4H).

[0084] Example 2: Synthesis of Intermediate A-2—N-(1,2,3,4-tetrahydroacridine-6-chloro-9-)-butane-1,4-diamine

[0085]

[0086] 2.5 g of 6-chloro-9-chloro-1,2,3,4-tetrahydroacridine, 0.29 g of sodium iodide and 9.4 g of phenol were weighed, mixed and heated at 90° C. for reaction for 1 hour, then 6.16 g of 1.4-butanediamine was added, the temperature was raised to 120° C. for reaction for 2 hours, and the reaction was cooled to room temperature. After the reaction was completed, 10 wt% NaOH solution was added to adjust the pH to 9-10, and the mixture was extracted twice with dichloromethane. The two extracts were combined, washed with water, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by silica gel column chromatography (eluent by volume: dichloromethane / methanol / triethylamine = 10:1:0.1) to obtain a brown oily product, which is the intermediate A—N-(1,2,3,4-tetrahydroacridine-6-chloro-9-)-butane-1,4-diamine 2.3 g, with a yield of 75%.

[0087] 1 H NMR(500MHz, CDCl3)8.03-8.10(m,1H),7.82-7.86(m,1H),7.32-7.35(m,1H),4.08(br,1H),3.15-3. 23(m,2H),2.97-3.02(m,2H),2.58-2.67(m,4H),2.01(br,2H),1.72-1.90(m,4H),1.46-1.59(m,4H).

[0088] Example 3: Synthesis of Intermediate B-1—2-Bromomethyl-3,5,6-trimethylpyrazine

[0089]

[0090] Weigh 5 g of 2,3,5,6-tetramethylpyrazine and 4.1 g of NBS in 20 mL of carbon tetrachloride. Under irradiation with 2 × 100 W incandescent lamps, reflux at 95°C for 1-2 hours, and concentrate under reduced pressure to obtain a crude product. Use vacuum distillation to obtain a colorless liquid. Cool at room temperature to obtain 4.3 g of white crystals, with a yield of 50%.

[0091] 1 H NMR(500MHz, CDCl3)4.54(s,2H),2.31(s,3H),2.23(s,3H),2.22(s,3H).

[0092] Example 4: Synthesis of Intermediate B-2—2-Bromomethyl-5-methoxypyrazine

[0093]

[0094] The synthesis process was the same as that in Example 3, except that 2-bromomethyl-3,5,6-trimethylpyrazine in Example 3 was replaced by 2-bromomethyl-5-methoxypyrazine, with a yield of 60%.

[0095] 1 H NMR (500MHz, CDCl3)8.23(s,1H),7.98(s,1H),4.44(s,2H),3.91(s,3H).

[0096] Example 5: Synthesis of target product I-1

[0097]

[0098] Weigh 6.4 g of intermediate A-1, 4.3 g of intermediate B-1, and 3 g of triethylamine in dichloromethane, react at 50°C for 5 hours, cool to room temperature, and concentrate under reduced pressure to obtain a crude product. The product was separated and purified by silica gel column chromatography (eluent, by volume: dichloromethane / methanol / triethylamine = 10:1:0.1) to obtain 6.9 g of a light yellow solid (target product I-1) with a yield of 75%.

[0099] 1 H NMR(500MHz, CDCl3)8.06(dd,1H),7.89(dd,1H),7.64(td,1H),7.39(ddd,1H),6.39(t,1H),4.10(d,2H),3.41(q,2H),3.00–2.90(m,2H),2.9 0–2.75(m,4H),2.32(s,3H),2.25(s,3H),2.22(s,3H),1.98(tt,1H),1 .93–1.81(m,4H),1.63(tt,2H),1.58–1.48(m,2H),1.41–1.28(m,4H).

[0100] Example 6: Synthesis of target product (I-2)

[0101]

[0102] The synthesis process is the same as that of Example 5, except that the intermediate A-1 (N-(1,2,3,4-tetrahydroacridine-9-)-hexane-1,6-diamine) in Example 5 is replaced by intermediate A-2 (N-(1,2,3,4-tetrahydroacridine-6-chloro-9-)-butane-1,4-diamine), and the intermediate B-1 (2-bromomethyl-3,5,6-trimethylpyrazine) is replaced by B-2 (2-bromomethyl-5-methoxypyrazine), with a yield of 60%.

[0103] 1 H NMR(500MHz, CDCl3)8.22(s,1H),7.96(s,1H),7.92(dd,1H),7.78(dd,1H),7.43(t,1H),6.46(t,1H),4.02(d,2H),3.91(s,3H), 3.38(q,2H),2.94–2.87(m,2H),2.87–2.80(m,2H),2.78(q,2H),2.08(tt,1H),1.92–1.81(m,4H),1.69(ttd,2H),1.56(ttd,2H).

[0104] Example 7: Synthesis of Intermediate C—9-(Piperazine-1-)-5,6,7,8-tetrahydroacridine

[0105]

[0106] 6.4 g of 9-chloro-1,2,3,4-tetrahydroacridine, 0.89 g of sodium iodide, and 28 g of phenol were weighed, mixed, and heated at 90°C for reaction for one hour. 17.6 g of piperazine was then added, and the temperature was raised to 120°C for reaction for two hours. After the reaction, the mixture was cooled to room temperature, and a 10 wt % NaOH solution was added to adjust the pH to 9-10. The mixture was extracted twice with dichloromethane. The two extracts were combined, washed with water, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by silica gel column chromatography (eluent, by volume: dichloromethane / methanol / triethylamine = 20:1:0.1) to obtain a brown oily product, namely, intermediate C, i.e., 5.9 g of 9-(piperazine-1-)-5,6,7,8-tetrahydroacridine, with a yield of 78%.

[0107] 1 H NMR(500MHz, CDCl3):8.19(dd,1H),7.86(dd,1H),7.64(td,1H),7.48(ddd,1H ),3.46–3.22(m,4H),3.11–2.94(m,4H),2.96–2.72(m,4H),1.99–1.77(m,5H).

[0108] Example 8: Synthesis of Intermediate D-1—9-(4-(2-hydroxy)ethyl-piperazine-1-)-5,6,7,8-tetrahydroacridine

[0109]

[0110] 2.67 g of intermediate C (9-(piperazine-1-)-5,6,7,8-tetrahydroacridine), 1.3 g of 2-bromoethanol, and 3 g of triethylamine were weighed and mixed in dichloromethane. The mixture was reacted at 50° C. for 3 hours, cooled to room temperature, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent, by volume: dichloromethane / methanol / triethylamine = 15:1:0.1) to obtain 2.1 g of a light yellow oil with a yield of 68%.

[0111] 1 H NMR(500MHz, CDCl3):8.19(dd,1H),7.86(dd,1H),7.64(td,1H),7.48(ddd,1H),3.62(dt,2H),3.32(t ,4H),3.13–3.05(m,1H),3.02–2.80(m,4H),2.71(t,2H),2.66(t,2H),2.55(t,2H),2.00–1.66(m,4H).

[0112] Example 9: Synthesis of Intermediate D-2—9-(4-(3-hydroxy)propyl-piperazine-1-)-5,6,7,8-tetrahydroacridine. The synthesis process was the same as in Example 8, except that 2-bromoethanol in Example 8 was replaced by 3-bromopropanol, with a yield of 65%.

[0113] 1 H NMR(500MHz, CDCl3)8.19(dd,1H),7.86(dd,1H),7.64(td,1H),7.48(ddd,1H),3.82–3.74(m,2H),3.40(t,1H), 3.32(t,4H),2.95–2.90(m,1H),2.93–2.83(m,3H),2.69(dt,4H),2.58(t,2H),1.93–1.80(m,4H),1.61(p,2H).

[0114]

[0115] Example 10: Synthesis of Intermediate D-3

[0116]

[0117] The synthesis process was the same as that of Example 8, except that 9-(piperazine-1-)-5,6,7,8-tetrahydroacridine in Example 8 was replaced by 2-methyl-9-(piperazine-1-)-5,6,7,8-tetrahydroacridine, and 2-bromoethanol was replaced by 3-bromopropanol. The yield was 65%.

[0118] 1H NMR(500MHz, CDCl3):7.87–7.81(m,1H),7.74(d,1H),7.70(dd,1H),3.84–3.72(m,2H),3.40(t,1H),3. 32(t,4H),2.97–2.82(m,4H),2.69(dt,4H),2.58(t,2H),2.46(s,3H),1.99–1.77(m,4H),1.61(p,2H).

[0119] Example 11: Synthesis of target product (II-1)

[0120]

[0121] The intermediate D-13.1 g, 2-bromomethyl-3,5,6-trimethylpyrazine were mixed and dissolved in acetonitrile. 2 g of K2CO3 was added and the mixture was stirred at room temperature for 10 hours. The insoluble matter was filtered and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent, by volume: dichloromethane / methanol / triethylamine = 15:1:0.1) to obtain 2.8 g of a light yellow solid with a yield of 64%.

[0122] 1 H NMR(500MHz, CDCl3):8.19(dd,1H),7.86(dd,1H),7.64(td,1H),7.48(ddd,1H),4.74(s,2H),3.64(t,2H),3 .32(t,4H),2.96–2.83(m,4H),2.75–2.65(m,6H),2.33(s,3H),2.27(s,3H),2.22(s,3H),1.92–1.81(m,4H).

[0123] Example 12: Synthesis of target product (II-2)

[0124]

[0125] The synthesis process is the same as that of Example 10, except that the intermediate D-1 in Example 10 is replaced by intermediate D-2, and 2-bromomethyl-3,5,6-trimethylpyrazine is replaced by 2-bromomethyl-5-methylpyrazine, with a yield of 61%.

[0126] 1H NMR(500MHz, CDCl3):8.27(d,2H),8.17(dd,1H),7.79(dd,1H),7.53–7.39(m,1H),4.70(s,2H),3.65–3.54(m, 2H),3.32(t,4H),2.97–2.79(m,4H),2.70(dt,4H),2.54(t,2H),2.41(s,3H),1.96–1.77(m,4H),1.74(p,2H).

[0127] Example 13: Synthesis of target product (II-3)

[0128]

[0129] The synthesis process is the same as that of Example 10, except that the intermediate D-1 in Example 10 is replaced by intermediate D-3, and 2-bromomethyl-3,5,6-trimethylpyrazine is replaced by 2-bromomethyl-5-methoxypyrazine, with a yield of 61%.

[0130] 1 H NMR(500MHz, CDCl3)8.22(s,1H),7.98(s,1H),7.84–7.83(m,1H),7.74(d,1H),7.72–7.67(m,1H),4.72(s,2H),3.9 1(s,3H),3.63(t,2H),3.32(t,4H),2.96–2.84(m,4H),2.72(dt,4H),2.65(t,2H),2.46(s,3H),1.92–1.80(m,4H).

[0131] Example 14: Synthesis of target product (III-1)

[0132]

[0133] 2.68 g of intermediate A-1 and 2.1 g of 3,5,6-trimethylpyrazine-2-carboxylic acid were weighed into a round-bottom flask. A suitable amount of tetrahydrofuran was added, followed by sonication for dissolution. 1.52 g of EDC·HCl and 0.48 g of DMAP were added. The reaction was placed in an ice-water bath and allowed to react at room temperature for 8-10 hours. The reaction solution was concentrated to dryness under reduced pressure, dissolved in dichloromethane, washed twice with 5% NaHCO₃ solution, twice with brine, dried over anhydrous Na₂SO₄, and filtered and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (eluent: dichloromethane / methanol / triethylamine = 15:1:0.1, by volume) afforded 2.8 g of a pale yellow solid in a 64% yield.

[0134] 1H NMR(500MHz, CDCl3)8.06(dd,1H),7.89(dd,1H),7.64(td,1H),7.48(t,1H),7.39(ddd,1H),6.47(t,1H),3.44–3.27(m, 4H),3.02–2.91(m,2H),2.89–2.77(m,2H),2.61(s,3H),2.33(s,3H),2.26(s,3H),1.97–1.80(m,4H),1.76–1.55(m,4H).

[0135] Example 15: Synthesis of target product (III-2)

[0136]

[0137] The synthesis process was the same as that of Example 13, except that N-(1,2,3,4-tetrahydroacridine-9-)-hexane-1,6-diamine in Example 13 was replaced by N-(1,2,3,4-tetrahydroacridine-6-chloro-9-)-pentane-1,5-diamine, and 3,5,6-trimethylpyrazine-2-carboxylic acid was replaced by 5-methylpyrazine-2-carboxylic acid, with a yield of 72%.

[0138] 1 H NMR(500MHz, CDCl3)8.84(s,1H),8.46(s,1H),7.92(dd,1H),7.83–7.75(m,2H),7.47–7.40(m,1H),6.38(t, 1H),3.37(dq,4H),2.94–2.77(m,4H),2.52(d,3H),1.93–1.81(m,4H),1.67–1.54(m,4H),1.41–1.32(m,2H).

[0139] Example 16: Assay for inhibition of acetylcholinesterase activity

[0140] Take a 1.5 mL PE tube and add 50 μL of acetylcholinesterase solution (0.2 U mL -1 ) and 50 μL sample solution (concentrations from 10 -3 mol·L -1Start), then add 0.1M phosphate buffer solution (pH=8.0) to 850μL. Incubate at 37℃ in a constant temperature oscillator for 15min, add 50μL 3mM iodinated thioacetylcholine (substrate), and incubate at 37℃ in a constant temperature oscillator for ten minutes, add 50μL 3% sodium dodecyl sulfate (SDS) to terminate the reaction, and finally add 50μL DNTB solution for color development, and measure the absorbance (OpticalDelnsity, OD) value of the sample at 412nm. In the standard group, an equal volume of PBS buffer was used to replace the sample solution to be tested, and an equal volume of prepared tacrine solution was used to replace the sample solution in the control group. In the blank group, an equal volume of PBS was used to replace the substrate and the sample solution to be tested. The final concentrations of the inhibitors were 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 - 7 M, 10 -8 M Calculation IC 50 Each compound was tested three times at each concentration, and 5-7 concentrations of the compound were selected to determine the enzyme inhibition rate. The inhibition rate was calculated according to the following formula, and the IC 50 (μM), and the experimental results are listed in Table 1.

[0141]

[0142] Example 17: ADP-induced platelet aggregation activity assay

[0143] Blood was collected from healthy New Zealand white rabbits via abdominal aorta cannulation. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation at 200 g for 10 min at room temperature using 3.8% sodium citrate as anticoagulant (blood to anticoagulant volume ratio of 9:1). 1 μL of target compound solution at various concentrations was precisely pipetted into 200 μL PRP. An equal volume of DMSO was added to the blank control group. After incubation at 37°C for 3 min, the mixture was adjusted to zero with PPP, and 5 μg mL -1 5 μL of adenosine diphosphate (ADP) solution was added and the maximum aggregation rate of PRP was observed within 5 minutes under stirring conditions before and after medication at 37°C. The platelet aggregation inhibition rate was calculated according to the following formula, and the half-maximal inhibitory concentration IC was calculated. 50 (μM), and the experimental results are listed in Table 1.

[0144]

[0145] Table 1 AChE and platelet aggregation inhibitory activities of target compounds

[0146]

[0147] The results in Table 1 show that the AChE and platelet aggregation inhibitory activities of tacrine-pyrazine derivatives obtained after structural modification of tacrine are still retained or slightly decreased. In comparison, compounds I-1 and II-2 show better AChE inhibitory activities, and their IC 50 The values ​​are similar to those of tacrine, which are around 200-300 nM; the inhibition results of platelet aggregation (IC 50 :0.45,0.38mM) are generally better than ligustrazine (0.49mM) and can be used as candidate compounds for further study.

[0148] Example 18: Effects of target products I-1 and II-2 on Tau protein hyperphosphorylation

[0149] SH-SY5Y cells were stimulated with 50 nM okadaic acid (OA) to establish a tau hyperphosphorylation model. Subsequently, SH-SY5Y cells were treated with a range of concentrations (1, 5, 10, 25, 50, 100, and 200 μM) of I-1 and II-2. Cell viability was assessed using CCK-8 assays. At concentrations of 10-50 μM, cell viability generally reached 92-99%.

[0150] Next, SH-SY5Y cells were stimulated with 50 nM O to establish a Tau protein hyperphosphorylation model, and intervention was performed with 25 μM and 50 μM of I-1, II-2, tacrine, ligustrazine, and the positive control drug Rember. ELISA was then used to detect the effects of I-1 and II-2 on the expression levels of p-Tau and Tau in the phosphorylated cell model, as shown in Table 2.

[0151] Table 2 Effects of compounds I-1 and II-2 on phosphorylation levels in SH-SY5Y cells

[0152]

[0153] ELISA test results showed that as the target products I-1 and II-2 acted on SH-SY5Y cells, the relative expression levels of p-Tau / Tau were reduced compared with the OA group, while intervention with tacrine or ligustrazine did not inhibit the hyperphosphorylation of Tau protein, indicating that I-1 and II-2 had a good improvement effect on the VaD-like cell model (P<0.05), and the effects of both were similar to those of the positive control Rember.

[0154] Example 18: Hepatotoxicity study of compounds in HepG2 cell line

[0155] HepG2 cell culture: Incubate in RPMI1640 medium (10% FPS and 1% double antibody) in a 5% CO2, 37% CO2 incubator. Seed the cells into 6- or 96-well plates to control the appropriate cell density.

[0156] Drug solution preparation: Tacrine, target compound I-1, and target compound II-2 were prepared in DMSO to prepare 0.2, 2, 20, and 200 μM stock solutions. During experiments, these solutions were diluted 1:200 (w / v) in culture medium and added to the culture plate. NAC was prepared in water to prepare a 0.5 M stock solution; DCFH-DA was prepared in DMSO to prepare a 10 mM stock solution; and DTNB was prepared in methanol to prepare a 10 mM stock solution. Store in a dark place and at a low temperature until needed.

[0157] (1) Evaluation of intracellular GSH activity

[0158] HepG2 cells were seeded into 6-well plates and cultured in a 5% CO2, 37% CO2 incubator for 24 hours. The old culture medium was discarded, and 1 mL of the NAC stock solution was diluted 100-fold with RPMI1640 medium and added to the corresponding wells as a negative control. Two hours later, 1 mL of each concentration gradient of tacrine was added, with a DMSO concentration of 5‰, and the cells were incubated in a 24-hour incubator. The cells were washed three times with PBS, and 40 μL / well of lysis buffer was added. After lysis on ice for 10 minutes, the cells were centrifuged at 4°C, 12,000 rpm, and 10 minutes. The supernatant was collected for GSH measurement and protein quantification (Coomassie Brilliant Blue method). A separate group was set up, in which the gradient concentrations were added directly without preincubation, but the other procedures were the same.

[0159] In a 96-well plate, add 10 μL of supernatant, 138 μL of tris-base (pH 8.0), and 2 μL of DTNB solution (10 mM) to set up blank control wells (no sample, DTNB). After mixing, shake at 37°C for 10 minutes and measure the absorbance at 412 nm on a microplate reader.

[0160] (2) DCFH-DA method to test ROS levels

[0161] Principle: DCFH-DA (2',7'-dichlorofluorescein diacetate) is used as a fluorescent probe to measure reactive oxygen species (ROS) production. DCFH-DA is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by esterases into DCFH2 (which cannot penetrate the cell membrane), making it easy for the probe to be loaded into the cell. Intracellular ROS can oxidize the non-fluorescent DCFH2 into a fluorescent DCFH2. Fluorescence intensity is measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm to assess intracellular ROS levels.

[0162] ②Methods: After incubation with 20μM DCFH-DA for 30 minutes, the old medium was discarded, the cells were washed twice with PBS, and then incubated with gradient concentrations of tacrine and compounds (I-1) and (II-2) for 3 hours, with a final DMSO concentration of 5‰. A solvent control group was set up. In another group, N-acetylcysteine ​​(NAC) was incubated for 1 hour before the gradient concentration of tacrine to investigate the effect of NAC. Before testing, the old medium was discarded, the cells were carefully washed with PBS, and then PBS or RPMI was added to measure the fluorescence intensity on a fluorescence microplate reader.

[0163] Experimental results: The intracellular ROS level in HepG2 cells increased in a concentration-dependent manner with tacrine (0.1-100 μM). After pre-incubation with NAC for 5 minutes, it could effectively resist the increase in intracellular ROS level. The intracellular GSH level in HepG2 cells decreased in a concentration-dependent manner with tacrine (0.1-100 μM). After pre-incubation with NAC for 5 minutes, the intracellular GSH level was significantly reversed. Compounds I-1 and I-2 did not cause an increase in the intracellular ROS level in HepG2 cells at 0.1-100 μM, and could cause an increase in the intracellular GSH level. Compared with compound I-1, compound II-2 increased GSH by 5.63% and 10.25% at 10 μM and 100 μM. The results of the hepatotoxicity study showed that compound II-2 had lower hepatotoxicity, so in vivo experiments were performed with compound II-2.

[0164] Example 19: Effects on neurological deficit scores, learning and memory, and oxidative damage in VaD rats

[0165] Grouping and Dosing: The VaD rat model was successfully established by permanent ligation of the bilateral carotid arteries. 40 rats with successful modeling were randomly divided into 5 groups, including the model control group, the high-, medium-, and low-dose groups of compound II-2, and the tacrine positive control group, each with 8 rats. If a rat died during the experiment, the number of rats was supplemented under parallel conditions. The positive control group: 10 mg·kg -1 ·d -1 The dose of tacrine raw material was gavaged with normal saline solution; compound II-2 was set up in high, medium and low dose groups: 5 mg·kg -1 ·d -1 , 10mg·kg -1 ·d -1 , 20mg·kg -1 ·d -1 The model control group and sham operation group were given the same amount of normal saline by gavage once a day for 28 days.

[0166] (1) Effect of Compound II-2 on Neurological Function Deficit Scoring in VaD Rats

[0167] After 28 days of drug administration, neurological deficit scores were performed.

[0168] Neurological deficits were scored according to the 5-level scoring method of ZeaLonga et al. The higher the score, the more severe the animal's behavioral disorder:

[0169] Level 1 (0 points): No signs of neurological deficit.

[0170] Level 2 (1 point): The lateral limb cannot be fully flexed.

[0171] Grade 3 (2 points): Limb weakness when the rat is pulled backward by its tail.

[0172] Level 4 (3 points): The rat spins in circles while dragging its tail.

[0173] Level 5 (4 points): Spontaneously spinning or falling over, unable to spontaneously walk.

[0174] (2) Study on the effects of compound II-2 on learning, memory, and oxidative damage in VaD rats

[0175] A radial eight-arm maze was used to investigate the spatial working memory and reference memory abilities of SD rats. Rats were fasted for 12 hours prior to each maze test, receiving only water and no food, to eliminate the effects of satiation. The entire experiment was conducted in a quiet environment to prevent noise from influencing the results.

[0176] Training period: Before training, rats were first adapted to the maze for 2 days, once a day. During the adaptation period, 3 to 4 rats were placed in the eight-arm maze at the same time, free to move and ingest food for 10 minutes. Subsequently, training was conducted twice a day, each time for 10 minutes, for a total of 7 days. During each training session, only 4 of the eight arms (1, 3, 5, and 7) were set up as working arms with 40-60 mg peanut bait, and the rest were reference arms. During the experiment, the rats were placed in the central area of ​​the maze. After 15 seconds, the small openings around them were opened. The rats could choose any arm to ingest food. If the rat entered the working arm and ingested food, it was a correct choice. Otherwise, it was an incorrect choice. The training was considered successful if the number of incorrect choices was 1 or 0 after 5 consecutive training sessions.

[0177] Test period: After preliminary training, modeling and drug administration, rats in each group began the eight-arm maze test at a fixed time before drug administration on the 28th to 29th day according to the preliminary training method.

[0178] The following parameters of rats were recorded in each experiment: re-entry into the control arm was called working memory error (WME), and entry into the non-control arm was called reference memory error (RME). WME and RME were recorded for each rat in each experiment within 10 minutes.

[0179] After the behavioral experiment of SD rats was completed, blood was collected from the abdominal aorta 2 hours after administration the next day, and PRP and PPP were prepared for ADP-induced platelet aggregation rate experiments.

[0180] After blood was collected from the abdominal aorta, the rats were quickly placed on an ice table and decapitated to remove the brain. The brain was homogenized under ice bath conditions and centrifuged. The supernatant was used for the determination of acetylcholinesterase, SOD, and MDA.

[0181] Table 3 lists the neurological deficit scores in VaD rats after 28 days of continuous drug administration. Compared with the sham-operated group, the neurological deficit scores in the model control group were significantly different (P < 0.01), indicating successful model establishment. Neurological deficits in the high- and medium-dose groups II-2 and the positive control group were significantly improved (P < 0.05). Neurological deficits in the low-dose group improved somewhat, but not statistically significantly (P > 0.05) (see Table 4-1).

[0182] Table 3II-2 Neurological deficit scores of rats after administration

[0183]

[0184] * P < 0.05 vs model group;

[0185] Table 4 lists the behavioral test results of VaD rats, and Tables 5 and 6 list the experimental results of rat acetylcholinesterase activity and platelet aggregation activity.

[0186] Table 4 Behavioral effects of compound II-2 on VaD rats

[0187]

[0188] * P<0.05, ** P<0.01 vs model group

[0189] On day 28 of continuous oral administration in VaD rats, the model control group showed significant differences in the total number of memory errors, WMEs, and RMEs compared to the sham-operated group. Compared to the model control group, the high-, medium-, and low-dose groups, as well as the positive control group, significantly reduced the total number of errors and RMEs. The low-dose group showed no significant reduction in WMEs. These results suggest that compound II-2 has a potent effect on improving cognitive function in VaD rats.

[0190] Table 5 Effects of compound II-2 on ACh content and AChE activity in the cerebral cortex of VaD rats

[0191]

[0192]

[0193] * P<0.05, ** P<0.01 vs model group

[0194] The results in Table 5 show that compared with the sham operation group, the AChE activity in the hippocampus of the model control group rats was significantly increased and the ACh content was significantly decreased; the high and medium dose groups of compound II-2 and the positive control group could significantly reduce the ACh activity and increase the ACh content in the hippocampus of the model rats; there was no significant difference in the low dose group.

[0195] Similarly, the effect of compound II-2 on ADP-induced platelet aggregation in VaD rats is shown in Table 6. The results in Table 6 show that there was no significant difference in platelet aggregation in the model control group compared with the sham group; compound II-2 in the high, medium, and low dose groups significantly inhibited ADP-induced platelet aggregation (P < 0.01, P < 0.05).

[0196] Table 6 Effect of compound II-2 on ADP-induced platelet aggregation in VaD rats

[0197]

[0198] * P<0.05, ** P<0.01 vs model group

[0199] Table 7 lists the effects of compound II-2 intervention on brain MDA content and SOD activity in VaD rats. Compared with the sham-operated group, the MDA content in hippocampal tissue homogenates of the model group was significantly increased, while SOD activity was significantly decreased (P < 0.01). MDA content in brain tissue of rats in the high- and medium-dose groups was significantly decreased, while SOD activity was significantly increased. There were no significant differences in SOD activity or MDA in the low-dose group (P > 0.05).

[0200] Table 7 Effects of compound II-2 on brain MDA content and SOD activity in VaD rats

[0201]

[0202] * P<0.05, ** P<0.01 vs model group

[0203] Example 20: Study on blood-brain barrier permeability of target product II-2

[0204] The experimental procedure was as follows: 72 Kunming mice, half male and half female, weighing 20±2g, were randomly divided into 12 groups, with 6 mice in each group. The first 6 groups served as the tacrine control group, which was administered with a 5mg / kg dose via tail vein injection. The last 6 groups served as the test group for the target compound II-2, which was administered with a 10.9mg / kg dose via tail vein injection (equimolar dose to tacrine). Plasma and brain tissue were collected from each group at 5min, 15min, 30min, 60min, 90min, and 180min after administration. The brain tissue was weighed, and 9 times its weight of normal saline was added to prepare a homogenate, which was then frozen at -80°C for later use.

[0205] Accurately aspirate 200 μL of plasma or brain homogenate, add 20 μL of internal standard solution, vortex mix, add 1 mL of methanol and vortex mix for 5 minutes for extraction, centrifuge at 4000 rpm for 10 minutes, take 1 μL of the supernatant and inject it into HPLC-MS, record the chromatogram and the peak areas of II-2, tacrine (As) and internal standard (Ai), and calculate the drug content in plasma and brain homogenate according to the linear equation.

[0206] The blood-brain barrier permeability of a drug is expressed as logBB. The definition of logBB is: the logarithm of the ratio of the drug concentration in the brain to the blood concentration when the drug distribution reaches a steady state. The mathematical expression is:

[0207] logBB =log( C brain C blood )

[0208] The blood and brain drug concentration data of II-2 at different time periods after administration are shown in Table 8.

[0209] After tail vein injection of II-2 in mice, drug concentrations in plasma and brain homogenates were calculated and found to be stable within 15 to 90 minutes after administration, with an average logBB of -1.10. Under similar conditions, the average logBB of tacrine was -1.25. These results indicate that the conversion of tacrine to a tacrine-pyrazine derivative maintains a relatively high blood-brain barrier penetration ability. The compound's metabolic rate in plasma is slowed, allowing for prolonged blood concentrations. Furthermore, the compound is rapidly transported from the blood into the brain and retained there for an extended period, suggesting its potential for effective brain targeting.

[0210] Table 8 Blood-brain barrier permeability study of target compound II-2

[0211]

[0212] Example 21: Acute toxicity test of target product II-2 in mice

[0213] Experimental animals: Kunming mice, weighing 18-22 g, half male and half female, were used for the experiment after adaptive feeding for 3-4 days.

[0214] Drug preparation: dissolve the target (II-2) in PEG 400 / EtOH, the maximum solubility can reach 180mg / ml.

[0215] Experimental method: The target product (II-2) (500, 350, 245, 172, 120, 84, 58.8, 41.2 mg / kg) was injected into the tail vein of mice respectively. The injection volume was calculated as 0.1 ml / 20 g body weight. The drugs were administered by single tail vein injection of equal volume and different concentrations. The appearance, mental state, behavior, food intake and other toxic reactions and death distribution of the animals were observed and recorded every day. The mice were observed continuously for 14 days.

[0216] Experimental results: The median lethal dose (LD50) was calculated using the Bliss method. 50 ) and its 95% confidence limit, the LD of the target product (II-2) injected into the tail vein of mice 50 The toxicity of the target product (II-2) is relatively low and its clinical use is relatively safe.

Claims

1. A tacrine-pyrazine derivative, characterized in that A compound selected from the following structures: 。 2. The use of the tacrine-pyrazine derivative according to claim 1, characterized in that: The tacrine-pyrazine derivative is used to prepare a pharmaceutical preparation, which is an AChE inhibitor and / or an antiplatelet aggregation drug and / or a Tau protein hyperphosphorylation inhibitor.

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