CN117050057A - Tacrine-pyrazine derivative and application thereof - Google Patents

Abstract

The invention discloses a tacrine-pyrazine derivative and application thereof, wherein the structure of the tacrine-pyrazine derivative is represented by the following general formula (I), general formula (II) and general formula (III):

Description

A tacrine-pyrazine derivative and its use

Technical Field

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

Background Art

Vascular dementia (VaD) is a brain dysfunction syndrome caused by cerebrovascular disease, characterized by cognitive impairment, psychiatric and behavioral symptoms, and decreased ability to live. Its incidence is second only to Alzheimer's disease (AD), and its risk and mortality are higher than AD. It has seriously affected the quality of life of patients and their families, and brought great psychological and economic burdens to patients. At present, there is no specific drug for the diagnosis and treatment of VaD. The "2019 Guidelines for the Diagnosis and Treatment of Vascular Cognitive Impairment in China (Full Version)" recommends the use of acetylcholinesterase inhibitors (AChEI), excitatory amino acid receptor (NDMA) antagonists, and calcium ion antagonists (such as nimodipine, etc.). In addition, antioxidants and traditional Chinese medicines (such as ginkgo and Chuanxiong) are used in combination to improve cognitive function, increase cerebral blood flow, and protect neurons. In summary, it is a dual treatment for dementia behavior 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 antiplatelet aggregation has been widely recognized.

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 However, it was withdrawn from the market due to liver toxicity caused by long-term use. Many studies have been conducted to reduce its liver toxicity and enhance or maintain its acetylcholinesterase activity. Tacrine derivatives such as thiol-tacrine hybrids, tacrine-caffeic acid mixtures, coumarin-tacrine, tacrine-diphenylesters, etc. are designed by modifying the groups or connecting active groups. [Novel tacrine-related drugs as potential candidates for the treatment of AD Alzheimer’s disease. Bioorg. Med. Chem. Lett., 2013, 23: 1916-1922; Research progress on multi-target drug design based on tacrine and donepezil and its anti-Alzheimer’s disease activity [Chinese Journal of Pharmacy, 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 multi-target and multi-action mechanism drug design, providing ideas and basis for the design of new anti-VaD drugs.

Ligustrazine is a traditional Chinese medicine commonly used in the clinical treatment of cerebrovascular diseases. It belongs to the pyrazine alkaloids and has the effects of dilating blood vessels, increasing arterial blood flow, and inhibiting platelet aggregation [Experimental study on the anti-platelet aggregation of ligustrazine. Laboratory Medicine, 2014, 9(9): 976-977]. In addition, ligustrazine injection can improve the learning and memory of rats with vascular dementia model [Study on the improvement of learning and memory of rats with vascular dementia model by ligustrazine injection 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

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, so as to better protect neurons and improve learning and memory impairment. It has the characteristics of low toxicity, high blood-brain barrier penetration rate, and easy preparation.

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, protecting neurons, etc., and can be applied to the prevention and treatment of VaD.

The tacrine-pyrazine derivative of the present invention is a composition in which tacrine and bromomethylpyrazine are connected via an alkyl diamine, and its structure is represented by the general formula (I):

In the general formula (I), R ₁ , R ₂ , R ₃ and R ₄ each independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ ; R ₅ , R ₆ and R ₇ each independently represent -H, -Me or -MeO, and n=1-7.

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

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), and continuing the reaction for 2 hours when the temperature rises to 120° C. to obtain intermediate A;

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

Here, R ₁ , R ₂ , R ₃ , and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO ₂ .

The general structural formula of the alkyl diamine is:

Where n=1~7.

The general structural formula of the intermediate A is:

The bromination reaction is to add substituted 2-methylpyrazine and N-bromosuccinimide (NBS) in a molar ratio of 1:0.9 to CCl ₄ , reflux for 1-3 hours under 100W light, and fractionate under vacuum to obtain brominated intermediate B;

The substituted 2-methylpyrazine has the general structural formula:

wherein R ₅ , R ₆ and R ₇ independently represent -H, -Me or -MeO.

The general structural formula of the intermediate B is:

wherein R ₅ , R ₆ and R ₇ independently represent -H, -Me or -MeO.

The substitution reaction is to add 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, reflux under stirring for 8-10 hours, and separate and purify by column chromatography (dichloromethane/methanol=20:1-15:1, V/V) to obtain the target product (I).

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

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

The reaction route is as follows:

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):

In the general formula (II), R ₁ , R ₂ , R ₃ and R ₄ each independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ ; R ₅ , R ₆ and R ₇ each independently represent -H, -Me or -MeO, and m=1 to 3.

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

The piperazine amination reaction is to mix benzene ring substituted 9-chloro-1,2,3,4-tetrahydroacridine, sodium iodide and phenol in a molar ratio of 1:0.2:(10-15), react at 90°C for 2 hours; then add 7 molar equivalents of piperazine (based on 9-chloro-1,2,3,4-tetrahydroacridine), wait for the temperature to rise to 120°C, and continue to react for 2 hours to obtain intermediate C;

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

Here, R ₁ , R ₂ , R ₃ , and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO, and -NO ₂ .

The general structural formula of the intermediate C is:

wherein R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ .

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;

The general structural formula of the bromohydrin is:

Among them, m=1-3.

The general structural formula of the intermediate D is:

wherein R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ ; and m=1-3.

The bromomethylpyrazine substitution reaction is to add intermediate D, substituted bromomethylpyrazine (intermediate B) and 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).

The general structural formula of the substituted bromomethylpyrazine is:

wherein R ₅ , R ₆ and R ₇ independently represent -H, -Me or -MeO.

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

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

The reaction route is as follows:

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):

In the general formula (III), R ₁ , R ₂ , R ₃ and R ₄ each independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ .

R ₅ and R ₆ each independently represent -H, -Br, -Cl, -Me or -MeO, and n=1-7.

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

The amination reaction is to mix benzene ring substituted 9-chloro-1,2,3,4-tetrahydroacridine, sodium iodide and phenol in a molar ratio of 1:0.2:10, react at 90°C for 2 hours, then add 7 molar equivalents of alkyl diamine (calculated as 9-chloro-1,2,3,4-tetrahydroacridine), raise the temperature to 120°C, and continue the reaction for 2 hours to obtain intermediate A.

The general structural formula of the intermediate A is:

wherein R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO or -NO ₂ , and n=1-7.

The amidation reaction is to add the 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, react at room temperature for 15-20 hours, and separate and purify by column chromatography (dichloromethane/methanol = 15:1-10:1, V/V) to obtain the target product (III).

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

R ₅ and R ₆ each independently represent -H, -Br, -Cl, -Me or -MeO.

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

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

The reaction route is as follows:

The use of the tacrine-pyrazine derivatives represented by the general formula (I), general formula (II) and general formula (III) of the present invention is to prepare a pharmaceutical preparation using the tacrine-pyrazine derivatives. The pharmaceutical preparation has multiple effects such as inhibiting AChE, resisting platelet aggregation, inhibiting Tau protein hyperphosphorylation, protecting neurons, and improving learning and memory impairment, and can be used as an improved AChE inhibitor or antiplatelet aggregation drug for the prevention and/or treatment of VaD.

The tacrine-pyrazine derivatives designed by the present invention show good AChE inhibition and anti-platelet aggregation effects, and also have the function of inhibiting Tau protein hyperphosphorylation, especially the derivatives of tacrine and pyrazine connected by alkyldiamine or piperazine (target products (I), (II)) have more prominent activity, and their AChE inhibitory activity is almost equivalent to that of tacrine, and enhances or retains the anti-platelet aggregation effect of ligustrazine. In addition, their Tau protein hyperphosphorylation inhibitory activity is almost equivalent to that of the positive control drug Rember (Tau protein aggregation inhibitor, Phase III clinical trial). More importantly, they have weak hepatotoxicity and high blood-brain barrier permeability.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. Compared with the existing drugs tacrine or ligustrazine (tetramethylpyrazine), the tacrine-pyrazine derivatives of the present invention retain or enhance the AChE inhibitory activity and antiplatelet aggregation activity, and the hepatotoxicity (intrinsic to tacrine) is greatly reduced. More importantly, this type of compound also exhibits a strong inhibitory activity on Tau protein hyperphosphorylation, and can ultimately intervene in the neuronal functional defects and learning and memory impairment caused by VaD in multiple ways.

2. The tacrine-pyrazine derivatives of the present invention have a 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 use of VaD.

DETAILED DESCRIPTION

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

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, then 18.3 g of 1.6-hexanediamine was added, the temperature was raised to 120°C and reacted for 2 hours, after the reaction was completed, the mixture was cooled to room temperature, 10 wt% NaOH solution was added to adjust the pH value to 9-10, and the mixture was extracted with dichloromethane twice, the two extracts were combined, washed with water, washed with saturated NaCl, dried with anhydrous Na ₂ SO ₄ , separated and purified by silica gel column chromatography (eluent by volume ratio: dichloromethane/methanol/triethylamine=15:1:0.1), and concentrated to obtain a brown oily product, which is intermediate A-1—N-(1,2,3,4-tetrahydroacridine-9-)-hexane-1,6-diamine 4.8 g, with a yield of 70%.

¹ H NMR (500MHz, CDCl ₃ )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,2H ),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 ).

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

Weigh 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, mix them, heat at 90°C for reaction for one hour, then add 6.16 g of 1.4-butanediamine, heat to 120°C for reaction for 2 hours, cool to room temperature after the reaction, add 10 wt % NaOH solution to adjust the pH value to 9-10, extract twice with dichloromethane, combine the two extracts, wash with water, wash with saturated NaCl, dry with anhydrous Na ₂ SO ₄ , separate and purify with silica gel column chromatography (eluent by volume ratio: 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%.

¹ H NMR (500MHz, CDCl ₃ )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).

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

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

¹ H NMR (500MHz, CDCl ₃ )4.54(s,2H),2.31(s,3H),2.23(s,3H),2.22(s,3H).

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

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

¹ H NMR (500MHz, CDCl ₃ )8.23(s,1H),7.98(s,1H),4.44(s,2H),3.91(s,3H).

Example 5: Synthesis of target product I-1

Weigh 6.4 g of intermediate A-1, 4.3 g of intermediate B-1, and 3 g of triethylamine, mix them in dichloromethane, react at 50°C for 5 hours, cool to room temperature, and concentrate under reduced pressure to obtain a crude product. Separate and purify by silica gel column chromatography (eluent by volume ratio: 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%.

¹ H NMR (500MHz, CDCl ₃ )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),2.90–2.75(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).

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

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%.

¹ H NMR (500MHz, CDCl ₃ )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).

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

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, then 17.6 g of piperazine was added, the temperature was raised to 120°C for reaction for 2 hours, cooled to room temperature after the reaction, 10 wt% NaOH solution was added to adjust the pH value to 9-10, extracted twice with dichloromethane, combined the two extracts, washed with water, washed with saturated NaCl, dried with anhydrous Na ₂ SO _4, separated and purified by silica gel column chromatography (eluent by volume ratio: dichloromethane/methanol/triethylamine=20:1:0.1) to obtain a brown oily product, which is intermediate C, i.e. 5.9 g of 9-(piperazine-1-)-5,6,7,8-tetrahydroacridine, with a yield of 78%.

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).

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

Weigh 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, mix them in dichloromethane, react at 50°C for 3 hours, cool to room temperature, and concentrate under reduced pressure to obtain a crude product, which is separated and purified by silica gel column chromatography (eluent by volume ratio: dichloromethane/methanol/triethylamine = 15:1:0.1) to obtain 2.1 g of a light yellow oil with a yield of 68%.

¹ H NMR (500MHz, CDCl ₃ ): 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).

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

¹ H NMR (500MHz, CDCl ₃ )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).

Example 10: Synthesis of Intermediate D-3

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

¹ H NMR (500MHz, CDCl ₃ ):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 ).

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

The intermediate D-13.1g, 2-bromomethyl-3,5,6-trimethylpyrazine were mixed and dissolved in acetonitrile, 2g K ₂ CO ₃ 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, which was separated and purified by silica gel column chromatography (eluent by volume: dichloromethane/methanol/triethylamine = 15:1:0.1) to obtain 2.8g of a light yellow solid with a yield of 64%.

¹ H NMR (500MHz, CDCl ₃ ):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).

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

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%.

¹ H 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).

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

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%.

¹ H NMR (500MHz, CDCl ₃ )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),3.91(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).

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

Weigh 2.68g of intermediate A-1 and 2.1g of 3,5,6-trimethylpyrazine-2-carboxylic acid into a round-bottom flask, add a certain amount of tetrahydrofuran, dissolve by ultrasonication, add 1.52g of EDC·HCl and 0.48g of DMAP, put into an ice-water bath for reaction, and then react at room temperature for 8-10 hours. After the reaction solution is concentrated to dryness under reduced pressure, it is dissolved in dichloromethane, washed twice with 5% NaHCO ₃ solution, washed twice with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a crude product. Separate and purify by silica gel column chromatography (eluent by volume ratio: dichloromethane/methanol/triethylamine = 15:1:0.1), and obtain 2.8g of light yellow solid with a yield of 64%.

¹ H NMR (500MHz, CDCl ₃ )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).

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

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

¹ H NMR (500MHz, CDCl ₃ )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).

Example 16: Assay for inhibition of acetylcholinesterase activity

Take a PE tube with a volume of 1.5mL, add 50μL of acetylcholinesterase solution (0.2U·mL ^-1 ) and 50μL of sample solution (concentration starts from 10 ^-3 mol·L _-1 ) to the tube, and then add 0.1M phosphate buffer solution (pH＝8.0) to 850μL. Incubate at 37℃ in a constant temperature oscillator for 15min, add 50μL of 3mM iodinated thioacetylcholine (substrate), and then incubate at 37℃ in a constant temperature oscillator for ten minutes, add 50μL of 3% sodium dodecyl sulfate (SDS) to terminate the reaction, and finally add 50μL of 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 tacrine solution was used to replace the sample solution in the control group, and an equal volume of PBS was used to replace the substrate and the sample solution to be tested in the blank group. _IC50 was calculated at the final concentrations of the inhibitors of ^10-3 M, ^10-4 M, ^10-5 M ^{, 10-6} M, ^10-7 M, and ^10-8 M. Each compound was tested three times at each concentration, and 5-7 concentrations of the compound were selected to determine the inhibition rate of the enzyme. The inhibition rate was calculated according to the following formula, and the _IC50 (μM) was calculated. The experimental results are listed in Table 1.

Example 17: ADP-induced platelet aggregation activity assay

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

Table 1 AChE and platelet aggregation inhibitory activities of target compounds

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

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

SH-SY5Y cells were stimulated with 50 nM okadaic acid (OA) to establish a Tau protein hyperphosphorylation cell model. Subsequently, a series of concentrations (1, 5, 10, 25, 50, 100, 200 μM) of I-1 and II-2 were used to act on SH-SY5Y cells, and cell viability was detected by CCK-8. It was found that when the compound concentration was 10-50 μM, the cell survival rate generally reached 92-99%;

Next, SH-SY5Y cells were stimulated with 50 nM OAs to establish a Tau protein hyperphosphorylation model, and intervened with 25 μM and 50 μM of I-1, II-2, tacrine, ligustrazine and the positive control drug Rember. Afterwards, the ELISA method was 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.

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

The results of ELISA test 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 the intervention of tacrine or ligustrazine did not inhibit the hyperphosphorylation of Tau protein, which indicated 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.

Example 18: Hepatotoxicity study of compounds in HepG2 cell line

HepG2 cell culture: Incubate in RPMI1640 medium (10% FPS and 1% double antibody) in a 5% CO ₂ , 37% incubator. Inoculate cells into 6- or 96-well plates to control the appropriate cell density.

Preparation of drug solution: Tacrine, target compound I-1, and target compound II-2 were prepared into 0.2, 2, 20, and 200 μM stock solutions using DMSO as solvent. During the experiment, the culture medium was diluted at 1:200 (w/v) and added to the culture plate; NAC was prepared into 0.5M stock solution using water as solvent; DCFH-DA was prepared into 10mM stock solution using DMSO as solvent; DTNB was prepared into 10mM stock solution using methanol as solvent. Store in a dark and low temperature for later use.

(1) Evaluation of intracellular GSH activity

HepG2 cells were inoculated into 6-well plates and cultured in a 5% CO ₂ , 37% incubator for 24 hours. The old culture medium was discarded, and the NAC stock solution was diluted 100 times with RPMI1640 culture medium, and 1 mL was added to the corresponding wells to set up a negative control. After 2 hours, 1 mL of each concentration of tacrine was added, and DMSO was controlled at 5‰, and placed in the incubator for 24 hours. The cells were washed 3 times with PBS, and 40 μL/well of lysis buffer was added. After lysis on ice for 10 minutes, centrifuged at 4°C and 12000 r/min for 10 minutes. The supernatant was taken for GSH determination and protein quantification (Coomassie Brilliant Blue method). Another group was set up, and the other operations were the same as those for adding gradient concentrations directly without pre-incubation.

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

(2) DCFH-DA method to test ROS levels

① Principle: DCFH-DA (2', 7'-dichlorofluorescein diacetate) is used as a fluorescent probe to test the generation of reactive oxygen species (ROS). DCFH-DA is non-fluorescent and can freely pass through the cell membrane. Once inside the cell, it can be hydrolyzed by esterase into DCFH ₂ (which cannot penetrate the cell membrane), so that the probe can be easily loaded into the cell. ROS inside the cell can oxidize non-fluorescent to fluorescent. The level of ROS inside the cell can be tested by measuring the fluorescence intensity at an excitation wavelength of 485nm and an emission wavelength of 530nm.

②Methods: After incubation with DCFH-DA 20 μM for 30 minutes, discard the old culture medium, wash the cells twice with PBS, and incubate with gradient concentrations of tacrine and compounds (I-1) (II-2) for 3 hours, and control the final DMSO concentration to 5‰. Set up a solvent control group. Set up another group, incubate with N-acetylcysteine (NAC) for 1 hour before giving gradient concentrations of tacrine, and investigate the effect of NAC. Before the test, discard the old culture medium, carefully wash with PBS, and add PBS or RPMI to test the fluorescence intensity under a fluorescence microplate reader.

Experimental results: The intracellular ROS level of HepG2 cells increased in a concentration-dependent manner with tacrine (0.1-100 μM). After pre-incubation with NAC for 5 minutes, the increase in intracellular ROS level can be effectively resisted. The intracellular GSH level of 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 of 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.

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

Grouping and administration: The VaD rat model was successfully established by permanent bilateral carotid artery ligation. 40 successfully modeled rats were randomly divided into 5 groups, including the model control group, compound II-2 high, medium and low dose groups, and tacrine positive control group, with 8 rats in each group. If a rat died during the experiment, the number of rats was supplemented under parallel conditions. Positive control group: gavage with tacrine raw material saline solution at a dose of 10 mg·kg ^-1 ·d ^-1 ; compound II-2 high, medium and low dose groups: 5 mg·kg ^-1 ·d ^-1 , 10 mg·kg ^-1 ·d ^-1 , 20 mg·kg ^-1 ·d ^-1 ; model control group and sham operation group: given an equal amount of saline. Gavage once a day for 28 consecutive days.

(1) Effect of Compound II-2 on neurological deficits in VaD rats

After 28 days of drug administration, neurological deficits were scored.

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

Grade 1 (0 points): No signs of neurological deficit.

Grade 2 (1 point): The lateral limb cannot be fully flexed.

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

Level 4 (3 points): The rat spins in circles while being pulled behind by its tail.

Level 5 (4 points): Spontaneous spinning or falling over, unable to walk spontaneously.

(2) Effects of compound II-2 on learning, memory and oxidative damage in VaD rats

The radial eight-arm maze was used to study the spatial working memory and reference memory ability of SD rats. Rats were fasted for 12 hours before the maze experiment each day, only drinking water but not eating, to eliminate the interference of animal satiation. The entire experiment was carried out in a quiet state to prevent noise from affecting the experimental results.

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 take 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, only 4 arms (1, 3, 5, and 7) of the eight arms were set 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 were opened. The rats could choose any arm to take food. If the rats entered the working arm and took food, it was a correct choice. Otherwise, it was a wrong choice. After 5 consecutive trainings, the number of wrong choices was 1 or 0, and the training was considered successful.

Test period: After preliminary training, modeling and medication were performed in each group of rats. According to the preliminary training method, the eight-arm maze test was started at a fixed time before medication on the 28th to 29th day.

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.

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 the ADP-induced platelet aggregation rate experiment.

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

Table 3 lists the neurological deficit scores of VaD rats after continuous administration for 28 days. Compared with the sham operation group, the neurological deficit score of the model control group was significantly different (P<0.01), indicating that the model was successfully established; the neurological deficits of the high and medium dose groups II-2 and the positive control group were significantly improved (P<0.05); the neurological deficits of the low dose group were improved but not statistically significant (P>0.05) (see Table 4-1).

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

^* P<0.05vs model group;

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

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

^* P<0.05, ^** P<0.01 vs model group

On the 28th day of continuous oral administration of VaD rats, the total number of memory errors, WMEs and RMEs in the model control group were significantly different from those in the sham operation group; compared with the model control group, the high, medium, low dose groups and the positive control group could significantly reduce the total number of errors and RMEs; the WME number in the low dose group did not decrease significantly. The results showed that compound II-2 has a good effect on improving the cognitive function of VaD rats.

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

^* P<0.05, ^** P<0.01 vs model group

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 in the hippocampus of the model rats and increase the ACh content; there was no significant difference in the low dose group.

Similarly, the effect of compound II-2 on ADP-induced platelet aggregation rate in VaD rats is listed in Table 6. From the results in Table 6, it can be seen that compared with the sham operation group, there was no significant difference in platelet aggregation rate in the model control group; the high, medium and low dose groups of compound II-2 could significantly inhibit ADP-induced platelet aggregation (P < 0.01, P < 0.05).

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

^* P<0.05, ^** P<0.01 vs model group

Table 7 lists the effects of compound II-2 intervention on the MDA content and SOD activity in the brain of VaD rats. Compared with the sham operation group, the MDA content in the hippocampal tissue homogenate of the model group rats was significantly increased, and the SOD activity was significantly decreased (P<0.01). The MDA content in the brain tissue of rats in the high and medium dose groups was significantly reduced, and the SOD activity was significantly enhanced. There was no significant difference in SOD activity and MDA in the low dose group (P>0.05).

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

^* P<0.05, ^** P<0.01 vs model group

Example 20: Study on the blood-brain barrier permeability of target product II-2

The experimental process is as follows: 72 Kunming mice, half male and half female, with a body weight range of 20±2g, were randomly divided into 12 groups, with 6 mice in each group. The first 6 groups were the tacrine control group, which was injected with 5 mg/kg of tail vein, and the last 6 groups were the target compound II-2 test group, which was injected with 10.9 mg/kg of tail vein (equimolar dose with tacrine). After administration, one group was taken from each group at 5min, 15min, 30min, 60min, 90min, and 180min respectively to obtain plasma and brain tissue. The brain tissue was weighed, and 9 times the weight of physiological saline was added to make a homogenate, which was frozen at -80℃ for later use.

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 min for extraction, centrifuge at 4000 rpm for 10 min, take 1 μL of 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.

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 drug concentration in the blood when the drug distribution reaches a steady state. The mathematical expression is:

logBB =log( C _brain C _blood )

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

After II-2 was injected into mice through the tail vein, the drug concentrations detected in plasma and brain homogenate were calculated and found to be in a steady state within 15 to 90 minutes after administration, with an average value of -1.10. Under similar conditions, the average logBB of tacrine was -1.25; the above results show that after tacrine was transformed into a tacrine-pyrazine derivative, its ability to penetrate the blood-brain barrier is still relatively high. The metabolic rate of the compound in plasma is slowed down, and the blood drug concentration can be maintained for a long time; at the same time, the compound is rapidly transported from the blood to the brain and retained in the brain for a long time, and it may also have a good brain targeting effect.

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

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

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

Drug preparation: The target (II-2) was dissolved in PEG ₄₀₀ /EtOH, and the maximum solubility could reach 180 mg/ml.

Experimental method: The target product (II-2) (500, 350, 245, 172, 120, 84, 58.8, 41.2 mg/kg) was injected through 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 for 14 consecutive days.

Experimental results: The median lethal dose (LD ₅₀ ) and its 95% confidence limit were calculated using the Bliss method. The LD ₅₀ of the target product (II-2) injected into the tail vein of mice was 318.23 mg/kg, and the 95% confidence interval was (228.46-575.28) mg/kg. This indicates that the target product (II-2) is relatively less toxic and relatively safe for clinical use.

Claims (9)

1. A tacrine-pyrazine derivative, characterized in that: The tacrine-pyrazine derivative is a composition in which tacrine and bromomethylpyrazine are connected through an alkyl diamine, and its structure is represented by the following general formula (I): In the general formula (I), R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO and -NO ₂ ; R ₅ , R ₆ , R ₇ each independently represents -H, -Me, -MeO, n=1 to 7. 2. The use of the tacrine-pyrazine derivative according to claim 1, characterized in that: The tacrine-pyrazine derivative is used to prepare pharmaceutical preparations, which are AChE inhibitors and/or anti-platelet aggregation drugs. 3. Use according to claim 2, characterized in that: The pharmaceutical preparation has multiple effects of inhibiting AChE, anti-platelet aggregation, inhibiting Tau protein hyperphosphorylation, protecting neurons, and improving impaired learning and memory. 4. A tacrine-pyrazine derivative, characterized in that: The tacrine-pyrazine derivative is a composition in which tacrine and bromomethylpyrazine are connected through piperazine-alkyl alcohol, and its structure is represented by the following general formula (II): In the general formula (II), R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO and -NO ₂ ; R ₅ , R ₆ , R ₇ independently represents -H, -Me, -MeO, m=1 to 3. 5. The use of the tacrine-pyrazine derivative according to claim 4, characterized in that: The tacrine-pyrazine derivative is used to prepare pharmaceutical preparations, which are AChE inhibitors and/or anti-platelet aggregation drugs. 6. Use according to claim 5, characterized in that: The pharmaceutical preparation has multiple effects of inhibiting AChE, anti-platelet aggregation, inhibiting Tau protein hyperphosphorylation, protecting neurons, and improving impaired learning and memory. 7. A tacrine-pyrazine derivative, characterized in that: The tacrine-pyrazine derivative is an amide composition in which tacrine and pyrazine carboxylic acid are connected through an alkyldiamine, and its structure is represented by the following general formula (III): In the general formula (III), R ₁ , R ₂ , R ₃ and R ₄ independently represent -H, -F, -Cl, -Br, -Me, -MeO and -NO ₂ ; R ₅ and R ₆ respectively represent Independently represents -H, -Br, -Cl, -Me, -MeO, n=1~7. 8. The use of the tacrine-pyrazine derivative according to claim 7, characterized in that: The tacrine-pyrazine derivative is used to prepare pharmaceutical preparations, which are AChE inhibitors and/or anti-platelet aggregation drugs. 9. Use according to claim 8, characterized in that: The pharmaceutical preparation has multiple effects of inhibiting AChE, anti-platelet aggregation, inhibiting Tau protein hyperphosphorylation, protecting neurons, and improving impaired learning and memory.