Hetero-diad compound taking indanone-tetrahydrothieno [3, 2-c] pyridine as matrix as well as preparation method and application of hetero-diad compound

Abstract

The invention discloses a hetero-diad compound taking indanone-tetrahydrothieno [3, 2-c] pyridine as a parent body and a preparation method and application thereof, the hetero-diad compound taking indanone-tetrahydrothieno [3, 2-c] pyridine as the parent body is selected from the compounds shown in the following general formulas (I), (II) and (III), the compound takes indanone of donepezil as a parent nucleus, and the compound is selected from the compounds shown in the general formulas (I), (II) and (III). And the compound is combined with an anti-platelet functional group tetrahydrothieno [3, 2-c] pyridine through a linking group. The compound has multiple action mechanisms (inhibiting AChE, resisting Abeta aggregation, resisting platelet aggregation and improving learning and memory impairment) and high blood-brain barrier permeability, and is suitable for preventing and treating vascular dementia.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis, and in particular relates to a heterodimer compound with indanone-tetrahydrothieno[3,2-c]pyridine as a parent, and a preparation method and application thereof. Background Art

[0002] Vascular dementia (VD) is a dementia syndrome caused by cerebrovascular disease (primarily vascular obstruction), with the primary clinical manifestation being memory impairment. Its core pathological mechanism is cerebral ischemia, defined as obstruction of blood flow to specific brain regions, leading to a significant decrease in cerebral blood flow (CBF). The onset and progression of VD involves the complex interaction and accumulation of multiple pathogenic factors and pathological processes at multiple levels and pathways, representing a dynamic evolutionary process. Specific mechanisms include, but are not limited to, cholinergic system dysfunction caused by cerebrovascular disease, persistently decreased CBF, neuronal apoptosis, neuroinflammatory responses, abnormal accumulation of β-amyloid protein (Aβ), and altered hippocampal microenvironment. These factors work together to ultimately lead to cognitive impairment in patients. Current clinical treatments primarily utilize drugs such as acetylcholinesterase inhibitors (AChEIs), N-methyl-D-aspartate receptor (NMDA) receptor antagonists, and calcium antagonists, or a combination of these drugs, in an effort to improve cognitive function, increase cerebral blood flow, and protect neurons. However, the overall effectiveness of existing therapies remains to be improved.

[0003] Donepezil is a second-generation specific reversible central acetylcholinesterase (AChE) inhibitor with higher selectivity and specificity for central cholinesterase. It is a mature clinical first-line drug for the treatment of Alzheimer's disease and mental illness. It is usually used to treat mild or moderate Alzheimer's disease. It has few adverse reactions and is a well-tolerated drug. However, it still has the disadvantages of requiring long-term medication and the need for continuous increase in drug dosage. Donepezil has a reversible inhibitory effect on central acetylcholinesterase without peripheral activity that may cause systemic adverse reactions. Its pharmacodynamics, pharmacokinetics and safety indicators are excellent, and it is well tolerated. The main adverse reactions are mild gastrointestinal reactions and cardiovascular reactions. Mishra et al. [1] Curcumin-donepezil hybrids were synthesized by linking the indone structure with various piperazine substituents via methylene groups, among which the hybrid IP-15 (eeAChE = 0.025 µmoL / L) showed better AChE inhibitory activity than donepezil; Wang et al. [2]A series of AChE inhibitors were designed and synthesized by introducing substituents on the benzene ring, among which compound 5c showed significant AChE inhibition (eeAChE=0.085µmoL / L) and Aβ aggregation inhibition ([1]MISHRA CB, KUMARI S, MANRAL A, et al. Design, synthesis, in-silico and biological evaluation of novel donepezilderivatives as multi-target-directed ligands for the treatment of Alzheimer's disease [J]. European journal of medicinal chemistry, 2017, 125: 736-750.[2]WANG ZM, CAI P, LIU QH, et al. Rational modification of donepezil asmultifunctional acetylcholinesterase inhibitors for the treatment of Alzheimer's disease [J]. European journal of medicinal chemistry, 2016, 123:282-297.).

[0004] Adenosine diphosphate (ADP) receptor inhibitors are primarily used for the preventive treatment of patients with acute coronary syndrome (ACS) or at risk for thromboembolism, myocardial infarction, or stroke. These drugs antagonize the P2Y12 platelet receptor, preventing the binding of ADP to the P2Y12 receptor, thereby reducing platelet aggregation and preventing thrombus formation. Clopidogrel and prasugrel, both members of this class of thienopyridine compounds, exhibit irreversible inhibition of P2Y12. They are prodrugs that must be converted into active metabolites in vivo to inhibit the P2Y12 receptor. The active metabolites of clopidogrel and prasugrel contain a highly reactive free sulfhydryl group that can form a covalent disulfide bond (-SS-) with specific cysteine ​​residues (primarily Cys97) on the platelet surface P2Y12 ADP receptor. This covalent binding is irreversible, resulting in permanent inhibition of the receptor, thereby blocking the ADP-mediated platelet activation and aggregation pathway. Although the thiol group is the part directly involved in covalent binding, the overall structure of the active metabolite, namely the modified thienopyridine skeleton, is crucial for identifying and locating the specific binding pocket on the P2Y12 receptor. This structural skeleton determines the selectivity of the active metabolite for the P2Y12 receptor, enabling it to specifically bind to the position containing the target cysteine ​​residue, creating conditions for the covalent reaction of the thiol group. It may participate in non-covalent binding such as hydrogen bonding and hydrophobic interactions, assisting the correct positioning of drug molecules. Liu Ying et al. [3] Starting from the structure of clopidogrel, we modified its structure and designed and synthesized a series of new compounds. We tested the activity of these compounds through animal experiments. Among them, the inhibition rates of compounds 6-8 on platelet aggregation in rats were 62.3%, 64.2% and 66.8% respectively; [4] In the study, the thienopyridine ring core was retained, and a piperazine-type hydrophobic side chain was introduced to improve bioavailability. Nine new thienotetrahydropyridine derivatives were designed and synthesized, and the biological activities of these compounds were tested through animal experiments. The results showed that all compounds had certain antiplatelet aggregation activity, among which compounds 9 and 10 had better platelet inhibition activity, with inhibition rates of 75.9% and 67.4%, respectively ([3] Liu Ying, Chen Ligong, Liao Shangteng, et al. Synthesis of new N-substituted tetrahydrothieno[3,2-c]pyridine derivatives and their antiplatelet aggregation activity [J]. Synthetic Chemistry, 2013, 21(04): 387-392. [4] Zhi Shuang, Zheng Guo, Liu Ying, et al. Synthesis of new piperazine-containing thienotetrahydropyridine derivatives and their antiplatelet aggregation activity [J]. Synthetic Chemistry, 2011, 19(6): 730-733.).

[0005] The goal of site-binding the main pharmacophores of two effective drugs, donepezil and a thienopyridine P2Y12 receptor antagonist, is to achieve dual acetylcholine inhibition and antiplatelet aggregation, inhibit β-amyloid protein aggregation, and fight neuroinflammation, while also possessing high blood-brain barrier penetration and ease of preparation. Therefore, we considered site-binding the acetylcholinesterase inhibitory indanones and piperidinones, and the antiplatelet aggregation drug tetrahydrothieno[3,2-c]pyridine, both of which are effective in improving cerebral circulation, to achieve a 1+1>2 effect. Summary of the Invention

[0006] The present invention aims to provide a heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine as a parent compound, a preparation method and application thereof. The technical problem to be solved is to select a suitable molecular structure so that it has better acetylcholinesterase (AChE) inhibition, anti-platelet aggregation activity, inhibition of β-amyloid protein aggregation and improvement of learning and memory impairment, and has the characteristics of high blood-brain barrier penetration rate and easy preparation.

[0007] The heterodimer compound of the present invention is a composition composed of indanone and tetrahydrothieno[3,2-c]pyridine connected by an alkyl group and a phenoxy group, and its structure is represented by the following general formula (I):

[0008] ;

[0009] wherein R1 and R2 independently represent -H, -OH or -OCH3.

[0010] The present invention provides a method for preparing a heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine, comprising the following unit processes: Williamson ether synthesis reaction, aldol condensation reaction, Hofmann alkylation reaction, and post-treatment:

[0011] Step 1: p-Hydroxybenzaldehyde or m-hydroxybenzaldehyde is placed in an acetone solution containing potassium carbonate, followed by the dropwise addition of 1,3-dibromopropane. The mixture is refluxed at 56°C for 4-5 hours. After completion of the reaction, the mixture is filtered, and the filtrate is evaporated under reduced pressure. The mixture is purified by silica gel column chromatography (methanol / dichloromethane = 1:20, v / v) to obtain a yellow oil, which is intermediate 1a or 2a.

[0012] The structure of the p- / m-hydroxybenzaldehyde is shown below:

[0013] .

[0014] The structures of the intermediates 1a and 2a are shown below:

[0015] .

[0016] In step 1, the molar ratio of p-hydroxybenzaldehyde or m-hydroxybenzaldehyde to 1,3-dibromopropane is 1:1-1.5, for example, 1:1, 1:1.2, or 1:1.5; wherein the best reaction effect is achieved when the molar ratio is 1:1.2.

[0017] Step 2: Dissolve the substituted 5,6-dimethoxy-1-indanone and intermediate 1a or 2a in ethanol. Slowly add 2.5 mol / L NaOH solution (10% w / w) dropwise to the solution in an ice-water bath while stirring. Remove the ice-water bath and stir at room temperature for 48 hours. After the reaction, quench the reaction mixture with dilute hydrochloric acid until the pH is neutral. Filter the precipitate, wash it with ethanol, and dry the solid product under vacuum to obtain intermediate 1b or 2b.

[0018] The structure of the substituted 5,6-dimethoxy-1-indanone is shown below:

[0019] .

[0020] The structures of the intermediates 1b and 2b are shown below:

[0021] .

[0022] wherein R1 and R2 independently represent -H, -OH or -OCH3.

[0023] In step 2, the molar ratio of intermediate 1a or 2a to 5,6-dimethoxy-1-indanone is 1:1-1.3; when the molar ratio is 1:1.2, the reaction effect is the best.

[0024] Step 3: Tetrahydrothieno[3,2-c]pyridine hydrochloride and intermediate 1b or 2b are placed in a round-bottom flask containing an organic solvent, and an equimolar amount of base is added. The mixture is stirred at 40°C for 6 h. After the reaction, the solvent is evaporated, the mixture is washed with water, extracted with chloroform, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography to obtain the target product (I).

[0025] The structure of the tetrahydrothieno[3,2-c]pyridine hydrochloride is shown below:

[0026] .

[0027] In step 3, the molar ratio of tetrahydrothieno[3,2-c]pyridine hydrochloride to intermediate 1b or 2b is 1:1-1.5, such as 1:1, 1:1.2, and 1:1.5; wherein the reaction effect is best when the molar ratio is 1:1.2.

[0028] In step 3, the base is selected from alkali metal carbonates, alkali metal bicarbonates, triethylamine or pyridine; among them, alkali metal carbonates are preferred, and in particular, anhydrous potassium carbonate has the best reaction effect.

[0029] In step 3, the organic solvent is selected from one or more of acetonitrile, ethanol, tetrahydrofuran, acetone, and dichloromethane, among which acetone has the best effect.

[0030] In step 3, the eluent used for purification by silica gel column chromatography is methanol: dichloromethane = 1:20 (v / v).

[0031] The reaction route is as follows:

[0032] .

[0033] The second heterodimer compound provided by the present invention is a composition composed of indanone and tetrahydrothieno[3,2-c]pyridine hydrochloride connected by a phenoxy group and a methyl chain with a carbonyl group, and its structure is represented by the following general formula (II):

[0034] .

[0035] Wherein R1 and R2 independently represent -H, -OH or -OCH3; n=1~3.

[0036] The present invention provides a method for preparing a heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine, comprising the following unit processes: halogenation reaction, Williamson ether synthesis reaction, Hofmann alkylation reaction, and post-treatment:

[0037] Step 1: Place 5,6-dimethoxy-1-indanone and copper bromide in a round-bottom flask containing ethyl acetate and chloroform, heat and reflux for 2-2.5 hours, filter, decolorize with activated carbon for 10 minutes, and concentrate to obtain intermediate A.

[0038] The structure of the intermediate A is shown below:

[0039] .

[0040] wherein R1 and R2 independently represent -H, -OH, and -OCH3.

[0041] In step 1, the molar ratio of 5,6-dimethoxy-1-indanone to copper bromide is 1:1.5-1:2, such as 1:1.5, 1:1.8, and 1:2, wherein the reaction effect is best when the molar ratio is 1:1.8; and the volume ratio of ethyl acetate to chloroform is 1:1.

[0042] Step 2: Tetrahydrothieno[3,2-c]pyridine hydrochloride and (2 / 3 / 4)-chloro-4'-hydroxyphenyl(ethyl / propyl / butyl)ketone were placed in a round-bottom flask containing an organic solvent. An equimolar amount of base was added and the mixture was stirred at 40°C for 6 h. After completion of the reaction, the solvent was evaporated, the mixture was washed with water, extracted with chloroform, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography (methanol / dichloromethane = 1:1) to obtain a yellow oil, which is intermediate 3b, 4b, or 5b.

[0043] The structure of the (2 / 3 / 4)-chloro-4'-hydroxyphenyl(ethyl / propyl / butyl)ketone is shown below:

[0044] n=1~3.

[0045] The structure of the intermediate 3b, 4b or 5b is shown below:

[0046] n=1~3.

[0047] In step 2, the molar ratio of tetrahydrothieno[3,2-c]pyridine hydrochloride to (2 / 3 / 4)-chloro-4'-hydroxyphenyl(ethyl / propyl / butyl)ketone is 1:1-1.5, such as 1:1, 1:1.2, and 1:1.5; wherein the reaction effect is best when the molar ratio is 1:1.2.

[0048] In step 2, the base is selected from alkali metal carbonates, alkali metal bicarbonates, triethylamine or pyridine; among them, alkali metal carbonates are preferred, and in particular, anhydrous potassium carbonate has the best reaction effect.

[0049] In step 2, the organic solvent is selected from one or more of acetonitrile, ethanol, tetrahydrofuran, acetone, and dichloromethane, among which acetone has the best effect.

[0050] In step 2, the eluent used for purification by silica gel column chromatography is petroleum ether / ethyl acetate = 1:1 (v / v).

[0051] Step 3: Intermediate 3b, 4b, or 5b is placed in an acetone solution containing potassium carbonate, followed by the addition of Intermediate A. The reaction mixture is refluxed at 56°C for 4-5 hours. After completion of the reaction, the mixture is filtered, and the filtrate is evaporated under reduced pressure. The mixture is washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography (methanol / dichloromethane = 1:1, v / v) to obtain a yellow oily substance, which is the target product (II).

[0052] In step 3, the molar ratio of intermediate A to intermediate 3b, 4b or 5b is 1:1-1.5, such as 1:1, 1:1.2, or 1:1.5; wherein the best reaction effect is achieved when the molar ratio is 1:1.2.

[0053] The reaction route is as follows:

[0054] .

[0055] The third heterodimer compound provided by the present invention is a composition composed of piperidindanone and tetrahydrothieno[3,2-c]pyridine hydrochloride modified at the α-position connected by a methylene group and a carbonyl group, and its structure is represented by the following general formula (III):

[0056] .

[0057] Wherein R1 and R2 independently represent -H, -OH or -OCH3; R3 represents -OCOCH3, -OCOCH=CHCH3 or -OCOCH=CHCH2CH3; and n=1~3.

[0058] The present invention provides a method for preparing a heterodimer compound based on piperidinone-tetrahydrothieno[3,2-c]pyridine, comprising the following unit processes: amide condensation reaction, Hofmann alkylation reaction, esterification reaction, and post-treatment:

[0059] Step 1: 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one hydrochloride and triethylamine were placed in a round-bottom flask containing tetrahydrofuran. The mixture was cooled to 0-5°C in an ice bath. The acid chloride was added thereto. The reaction mixture was stirred at room temperature for 1 hour and filtered. The filtrate was rotary evaporated, washed with water, and extracted with ethyl acetate. The ethyl acetate layer was separated, washed with brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The mixture was purified by silica gel column chromatography (methanol / dichloromethane = 1:12, v / v) to obtain a yellow oil, which was intermediate B1.

[0060] The structure of the 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one hydrochloride is shown below:

[0061] .

[0062] The structure of the intermediate B1 is shown below:

[0063] .

[0064] In step 1, the molar ratio of 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one hydrochloride and acyl chloride is 1:1.2-1:1.5, such as 1:1.2, 1:1.4, and 1:5, wherein the reaction effect is best when the molar ratio is 1:1.2; and the volume ratio of ethyl acetate to chloroform is 1:1.

[0065] Step 2: Tetrahydrothieno[3,2-c]pyridine hydrochloride and intermediate B1 were placed in a round-bottom flask containing an organic solvent. An equimolar amount of base was added and the mixture was stirred at 40°C for 6 h. After the reaction, the solvent was evaporated, the mixture was washed with water, extracted with chloroform, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography (methanol / dichloromethane = 1:12, v / v) to obtain intermediate B2.

[0066] The structure of the intermediate B2 is shown below:

[0067] .

[0068] In step 2, the molar ratio of tetrahydrothieno[3,2-c]pyridine hydrochloride to intermediate B1 is 1:1-1.5, such as 1:1, 1:1.2, and 1:1.5; wherein the reaction effect is best when the molar ratio is 1:1.2.

[0069] In step 2, the base is selected from alkali metal carbonates, alkali metal bicarbonates, triethylamine or pyridine; among them, alkali metal carbonates are preferred, and in particular, anhydrous potassium carbonate has the best reaction effect.

[0070] In step 2, the organic solvent is selected from one or more of acetonitrile, ethanol, tetrahydrofuran, acetone, and dichloromethane, among which acetone has the best effect.

[0071] In step 2, the eluent used for purification by silica gel column chromatography is methanol:dichloromethane = 1:15 (v / v).

[0072] Step 3: Place intermediate B2 in a round-bottom flask containing dichloromethane, and add triethylamine, EDCI, DMAP, glacial acetic acid / crotonic acid / (E)-pent-2-enoic acid in sequence. Stir at 20-25°C for 4-5 hours, evaporate the solvent under reduced pressure, wash with water, extract with dichloromethane, dry over anhydrous magnesium sulfate, filter, evaporate the filtrate under reduced pressure, and purify by silica gel column chromatography to obtain product (III).

[0073] In step 3, the molar ratio of intermediate B2 to glacial acetic acid / crotonic acid / (E)-pent-2-enoic acid is 1:1-1.5, such as 1:1, 1:1.2, and 1:1.5; among which, when the molar ratio is 1:1.2, the reaction effect is the best.

[0074] In step 3, the eluent used for purification by silica gel column chromatography is methanol:dichloromethane = 1:15 (v / v).

[0075] The reaction route is as follows:

[0076] .

[0077] The invention discloses an application of a heterodimer compound with indanone-tetrahydrothieno[3,2-c]pyridine as a parent in the preparation of a pharmaceutical preparation.

[0078] The pharmaceutical preparation has both acetylcholinesterase inhibition and platelet aggregation effects, and further has beta-amyloid protein aggregation inhibition and oxidative stress resistance effects, thereby more effectively preventing and treating vascular dementia.

[0079] Compared with combined medication, the drug preparation has increased blood-brain barrier permeability and has better brain targeting effect, which is conducive to reducing the dosage of the drug and reducing the metabolic load and safety risks of long-term medication.

[0080] The present invention is based on an indanone structure with acetylcholinesterase inhibition and tetrahydrothieno[3,2-c]pyridine with antiplatelet aggregation effect, and is designed through different connecting groups to have the dual effects of acetylcholinesterase inhibition and antiplatelet aggregation, can inhibit β-amyloid protein aggregation, resist oxidative stress, and has the purposes of high blood-brain barrier penetration rate and easy preparation.

[0081] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0082] 1. Compared with the existing drugs donepezil or clopidogrel, the indanone-tetrahydrothieno[3,2-c]pyridine derivative of the present invention retains the cholinesterase inhibitory effect and anti-platelet aggregation effect of the two parent core structures. In addition, it also increases the inhibition of β-amyloid protein aggregation and anti-oxidative stress effects, thereby more effectively preventing and treating vascular dementia.

[0083] 2. Compared with existing combination drugs, the piperidinone-tetrahydrothieno[3,2-c]pyridine derivatives of the present invention exhibit better pharmacokinetic properties, such as increased blood-brain barrier permeability and better brain targeting, which is conducive to reducing the dosage of the drug and is more conducive to reducing the burden on metabolic organs and potential toxicity risks caused by long-term drug use. DETAILED DESCRIPTION

[0084] Example 1: Synthesis of Intermediate 1a - 4-(3-bromopropoxy)benzaldehyde

[0085]

[0086] 5 mmol of p-hydroxybenzaldehyde was placed in an acetone solution containing 6 mmol of potassium carbonate. 6 mmol of 1,3-dibromopropane was added with stirring, and the mixture was refluxed at 56°C for 4 h. After the reaction, the reaction was filtered, and the filtrate was evaporated under reduced pressure. The product was washed with water, extracted with dichloromethane, and concentrated. The product was purified by column chromatography (eluent: dichloromethane / methanol = 20:1 by volume) to obtain a yellow oil, which was intermediate 1a, with a yield of 70%.

[0087] 1 H NMR (600 MHz, CDCl3) δ 2.10 (2H, tt, J = 7.4, 6.5 Hz), 3.17 (2H,t, J = 6.5 Hz), 4.22 (2H, t, J = 7.4 Hz), 7.05 (2H, ddd, J = 8.3, 1.2, 0.4Hz), 7.96 (2H, ddd, J = 8.3, 1.6, 0.4 Hz), 9.92 (1H, s).

[0088] Example 2: Synthesis of Intermediate 2a - 3-(3-bromopropoxy)benzaldehyde

[0089]

[0090] The synthesis method is the same as that in Example 1, except that the p-hydroxybenzaldehyde in Example 1 is replaced by m-hydroxybenzaldehyde, and the yield is 62%.

[0091] 1 H NMR (600 MHz, CDCl3) δ 2.10 (2H, tt, J = 7.4, 6.5 Hz), 3.17 (2H,t, J = 6.5 Hz), 4.22 (2H, t, J = 7.4 Hz), 7.05 (2H, ddd, J = 8.3, 1.2, 0.4Hz), 7.96 (2H, ddd, J = 8.3, 1.6, 0.4 Hz), 9.92 (1H, s).

[0092] Example 3: Synthesis of Intermediate 1b - (E)-2-(4-(3-bromopropyloxy)benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0093]

[0094] 5,6-Dimethoxy-1-indanone and intermediate 1a (substance ratio 1:1.2) were dissolved in ethanol. A 10% w / w aqueous sodium hydroxide solution was added dropwise to the solution with stirring. The reaction mixture was stirred at room temperature for 48 hours. The precipitate was filtered and washed with 50% v / v aqueous ethanol. The solid product was dried under vacuum to obtain a pale yellow powder, intermediate 1b, in a 65% yield.

[0095] 1H NMR (600 MHz, CDCl3) δ 2.10 (2H, tt, J = 7.5, 6.5 Hz), 3.17 (2H,t, J = 6.5 Hz), 3.56-3.79 (5H, 3.63 (d, J = 15.0 Hz), 3.74 (s)), 3.94 (3H,s), 4.25 (2H, t, J = 7.5 Hz), 6.74 (1H, d, J = 0.4 Hz), 7.10-7.26 (4H, 7.16(ddd, J = 8.8, 1.8, 0.5 Hz), 7.19 (ddd, J = 8.8, 1.2, 0.5 Hz)), 7.43 (1H, d,J = 0.4 Hz), 7.65 (1H, s).

[0096] Example 4: Synthesis of Intermediate 2b - (E)-2-(3-(3-bromopropyloxy)benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0097]

[0098] The synthesis method is the same as that of Example 3, except that the intermediate 1a in Example 3 is replaced by intermediate 2a, and the yield is 62%.

[0099] 1 H NMR (600 MHz, CDCl3) δ 2.09 (2H, tt, J = 7.5, 6.5 Hz), 3.16 (2H,t, J = 6.5 Hz), 3.69-3.85 (5H, 3.74 (s), 3.78 (d, J = 15.0 Hz)), 3.94 (3H,s), 4.08 (2H, t, J = 7.5 Hz), 6.74 (1H, d, J = 0.4 Hz), 6.96 (1H, dt, J =8.2, 1.4 Hz), 7.29 (1H, ddd, J = 8.2, 7.5, 0.5 Hz), 7.37-7.60 (3H, 7.44 (ddd,J = 7.5, 1.7, 1.4 Hz), 7.44 (d, J = 0.4 Hz), 7.54 (ddd, J = 1.7, 1.4, 0.5Hz)), 7.67 (1H, s).

[0100] Example 5: Synthesis of Intermediate A - 2-Bromo-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0101]

[0102] Place 5,6-dimethoxy-1-indanone and copper bromide in a round-bottom flask containing 25 mL of ethyl acetate / chloroform (volume ratio 1:1). Heat and reflux for 2-2.5 hours. Filter, decolorize with activated carbon for 10 minutes, and concentrate to obtain intermediate A with a yield of 72%.

[0103] 1 H NMR (600 MHz, CDCl3) δ 3.36 (2H, dd, J = 15.7, 6.1 Hz), 3.73 (3H,s), 3.96 (3H, s), 5.55 (1H, dd, J = 8.1, 4.1 Hz), 6.81 (1H, d, J = 0.5 Hz),7.46 (1H, d, J = 0.5 Hz).

[0104] Example 6: Synthesis of Intermediate 3b - 2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-1-(4-hydroxyphenyl)ethan-1-one

[0105]

[0106] 2-Chloro-4'-hydroxyacetophenone was added to a round-bottom flask containing dichloromethane. Triethylamine was added with stirring, and 4,5,6,7-tetrahydrothiophen[3,2-c]pyridine hydrochloride was added portionwise. After the addition was complete, the reaction was refluxed for 6 h. After the reaction, the solvent was evaporated under reduced pressure, the product was washed with water, extracted with dichloromethane, and concentrated. The light yellow oil was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1:1 by volume) to obtain intermediate 3b in a yield of 68%.

[0107] 1 H NMR (600 MHz, CDCl3) δ 2.74-2.99 (4H, 2.82 (ddd, J = 10.5, 7.0,3.0 Hz), 2.91 (ddd, J = 14.2, 7.1, 2.9 Hz)), 3.40 (2H, d, J = 13.0 Hz), 3.74(2H, s), 6.93 (2H, ddd, J = 8.3, 1.1, 0.4 Hz), 7.07 (1H, d, J = 6.1 Hz), 7.29(1H, d, J = 6.1 Hz), 8.00 (2H, ddd, J = 8.3, 1.8, 0.4 Hz).

[0108] Example 7: Synthesis of Intermediate 4b - 3-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-1-(4-hydroxyphenyl)propan-1-one

[0109]

[0110] The synthesis method is the same as that of Example 7, except that the 2-chloro-4'-hydroxyacetophenone in Example 7 is replaced by 3-chloro-1-(4-hydroxyphenyl)propan-1-one, with a yield of 65%.

[0111] 1 H NMR (600 MHz, CDCl3) δ 2.51 (2H, t, J = 3.9 Hz), 2.70 (2H, ddd, J= 13.5, 7.0, 3.0 Hz), 2.82-2.98 (4H, 2.90 (ddd, J = 14.2, 7.1, 2.9 Hz), 2.92(t, J = 3.9 Hz)), 3.37 (2H, d, J = 11.4 Hz), 6.91-7.12 (3H, 6.98 (ddd, J =8.3, 1.1, 0.4 Hz), 7.06 (d, J = 6.1 Hz)), 7.29 (1H, d, J = 6.1 Hz), 7.99 (2H,ddd,J = 8.3, 1.8, 0.4 Hz).

[0112] Example 8: Synthesis of Intermediate 5b - 4-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-1-(4-hydroxyphenyl)butanone

[0113]

[0114] The synthesis method is the same as that of Example 6, except that the 2-chloro-4'-hydroxyacetophenone in Example 7 is replaced with 4-chloro-4'-hydroxybutylphenyl ketone, and the yield is 61%.

[0115] 1H NMR (600 MHz, CDCl3) δ 0.77 (6H, q, J = 8.1 Hz), 1.92 (2H, tt, J =7.4, 2.7 Hz), 2.42-2.54 (2H, 2.48 (t, J = 7.4 Hz), 2.48 (t, J = 7.4 Hz)),2.61-2.77 (4H, 2.69 (ddd, J = 11.5, 7.0, 3.0 Hz), 2.69 (t, J = 2.7 Hz)), 2.90(2H, ddd, J = 14.2, 7.1, 2.9 Hz), 3.38 (2H, d, J = 11.9 Hz), 6.97 (1H, ddd, J= 8.3, 1.1, 0.4 Hz), 7.18 (2H, d, J = 6.1 Hz), 7.48 (2H, ddd, J = 8.3, 1.1, 0.8 Hz), 7.99 (1H, ddd, J = 8.3, 1.8, 0.4 Hz).

[0116] Example 9: Synthesis of Intermediate B1 - 5-(2-chloroacetyl)-5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one

[0117]

[0118] Dissolve 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one and triethylamine in an ice-cold solution of tetrahydrofuran. Add chloroacetyl chloride and stir the reaction mixture at room temperature for 1 hour. After the reaction, filter and evaporate the solvent under reduced pressure. Wash with water, extract with ethyl acetate, and concentrate to obtain a pale yellow oil, Intermediate B1, in a 69% yield.

[0119] 1 H NMR (600 MHz, CDCl3) δ 2.01 (2H, dddd, J = 13.5, 6.8, 6.6, 2.8Hz), 3.46 (2H, dddd, J = 13.8, 6.6, 2.8 Hz), 4.03-4.21 (4H, 4.10 (d, J = 17.0Hz), 4.16 (s)), 4.49 (1H, dd, J = 10.2, 3.4 Hz), 6.02 (1H, s).

[0120] Example 10: Synthesis of Intermediate B2 - 5-(2-(4-(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)acetyl)-5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one

[0121]

[0122] Piperidinone and Intermediate 6a were placed in a round-bottom flask containing acetone. Potassium carbonate was added and the mixture was allowed to react at 40°C for 12 hours. After completion of the reaction, the solvent was evaporated, the mixture was washed with water, extracted with chloroform, and dried over anhydrous sodium sulfate. Purification by column chromatography (eluent: methanol / dichloromethane = 1:15, v / v) afforded Intermediate B2 as a yellow oil in a 66% yield.

[0123] 1H NMR (600 MHz, CDCl3) δ 1.42-1.72 (5H, 1.50 (dtd, J = 12.8, 10.2,2.6 Hz), 1.50 (dtd, J = 12.8, 10.2, 2.6 Hz), 1.54 (ttt, J = 10.2, 7.0, 3.3Hz), 1.64 (dddd, J = 12.8, 3.3, 2.9, 2.7 Hz), 1.64 (dddd, J = 12.8, 3.3, 2.9,2.7 Hz)), 1.86 (1H, dtd, J = 13.5, 10.2, 2.9 Hz), 2.15 (1H, dddd, J = 13.5,3.4, 3.0, 2.7 Hz), 2.32-2.65 (6H, 2.39 (ddd, J = 14.3, 2.9, 2.6 Hz), 2.39(ddd, J = 14.3, 2.9, 2.6 Hz), 2.52 (ddd, J = 14.3, 10.3, 2.7 Hz), 2.52 (ddd,J = 14.3, 10.3, 2.7 Hz), 2.59 (dd, J = 7.0, 6.1 Hz), 2.59 (dd, J = 7.0, 6.1Hz)), 3.05-3.36 (3H, 3.13 (dd, J = 15.7, 8.1 Hz), 3.16 (dd, J = 15.7, 4.1Hz), 3.29 (ddd, J = 13.8, 2.9, 2.7 Hz)), 3.46 (1H, dtd, J = 8.1, 6.1, 4.1Hz), 3.53-3.78 (5H, 3.58 (s), 3.58 (s), 3.68 (ddd, J = 13.8, 10.3, 3.0 Hz),3.73 (s)), 3.83-4.00 (4H, 3.88 (s), 3.95 (s)), 4.12 (2H, d, J = 16.3 Hz),4.49 (1H, dd, J = 10.2, 3.4 Hz), 6.01 (1H, s), 6.69 (1H, d, J = 0.4 Hz), 7.44(1H, d, J = 0.4 Hz).

[0124] Example 11: Synthesis of target product C1 - ((E)-2-(4-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-ylpropoxy)benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0125]

[0126] 4,5,6,7-Tetrahydrothieno[3,2-c]pyridine, intermediate 1b, and potassium carbonate were mixed in acetonitrile. The reaction mixture was stirred at room temperature for 18 h, filtered, and the filtrate was evaporated under reduced pressure, washed with water, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The target product C1 was isolated and purified by column chromatography (eluent: methanol / dichloromethane = 1:20, v / v) to obtain the target product C1.

[0127] Yellow solid, yield 60%, molecular formula C 28 H 31 NO4S, theoretical relative molecular mass 477.2, molecular ion peak m / z 478.20 is [MH] - .

[0128] 1 H NMR (600 MHz, CDCl3) δ 1.96 (2H, quint, J = 2.7 Hz), 2.56 (2H, t,J = 2.7 Hz), 2.69 (2H, ddd, J = 11.4, 7.0, 3.0 Hz), 2.90 (2H, ddd, J = 14.2,7.1, 2.9 Hz), 3.37 (2H, d, J = 11.8 Hz), 3.61 (2H, d, J = 15.0 Hz), 3.74 (3H,s), 3.94 (3H, s), 4.17 (2H, t, J = 2.7 Hz), 6.74 (1H, d, J = 0.4 Hz), 7.01-7.35 (6H, 7.06 (d, J = 6.1 Hz), 7.16 (ddd, J = 8.8, 1.8, 0.5 Hz), 7.19 (ddd,J = 8.8, 1.2, 0.5 Hz), 7.29 (d, J = 6.1 Hz)), 7.43 (1H, d, J = 0.4 Hz), 7.65(1H, s).

[0129] Example 12: Synthesis of target product C2 - (E)-2-(3-(3-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-ylpropoxy)benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0130]

[0131] The synthesis process is the same as that of Example 1, except that the intermediate 1b in Example 1 is replaced by intermediate 2b.

[0132] Yellow solid, yield 65%, molecular formula C 28 H 31 NO4S, theoretical relative molecular mass 475.18, molecular ion peak m / z 476.19 is [MH] - .

[0133] 1H NMR (600 MHz, CDCl3) δ 1.95 (2H, tt, J = 7.4, 2.7 Hz), 2.55 (2H,t, J = 2.7 Hz), 2.69 (2H, ddd, J = 11.4, 7.0, 3.0 Hz), 2.90 (2H, ddd, J =14.2, 7.1, 2.9 Hz), 3.37 (2H, d, J = 11.8 Hz), 3.69-3.85 (5H, 3.74 (s), 3.78(d, J = 15.5 Hz)), 3.89-4.05 (5H, 3.94 (s), 3.99 (t, J = 7.4 Hz)), 6.74 (1H,d, J = 0.4 Hz), 6.90-7.12 (2H, 6.96 (dt, J = 8.2, 1.4 Hz), 7.06 (d, J = 6.1Hz)), 7.22-7.36 (2H, 7.29 (ddd, J = 8.2, 7.5, 0.5 Hz), 7.29 (d, J = 6.1 Hz)), 7.37-7.60 (3H, 7.44 (ddd, J = 7.5, 1.7, 1.4 Hz), 7.44 (d, J = 0.4 Hz), 7.54(ddd, J = 1.7, 1.4, 0.5 Hz)), 7.67 (1H, s).

[0134] Example 13: Synthesis of target compound C3 - 2-(4-(2-(6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetyl)phenoxy)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0135]

[0136] Intermediate A and intermediate 3b were placed in a round-bottom flask containing acetonitrile and refluxed in the presence of potassium carbonate for 2-3 h. After completion of the reaction, the solvent was evaporated under reduced pressure, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1:1, v / v, 3%-5% dichloromethane as solubilizer) to obtain the target product C3.

[0137] Yellow solid, yield 61%, molecular formula C 26 H 25 NO5S, theoretical relative molecular mass 463.15, molecular ion peak m / z 464.15 is [MH] - .

[0138] 1 H NMR (600 MHz, CDCl3) δ 2.74-2.99 (4H, 2.82 (ddd, J = 10.5, 7.0,3.0 Hz), 2.91 (ddd, J = 14.2, 7.1, 2.9 Hz)), 3.24-3.46 (4H, 3.32 (dd, J =15.7, 6.1 Hz), 3.40 (d, J = 13.1 Hz)), 3.68-3.79 (5H, 3.73 (s), 3.74 (s)), 3.96 (3H, s), 5.90 (1H, dd, J = 8.1, 4.1 Hz), 6.83 (1H, d, J = 0.5 Hz), 6.96-7.12 (3H, 7.02 (ddd, J = 8.3, 1.2, 0.4 Hz), 7.07 (d, J = 6.1 Hz)), 7.29 (1H,d, J = 6.1 Hz), 7.47 (1H, d, J = 0.5 Hz), 8.00 (2H, ddd, J = 8.3, 1.8, 0.4Hz).

[0139] Example 14: Synthesis of target compound C4 - 2-(4-(3-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-ylpropionyl)phenoxy)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0140]

[0141] The synthesis process is the same as that of Example 13, except that the intermediate 3b in Example 13 is replaced by intermediate 4b.

[0142] Yellow solid, yield 61%, molecular formula C 27 H 27 NO5S, theoretical relative molecular mass 477.16, molecular ion peak m / z 478.16 is [MH] - .

[0143] 1 H NMR (600 MHz, CDCl3) δ 2.50 (2H, t, J = 4.0 Hz), 2.70 (2H, ddd, J= 10.0, 7.0, 3.0 Hz), 2.82-2.98 (4H, 2.90 (ddd, J = 14.2, 7.1, 2.9 Hz), 2.92(t, J = 4.0 Hz)), 3.24-3.43 (4H, 3.32 (dd, J = 15.7, 6.1 Hz), 3.37 (d, J =11.4 Hz)), 3.73 (3H, s), 3.96 (3H, s), 5.90 (1H, dd, J = 8.1, 4.1 Hz), 6.83(1H, d, J = 0.5 Hz), 6.95-7.12 (3H, 7.01 (ddd, J = 8.3, 1.2, 0.4 Hz), 7.07(d, J = 6.1 Hz)), 7.29 (1H, d, J = 6.1 Hz), 7.47 (1H, d, J = 0.5 Hz), 7.98(2H, ddd, J = 8.3, 1.8, 0.4 Hz).

[0144] Example 15: Synthesis of target compound C5 - 2-(4-(4-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)butanoyl)phenoxy)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

[0145]

[0146] The synthesis process is the same as that of Example 13, except that the intermediate 3b in Example 13 is replaced by intermediate 5b.

[0147] Yellow solid, yield 60%, molecular formula C 28 H 29NO5S, theoretical relative molecular mass 491.18, molecular ion peak m / z 492.18 is [MH] - .

[0148] 1 H NMR (600 MHz, CDCl3) δ 1.92 (2H, tt, J = 7.4, 2.7 Hz), 2.45 (2H,t, J = 7.4 Hz), 2.61-2.77 (4H, 2.69 (ddd, J = 11.5, 7.0, 3.0 Hz), 2.69 (t, J= 2.7 Hz)), 2.90 (2H, ddd, J = 14.2, 7.1, 2.9 Hz), 3.24-3.44 (4H, 3.32 (dd, J= 15.7, 6.1 Hz), 3.38 (d, J = 11.9 Hz)), 3.73 (3H, s), 3.96 (3H, s), 5.90(1H, dd, J = 8.1, 4.1 Hz), 6.83 (1H, d, J = 0.5 Hz), 6.95-7.12 (3H, 7.01(ddd, J = 8.3, 1.2, 0.4 Hz), 7.06 (d, J = 6.1 Hz)), 7.29 (1H, d, J = 6.1 Hz), 7.47 (1H, d, J = 0.5 Hz), 7.98 (2H, ddd, J = 8.3, 1.8, 0.4 Hz).

[0149] Example 16: Synthesis of target compound C6 - 5-(2-(4-(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)acetyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate

[0150]

[0151] Intermediate B2 was added to a round-bottom flask containing dichloromethane, and triethylamine, EDCI, DMAP, and glacial acetic acid were added in sequence. The mixture was stirred at 20-25°C for 3-4 hours. After the reaction, the solvent was evaporated under reduced pressure, and the mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by column chromatography (eluent: methanol / dichloromethane = 1:15, v / v) to obtain the target product C6.

[0152] Yellow solid, yield 59%, molecular formula C 28 H 34N2O6S, theoretical relative molecular mass 526.21, molecular ion peak m / z 527.22 is [MH] - .

[0153] 1 H NMR (600 MHz, CDCl3) δ 1.47-1.66 (5H, 1.54 (ttt, J = 10.2, 7.0,3.3 Hz), 1.57 (dddd, J = 12.8, 6.7, 6.6, 2.7 Hz)), 2.11 (3H, s), 2.38-2.65(6H, 2.46 (ddd, J = 14.3, 6.6, 2.7 Hz), 2.59 (dd, J = 7.0, 6.1 Hz)), 2.93(2H, ddd, J = 14.2, 7.2, 3.1 Hz), 3.14 (2H, dd, J = 15.7, 6.1 Hz), 3.39-3.78(8H, 3.46 (dtd, J = 8.1, 6.1, 4.1 Hz), 3.61 (ddd, J = 13.7, 7.2, 3.1 Hz), 3.58 (s), 3.73 (s)), 3.95 (3H, s), 4.45 (2H, d, J = 17.2 Hz), 6.69 (1H, d, J = 0.4 Hz), 6.81 (1H, s), 7.44 (1H, d, J = 0.4 Hz).

[0154] Example 17: Synthesis of target compound C7 - 5-(2-(4-((5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)acetyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl(E)-2-butyrate

[0155]

[0156] The synthesis process is the same as that of Example 16, except that the glacial acetic acid in Example 16 is replaced by crotonic acid.

[0157] Yellow solid, yield 66%, molecular formula C 30 H 36 N2O6S, theoretical relative molecular mass 552.23, molecular ion peak m / z 553.23 is [MH] - .

[0158] 1H NMR (600 MHz, CDCl3) δ 1.47-1.66 (5H, 1.54 (ttt, J = 10.2, 7.0,3.3 Hz), 1.57 (dddd, J = 12.8, 6.7, 6.6, 2.7 Hz)), 1.88 (3H, d, J = 6.9 Hz), 2.38-2.65 (6H, 2.46 (ddd, J = 14.3, 6.6, 2.7 Hz), 2.59 (dd, J = 7.0, 6.1Hz)), 2.94 (2H, ddd, J = 14.2, 7.2, 3.1 Hz), 3.14 (2H, dd, J = 15.7, 6.1 Hz),3.39-3.78 (8H, 3.46 (dtd, J = 8.1, 6.1, 4.1 Hz), 3.61 (ddd, J = 13.6, 7.2,3.1 Hz), 3.58 (s), 3.73 (s)), 3.95 (3H, s), 4.45 (2H, d, J = 17.2 Hz), 6.15(1H, d, J = 15.8 Hz), 6.69 (1H, d, J = 0.4 Hz), 6.82 (1H, s), 7.12 (1H, dq, J= 15.8, 6.9 Hz), 7.44 (1H, d, J = 0.4 Hz).

[0159] Example 18: Synthesis of target compound C8 - 5-(2-(4-((5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)acetyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl(E)-2-pentenoate

[0160]

[0161] The synthesis process is the same as that of Example 16, except that the glacial acetic acid in Example 16 is replaced by (E)-pent-2-enoic acid.

[0162] Yellow solid, yield 62%, molecular formula C 31 H 38 N2O6S, theoretical relative molecular mass 566.25, molecular ion peak m / z 567.25 is [MH] - .

[0163] 1H NMR (600 MHz, CDCl3) δ 1.11 (3H, t, J = 7.1 Hz), 1.47-1.66 (5H,1.54 (ttt, J = 10.2, 7.0, 3.3 Hz), 1.58 (dddd, J = 12.8, 6.7, 6.6, 2.7 Hz)), 2.26 (2H, qd, J = 7.1, 6.8 Hz), 2.38-2.65 (6H, 2.46 (ddd, J = 14.3, 6.6, 2.7Hz), 2.59 (dd, J = 7.0, 6.1 Hz)), 2.93 (2H, ddd, J = 14.2, 7.2, 3.1 Hz), 3.14(2H, dd, J = 15.7, 6.1 Hz), 3.39-3.78 (8H, 3.46 (dtd, J = 8.1, 6.1, 4.1 Hz), 3.61 (ddd, J = 13.6, 7.2, 3.1 Hz), 3.58 (s), 3.73 (s)), 3.95 (3H, s), 4.45 (2H, d, J = 17.2 Hz), 6.16 (1H, d, J = 15.8 Hz), 6.69 (1H, d, J = 0.4 Hz), 6.82 (1H, s), 7.24 (1H, dt, J = 15.8, 6.8 Hz), 7.44 (1H, d, J = 0.4 Hz).

[0164] Example 19: Assay for inhibition of acetylcholinesterase activity

[0165] The acetylcholinesterase (AChE) inhibitory activity of the compounds was determined using a modified Ellman method. In a 96-well plate, 280 μL of phosphate-buffered saline (PBS, pH 8.0) and 40 μL of acetylcholinesterase solution (50 U / L, dissolved in PBS) were added to each well. Subsequently, 40 μL of the test sample solution (dissolved in PBS containing 1% DMSO, with final concentrations of 1000, 100, 10, 1, and 0.1 μM, respectively) was added. The reaction was initiated by the addition of 40 μL of iodothioacetylcholine (ATCh, 1.2 mmol / L) substrate solution. The reaction was incubated at 37°C for 30 minutes and then terminated by the addition of 40 μL of 4% (w / w) sodium dodecyl sulfate (SDS) solution. Finally, 40 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 3.4 mmol / L) color developer was added, and the absorbance of the reaction system at a wavelength of 412 nm was immediately measured.

[0166] The percentage inhibition rate is calculated by the formula I= (A 对照 -A 实验 ) / (A 对照 -A 空白 ) × 100%, where A 实验 For the drug-added group, A 对照 A is the control group without drug addition, 空白 The blank group was not added with enzyme and drug.

[0167] The IC of the bold compound was calculated by calculating the inhibition rate of acetylcholinesterase at different sample concentrations. 50 Each experiment was repeated 3 times, and the IC 50 (mM), the experimental results are listed in Table 1.

[0168] Example 20: ADP-induced platelet aggregation activity assay

[0169] The in vitro antiplatelet aggregation activity of the target compounds was determined using the Born turbidimetric method. Platelets in platelet-rich plasma (PRP) are uniformly dispersed, and their turbidity is positively correlated with platelet number. Adding adenosine diphosphate (ADP) to PRP with continuous stirring induces platelet aggregation, resulting in a decrease in plasma turbidity (decreased absorbance). The higher the platelet aggregation, the more significant the decrease in turbidity. When the aggregation reaction reaches the plateau phase (absorbance stabilizes), platelet-poor plasma (PPP) is used as a blank control. The absorbance change (platelet aggregation curve) is automatically recorded using a platelet aggregation analyzer, and the platelet aggregation rate is calculated. ADP is a key agonist stored within platelet dense granules. It is released upon platelet activation and plays a crucial role in hemostasis and thrombosis by amplifying the effects of other agonists. Therefore, ADP was selected as the platelet aggregation inducer in this study.

[0170] The platelet aggregation inhibition rate was calculated by the formula I = [(maximum aggregation rate of blank control group - maximum aggregation rate of drug-treated group) / maximum aggregation rate of blank control group] × 100%, and the half-maximal inhibitory concentration IC was calculated. 50 (µM), the experimental results are shown in Table 1:

[0171]

[0172] Table 1. Results indicate that the target compounds obtained through structural modification retain or slightly decrease the acetylcholinesterase activity of donepezil, while exhibiting slightly enhanced antiplatelet aggregation activity. Compounds C7 and C8 exhibited both excellent anti-acetylcholinesterase and anti-platelet aggregation activities and were selected as candidate compounds for further investigation.

[0173] Example 21: Effects of Compounds C7 and C8 on H2O2-Induced Oxidative Damage to PC12

[0174] 1. Cell culture

[0175] PC12 cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified, 5% CO2 incubator. The medium was replaced every 48 hours, and cells were passaged the following day. Cells in the logarithmic growth phase were used for subsequent experiments.

[0176] 2. Establishment of H2O2 oxidative damage model in PC12 cells

[0177] PC12 cells in the logarithmic growth phase were cultured at a rate of 1.0×10 5 Cells were seeded in 96-well plates and grouped as blank, 100, 150, 200, 250, and 300 µmoL / L, with five replicates per group. 100 µL of cell suspension was added to each well, and sterile PBS was added to the peripheral wells. The 96-well plates were then placed in a cell culture incubator (37°C, 5% CO2) for continued incubation. After 24 hours of incubation, the 96-well plates seeded with PC12 cells were removed, the original culture medium aspirated, and DMEM medium containing varying concentrations of H2O2 was added to each well for an additional 12 hours. The original culture medium was aspirated, and 100 µL of DMEM medium was added to each well. Subsequently, 20 µL of MTT solution with a final concentration of 5 mg / mL was added to each well and incubated for an additional 4 hours. The incubation was terminated, the culture medium aspirated, and 150 µL of dimethyl sulfoxide was added to each well. The cells were shaken on a shaker at low speed for 10 minutes.

[0178] Results: When treated with a series of H2O2 concentrations for 24 hours, H2O2 inhibited PC12 cell proliferation in a dose-dependent manner. At a H2O2 concentration of 150 μmol / L, cell damage was minimal, with a cell survival rate of approximately 72%, which did not meet the requirements for modeling. Concentrations greater than 200 μmol / L were too potent, with a moderate cell survival rate of approximately 50% at 200 μmol / L, a statistically significant difference (P < 0.001). Therefore, the final H2O2 treatment condition for modeling was 200 μmol / L for 24 hours.

[0179] 3. Effects of compounds C7 and C8 on H2O2-induced PC12 cell damage

[0180] PC12 cells in the logarithmic growth phase were cultured at a rate of 1.0×10 5 Cells were seeded in 96-well plates and divided into five replicates: blank, model (H2O2), low-dose (H2O2 + 20 µmoL / L compound), medium-dose (H2O2 + 80 µmoL / L compound), and high-dose (H2O2 + 120 µmoL / L compound). 100 µL of cell suspension was added to each well, and sterile PBS was added to the peripheral wells. The 96-well plates were then incubated in a cell culture incubator (37°C, 5% CO2) for 24 hours. After 24 hours of incubation, the 96-well plates seeded with PC12 cells were removed, the original culture medium was aspirated, and DMEM medium containing different concentrations of compound was added to each group. Culture was continued for 12 hours. The original culture medium was aspirated, and 100 μL of DMEM medium was added to each well. Then, 20 μL of MTT solution with a final concentration of 5 mg / mL was added to each well and cultured for 4 h. The culture was terminated, the culture medium in the well was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The wells were shaken at low speed for 10 min to fully dissolve the crystals. The OD value of each well was measured using a microplate reader. 490 .

[0181] Experimental results: When compounds C7 and C8 were applied to H2O2-damaged PC12 cells for 24 hours, the cell viability of the model group was significantly reduced compared with the blank control group (P < 0.001). However, the drug-treated group at three concentrations of 20, 80, and 120 μmol / L significantly improved the survival rate of the oxidatively damaged cell model (P < 0.001).

[0182] Example 22: Study on the blood-brain barrier permeability of compounds C7 and C8

[0183] 1. Animal grouping, drug administration and treatment

[0184] Grouping: 68 healthy Kunming mice (50 / 50 male, 20 ± 2 g) were housed in an SPF-grade barrier facility. The temperature was 20 ± 2°C, the humidity was 45%-55%, the pressure gradient was 20 Pa-50 Pa, and the mice were housed on a 12 h / 12 ​​h free light cycle. The entire housing process was conducted and managed in strict accordance with standard operating procedures of the barrier facility.

[0185] The patients were divided into 3 groups according to the random unit group design method. Group A: Compound C7 group; Group B: Compound C8 group; Group C: Donepezil group;

[0186] After the start of the experiment, the mice's condition was observed and recorded daily. Dosing was performed approximately three times per week, and mice were weighed before and after each administration. The experimental and control groups received an equimolar dose of 0.032 mmol / kg via the tail vein. After administration, mice were sacrificed at 10, 30, 90, 180, and 480 minutes, and plasma and brain tissue were collected. Brain tissue was weighed, homogenized with 3 volumes of normal saline, and stored frozen at -80°C until further use.

[0187] 180 μL of plasma or brain homogenate was accurately aspirated, 20 μL of internal standard solution was added, and the mixture was mixed. 1 mL of methanol was added and vortexed for 6 min for extraction. The mixture was centrifuged at 4000 rpm for 12 min, and 2 μL of the supernatant was injected into HPLC-MS. The chromatogram and the peak areas of the compound, donepezil (As), and internal standard (Ai) were recorded. The drug content in plasma and brain homogenate was calculated according to the linear equation.

[0188] Log BB represents the blood-brain barrier permeability of a drug. It is defined as the logarithm of the ratio of the drug concentration in the brain (ng / g) to the blood concentration (ng / mL) when the drug distribution reaches steady state. The mathematical expression is:

[0189] Log BB = Log (C brain / C blood ).

[0190] 2. Experimental results

[0191]

[0192] After mice were orally administered with donepezil, the drug concentration detected in the brain homogenate was low, and its Log BB was steady-state within 30-180 minutes after administration, with an average value of 0.301. However, the Log BB of compounds C7 and C8 was relatively steady-state within 30-180 minutes or 30-480 minutes after administration, with an average value of 0.314 and 0.308, respectively.

[0193] The results in Table 2 indicate that the introduction of an ester bond at the α-position of tetrahydrothieno[3,2-c]pyridine, i.e., conversion to an ester derivative, enhances blood-brain barrier penetration. Compounds C7 and C8 also exhibit somewhat higher steady-state log BB values ​​compared to nepezil. Compound C7 is rapidly transported from the blood into the brain, and brain concentrations are maintained for extended periods (relatively stable BB values ​​at 180 or 480 minutes), suggesting its potential for improved blood-brain barrier penetration. Therefore, we conducted behavioral studies on compound C7 in mice.

[0194] Example 23: Compound C7 improves learning and memory impairment in VD rats

[0195] 1. Modeling, animal grouping, drug administration and treatment

[0196] A modified bilateral common carotid artery ligation method was used to establish a VD rat model. For the first week, rats were deprived of food and water before surgery and anesthetized intraperitoneally with 3% sodium pentobarbital (1 ml / kg). A 0.5 cm midline incision was made, and the left common carotid artery was bluntly and gradually isolated, avoiding the vagus nerve. The artery was then double-ligated with sterile surgical sutures, sutured with iodine disinfection, and 300,000 units of penicillin sodium were injected intramuscularly. The right common carotid artery was ligated as described above in the second week. In the sham-operated group, only the common carotid artery was isolated but not ligated. The rats with successfully established models were randomly divided into five groups: a sham-operated group, a 2-VO model group, and low-, medium-, and high-dose groups of compound C7. All rats were fed for four consecutive weeks and then sacrificed.

[0197] The Morris water maze (MWM) test was used to evaluate whether the 2-VO model was successfully established. The MWM experiment was performed seven days after model establishment to evaluate the modeling status by testing the learning and memory abilities of the rats.

[0198] 2. Animal behavior testing experiment

[0199] Morris (MWM) test

[0200] Cognitive function in 2-VO rats was assessed using the MWM before and after the treatment period. The water maze experiment lasted five days. The maze consisted of a 120 cm diameter, circular black plastic pool divided into four quadrants. The water temperature was maintained at 23 ± 2°C and the water was kept opaque using white ink. A 10 cm diameter escape platform was placed in the center of the fourth quadrant, with the water level covering the platform by 1 cm. During the first four days of the orientation and navigation experiment, during training, rats were placed facing the pool wall into a random quadrant. The time from entry to finding the submerged platform was recorded (escape latency) in seconds. After finding the platform, the rat was instructed to remain on the platform for 10 seconds. If the rat did not find the platform within 120 seconds after entering the water, it was guided with a stick to find the platform and remain there for 10 seconds. The escape latency in this case was recorded as 120 seconds. After the training experiment, the rat was removed from the platform, dried, and placed under an incandescent lamp to dry. The rat was then returned to its cage. Each rat underwent four experiments daily. On the fifth day, the platform was removed and a 120-second spatial exploration experiment was conducted to assess the rats' spatial memory and model development. The rats were placed in the quadrant opposite the original platform location as the starting position. The number of times they crossed the original platform location and the time spent in the target quadrant were recorded. Data were recorded and analyzed using SPSS 29.0 statistical software.

[0201] Experimental results: Compound C7 improves spatial learning and memory deficits in 2-VO model rats

[0202] Compared with the sham-operated group, the escape latency and number of platform crossings in all established model rats were significantly reduced (P < 0.01), indicating a significant reduction in the learning and memory abilities of the model rats, suggesting successful model establishment (Table 3). Compared with the sham-operated group, the number of platform crossings in the 2-VO model group was significantly reduced (P < 0.05). Compared with the model group, the escape latency of mice in the low- and medium-dose compound C7 groups was significantly reduced (P < 0.01), as shown in Table 4. These experimental results demonstrate that treatment with compound C7 significantly improves learning and memory in rats.

[0203]

[0204]

[0205] 3. Quantitative experiment of β-amyloid protein (Aβ) in mouse brain

[0206] Aβ40 and Aβ42 protein levels were measured according to the ELISA kit instructions. Standards were prepared at concentrations of 160, 80, 40, 20, and 10 ng / L to generate a reference curve. A 96-well plate was opened and 50 µL of the diluted standard was added to the standard, along with 40 µL of diluent and 10 µL of diluent. The plate was sealed with a semipermeable membrane. The plate was incubated at 37°C for 30 min, washed, and 50 µL of enzyme labeling reagent was added. The plate was incubated at 37°C for 30 min, washed, and 50 µL each of colorimetric solutions A and B were added. The plate was incubated in a dark, water-incubated chamber at room temperature for 10 min before the assay was completed. The plate was read at 450 mm using a microplate reader. All measurements were performed in duplicate. The measured Aβ protein levels in the transgenic mice, corrected for the dilution factor, represent the total protein levels in brain tissue and are reported as µg(Aβ) / g (total protein).

[0207] Compared with the immunohistochemical OD value of the normal group (0.00±0.00), the model group (70356.50±3961.77) showed a significant increase, with obvious Aβ40 and Aβ42 plaque deposition in brain tissue (P<0.01). Compared with the model group, the immunohistochemical OD value of the model-treated group (4529.42±468.35) was significantly reduced, with a significant decrease in Aβ40 and Aβ42 plaque deposition in brain tissue (P<0.01). This indicates that compound C7 can effectively reduce the content of Aβ protein in the mouse brain.

Claims

1. A heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine, characterized in that Selected from the compounds represented by the following general formulas (I), (II), and (III): ； Wherein R1 and R2 independently represent -H, -OH or -OCH3; R3 represents -OCOCH3, -OCOCH=CHCH3 or -OCOCH=CHCH2CH3; and n=1~3.

2. The method for preparing the heterodimer compound (I) according to claim 1, characterized in that The synthetic route is as follows: 。 3. The method for preparing the heterodimer compound (II) according to claim 1, characterized in that The synthetic route is as follows: 。 4. The method for preparing the heterodimer compound (III) according to claim 1, characterized in that The synthetic route is as follows: 。 5. Use of the heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine as claimed in claim 1 in the preparation of a pharmaceutical preparation, characterized in that: The pharmaceutical preparation has both acetylcholinesterase inhibition and platelet aggregation effects, and also has beta-amyloid protein aggregation inhibition and oxidative stress resistance effects.

6. Use of the heterodimer compound based on indanone-tetrahydrothieno[3,2-c]pyridine as claimed in claim 1 in the preparation of a pharmaceutical preparation for preventing and / or treating vascular dementia.

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