Tacrine-sulfur-containing compound spliced compound as well as preparation method and application thereof

Abstract

The invention provides a tacrine-sulfur-containing compound combined compound as well as a preparation method and application thereof, and belongs to the field of medical chemistry. The compound 1 and the compound 2 prepared by the invention can inhibit cholinesterase and effectively reduce hepatotoxicity of a parent compound tacrine, can be used as a drug for inhibiting cholinesterase, and have a wide application prospect in preparation of drugs for treating Alzheimer's disease. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a tacrine-sulfur-containing compound conjugate, a preparation method thereof and uses thereof. Background Art

[0002] Alzheimer disease (AD) is a serious neurodegenerative disease of the brain, characterized by memory loss and cognitive deficits. Since Alzheimer disease was first reported, its pathogenesis remains unclear. Based on its pathogenic factors, several hypotheses have been proposed, including the cholinergic hypothesis, the amyloid hypothesis, the neuroinflammation hypothesis, the tau protein hyperphosphorylation hypothesis, the oxidative stress hypothesis, etc. Currently, the cholinergic hypothesis still occupies a major position, and cholinesterase inhibitors are also used as the mainstream drugs for clinical treatment of AD.

[0003] Tacrine is the first acetylcholinesterase inhibitor approved for the treatment of Alzheimer disease. However, due to its strong hepatotoxicity, tacrine has been withdrawn by the US Food and Drug Administration. On the other hand, the effect of tacrine in treating AD still needs to be further improved. Therefore, it is of great significance to develop new drugs with both cholinesterase inhibitory activity and low hepatotoxicity.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a tacrine-sulfur-containing compound conjugate, a preparation method thereof and uses thereof.

[0006] The present invention provides the following compounds, their stereoisomers or their pharmaceutically acceptable salts:

[0007]

[0008] The present invention also provides a method for preparing the above compounds, their stereoisomers or their pharmaceutically acceptable salts, and the method is selected from Method A or Method B:

[0009] Method A:

[0010] (1) Thiopronine reacts with a mercapto protecting group to obtain Intermediate A;

[0011] (2) Intermediate A reacts with Intermediate X to obtain Intermediate B;

[0012] (3) The mercapto protecting group of Intermediate B is removed to obtain Compound 1;

[0013] The reaction route of Method A is:

[0014]

[0015] Method B:

[0016] (a) S-propargylcysteine reacts with an amino protecting group to obtain intermediate C;

[0017] (b) Intermediate C reacts with intermediate X to obtain intermediate D;

[0018] (c) The amino protecting group is removed from intermediate D to obtain compound 2;

[0019] The reaction route of Method B is as follows:

[0020]

[0021] Among them, n = 6.

[0022] Furthermore, in step (1) of Method A, the thiol protecting group is triphenylmethyl chloride; the reaction is carried out under the action of a base, and the base is preferably potassium carbonate; the molar ratio of tiopronin, the thiol protecting group and the base is 1:1 - 1.5:1, preferably 1:1.2:1; the solvent for the reaction is an organic solvent, preferably tetrahydrofuran; the reaction conditions are: under the protection of an inert gas, reacting at 50 - 80 °C for 2 - 6 hours, preferably: under the protection of an inert gas, reacting at 66 °C for 4 hours;

[0023] In step (2) of Method A, the reaction is carried out under the action of a condensing agent and an activating agent. The condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; the activating agent is 1-hydroxybenzotriazole; the molar ratio of intermediate A, intermediate X, the condensing agent and the activating agent is 0.5 - 1:0.5 - 1:1 - 1.5:1 - 1.5, preferably 0.7:0.7:1.1:1.1; the solvent for the reaction is an organic solvent, preferably a mixture of dichloromethane and N,N-dimethylformamide; the reaction conditions are: reacting at 10 - 40 °C for 6 - 12 hours, preferably: reacting at 15 - 35 °C for 8 - 9 hours;

[0024] In step (3) of Method A, the reaction is carried out under the action of a reducing agent and an acid. The reducing agent is preferably triisopropylsilane; the acid is preferably trifluoroacetic acid; the molar ratio of intermediate B and the reducing agent is 1:0.5 - 1.5, preferably 1:1; the solvent for the reaction is an organic solvent, preferably dichloromethane; the reaction conditions are: reacting at 10 - 40 °C for 0.5 - 2 hours, preferably: reacting at 15 - 35 °C for 1 hour;

[0025] In step (a) of Method B, the amino protecting group is di-tert-butyl dicarbonate; the reaction is carried out under the action of a base, and the base is preferably sodium carbonate; the molar ratio of S-propargyl cysteine, the amino protecting group and the base is 1:2:2 to 4, preferably 1:2:3; the solvent for the reaction is a mixture of an organic solvent and water, preferably a mixture of acetone and water; the reaction conditions are: under the protection of an inert gas, reacting at 10 to 40 °C for 2 to 6 hours, preferably: under the protection of an inert gas, reacting at 15 to 35 °C for 4 hours;

[0026] In step (b) of Method B, the reaction is carried out under the action of a condensing agent and an activator. The condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; the activator is 1-hydroxybenzotriazole; the molar ratio of intermediate C, intermediate X, the condensing agent and the activator is 1:1:1 to 2:1 to 2, preferably 1:1:1.5:1.5; the solvent for the reaction is an organic solvent, preferably a mixture of dichloromethane and N,N-dimethylformamide; the reaction conditions are: reacting at 10 to 40 °C for 6 to 12 hours, preferably: reacting at 15 to 35 °C for 8 to 9 hours;

[0027] In step (c) of Method B, the reaction is carried out under the action of an acid, and the acid is preferably trifluoroacetic acid; the solvent for the reaction is an organic solvent, preferably dichloromethane; the reaction conditions are: reacting at 10 to 40 °C for 0.5 to 2 hours, preferably: reacting at 15 to 35 °C for 1 hour.

[0028] Furthermore, after the reaction in step (1) of Method A is completed, the following purification steps are also included: removing the solvent in the reaction solution, adjusting the pH to acidic, extracting and then purifying by silica gel column chromatography to obtain intermediate A;

[0029] After the reaction in step (a) of Method B is completed, the following purification steps are also included: removing the solvent in the reaction solution, extracting, adjusting the pH of the aqueous layer to acidic, extracting again and purifying by silica gel column chromatography to obtain intermediate C.

[0030] Furthermore, the preparation method of the intermediate X includes the following steps:

[0031]

[0032] Reacting intermediate Y with 1,6-hexanediamine to obtain intermediate X; where n = 6.

[0033] Further, the reaction is carried out under the action of a catalyst, and the catalyst is preferably potassium iodide; the molar ratio of intermediate Y, 1,6 - hexanediamine and the catalyst is 1:8 - 12:0.1 - 1, preferably 1:10:0.5; the solvent for the reaction is an organic solvent, preferably phenol; the reaction conditions are: adding 1,6 - hexanediamine at 10 - 40 °C and stirring for 5 - 15 minutes, then reacting at 150 - 200 °C for 4 - 8 hours, preferably: adding 1,6 - hexanediamine at 15 - 35 °C and stirring for 10 minutes, then reacting at 170 °C for 6 hours;

[0034] Further, after the reaction is completed, the following purification steps are also included: adjusting the pH of the reaction solution to alkaline, extracting and then purifying by silica gel column chromatography to obtain intermediate X.

[0035] Further, the preparation method of intermediate Y includes the following steps:

[0036]

[0037] Reacting cyclohexanone with 5 - amino - 5 - chlorobenzoic acid to obtain intermediate Y.

[0038] Further, the reaction is carried out under the action of a chlorinating agent, and the chlorinating agent is preferably phosphorus oxychloride; the molar ratio of cyclohexanone and 5 - amino - 5 - chlorobenzoic acid is 5 - 9:5 - 6, preferably 7:5.8; the reaction conditions are: adding the chlorinating agent at - 5 - 5 °C and stirring for 5 - 15 minutes, then reacting at 100 - 110 °C for 2 - 6 hours under the protection of an inert gas, preferably: adding the chlorinating agent at 0 °C and stirring for 10 minutes, then reacting at 104 °C for 4 hours under the protection of an inert gas;

[0039] Further, after the reaction is completed, the following purification steps are also included: quenching with water, adjusting the pH of the reaction solution to alkaline, extracting and then purifying by silica gel column chromatography to obtain intermediate Y.

[0040] The present invention also provides the use of the above - mentioned compound, its stereoisomer or its pharmaceutically acceptable salt in the preparation of a cholinesterase inhibitor.

[0041] Further, the cholinesterase inhibitor is an acetylcholinesterase inhibitor and / or a butyrylcholinesterase inhibitor.

[0042] Further, the cholinesterase inhibitor is a drug for preventing and / or treating cognitive impairment.

[0043] Further, the cholinesterase inhibitor is a drug for preventing and / or treating Alzheimer's disease.

[0044] The present invention also provides a pharmaceutical composition, which is a preparation made from the above-mentioned compound as the active ingredient and pharmaceutically acceptable excipients.

[0045] The present invention has achieved the following beneficial effects:

[0046] (1) The compound represented by Formula I provided by the present invention has both the effects of inhibiting cholinesterase and reducing the hepatotoxicity of parent tacrine, and can be used as a drug for inhibiting cholinesterase, having broad application prospects in the preparation of drugs for multi-target treatment of Alzheimer's disease.

[0047] (2) Neither tiopronin nor S-propargylcysteine can effectively inhibit the activities of acetylcholinesterase and butyrylcholinesterase. Tacrine has certain inhibitory activities on acetylcholinesterase and butyrylcholinesterase. However, the tacrine-tiopronin / S-propargylcysteine conjugate of the present invention has better inhibitory activity on butyrylcholinesterase than tacrine. Among them, the butyrylcholinesterase inhibitory activity of the compound in Example 2 (tacrine-S-propargylcysteine) is significantly higher than that of tacrine, and the IC 50 value of tacrine is 21.4 times that of it. The present invention has achieved unexpected technical effects.

[0048] (3) Tacrine was withdrawn from the market due to its strong hepatotoxicity, while the tacrine-tiopronin conjugate of the present invention significantly reduces the toxicity to two types of hepatocytes. The present invention has achieved unexpected technical effects.

[0049] (4) The preparation method of the tacrine-tiopronin / S-propargylcysteine conjugate of the present invention is simple and feasible, and is suitable for industrial production.

[0050] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can also be made.

[0051] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 shows (A) the activity trajectory diagram of mice in the spatial exploration experiment of the compound 1 group; (B) the frequency of crossing the platform during the spatial exploration experiment of the compound 1 group; (C) the duration of activity in the target quadrant during the spatial exploration experiment of the compound 1 group; (D) the latency during the spatial exploration experiment of the compound 1 group; (E) the percentage of the duration of activity in the target quadrant during the spatial exploration experiment of the compound 1 group accounting for the total activity time.

[0053] Figure 2 (A) Activity trajectory diagram of mice in the spatial exploration experiment of Compound 2 group; (B) Frequency of crossing the platform during the spatial exploration experiment of Compound 2; (C) Duration of activity in the target quadrant during the spatial exploration experiment of Compound 2 group; (D) Latency during the spatial exploration experiment of Compound 2 group; (E) Percentage of the duration of activity in the target quadrant during the spatial exploration experiment of Compound 2 group in the total activity time. Detailed implementation mode

[0054] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0055] "Room temperature / normal temperature" referred to in the present invention means 25 ± 10 °C.

[0056] Example 1: Synthesis of tacrine-thiopronine conjugate

[0057] The synthetic route of tacrine-thiopronine conjugate is shown as follows:

[0058]

[0059] 1. Synthesis of intermediate Y

[0060] Take 1 g (5.8 mmol) of 5-amino-5-chlorobenzoic acid and 725 μl (7 mmol) of cyclohexanone in a two-necked flask. At 0 °C, add 2 ml of phosphorus oxychloride dropwise while stirring. Stir at 0 °C for about 10 minutes, then transfer to room temperature and gradually heat up to 105 °C. Under the protection of argon, the mixed suspension is refluxed for 4 hours to obtain a dark yellow solution, which is then quenched with ice water, adjusted to alkaline pH with NaOH. The reaction solution is extracted with ethyl acetate, the organic layers are combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The ethyl acetate is removed by distillation under reduced pressure, and the residue is purified by silica gel column chromatography (petroleum ether:ethyl acetate = 55:1 - 40:1) to obtain intermediate Y.

[0061] 2. Synthesis of intermediate X

[0062] Dissolve 500 mg (2 mmol) of intermediate Y in ultradry phenol, add potassium iodide (KI) (166.2 mg, 1 mmol), add 1,6-hexanediamine (2.6 ml, 20 mmol) at room temperature, stir for about 10 minutes, gradually heat up to 170 °C, and continue the reaction for 6 hours. The reaction is terminated, adjusted to alkaline pH with NaOH, the reaction solution is extracted with dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. The dichloromethane is removed by distillation under reduced pressure, and the residue is purified by silica gel column chromatography (dichloromethane:methanol = 10:1 - 2:1) to obtain intermediate X.

[0063] 3. Synthesis of intermediate A

[0064] Dissolve 800 mg (5 mmol) of tiopronin in tetrahydrofuran, add K2CO3 (691 mg, 5 mmol), then add triphenylmethyl chloride (1.7 g, 6 mmol). Under the protection of argon, the mixed suspension is refluxed at 66 °C for 4 hours to obtain a white suspension. The reaction is terminated, and tetrahydrofuran is removed by distillation under reduced pressure. The pH is adjusted to acidic with dilute hydrochloric acid. The reaction solution is extracted with dichloromethane and dried over anhydrous sodium sulfate. Dichloromethane is removed by distillation under reduced pressure, and the residue is purified by silica gel column chromatography (dichloromethane:methanol = 100:1 - 80:1) to obtain intermediate A.

[0065] 4. Synthesis of Intermediate B

[0066] Dissolve 300 mg (0.7 mmol) of intermediate A in ultra-dry dichloromethane and ultra-dry N,N-dimethylformamide (DMF). At 0 °C, add 212.8 mg (1.1 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 148.6 mg (1.1 mmol) of 1-hydroxybenzotriazole (HOBT). Transfer to room temperature and stir for about 1.5 h. Add 208.6 mg (0.7 mmol) of intermediate X and continue the reaction for 8 hours to obtain a yellow solution. The reaction is terminated. Dichloromethane is removed by distillation under reduced pressure. The reaction is quenched with saturated brine. The reaction solution is extracted with ethyl acetate, the organic layers are combined, washed with saturated brine, and dried over anhydrous sodium sulfate. Ethyl acetate is removed by distillation under reduced pressure, and the residue is purified by silica gel column chromatography (dichloromethane:methanol = 60:1 - 30:1) to obtain intermediate B.

[0067] 5. Synthesis of Compound 1 (tacrine - tiopronin conjugate)

[0068] Dissolve 300 mg (0.4 mmol) of intermediate B in dichloromethane, add 90 μl (0.4 mmol) of triisopropylsilane. While stirring at 0 °C, slowly add 3 ml of trifluoroacetic acid (TFA) dropwise, stir for about 10 minutes, transfer to room temperature and react for 1 h to obtain a yellow reaction solution. Dichloromethane and trifluoroacetic acid are removed by distillation under reduced pressure. The residue is purified by silica gel column chromatography (dichloromethane:methanol = 40:1 - 20:1) to obtain compound 1. Its structure and characterization are as follows:

[0069]

[0070] Pale yellow solid (150 mg), yield 70%.

[0071] 11H NMR (400 MHz, MeOD) δ 8.32 (dd, J = 9.3, 5.7 Hz, 1H), 7.72 (dd, J = 4.0, 2.1 Hz, 1H), 7.51 (d, J = 9.1 Hz, 1H), 4.02–3.66 (m, 4H), 3.30–3.24 (m, 1H), 3.15 (t, J = 6.8 Hz, 2H), 3.05–2.83 (m, 3H), 2.62 (d, J = 6.1 Hz, 2H), 1.93–1.88 (m, 3H), 1.82–1.75 (m, 2H), 1.48 (t, J = 7.0 Hz, 2H), 1.41 (d, J = 7.0 Hz, 3H), 1.37–1.18 (m, 6H); 13 13C NMR (101 MHz, MeOD) δ 176.8, 171.4, 157.7, 152.2, 140.6, 140.0, 128.7, 126.7, 119.2, 115.4, 113.4, 43.5, 40.1, 37.7, 31.2, 30.7, 30.1, 29.4, 27.3, 27.2, 24.8, 22.9, 22.0, 21.8; HRESIMS m / z 477.2125 [M+H] + , calcd for C 24 H 34 ClN4O2S , 477.2091.

[0072] Example 2. Synthesis of Tacrine-S-propargylcysteine conjugate

[0073] The synthetic route of Tacrine-S-propargylcysteine conjugate is as follows:

[0074]

[0075] 1. Synthesis of Intermediate Y

[0076] As described in "Synthesis of Intermediate Y" in Example 1.

[0077] 2. Synthesis of Intermediate X

[0078] As described in "Synthesis of Intermediate X" in Example 1.

[0079] 3. Synthesis of Intermediate C

[0080] Dissolve 1 g (4.7 mmol) of S-propargylcysteine in acetone and water, add Na2CO3 (1.5 g, 14 mmol), then add di-tert-butyl dicarbonate (2 g, 9.4 mmol). Under argon protection, react at room temperature for 4 hours, then terminate the reaction. Evaporate acetone under reduced pressure. Extract the reaction solution with ethyl acetate, discard the organic phase. Adjust the pH of the aqueous layer to acidic with KHSO4, then extract with ethyl acetate again and dry over anhydrous sodium sulfate. Evaporate ethyl acetate under reduced pressure. Purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 - 1:1) to obtain intermediate C.

[0081] 4. Synthesis of Intermediate D

[0082] Dissolve 300 mg (1.2 mmol) of intermediate C in ultradry dichloromethane and ultradry N,N-dimethylformamide (DMF). At 0 °C, add EDCI 345.1 mg (1.8 mmol) and HOBT 243.2 mg (1.8 mmol). Transfer to room temperature and stir for about 1.5 h. Add intermediate X 357.6 mg (1.2 mmol) and continue the reaction for 8 hours to obtain a yellow solution, then terminate the reaction. Evaporate dichloromethane under reduced pressure. Quench the reaction by adding saturated brine. Extract the reaction solution with ethyl acetate, combine the organic layers, wash with saturated brine, and dry over anhydrous sodium sulfate. Evaporate ethyl acetate under reduced pressure. Purify the residue by silica gel column chromatography (dichloromethane:methanol = 60:1 - 30:1) to obtain intermediate D.

[0083] 5. Synthesis of Compound 2 (tacrine-S-propargylcysteine conjugate)

[0084] Dissolve 300 mg (0.5 mmol) of intermediate D in dichloromethane. At 0 °C, add 3 ml of trifluoroacetic acid dropwise with stirring and stir for about 10 minutes. Transfer to room temperature and react for 1 h to obtain a yellow reaction solution. Evaporate dichloromethane and trifluoroacetic acid under reduced pressure. Purify the residue by silica gel column chromatography (dichloromethane:methanol = 40:1 - 15:1) to obtain Compound 2. Its structure and characterization are as follows:

[0085]

[0086] Yellow solid (150 mg), yield 70%.

[0087] 11H NMR (400 MHz, CDCl3) δ 7.92 (dd, J = 5.6, 3.4 Hz, 2H), 7.42 (s, 1H), 7.28 (d, J = 2.2 Hz, 1H), 3.58 (dd, J = 8.3, 3.9 Hz, 1H), 3.53 (t, J = 7.2 Hz, 2H), 3.28–3.19 (m, 6H), 3.04 (q, J = 3.9 Hz, 3H), 2.88–2.82 (m, 1H), 2.69–2.59 (m, 3H), 2.25 (t, J = 2.6 Hz, 2H), 1.92 - 1.87 (m, 6H), 1.26 - 1.23 (m, 1H), 1.25 (s, 4H); 13 13C NMR (101 MHz, CDCl3) δ 173.1, 158.6, 151.4, 146.9, 134.7, 126.4, 124.8, 124.5, 117.8, 115.1, 79.9, 71.6, 53.9, 49.2, 38.9, 37.9, 33.2, 29.7, 29.5, 26.5, 26.4, 24.5, 22.8, 22.4, 19.8; HRESIMS m / z 473.2112 [M + H] + , calcd for C 45 H 67 N3O 6, 473.2142.

[0088] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0089] Experimental Example 1: Determination of anti - acetylcholinesterase and anti - butyrylcholinesterase activities

[0090] 1. Experimental method

[0091] The modified Ellmann method was used to determine the activities of the compounds against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

[0092] 2.5 mg (0.5 U / mL) of acetylcholinesterase was dissolved in 1 mL of pH 8.0 phosphate buffer to prepare the enzyme solution. The stock solutions of the test compounds (i.e., Compound 1 and Compound 2) were prepared with DMSO and diluted to the final concentration with phosphate buffer. In a 96 - well plate, 140 μL of phosphate buffer, 10 μL of the enzyme solution, and 10 μL of the test compounds at different concentrations were added and incubated at 37 °C for 20 minutes. Then, 10 μL of 5,5'-dithiobis(2 - nitrobenzoic acid) (0.75 mM) and 10 μL of acetylthiocholine iodide (1.5 mM) were added and incubated at 37 °C for 20 min. The absorbance was measured at a wavelength of 405 nm using a microplate reader. Each group was measured at least three times in duplicate.

[0093] The enzyme solution was prepared by dissolving 1.0 mg (0.5 U / mL) of butyrylcholinesterase in 20 mL of phosphate buffer at pH 8.0. The stock solution of the compound to be tested was prepared with DMSO and diluted to the final concentration with phosphate buffer. In a 96-well plate, 140 μL of phosphate buffer, 10 μL of the enzyme solution, and 10 μL of the compound to be tested at different concentrations were added and incubated at 37 °C for 20 minutes. Then, 10 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (0.75 mM) and 10 μL of acetylthiocholine iodide (1.5 mM) were added and incubated at 37 °C for 20 min. The absorbance was measured at a wavelength of 405 nm using a microplate reader. Each group was measured at least three times in duplicate.

[0094] According to the absorbance values, the inhibition rates of the compounds at different concentrations on acetylcholinesterase and butyrylcholinesterase were calculated, and the IC 50 values of the compounds were calculated using software. Tiopronin and S-propargylcysteine were used as controls, and tacrine was used as a positive control.

[0095] 2. Experimental results

[0096] The IC 50 values of the tested compounds against acetylcholinesterase and butyrylcholinesterase are shown in Table 1

[0097]

[0098]

[0099] As can be seen from Table 1, neither tiopronin nor S-propargylcysteine could effectively inhibit the activities of acetylcholinesterase and butyrylcholinesterase. The compounds obtained in Examples 1 and 2 of the present invention could effectively inhibit the activities of acetylcholinesterase and butyrylcholinesterase. Among them, the inhibitory activities of the compounds obtained in Examples 1 and 2 against acetylcholinesterase were comparable to that of tacrine; the inhibitory activities of the compounds obtained in Examples 1 and 2 against butyrylcholinesterase were significantly better than that of tacrine. Among them, the compound obtained in Example 2 (tacrine-S-propargylcysteine) had the best inhibitory activity against butyrylcholinesterase, and the IC 50 value of tacrine was 21.4 times that of it.

[0100] Experimental Example 2. Mouse behavioral test

[0101] 1. Experimental method

[0102] The Morris water maze experiment was used to verify that compounds 1 and 2 reversed the cognitive impairment induced by the scopolamine model. Forty-four mice were randomly divided into 9 groups: control group, scopolamine model group (scopolamine, 3 mg / kg), tacrine positive drug group (tacrine, 5 mg / kg + scopolamine), compound 1 administration groups (compound1 5 mg / kg + scopolamine, compound1 15 mg / kg + scopolamine, compound1 45 mg / kg + scopolamine), and compound 2 administration groups (compound2 5 mg / kg + scopolamine, compound2 15 mg / kg + scopolamine, compound2 45 mg / kg + scopolamine). The tacrine positive drug group and the compound 1 and 2 administration groups were continuously administered according to the dose for 21 days, and the control group and the scopolamine model group were intragastrically administered with normal saline according to body weight. In the last 7 days, scopolamine 3 mg / kg was intraperitoneally injected half an hour before each water maze test for modeling.

[0103] The Morris water maze experiment was divided into three stages: visible platform experiment, hidden platform experiment (training period), and no-platform exploration experiment (testing period).

[0104] ① Visible platform experiment

[0105] (1) According to the experimental requirements, the Morris water maze experimental facility was built, the water level was adjusted to be about 1 cm lower than the platform, and orientation cues were pasted inside the pool.

[0106] (2) The pool was divided into four quadrants, named N, S, W, and E respectively. In addition, 8 positions for putting the mice into the water were set, starting from position 1 at 9 o'clock and counterclockwise to position 8 at 10 o'clock.

[0107] (3) Each mouse was subjected to 4 experiments per day. The platforms for the first two and the last two times were placed at two different positions respectively. As Figure 2 shown, the positions for putting the mice were on both sides of the quadrant opposite to the quadrant where the platform was located. For example, if the platform was at position N, the positions for putting the mice were at 2 and 4.

[0108] ② Hidden platform experiment (5 days)

[0109] (1) According to the experimental requirements, the Morris water maze experimental facility was built, the water level was adjusted to be nearly 1 cm higher than the platform surface, titanium dioxide was added until the platform was invisible, and the platform position was fixed during all hidden platform periods.

[0110] (2) Gently place the mice into the water facing the pool wall. For the four experiments on each mouse every day, the mice are randomly placed from the positions 2, 3, 8, and 7.

[0111] (3) Conduct 4 experiments on each mouse every day. The platforms are placed at two different positions for the first two and the last two experiments respectively. The positions where the mice are placed are on both sides of the opposite quadrant of the quadrant where the platform is located.

[0112] (4) The mice are allowed to freely explore in the water for 1 minute. If the platform is not found after more than 1 minute, guide the mice to the platform and let the mice stay on the platform for 15 s.

[0113] (5) Record all the video data of the process for subsequent analysis.

[0114] ③ Experiment in the period of platform-free exploration (1 day)

[0115] This experiment is carried out after the experiment of platform-hidden period. Remove the platform and place the mice from the farthest position (position 1), and record the movement process of the mice for subsequent analysis.

[0116] 2. Experimental results

[0117] As can be seen from Figures 1 and 2, during the spatial exploration experiment period, both the platform-crossing frequency and the cumulative duration in the target quadrant of the scopolamine group decreased, and the latency increased. After administration, it recovered somewhat. It is proved that Compounds 1 and 2 can restore the memory ability of the scopolamine model. Through the above experiments, it is proved that Compounds 1 and 2 can improve cognitive impairment.

[0118] Experimental Example 3. Determination of hepatotoxicity of cells

[0119] 1. Experimental method

[0120] The MTT method is used to determine the cytotoxicity. The principle of this method is as follows: MTT can be reduced by succinate dehydrogenase in the mitochondria of living cells to generate formazan, a blue-violet crystal. This formazan can be dissolved by DMSO and has an absorbance value at a wavelength of 492 nm. In addition, the amount of formazan generated is proportional to the number of living cells within a certain cell range. The specific steps are as follows:

[0121] a. Seeding: Wait for the liver cancer cells (HepG2 cells) / normal liver cells (LO2 cells) to grow to the logarithmic phase, digest the cells, collect them in a 15 mL centrifuge tube for counting, and inoculate them into a 96-well plate at 8×10 4 cells / mL (100 μL per well), and culture them in an incubator at 37 °C.

[0122] b. Administration: After 24 h, discard the culture medium and perform according to the experimental settings. Blank group (no cells): Culture medium (100 μL / well). Control group: Culture medium (100 μL / well). Drug group: Compound solution (100 μL / well). Set 6 replicate wells for each group.

[0123] c. MTT assay: After 24 h of drug treatment, discard the culture medium, add MTT solution (20 μL / well), continue culturing for 4 h, discard the culture medium, add DMSO (150 μL / well), gently shake on a shaker for 5 - 10 min, and measure the absorbance OD value at 492 nm.

[0124] d. Calculate the cell survival rate.

[0125] Cell survival rate (%) = (ODdrug group - ODblank group) / (ODcontrol group - ODblank group) × 100%

[0126] 2. Experimental results

[0127] Table 2 Effects of Compounds 1 and 2 on the Survival Rate of LO2 Cells

[0128]

[0129] Table 3 Effects of Compounds 1 and 2 on the Survival Rate of HepG2 Cells

[0130]

[0131]

[0132] As can be seen from Tables 2 and 3, the compounds obtained in Examples 1 and 2 of the present invention can reduce the toxicity of parent tacrine to the two types of hepatocytes. Among them, Compound 1 obtained in Example 1 is still basically non-toxic to the two types of hepatocytes when the concentration rises to 100 μM.

[0133] In summary, the present invention provides a tacrine-sulfur-containing compound conjugate, its preparation method and use. The present invention prepares a conjugate of tacrine and a sulfur-containing compound, which can inhibit cholinesterase and effectively reduce the hepatotoxicity of the parent compound tacrine, and can be used as a drug for inhibiting cholinesterase, having broad application prospects in the preparation of drugs for treating Alzheimer's disease.

Claims

1. The following compound, its stereoisomer or its pharmaceutically acceptable salt:

2. A method for preparing the compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The method is selected from method A or method B: Method A: (1) thiopronin reacts with a thiol protecting group to obtain intermediate A; (2) Intermediate A reacts with intermediate X to obtain intermediate B; (3) removing the thiol protecting group from intermediate B to obtain compound 1; The reaction route of method A is: Method B: (a) S-propargylcysteine ​​reacts with an amino protecting group to obtain intermediate C; (b) intermediate C reacts with intermediate X to obtain intermediate D; (c) removing the amino protecting group from intermediate D to obtain compound 2; The reaction route of method B is: Among them, n=6.

3. The method according to claim 2, characterized in that In step (1) of method A, the thiol protecting group is triphenylmethane; the reaction is carried out under the action of a base, and the base is preferably potassium carbonate; the molar ratio of thiopronine, the thiol protecting group and the base is 1:1-1.5:1, preferably 1:1.2:1; the solvent of the reaction is an organic solvent, preferably tetrahydrofuran; the reaction conditions are: under the protection of an inert gas, the reaction is carried out at 50-80°C for 2-6 hours, preferably: under the protection of an inert gas, the reaction is carried out at 66°C for 4 hours; In step (2) of method A, the reaction is carried out under the action of a condensing agent and an activating agent, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; the activating agent is 1-hydroxybenzotriazole; the molar ratio of the intermediate A, the intermediate X, the condensing agent and the activating agent is 0.5-1:0.5-1:1-1.5:1-1.5, preferably 0.7:0.7:1.1:1.1; the solvent of the reaction is an organic solvent, preferably a mixture of dichloromethane and N,N-dimethylformamide; the reaction conditions are: reaction at 10-40°C for 6-12 hours, preferably: reaction at 15-35°C for 8-9 hours; In step (3) of method A, the reaction is carried out under the action of a reducing agent and an acid, the reducing agent is preferably triisopropylsilane; the acid is preferably trifluoroacetic acid; the molar ratio of the intermediate B to the reducing agent is 1:0.5-1.5, preferably 1:1; the solvent of the reaction is an organic solvent, preferably dichloromethane; the reaction conditions are: reacting at 10-40°C for 0.5-2 hours, preferably: reacting at 15-35°C for 1 hour; In step (a) of method B, the amino protecting group is di-tert-butyl dicarbonate; the reaction is carried out under the action of a base, and the base is preferably sodium carbonate; the molar ratio of S-propargylcysteine, the amino protecting group and the base is 1:2:2-4, preferably 1:2:3; the solvent of the reaction is a mixture of an organic solvent and water, preferably a mixture of acetone and water; the reaction conditions are: under the protection of an inert gas, the reaction is carried out at 10-40°C for 2-6 hours, preferably: under the protection of an inert gas, the reaction is carried out at 15-35°C for 4 hours; In step (b) of method B, the reaction is carried out under the action of a condensing agent and an activating agent, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; the activating agent is 1-hydroxybenzotriazole; the molar ratio of the intermediate C, the intermediate X, the condensing agent and the activating agent is 1:1:1-2:1-2, preferably 1:1:1.5:1.5; the solvent of the reaction is an organic solvent, preferably a mixture of dichloromethane and N,N-dimethylformamide; the reaction conditions are: reacting at 10-40°C for 6-12 hours, preferably: reacting at 15-35°C for 8-9 hours; In step (c) of method B, the reaction is carried out under the action of an acid, preferably trifluoroacetic acid; the solvent of the reaction is an organic solvent, preferably dichloromethane; the reaction conditions are: reaction at 10-40°C for 0.5-2 hours, preferably: reaction at 15-35°C for 1 hour.

4. The method according to claim 3, characterized in that The preparation method of the intermediate X comprises the following steps: The intermediate Y and 1,6-hexanediamine are reacted to obtain the intermediate X, wherein n=6.

5. The method according to claim 4, characterized in that The preparation method of the intermediate Y comprises the following steps: Cyclohexanone is reacted with 5-amino-5-chlorobenzoic acid to obtain intermediate Y.

6. Use of the compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a cholinesterase inhibitor.

7. The use according to claim 6, characterized in that The cholinesterase inhibitor is an acetylcholinesterase inhibitor and / or a butyrylcholinesterase inhibitor.

8. The use according to claim 7, characterized in that The cholinesterase inhibitor is a drug for preventing and / or treating cognitive impairment.

9. The use according to claim 7, characterized in that: The cholinesterase inhibitor is a drug for preventing and / or treating Alzheimer's disease.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition is a preparation prepared with the compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable excipients.

Images