A small molecule proteasome agonist and its preparation method and application

Abstract

The present invention discloses a small molecule proteasome agonist, a preparation method thereof and an application. The present invention uses cyclohexylamine as a starting material and prepares 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide through five-step reactions. The two compounds provided by the present invention have good 20S proteasome agonist activity and ischemic injury cardiomyocyte protection activity, and have no obvious cytotoxicity, and are expected to become an effective option for ischemic cardiomyopathy and other proteotoxic diseases.

Description

Technical Field

[0001] The present invention relates to the field of drugs. Specifically, the present invention relates to a class of novel small molecule proteasome agonists, their preparation methods and applications, and the use of the compounds in the preparation of drugs for the treatment of protein-toxic diseases. Background Art

[0002] The proteasome is a large protease system composed of multiple subunits, which undertakes the main task of protein degradation in eukaryotes. Given the key role played by this catalytic complex in maintaining protein homeostasis, its dysfunction is closely related to the development of various pathological conditions, including cancer, neurodegenerative diseases, and cardiovascular diseases. With the increase of age, the function and content of the proteasome generally decline, resulting in the obstruction of protein degradation, and then leading to the accumulation and aggregation of proteins. These abnormally aggregated proteins produce protein toxicity, which may trigger a series of protein-toxic diseases, including neurodegenerative diseases and cardiovascular diseases, such as Alzheimer's disease, Parkinson's disease, primary lateral sclerosis, coronary heart disease, cardiomyopathy, thromboembolic diseases, etc. Proteasome agonists can enhance its activity and help restore protein degradation function, thus showing great potential in slowing down or treating related diseases.

[0003] Although the concept of 20S proteasome activators is relatively new and the number of currently known proteasome activators is limited, related research is steadily advancing, providing new directions for the development of future treatment strategies. Summary of the Invention

[0004] In view of this, the embodiments of the present application provide a small molecule proteasome agonist, its preparation method and application.

[0005] According to the embodiments of the present application, a small molecule proteasome agonist is provided, and its structural formula is shown as formula (1) or (2):

[0006]

[0007] The chemical name of formula (1) is 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide, and the chemical name of formula (2) is N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide.

[0008] The preparation method of the above-mentioned small molecule proteasome agonist is realized through the following synthetic route:

[0009]

[0010] The above-mentioned 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide, and the preparation method thereof comprises the following steps:

[0011] (1) Cyclohexylamine, triethylamine and acryloyl chloride react in dichloromethane under an ice bath to form N -cyclohexylacrylamide (Compound 3);

[0012] (2) N -cyclohexylacrylamide and tert-butyl hydrazinecarboxylate react at a high temperature in isopropanol to form tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (Compound 4);

[0013] (3) Tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate is condensed with benzo[d]thiazole-2-carboxylic acid and 2-quinolinecarboxylic acid respectively in the presence of a condensing agent, and tert-butyl 2-(benzo[d]thiazole-2-carbonyl)-2-3-((cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (Compound 5) and tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)-2-(quinoline-2-carbonyl)hydrazine-1-carboxylate (Compound 6) are formed at room temperature;

[0014] (4) Tert-butyl 2-(benzo[d]thiazole-2-carbonyl)-2-3-((cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate and tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)-2-(quinoline-2-carbonyl)hydrazine-1-carboxylate are deprotected with trifluoroacetic acid to form 3-(1-(benzo[d]thiazole-2-carbonyl)hydrazono)- N -cyclohexylpropanamide (Compound 7) and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propanamide (Compound 8);

[0015] (5) 3-(1-(benzo[d]thiazole-2-carbonyl)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propanamide react with furan-2-carbaldehyde at a high temperature respectively to form the target compounds 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide (Compound 1) and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide (Compound 2).

[0016] Specifically, the preparation method may include the following steps:

[0017] (1) Add cyclohexylamine and triethylamine to a reaction flask, dissolve them in dichloromethane, cool in an ice bath, and slowly add acryloyl chloride dropwise. After the addition is complete, remove the ice bath and continue the reaction. When TLC plate shows complete reaction, add saturated ammonium chloride aqueous solution, extract, take the organic layer, extract the aqueous layer with dichloromethane twice more, combine the organic layers, and concentrate by rotary evaporation to obtain product 3;

[0018] (2) Add compound 3 and tert-butyl carbazate to a sealed tube, dissolve them in isopropanol, heat the reaction system to a high temperature for reaction. Cool to room temperature, concentrate the system by rotary evaporation, add dichloromethane and water, extract, concentrate the organic layer by rotary evaporation, and purify the crude product by column chromatography to obtain product 4;

[0019] (3) Add benzothiazole-2-carboxylic acid and 2-quinolinecarboxylic acid to two round-bottom flasks respectively, dissolve them in dichloromethane, add HATU and DIPEA to each flask, stir at room temperature for 15 min, then add compound 4 to each, and continue the reaction at room temperature. When TLC plate shows complete reaction, add saturated ammonium chloride aqueous solution to quench each reaction system, extract, concentrate the organic layer by rotary evaporation, and purify the crude product by column chromatography to obtain product 5 and product 6;

[0020] (4) Add compound 5 and compound 6 to two round-bottom flasks respectively, dissolve them in dichloromethane, add trifluoroacetic acid to each, react at room temperature. When TLC plate shows complete reaction, concentrate the solvent and trifluoroacetic acid to obtain product 7 and 8;

[0021] (5) Add compound 7 and compound 8 to sealed tubes respectively, dissolve them in isopropanol, then add acetic acid and furan-2-carbaldehyde to each, heat the two reaction systems to a high temperature and react overnight. When TLC plate shows complete reaction, concentrate the solvent, add ether to each for slurrying, filter, wash the filter cake with ether again, and dry the obtained solid under vacuum to obtain product 1 and 2.

[0022] In the above technical solution, further, the high temperature in (2) is 120 °C;

[0023] Further, the high temperature in (5) is 80 °C.

[0024] The above 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide in the use for preparing drugs for protein-toxic diseases.

[0025] The small molecule proteasome agonist described above can be a drug for treating neurodegenerative diseases, cardiovascular diseases, diabetes, etc., to activate the activity of the 20S proteasome and exert the protein degradation function.

[0026] This invention has been confirmed by molecular and cell experiments: 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide have good 20S proteasome agonist activity, have the effect of repairing hypoxia-induced loss of cardiomyocytes, and have no obvious cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a test result diagram of the protective effect of compounds 1 and 2 on hypoxia-injured cardiomyocytes. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following examples are only illustrative of the invention and do not limit the invention in any way.

[0029] Example 1 Preparation of 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide

[0030]

[0031] (1) N Preparation of -cyclohexylacrylamide (Compound 3)

[0032] Dissolve cyclohexylamine (2 g, 20.2 mmol) and triethylamine (4.09 g, 40.4 mmol) in 10 mL of dichloromethane, cool in an ice bath, and dropwise add a solution of acryloyl chloride (1.83 g, 20.2 mmol) in dichloromethane (5 mL). After the addition is complete, remove the ice bath and continue the reaction for 5 - 10 min. After TLC plate shows complete reaction, add 50 ml of saturated ammonium chloride aqueous solution to quench, extract, take the organic layer, and extract the aqueous layer with dichloromethane (10 ml) twice more. Combine the organic layers and evaporate to dryness to obtain the white solid product 3 (2.94 g, 95.1%). 1 H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 6.36 (dd, J = 16.8, 10.6 Hz,1H), 6.13 (dd, J= 16.8, 1.4 Hz, 1H), 5.43 (dd, J = 10.6, 1.4 Hz, 1H), 3.62 (td, J = 14.6, 7.3 Hz, 1H), 1.90 - 1.85 (m, 2H), 1.69 -1.62 (m, 2H), 1.58 (dd, J =9.1, 3.8 Hz, 1H), 1.29 (td, J = 12.2, 3.4 Hz, 2H), 1.19 - 1.11 (m, 3H), ESI-MS:m / z =154.1221[M+H] + .

[0033] (2) Preparation of tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (Compound 4)

[0034] Compound 3 (2.94 g, 19.2 mmol) and tert-butyl hydrazinecarboxylate (5.07 g, 38.4 mmol) were dissolved in 100 mL of a sealed tube containing isopropanol (30 mL). The reaction system was heated to 120 °C and reacted for 24 h. After cooling to room temperature, the isopropanol was evaporated. Dichloromethane (30 mL) and water (100 mL) were added, and extraction was performed. The aqueous layer was yellow and was discarded. The aqueous layer was extracted two more times with water (100 mL) until the aqueous layer was colorless. The organic layer was evaporated, and the crude product was separated and purified by column chromatography to obtain the white solid product 4 (3.1 g, 56.6%). 1 HNMR (400 MHz, CDCl3) δ 6.69 (s, 1H), 6.15 (s, 1H), 3.81 - 3.71 (m, 1H), 3.10(t, J = 6.1 Hz, 2H), 2.33 (t, J = 6.1 Hz, 2H), 1.89 (dd, J = 12.4, 3.2 Hz, 2H),1.73 - 1.64 (m, 2H), 1.62 - 1.56 (m, 1H), 1.45 (s, 9H), 1.41 - 1.30 (m, 2H),1.30 – 1.09 (m, 4H), ESI-MS: m / z =286.2124 [M+H] + .

[0035] Preparation of tert-butyl 2-(benzo[d]thiazole-2-carbonyl)-2-3-((cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (Compound 5) and tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)-2-(quinoline-2-carbonyl)hydrazine-1-carboxylate (Compound 6)

[0036] Add benzothiazole-2-carboxylic acid (197.1 mg, 1.1 mmol) and 2-quinolinecarboxylic acid (190.5 mg, 1.1 mmol) respectively to two 25 ml round-bottom flasks containing dichloromethane (5 ml). Add HATU (570.4 mg, 1.5 mmol) and DIPEA (387 mg, 3.0 mmol) to each flask, stir at room temperature for 15 min, and then add Compound 4 (285.4 mg, 1.0 mmol) to each. Continue stirring at room temperature for 2 h. After TLC plate monitoring shows complete reaction, quench each reaction system by adding 20 ml of saturated ammonium chloride aqueous solution, extract, take the organic layer, and extract the aqueous layer twice with dichloromethane (5 ml). Combine the organic layers, evaporate to dryness, and purify the crude product by column chromatography to obtain white solid products 5 (361.7 mg, 81.0%) and 6 (349.2 mg, 79.3%). Compound 5 1 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J J = 8.0 Hz, 1H), 7.95 (d, J J = 7.8 Hz, 1H), 7.76(s, 1H), 7.51 (dt, J J = 14.8, 7.1 Hz, 2H), 4.04 (t, J J = 5.7 Hz, 2H), 3.82 - 3.68(m, 1H), 2.63 (t, J J = 6.1 Hz, 2H), 1.88 (d, J J = 10.4 Hz, 2H), 1.63 - 1.49 (m,3H), 1.35 (s, 9H), 1.33 - 1.23 (m, 2H), 1.19 - 1.05 (m, 3H), ESI-MS: m / z =447.2061 [M+H] + ; Compound 6 1 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J J = 8.5 Hz, 1H), 8.07(d, J J = 8.5 Hz, 1H), 7.86 - 7.80 (m, 2H), 7.76 (dd,J = 13.9, 5.9 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 4.07 (t, J = 6.2 Hz, 2H), 3.85 - 3.75 (m, 1H), 2.65 (t, J = 6.4 Hz, 2H), 1.93 (d, J = 9.5 Hz, 2H), 1.74 - 1.67 (m, 2H), 1.60 (d, J = 12.7 Hz, 1H), 1.36 (d, J = 12.7 Hz, 2H), 1.15 (d, J = 12.1 Hz, 3H), 1.04 (s, 9H), ESI-MS: m / z = 441.2502 [M+H] + .

[0037] (4)3-(1-(Benzo[d]thiazole-2-carbonyl)hydrazinyl)- N -cyclohexylpropanamide (Compound 7) and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazinyl)propanamide (Compound 8) Preparation

[0038] Compound 5 (361.7 mg, 0.81 mmol) and Compound 6 (349.2 mg, 0.79 mmol) were separately added to two 25 ml round-bottom flasks containing dichloromethane (5 ml), trifluoroacetic acid (2 ml) was added to each, and the reaction was carried out at room temperature for 1 h. After TLC plate showed complete reaction, the solvent and trifluoroacetic acid were rotary evaporated to obtain Products 7 and 8.

[0039] (5)3-(1-(Benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazinyl)- N -cyclohexylpropanamide (Compound 1) and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazinyl)propanamide (Compound 2) Preparation

[0040] Compound 7 (173.3 mg, 0.5 mmol) and compound 8 (170.2 mg, 0.5 mmol) were separately added to a 25 ml sealed tube containing isopropanol (3 ml). Subsequently, acetic acid (100 μl) and furan-2-carbaldehyde (57.7 mg, 0.6 mmol) were added to each. The two reaction systems were heated to 80 °C and reacted overnight. After TLC plate showed complete reaction, the solvent was evaporated to dryness. Each was slurried with diethyl ether (3 ml), filtered by suction, and the filter cake was washed 3 - 5 times with diethyl ether (3 ml). The resulting solid was dried under vacuum to obtain product 1 (157.1 mg, 74.0%) and 2 (148.0 mg, 70.7%). Compound 1 1 H NMR (400 MHz, DMSO) δ 8.24 - 8.15 (m, 3H),7.90 (s, 2H), 7.58 (t, J = 7.0 Hz, 2H), 7.02 (s, 1H), 6.67 (s, 1H), 4.34 (s,2H), 3.48 (d, J = 7.0 Hz, 1H), 2.47 - 2.42 (m, 2H), 1.63 (dd, J = 25.0, 10.5 Hz,4H), 1.49 (d, J = 8.9 Hz, 1H), 1.23 - 1.14 (m, 2H), 1.06 (dd, J = 22.3, 11.4 Hz,3H), ESI-MS: m / z =425.1640 [M+H] + ; Compound 2 1 H NMR (400 MHz, CDCl3) δ 8.21 (dd, J =13.8, 8.6 Hz, 2H), 7.95 (s, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 8.2 Hz,1H), 7.60 (t, J = 7.8 Hz, 2H), 7.33 (s, 1H), 6.45 (s, 1H), 6.33 (s, 1H), 6.07(d, J = 7.0 Hz, 1H), 4.45 (t, J = 6.6 Hz, 2H), 3.78 - 3.66 (m, 1H), 2.66 (t, J =6.8 Hz, 2H), 1.84 (dd,J = 12.2, 2.8 Hz, 2H), 1.65 (dd, J = 9.6, 3.7 Hz, 2H),1.56 (dd, J = 9.4, 3.5 Hz, 1H), 1.27 (d, J = 9.7 Hz, 2H), 1.17 - 1.07 (m, 3H), ESI-MS: m / z = 419.2074 [M+H] + .

[0041] Example 2 (Activity Evaluation) Evaluation of 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide for 20S proteasome agonist activity and protective effect on hypoxic damaged cardiomyocytes

[0042] Test for 20S proteasome agonist activity

[0043] Experimental method: The activity was detected using the fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC, and the activation of the enzyme by different compounds was observed to preliminarily evaluate the activation effect of the compounds. The 20S proteasome hydrolyzes the Tyr-AMC sequence in the substrate, releasing AMC, and the fluorescence absorption value of the hydrolyzed product AMC can be detected under the conditions of excitation light at 355 nm and emission light at 460 nm to observe the activation of the enzyme activity by the compounds. The results are shown in Table 1.

[0044] Test for protective effect on hypoxic damaged cardiomyocytes

[0045] Experimental method: The cell viability was detected by MTT analysis. That is, H9C2 cells (1×104 / well) were seeded in 96-well plates and incubated overnight, and then treated with the test compounds (0, 0.25, 0.5, 1, and 10 μmol / L) for 48 hours. 10 μL of MTT solution (5 mg / ml) was added to each well, and after incubation for 4 hours, the supernatant was aspirated, and 150 μl of DMSO was added to dissolve the resulting formazan crystals. The absorbance value at 570 nm was measured using an enzyme-linked immunosorbent assay reader (Labsystems, Finland). The cell viability was calculated as follows: Cell viability (%) = OD of the test group / OD of the control group x 100%. The cell viability values were plotted against the different concentrations of the agonist, see Figure 1 .

[0046] Table 1 Agonist activity of compounds on 20S proteasome

[0047]

[0048] Activity (%) @ 20 μM: The agonist fold increase of the 20S proteasome hydrolysis activity of the compound at a concentration of 20 μM; Max Fold increase: The maximum agonist fold of the compound on the 20S proteasome;

[0049] Two compounds of the present invention have good 20S proteasome agonist activity, have significant protective activity on hypoxic damaged cardiomyocytes, and have no obvious cytotoxicity. The above experiments show that this type of compound has excellent disease treatment application prospects and thus has good commercial value.

Claims

1. A small molecule proteasome agonist, characterized in that, Its structural formula is shown in Formula (1) or (2): ， The chemical formula name of compound (1) is 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide, and the chemical formula name of compound (2) is N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide.

2. The preparation method of the small molecule proteasome agonist according to claim 1, characterized in that, It includes the following steps: (1) Cyclohexylamine, triethylamine and acryloyl chloride react in dichloromethane under an ice bath to form N - cyclohexylacrylamide; (2) N - Cyclohexylacrylamide and tert-butyl hydrazinecarboxylate react in isopropanol at 120 °C to form tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate; (3) tert-Butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate is condensed with benzothiazole-2-carboxylic acid and 2-quinolinecarboxylic acid respectively in the presence of a condensing agent to form tert-butyl 2-(benzo[d]thiazole-2-carbonyl)-2-3-((cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate and tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)-2-(quinoline-2-carbonyl)hydrazine-1-carboxylate at room temperature; (4) tert-Butyl 2-(benzo[d]thiazole-2-carbonyl)-2-3-((cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate and tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)-2-(quinoline-2-carbonyl)hydrazine-1-carboxylate are deprotected with trifluoroacetic acid to form 3-(1-(benzo[d]thiazole-2-carbonyl)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propanamide; (5) 3-(1-(benzo[d]thiazole-2-carbonyl)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propanamide were respectively reacted with furan-2-carbaldehyde at 80 °C to form the target compounds 3-(1-(benzo[d]thiazole-2-carbonyl)-2-(furan-2-ylmethylene)hydrazono)- N -cyclohexylpropanamide and N -cyclohexyl-3-(2-(furan-2-ylmethylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide.

3. The preparation method of the small molecule proteasome agonist according to claim 2, characterized in that, The condensing agent in (3) is HATU.

4. Use of the small molecule proteasome agonist according to claim 1 in the preparation of a drug for repairing myocardial cell hypoxic injury.

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