Small-molecular proteasome agonist as well as preparation method and application thereof

Abstract

The invention discloses a small molecular proteasome agonist as well as a preparation method and application thereof. According to the invention, cyclohexylamine is used as an initial raw material, and 3-(1-(benzo [D] thiazole-2-carbonyl)-2-(furan-2-methylene) hydrazone group)-N-cyclohexylpropanamide and N-cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl) hydrazone group) propanamide are prepared through a five-step reaction. The two compounds provided by the invention have good 20S proteasome agonistic activity and ischemic injury myocardial cell protection activity, do not have obvious cytotoxicity, and are expected to become effective choices for ischemic cardiomyopathy and other proteotoxic diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular, to a new class of small molecule proteasome agonists, preparation methods and applications thereof, and application of the compounds in the preparation of drugs for treating protein toxic diseases. Background Art

[0002] The proteasome is a large protease system composed of multiple subunits. It is responsible for 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 a variety of pathological conditions, including cancer, neurodegenerative diseases, and cardiovascular diseases. With age, the function and content of the proteasome generally decline, resulting in the obstruction of protein degradation, which in turn causes the accumulation and aggregation of proteins. These abnormally aggregated proteins produce protein toxicity and may cause 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 their 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 known proteasome activators is limited, related research is progressing steadily, providing new directions for the development of future therapeutic strategies. Summary of the invention

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

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

[0006] The chemical formula of formula (1) is 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -cyclohexylpropionamide, the chemical formula name of formula (2) is N -cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide.

[0007] The preparation method of the above-mentioned small molecule proteasome agonist is achieved by the following synthetic route:

[0008] The above-mentioned 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -Cyclohexylpropionamide and N -cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide, the preparation method comprising the following steps: (1) Cyclohexylamine, triethylamine and acryloyl chloride react in dichloromethane under ice bath to generate N -cyclohexylacrylamide (compound 3); (2) N - Cyclohexyl acrylamide and tert-butyl carbazate react in isopropanol at high temperature to generate tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (compound 4); (3) tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate is condensed with benzothiazole-2-carboxylic acid and 2-quinolinecarboxylic acid in the presence of a condensation reagent to generate 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) 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 were deprotected by trifluoroacetic acid to generate 3-(1-(benzo[D]thiazole-2-carbonyl)hydrazine)- N -cyclohexylpropionamide (compound 7) and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propionamide (compound 8); (5) 3-(1-(Benzo[D]thiazole-2-carbonyl)hydrazine)- N -Cyclohexylpropionamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propionamide and furan-2-carboxaldehyde were reacted at high temperature to generate the target compound 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazono)- N -cyclohexylpropionamide (compound 1) and N -cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propanamide (Compound 2).

[0009] Specifically, the preparation method may include the following steps: (1) Add cyclohexylamine and triethylamine to the reaction flask, dissolve in dichloromethane, place in an ice bath, add acryloyl chloride dropwise, remove the ice bath after the addition is complete, and continue the reaction. After the TLC plate shows complete reaction, add saturated ammonium chloride aqueous solution, extract, take the organic layer, extract the aqueous layer twice with dichloromethane, combine the organic layers, and spin dry to obtain product 3; (2) Add compound 3 and tert-butyl carbazate to a sealed tube, dissolve in isopropanol, and heat the reaction system to a high temperature for reaction. Cool to room temperature, spin dry the system, add dichloromethane and water, extract, spin dry the organic layer, and separate and purify the crude product by column chromatography to obtain product 4; (3) Add benzothiazole-2-carboxylic acid and 2-quinolinecarboxylic acid to two round-bottom flasks respectively, dissolve in dichloromethane, add HATU and DIPEA to each flask, stir at room temperature for 15 min, then add compound 4 to each flask, and continue to react at room temperature. After the TLC plate shows complete reaction, add saturated ammonium chloride aqueous solution to each of the two reaction systems for quenching, extract, spin dry the organic layer, and separate and purify the crude products by column chromatography to obtain product 5 and product 6; (4) Compound 5 and compound 6 were added to two round-bottom flasks respectively, dissolved in dichloromethane, and trifluoroacetic acid was added to each, and reacted at room temperature. After the TLC plate showed complete reaction, the solvent and trifluoroacetic acid were dried to obtain products 7 and 8; (5) Compound 7 and compound 8 were added to a sealed tube, dissolved in isopropanol, and then acetic acid and furan-2-carboxaldehyde were added, respectively. The two reaction systems were heated to a high temperature and reacted overnight. After the TLC plate showed complete reaction, the solvent was dried by spin drying, ether was added to each of the two systems for slurrying, and the filter cakes were washed with ether. The solids were vacuum dried to obtain products 1 and 2.

[0010] In the above technical solution, further, the high temperature described in (2) is 120°C; Furthermore, the high temperature described in (5) is 80°C.

[0011] The above 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazonoyl)- N -Cyclohexylpropionamide and N -Use of cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide in preparing drugs for protein toxic diseases.

[0012] The small molecule proteasome agonist can be a drug for treating neurodegenerative diseases, cardiovascular diseases, diabetes, etc., to stimulate the activity of 20S proteasome and activate the protein degradation function.

[0013] The present invention has been confirmed by molecular and cell experiments: 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -Cyclohexylpropionamide and N -Cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide has good 20S proteasome agonist activity, has the effect of repairing hypoxia loss in cardiomyocytes, and has no obvious cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the test results of the protective effects of compounds 1 and 2 on hypoxic damaged cardiomyocytes. DETAILED DESCRIPTION

[0015] The following examples are merely illustrative of the present invention and are not intended to limit the present invention in any way.

[0016] Example 1 3-(1-(Benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -Cyclohexylpropionamide and N Preparation of cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide

[0017] (1) N - Preparation of cyclohexyl acrylamide (Compound 3) Cyclohexylamine (2 g, 20.2 mmol) and triethylamine (4.09 g, 40.4 mmol) were dissolved in 10 mL of dichloromethane, and then added dropwise in an ice bath to a dichloromethane (5 mL) solution of acryloyl chloride (1.83 g, 20.2 mmol). After the addition was complete, the ice bath was removed and the reaction was continued for 5-10 min. After the TLC plate showed complete reaction, 50 ml of saturated ammonium chloride aqueous solution was added to quench the reaction, and the organic layer was extracted. The aqueous layer was extracted twice with dichloromethane (10 ml), and the organic layers were combined and dried to obtain a 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] + . (2) Preparation of tert-butyl 2-(3-(cyclohexylamino)-3-oxopropyl)hydrazine-1-carboxylate (Compound 4) Compound 3 (2.94 g, 19.2 mmol) and tert-butyl carbazate (5.07 g, 38.4 mmol) were dissolved in a 100 mL sealed tube filled with isopropanol (30 ml), and the reaction system was heated to 120 °C for 24 h. After cooling to room temperature, the isopropanol was dried by spin drying, and dichloromethane (30 mL) and water (100 mL) were added for extraction. The water layer was yellow, and the water layer was discarded. The mixture was extracted twice with water (100 mL) until the water layer was colorless. The organic layer was spin dried, and the crude product was separated and purified by column chromatography to obtain a 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] + . (3) 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) Benzothiazole-2-carboxylic acid (197.1 mg, 1.1 mmol) and 2-quinolinecarboxylic acid (190.5 mg, 1.1 mmol) were added to two 25 ml round-bottom flasks filled with dichloromethane (5 ml), HATU (570.4 mg, 1.5 mmol) and DIPEA (387 mg, 3.0 mmol) were added to each flask, and stirred at room temperature for 15 min, followed by adding compound 4 (285.4 mg, 1.0 mmol) and continuing stirring at room temperature for 2 h. After TLC plate showed complete reaction, 20 ml of saturated ammonium chloride aqueous solution was added to each of the two reaction systems to quench, extract, take the organic layer, extract the aqueous layer twice with dichloromethane (5 ml), combine the organic layers, spin dry, and separate 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 H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.76(s, 1H), 7.51 (dt, J = 14.8, 7.1 Hz, 2H), 4.04 (t, J = 5.7 Hz, 2H), 3.82 - 3.68(m, 1H), 2.63 (t, J = 6.1 Hz, 2H), 1.88 (d, 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 H NMR (400 MHz, CDCl3) δ 8.23 ​​(d, J = 8.5 Hz, 1H), 8.07(d, 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] + . (4) 3-(1-(Benzo[D]thiazole-2-carbonyl)hydrazine)- N -cyclohexylpropionamide (compound 7) and N Preparation of cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propionamide (Compound 8) Compound 5 (361.7 mg, 0.81 mmol) and compound 6 (349.2 mg, 0.79 mmol) were added to two 25 ml round-bottom flasks filled with dichloromethane (5 ml), and trifluoroacetic acid (2 ml) was added to each. The reaction was carried out at room temperature for 1 h. After the TLC plate showed complete reaction, the solvent and trifluoroacetic acid were dried to obtain products 7 and 8.

[0018] (5) 3-(1-(Benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -cyclohexylpropionamide (compound 1) and N Preparation of cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide (Compound 2) Compound 7 (173.3 mg, 0.5 mmol) and compound 8 (170.2 mg, 0.5 mmol) were added to a 25 ml sealed tube containing isopropanol (3 ml), followed by acetic acid (100 µl) and furan-2-carboxaldehyde (57.7 mg, 0.6 mmol), and the two reaction systems were heated to 80 °C for overnight reaction. After TLC plate showed complete reaction, the solvent was dried by spin drying, ether (3 ml) was added to each, slurried, filtered, and the filter cake was washed with ether (3 ml) for 3-5 times. The obtained solid was vacuum dried to obtain products 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] + . Example 2 (Activity Evaluation) Evaluation of 3-(1-(Benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -Cyclohexylpropionamide and N20S proteasome agonist activity and protective effect of cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide on hypoxic cardiomyocytes 20S proteasome agonist activity test Experimental method: The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC was used to detect the activity and observe the activation of the enzyme by different compounds to preliminarily evaluate the activation effect of the compound. The 20S proteasome hydrolyzes the Tyr-AMC sequence in the substrate to release AMC. Under the conditions of excitation light 355nm and emission light 460nm, the fluorescence absorption value of the hydrolysis product AMC can be detected to observe the activation of the enzyme activity by the compound. The results are shown in Table 1.

[0019] Test of the protective effect on myocardial cells damaged by hypoxia Experimental methods: MTT assay was used to detect cell viability, that is, H9C2 cells (1×104 / well) were plated in 96-well plates, incubated overnight, and then treated with the test compound (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. After incubation for 4 hours, the supernatant was discarded, 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 different concentrations of the agonist, see Figure 1 .

[0020] Table 1 Agonist activity of compounds on 20S proteasome

[0021] Activity (%) @ 20 μM: The 20S proteasome hydrolysis activity activation fold of the compound at 20 μM concentration; Max Fold increase: The maximum activation fold of the compound on the 20S proteasome; The two compounds of the present invention have good 20S proteasome stimulating activity, have significant protective activity against hypoxic damaged cardiomyocytes, and have no obvious cytotoxicity. The above experiments show that the compounds have excellent application prospects for disease treatment and thus have 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 of compound (1) is 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazine)- N -cyclohexylpropionamide, the chemical formula of compound (2) is N -cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide.

2. The method for preparing a small molecule proteasome agonist according to claim 1, characterized in that: The following steps are involved: (1) Cyclohexylamine, triethylamine and acryloyl chloride react in dichloromethane under ice bath to generate N - Cyclohexylacrylamide; (2) N - Cyclohexyl acrylamide and tert-butyl carbazate react in isopropanol at high temperature to produce 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 in the presence of a condensation reagent to generate 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 were deprotected by trifluoroacetic acid to generate 3-(1-(benzo[D]thiazole-2-carbonyl)hydrazine)- N -Cyclohexylpropionamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propionamide; (5) 3-(1-(Benzo[D]thiazole-2-carbonyl)hydrazine)- N -Cyclohexylpropionamide and N -cyclohexyl-3-(1-(quinoline-2-carbonyl)hydrazono)propionamide and furan-2-carboxaldehyde were reacted at high temperature to generate the target compound 3-(1-(benzo[D]thiazole-2-carbonyl)-2-(furan-2-methylene)hydrazono)- N -Cyclohexylpropionamide and N -cyclohexyl-3-(2-(furan-2-methylene)-1-(quinoline-2-carbonyl)hydrazono)propionamide.

3. The method for preparing a small molecule proteasome agonist according to claim 2, characterized in that: The high temperature in (2) is 120°C; the condensation reagent in (3) is HATU; and the high temperature in (5) is 80°C.

4. Use of the small molecule proteasome agonist according to claim 1 in the preparation of drugs for protein toxic diseases.

5. The use according to claim 4, characterized in that: The diseases are neurodegenerative diseases, cardiovascular diseases and diabetes.

6. The use according to claim 5, characterized in that: The neurodegenerative diseases are Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia, and myasthenia gravis; the cardiovascular diseases are coronary heart disease, stroke, heart failure, cardiomyopathy, rheumatic heart disease, abnormal heart rhythm, congenital heart disease, myocarditis, thromboembolic disease, and venous thrombosis.

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