N-phenethyl amide compound as well as preparation method and application thereof

Abstract

The invention provides an N-phenethyl amide compound as well as a preparation method and application thereof. The structural formula of the compound is shown as a formula (I) or a formula (II). The compound provided by the invention can be used as an AC inhibitor, shows a very strong inhibition effect in the aspects of inhibiting AC, melanoma or breast cancer proliferation and the like, has an inhibition effect superior to that of carbamofluorine, and has the potential of being developed into a novel anti-cancer treatment drug. Moreover, the preparation method of the compound provided by the invention is simple, has low requirements on equipment, is easy to operate, does not need to adopt dangerous reagents, can reduce the production cost, and is suitable for industrial production. The invention provides a new direction for the field of anticancer treatment.

Description

Technical Field

[0001] This invention relates to the field of antitumor compound preparation, and more specifically to a... N - Phenethylamide compounds, their preparation methods, and applications. Background Technology

[0002] Acid ceramidinase (AC) is a key regulator of sphingolipid metabolism, and its abnormally high expression is often associated with cancer drug resistance and tumor growth. Acid ceramidinase inhibitors (ACi) are a class of compounds that can specifically block the activity of acid ceramidinase (AC). Their core mechanism lies in "restoring" the toxicity of ceramide, thereby inducing apoptosis in cancer cells.

[0003] Existing ceramide (AC) inhibitors are structurally limited, primarily revolving around substrates or carmofluridine derivatives, and generally suffer from poor metabolic stability and low oral bioavailability. Studies have shown that adding the substrate analog De-MAPP as an AC inhibitor to cells leads to a time-dependent increase in endogenous ceramide levels 24 hours after treatment, indicating De-MAPP's inhibitory activity. However, De-MAPP is poorly metabolized and therefore not a suitable AC inhibitor. Carmofluridine and its derivatives block AC by forming covalent adducts with catalytic Cys143, but covalent inhibitors also carry potential off-target effects and immunogenicity risks.

[0004] Therefore, there is a need to develop novel AC inhibitors with higher enzyme inhibition and anticancer activity to provide more options for the development of AC-targeted drugs. Summary of the Invention

[0005] One of the objectives of this invention is to provide a N - Phenethylamide compounds or their pharmaceutically acceptable salts to address the problem of low efficacy of existing AC inhibitors.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned compounds and their pharmaceutical salts.

[0007] A further object of the present invention is to provide the use of the above-mentioned compounds and their pharmaceutical salts in the preparation of antitumor drugs.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A sort of N - A phenylethylamide compound or a pharmaceutically acceptable salt thereof, the general structural formula of which is shown in formula (I) or (II): Wherein, R1 is any one of furanyl, thienyl, pyridinyl, 3-methyl-1H-pyrazolyl, 2-aminothiazolyl, 2-methylthiazolyl, 3-ethyl-3H-indolyl, chlorophenyl, 3-(difluoromethyl)-1-methyl-1H-pyrazolyl, morpholinyl, 4-aminopiperidinyl, 4-aminocyclohexanolyl, piperazinyl; and R2 is any one of pyrimidinyl, pyridinyl, isoxazolyl, phenyl, 4-formylphenyl, 4-chlorophenyl, 4-methylphenyl, pyrazolyl.

[0009] Preferably, R1 is , , , , , , , , , , , , , , Any one of them; R2 is , , Any one of them.

[0010] More preferably, the compound is selected from one of the following structures: .

[0011] The pharmaceutical salts of the compounds include, but are not limited to, acetates, ascorbic acid salts, benzoates, benzenesulfonates, citrates, fumarates, hydrochlorides, hydrobroms, maleates, and mesylates.

[0012] The present invention also provides a method for preparing the above-mentioned compound, the synthetic route and steps of which are as follows.

[0013] The synthetic route for the compound shown in formula (I) is as follows:

[0014] or

[0015] The specific steps for preparing the compound shown in formula (I) are as follows: (a) The compound having R1 undergoes a condensation reaction with p-bromophenethylamine to form compound A; (b) Compound A undergoes Suzuki coupling with R2B(OH)2 to produce the compound shown in formula (I); or, (a') p-Bromophenylethylamine undergoes Suzuki coupling with R2B(OH)2 to form compound B; (b') Compound B and phenyl chloroformate undergo a condensation reaction to form compound C; (c') Compound C reacts with a compound having R1, and part of the compound undergoes deprotection and salt formation to produce the compound shown in formula (I).

[0016] Specifically, in step (a), the compound having R1, p-bromophenethylamine, PyBOP, and triethylamine are... N , N In a dimethylformamide solution, the reaction was carried out in an ice bath for 2 h to generate compound A. The molar ratio of the compound with R1, p-bromophenylethylamine, PyBOP and triethylamine was 1:1.1:1:1.

[0017] Specifically, in step (b), compound A and R2B(OH)2 are heated to reflux at 85°C for 7 h in a mixed solvent of 1,4-dioxane and water (volume ratio 4:1) under N2 protection to generate compound (Ⅰ), wherein the molar ratio of compound A to R2B(OH)2 is 1:1.5.

[0018] Specifically, in step (a'), p-bromophenylethylamine and R2B(OH)2 are heated to reflux at 85°C for 7 h under N2 protection in a mixed solvent of 1,4-dioxane and water (volume ratio 4:1) to generate compound B. The molar ratio of p-bromophenylethylamine to R2B(OH)2 is 1:1.5.

[0019] Specifically, in step (b'), compound B, phenyl chloroformate, and triethylamine are reacted in a dichloromethane solution in an ice bath for 5 h to generate compound C. The molar ratio of compound B, phenyl chloroformate, and triethylamine is 1:1.1:1.5.

[0020] Specifically, in step (c'), compound C, the compound having R1, DIPEA and triethylamine undergo ammonolysis in dichloromethane solution, and some compounds undergo deprotection and salt formation to generate compound (Ⅰ). The molar ratio of compound C, the compound having R1, DIPEA and triethylamine is 1:1.2:2:2.

[0021] The synthetic route for the compound shown in formula (II) is as follows:

[0022] The specific steps for preparing the compound shown in formula (II) are as follows: (1) p-Bromophenylethylamine and oxaloyl chloride monoethyl ester undergo a condensation reaction to form compound D; (2) Compound D undergoes a hydrolysis reaction with sodium hydroxide to produce compound E; (3) Compound E undergoes a condensation reaction with a compound having R1, and some of the compounds undergo deprotection and salt formation to form compound F; (4) Compound F undergoes Suzuki coupling with R2B(OH)2 to produce the compound shown in formula (II).

[0023] Specifically, in step (1), p-bromophenylethylamine, oxaloyl chloride monoethyl ester and triethylamine are reacted in dichloromethane in an ice bath for 2 h to generate compound D. The molar ratio of p-bromophenylethylamine, oxaloyl chloride monoethyl ester and triethylamine is 1:1:1.

[0024] Specifically, in step (2), compound D reacts with sodium hydroxide in an ethanol:water ratio of 2:1 system at room temperature for 2 h to produce compound E.

[0025] Specifically, in step (3), compound E, the compound having R1, TBTU and triethylamine are reacted in dichloromethane at room temperature for 4 h to generate compound F. The molar ratio of compound E, the compound having R1, TBTU and triethylamine is 1:1.2:1.2:2.

[0026] Specifically, in step (4), compound F and R2B(OH)2 are heated to 85°C and refluxed for 7 h in a mixed solvent of 1,4-dioxane and water (volume ratio 4:1) under N2 protection to generate the compound shown in formula (II). The molar ratio of compound F to R2B(OH)2 is 1:1.5.

[0027] The present invention also provides the above. N - The use of phenylethylamide compounds or their pharmaceutically acceptable salts in the preparation of antitumor drugs.

[0028] The tumor is melanoma, breast cancer, liver cancer, or colon cancer.

[0029] The present invention also provides a pharmaceutical composition comprising the compound described herein or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers and / or excipients.

[0030] This invention synthesizes a novel class of small molecule drugs targeting AC. Some of the synthesized compounds exhibit very strong inhibitory effects on the proliferation of AC, melanoma, or breast cancer, with inhibitory effects superior to camofluoride, and have the potential to be developed into novel anticancer therapeutic drugs.

[0031] The compound reaction steps provided by this invention are simple, require low-end equipment, and are easy to operate, significantly improving reaction yield and further reducing production costs. It eliminates the need for dangerous, flammable, and explosive reagents, ensuring good catalytic effects and reducing production costs while achieving process simplification, cost savings, and convenient post-processing, making it suitable for industrial production. Detailed Implementation

[0032] In the following embodiments, the various processes and methods not described in detail are conventional methods known in the art, and the reagents used, unless otherwise specified, are commercially available analytical grade or chromatographic grade.

[0033] Example 1: Preparation of compounds 1-11 (a series of compounds shown in formula (I)) (1) Weigh 6.93 mmol of different types of acids or acyl chlorides with the R1 structure and 1.52 g (7.6 mmol) of p-bromophenylethylamine, and place them in a 50 mL round-bottom flask containing 20 mL of N,N-dimethylformamide. Then add 3.6 g (6.91 mmol) of PyBOP and 0.7 g (6.91 mmol) of triethylamine, and stir the mixture in an ice bath for 2 h. The reaction endpoint was detected by TCL, the reaction was quenched with water, and the mixture was extracted three times with dichloromethane. The organic phases were combined, extracted three times with saturated brine, and the organic phases were combined and dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, 0.5 g of a white or brown solid (compound A) was obtained, with a yield of 56.9-78.8%.

[0034] (2) 0.4 g (1.12 mmol) of compound A and 1.61 mmol of different types of boric acids with the R2 structure were placed in a reaction flask. 0.54 g (1.68 mmol) of cesium carbonate and 0.06 g (0.05 mmol) of tetrakis(triphenylphosphine)palladium were added, followed by the addition of a mixed solution of 1,4-dioxane and water in a volume ratio of 4:1 to dissolve the compounds. The reaction flask was then purged with nitrogen, and the reaction was stirred under nitrogen and 85°C. The reaction endpoint was determined by TCL, and the mixture was extracted with water and dichloromethane. The extracted dichloromethane layer was collected and placed in a 250 mL conical flask, and then dried with an appropriate amount of anhydrous sodium sulfate. The mixture was separated by vacuum filtration and silica gel column chromatography (eluent: dichloromethane:methanol = 60:1) to obtain 0.8 g of white solid, which was part of the series of compounds shown in formula (I), with a yield of approximately 50-69%.

[0035] The different types of acids or acyl chlorides with R1 structures used in step (1) and the boric acids with different types of R2 structures used in step (2), as well as the specific structures of the corresponding compounds obtained, are shown in Table 1.

[0036] Table 1

[0037] The characterization data of compounds 1-11 are as follows: Compound 1: 1 H NMR (600 MHz, Chloroform- d ) δ 9.20 (s, 1H), 8.95 (s, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 3H), 7.12 (d, J = 3.5 Hz, 1H), 6.51– 6.47 (m, 1H), 6.43 (s, 1H), 3.73 (dt, J = 13.3, 7.0 Hz, 2H), 2.99 (t, J = 7.0Hz, 2H). 13 C NMR (151 MHz, Chloroform- d ) δ 158.56, 157.56, 154.94, 148.07, 144.00, 140.11, 134.18, 132.69, 130.06, 127.40, 114.42, 112.33, 40.34, 35.74.

[0038] Compound 2: 1 H NMR (600 MHz, Chloroform- d ) δ 9.20 (s, 1H), 8.94 (s, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.50 – 7.43 (m, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.08 –7.05 (m, 1H), 6.06 (s, 1H), 3.74 (dt, J = 13.6, 7.0 Hz, 2H), 3.01 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, Chloroform- d) δ 162.06, 157.55, 154.92, 140.16, 138.90, 134.15, 132.73, 130.11, 130.06, 128.18, 127.80, 127.41, 41.18, 35.67。

[0039] Compound 3: 1 H NMR (600 MHz, Chloroform- d ) δ 9.20 (s, 1H), 8.94 (s, 2H), 7.91 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 5.98 (s, 1H), 3.72 (dt, J = 13.5, 7.0 Hz, 二氢), 3.00 (t, J = 7.0 Hz, 2H), 2.72 (s, 3H)。 13 C NMR(151 MHz, Chloroform- d ) δ 170.29, 160.74, 157.56, 154.89, 142.85, 139.96, 134.16, 134.07, 132.77, 130.06, 127.41, 41.21, 35.54, 19.74。

[0040] Compound 4: 1 H NMR (600 MHz, Chloroform- d ) δ 9.19 (s, 1H), 8.93 (s, 2H), 8.90 (d, J = 2.3 Hz, 1H), 8.75 – 8.67 (m, 1H), 8.13 – 8.07 (m, 1H), 7.56 (d, J =8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.40 – 7.37 (m, 1H), 6.32 (s, 1H), 3.80(dt, J = 13.4, 7.0 Hz, 2H), 3.04 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, Chloroform- Note: In the translation of "3.72 (dt, = 13.5, 7.0 Hz, 二氢)", "二氢" might be a wrong or unclear term in the original. I've just translated it as "2H" according to the context, but it needs to be double-checked with the original source for accuracy.d ) δ 165.71, 157.57, 154.90, 152.27, 147.67, 139.96, 135.48, 134.04, 132.83,130.43, 130.05, 127.46, 123.80, 41.28, 35.49.

[0041] Compound 5: 1 H NMR (600 MHz, DMSO- d 6) δ 9.17 (s, 1H), 9.13 (s, 2H), 8.20(t, J = 5.6 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.58 (s, 1H), 7.42 (s, 2H), 7.39(d, J = 8.2 Hz, 2H), 3.43 (dt, J = 13.9, 7.0 Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H). 13 CNMR (151 MHz, DMSO- d 6) δ 171.45, 160.92, 157.08, 154.52, 141.46, 140.59,133.06, 131.57, 129.66, 126.87, 121.67, 40.38, 34.94.

[0042] Compound 6: 1 H NMR (600 MHz, DMSO- d 6) δ 10.75 (s, 1H), 9.17 (s, 1H), 9.11 (s, 2H), 7.93 (t, J = 5.6 Hz, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 2.4 Hz, 1H),7.07 – 7.04 (m, 1H), 6.97 (d, J= 7.9 Hz, 1H), 3.32 – 3.28 (m, 2H), 2.91 (t, J =7.7 Hz, 2H), 2.75 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 7.7 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 172.34, 157.54, 154.96, 141.10, 136.72, 133.54, 131.94, 130.13,127.52, 127.23, 122.60, 121.33, 118.83, 118.56, 114.31, 111.75, 40.52, 36.74, 35.24, 21.50.

[0043] Compound 7: 1 H NMR (600 MHz, Chloroform- d ) δ 9.19 (s, 1H), 8.93 (s, 2H), 7.64 (m, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.39 – 7.29 (m,3H), 6.28 (s, 1H), 3.80 (dt, J = 13.56Hz, 6.7 Hz, 2H), 3.05 (t, J = 6.7 Hz, 2H). 13 C NMR (151 MHz, Chloroform- d ) δ 166.65, 157.43, 154.89, 140.11, 135.10,134.17, 132.67, 131.49, 130.69, 130.38, 130.29, 130.14, 127.37, 127.26, 41.28, 35.39.

[0044] Compound 8: 1 H NMR (600 MHz, Chloroform- d ) δ 9.19 (s, 1H), 8.94 (s, 2H), 7.90 (s, 1H), 7.53 (d, J= 7.7 Hz, 2H), 7.39 (d, J = 7.7 Hz, 2H), 6.98 – 6.62 (m,1H), 6.40 (s, 1H), 3.72 (dt, J =13.67 Hz, 7.1 Hz, 2H), 2.98 (t, J = 7.1 Hz, 2H). 13 C NMR (151 MHz, Chloroform- d ) δ 161.29, 157.49, 154.92, 142.74, 142.55,142.36, 140.07, 135.54, 134.23, 132.60, 130.05, 127.30, 116.87, 113.51,111.97, 110.42, 40.97, 39.61, 35.47.

[0045] Compound 9: 1 H NMR (600 MHz, Chloroform- d ) δ 9.19 (s, 1H), 8.94 (s, 2H), 7.53 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H), 7.26 (s, 2H), 6.91 (s, 1H), 6.56 (s, 1H), 3.72 (dt, J = 13.89 Hz, 7.1 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H), 2.35 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 161.89, 157.06, 154.51, 147.04, 140.64, 139.64, 133.04, 131.50, 129.60, 126.84, 103.92, 40.06, 34.86, 10.31.

[0046] Compound 10: 1 H NMR (600 MHz, Chloroform- d ) δ 8.81 (d, J= 2.4 Hz, 1H),8.58 – 8.55 (m, 1H), 7.85 (dd, J = 7.9, 2.0 Hz, 1H), 7.54 – 7.47 (m, 2H), 7.35(dd, J = 7.9, 2.0 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.02 (s, 1H), 6.55 (s, 1H), 3.70 (dt, J = 13.9, 7.0 Hz, 2H), 2.95 (t, J = 7.1 Hz, 2H), 2.32 (s, 3H). 13 C NMR (151 MHz, Chloroform- d ) δ 162.50, 148.29, 148.17, 139.33, 136.60, 135.97,134.50, 129.70, 127.42, 123.78, 104.96, 40.45, 35.71, 11.22.

[0047] Compound 11: 1 H NMR (600 MHz, DMSO- d 6) δ 12.84 (s, 1H), 9.40 (s, 1H), 9.13 (s, 1H), 8.02 (t, J = 5.9 Hz, 1H), 7.74 – 7.49 (m, 2H), 7.47 – 7.10 (m, 2H), 6.33 (s, 1H), 3.46 (dt, J = 13.67 Hz, 7.4 Hz, 2H), 2.83 (t, J = 7.4 Hz, 2H), 2.24 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 161.85, 154.66, 148.20, 147.04, 139.63,139.28, 129.31, 129.19, 128.06, 126.26, 126.20, 120.62, 103.91, 34.89, 21.99,10.31.

[0048] Example 2: Preparation of compounds 12-15 (a series of compounds shown in formula (I)) (1) 0.5 g (2.5 mmol) of p-bromophenylethylamine and 0.47 g (3.75 mmol) of boric acid with the R2 structure were added to a reaction flask. 1.218 g (3.75 mmol) of cesium carbonate and 0.058 g (0.05 mmol) of tetrakis(triphenylphosphine)palladium were added, followed by dissolution in a 4:1 mixture of 1,4-dioxane and water. The reaction flask was then purged with nitrogen, and the mixture was stirred under nitrogen and 85°C. The reaction endpoint was determined by TCL. The mixture was extracted with water and dichloromethane, and then dried with an appropriate amount of anhydrous sodium sulfate. The product was separated by vacuum filtration and silica gel column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 0.4 g of white powder (compound B), with a yield of approximately 70-85%.

[0049] (2) Weigh 2.9 g (14.6 mmol) of compound B and 2.3 g (21.9 mmol) of triethylamine into a 100 mL flask containing 30 mL of dichloromethane. Under ice bath stirring, slowly add 2.51 g (16.06 mmol) of phenyl chloroformate. After 5 h of reaction, use a TCL to detect the reaction endpoint. Quench the reaction with water, extract three times with dichloromethane (50 mL), combine the organic phases, and dry them with anhydrous Na2SO4. Separate and purify by silica gel column chromatography (eluent: petroleum ether and ethyl acetate = 1:1) to give 2 g of white solid (compound C), with a yield of 55-73%.

[0050] (3) Weigh 0.4 g (1.2 mmol) of (4-(pyridin-3-yl)phenethylcarbamate (compound C), 1.44 mmol of different types of amines with R1 structure, 0.243 g (2.4 mmol) of triethylamine, and 0.31 g (2.4 mmol) of N,N-diisopropylethylamine and add them to a 100 mL round-bottom flask containing 30 mL of dichloromethane. Stir overnight and detect the reaction endpoint with TCL. Quench the reaction with water, extract three times with dichloromethane (50 mL), combine the organic phases, and dry with anhydrous Na2SO4. Separate and purify by silica gel column chromatography (eluent: petroleum ether and ethyl acetate = 1:1) to obtain 0.17 g of white solid (intermediate). Weigh 0.22 g of the intermediate and stir at room temperature for 2 minutes in a reaction system of dichloromethane and trifluoroacetic acid (volume ratio 15:1). h, TCL was used to determine the reaction endpoint. The solvent was removed by vacuum distillation, the reaction was quenched with water, and the pH was adjusted to alkaline by adding an appropriate amount of saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (50 mL), and the combined organic phases were dried over anhydrous Na₂SO₄. The solvent was removed by vacuum distillation, and the mixture was dissolved in a small amount of methanol. Hydrochloric acid was added dropwise to adjust the pH to acidic. The solvent was removed by vacuum distillation, and the mixture was crystallized from methanol and ethyl acetate to give 0.10 g of a white solid, which is part of the series of compounds shown in formula (I), with yields ranging from 43% to 67%.

[0051] The different types of amines with R1 structure used in step (3) and the boric acid with R2 structure in step (1), as well as the specific structures of the corresponding compounds obtained, are shown in Table 2.

[0052] Table 2

[0053] The characterization data of compounds 12-15 are as follows: Compound 12: 1 H NMR (600 MHz, Chloroform- d δ 8.84 (s, 1H), 8.58 (d, J =4.9 Hz, 1H), 7.88 (dt, J = 7.9, 2.0 Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.38 (dd, J = 7.9, 4.8 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 4.54 (t, J = 5.7 Hz, 1H), 3.66 (t, J= 4.9 Hz, 3H), 3.54 (q, J = 6.6 Hz, 2H), 3.30 (t, J = 4.9 Hz, 3H), 2.89 (t, J = 7.0Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 157.54, 148.26, 147.50, 139.96, 135.45,134.78, 133.88, 129.47, 126.78, 123.85, 65.94, 43.82, 41.74, 39.53, 35.58.

[0054] Compound 13: 1 H NMR (600 MHz, Methanol- d 4) δ 8.87 (d, J = 2.0 Hz, 1H), 8.57(d, J = 5.1 Hz, 1H), 8.27 (dd, J = 8.2, 2.0 Hz, 1H), 7.68 – 7.66 (m, 1H), 7.65(d, J = 7.9 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H), 4.10 (dt, J = 14.1, 2.8 Hz, 2H), 3.41 (t, J = 7.5 Hz, 2H), 3.36 – 3.32 (m, 1H), 2.97 – 2.78 (m, 4H), 2.04 – 1.92(m, 2H), 1.57 – 1.38 (m, 2H). 13 C NMR (151 MHz, Methanol- d 4) δ 159.66, 146.82,146.49, 141.97, 139.29, 138.38, 135.67, 130.95, 128.23, 126.23, 49.81, 43.40,43.27, 39.53, 37.11, 30.96.

[0055] Compound 14: 1 H NMR (600 MHz, DMSO- d6) δ 9.19 (d, J J = 2.0 Hz, 1H), 8.97 (s,2H), 8.82 (d, J J = 4.8 Hz, 1H), 8.75 (dd, J J = 8.0, 2.0 Hz, 1H), 8.01 (dd, J J = 8.0,4.8 Hz, 1H), 7.80 (d, J J = 7.9 Hz, 2H), 7.41 (d, J J = 7.9 Hz, 2H), 6.41 (s, 1H),5.97 (s, 1H), 3.65 – 3.62 (m, 1H), 3.27 (t, J J = 7.2 Hz, 2H), 3.23 – 3.13 (m,2H), 3.00 – 2.86 (m, 2H), 2.76 (t, J J = 7.2 Hz, 2H), 1.96 – 1.78 (m, 2H), 1.61 –1.45 (m, 2H). 13 C NMR (151 MHz, Methanol- d d4) δ 159.66, 146.82, 146.49, 141.97,139.29, 138.38, 135.67, 130.95, 128.23, 126.23, 49.81, 49.03, 43.40, 43.27,37.11, 30.96。

[0056] Compound 15: 1 H NMR (600 MHz, Methanol- d d4) δ 8.77 (d, J J = 2.0 Hz, 1H), 8.49(d, J J = 4.8 Hz, 1H), 8.07 (dt, J J = 8.0, 2.0 Hz, 1H), 7.59 (d, J J = 7.8 Hz, 2H), 7.50(dd, J J = 8.0, 4.8 Hz, 1H), 7.36 (d, J= 7.8 Hz, 2H), 3.81 – 3.78 (m, 1H), 3.78 –3.74 (m, 2H), 3.43 – 3.38 (m, 2H), 3.04 – 2.95 (m, 2H), 2.85 (t, J = 7.4 Hz, 2H), 1.85 – 1.75 (m, 2H), 1.43 – 1.35 (m, 2H). 13 C NMR (151 MHz, Methanol- d 4) δ150.42, 138.96, 138.63, 132.03, 129.01, 126.89, 121.39, 118.61, 115.98,58.80, 49.03, 33.84, 33.17, 27.63, 25.48.

[0057] Example 3: Preparation of the series of compounds shown in formula (II) (1) Weigh 0.5 g (2.5 mmol) of p-bromophenylethylamine, 0.34 g (2.5 mmol) of oxaloyl chloride monoethyl ester, and 0.25 g (2.5 mmol) of triethylamine, and place them in a 50 mL round-bottom flask containing 20 mL of dichloromethane. After reacting in an ice bath for 2 h, the reaction endpoint was detected by TCL. The reaction was quenched with water, extracted three times with dichloromethane, and the organic phases were combined. The organic phases were then extracted three times with saturated brine, and the organic phases were combined. The organic solvent was removed under reduced pressure, and 1 mL of dichloromethane and 20 mL of petroleum ether were added for recrystallization to obtain 0.61 g of white powder (compound D), with a yield of approximately 69-81%.

[0058] (2) Weigh 0.54 g (1.8 mmol) of ethyl 2-((4-bromophenylethyl)amino)-2-oxoethyl acetate (compound D) and place it in a 100 mL round-bottom flask containing 30 mL of ethanol and water (volume ratio 2:1). Add 1 mL of 4 N NaOH dropwise while stirring. After 2 h of reaction, use a TCL to detect the reaction endpoint. Adjust the pH to acidic, and a white solid precipitates. Filter to obtain 0.32 g of white solid (compound E), with a yield of 60-71%.

[0059] (3) Weigh 0.5 g (1.8 mmol) of 2-((4-bromophenylethyl)amino)-2-oxoacetic acid (compound E), 2.16 mmol of different types of amines with the R1 structure, 0.37 g (3.6 mmol) of triethylamine, and 0.7 g (2.16 mmol) of TBTU and place them in a 50 mL round-bottom flask containing 20 mL of dichloromethane. After reacting in an ice bath for 4 h, the reaction endpoint was detected by TCL. The reaction was quenched with water, extracted three times with dichloromethane, and the organic phases were combined. Then, an appropriate amount of anhydrous sodium sulfate was added for dehydration and drying. The mixture was separated by vacuum filtration and silica gel column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:1) to obtain 0.2 g of intermediate white powder. Weigh 0.44 g of the intermediate and stir it in a reaction system of dichloromethane and trifluoroacetic acid (volume ratio 15:1) at room temperature for 2 h. The reaction endpoint was detected by TCL. The solvent was removed by vacuum distillation, the reaction was quenched with water, and the pH was adjusted to alkaline by adding an appropriate amount of saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (50 mL), and the combined organic phases were dried over anhydrous Na₂SO₄. The solvent was then removed by vacuum distillation, and the mixture was dissolved in a small amount of methanol. Hydrochloric acid was added dropwise to adjust the pH to acidic. The solvent was removed by vacuum distillation, and the mixture was crystallized from methanol and ethyl acetate to give 0.20 g of a white solid (compound F), with a yield of approximately 80-95%.

[0060] (4) Weigh 0.4 g (1.1 mmol) of compound F and 0.20 g (1.65 mmol) of boric acid with R2 structure pyridine and add them to the reaction flask. Add 0.32 g (2.3 mmol) of potassium carbonate and 0.016 g (0.01 mmol) of tetrakis(triphenylphosphine)palladium. Then add a mixed solution of 1,4-dioxane and water in a volume ratio of 4:1 to dissolve them. Then purge the reaction apparatus with nitrogen and stir the reaction under nitrogen and 85°C. Detect the reaction endpoint with TCL. Extract with water and dichloromethane, and then add an appropriate amount of anhydrous sodium sulfate to remove water and dry. Separate by vacuum filtration and silica gel column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain 0.2 g of yellow oil, which is the series of compounds shown in formula (II), with a yield of about 70-95%.

[0061] The different types of amines with R1 structure used in step (3), the boric acid with pyridine as R2 in step (4), and the specific structures of the corresponding compounds are shown in Table 3.

[0062] Table 3

[0063] The characterization data of compounds 16-20 are as follows: Compound 16: 1 H NMR (600 MHz, Chloroform-d ) δ 4.54 (t, J J = 5.8 Hz, 1H), 3.66 (t, J J = 4.9 Hz, 4H), 3.54 (q, J J = 6.6 Hz, 2H), 3.30 (t, J J = 4.9 Hz, 4H), 2.89 (t, J J = 7.0 Hz, 2H). 13 C NMR (151 MHz, Chloroform- d ) δ 160.98, 160.52, 148.56, 148.31, 138.52, 136.42, 136.38, 134.37, 129.64, 127.59, 123.72, 77.06, 67.33, 66.87, 47.14, 43.71, 40.63, 35.22。

[0064] Compound 17: 1 H NMR (600 MHz, DMSO- d 6) δ 8.90 (d, J J = 2.4 Hz, 1H), 8.82 (t, J J = 5.7 Hz, 1H), 8.57 (dd, J J = 4.8, 1.6 Hz, 1H), 8.23 (s, 3H), 8.10 (dt, J J = 8.0, 2.0 Hz, 1H), 7.67 (d, J J = 8.1 Hz, 2H), 7.51 (dd, J J = 8.0, 4.8 Hz, 1H), 7.37 (d, J J = 8.1 Hz, 2H), 4.24 (dq, J J = 13.3, 2.5 Hz, 1H), 3.58 (d, J J = 14.1 Hz, 1H), 3.44 (q, J J = 6.8 Hz, 2H), 3.25 (dq, J J = 10.8, 5.5 Hz, 2H), 3.00 (ddd, J J = 14.1, 11.9, 2.8 Hz, 1H), 2.83 (t, J= 7.1 Hz, 2H), 2.79 – 2.71 (m, 1H), 1.99 – 1.93 (m, 1H), 1.89 – 1.81 (m, 1H), 1.48 – 1.32 (m, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 163.42, 163.19, 147.90, 147.09, 139.32, 135.53, 134.81, 134.38, 129.57, 126.77, 124.07, 47.13, 43.43, 39.52, 38.69, 34.20, 29.97, 29.05。

[0065] Compound 18: 1 H NMR (600 MHz, DMSO- d 6) δ 9.40 (s, 2H), 8.92 – 8.83 (m, 2H), 8.56 (dd, J = 4.8, 2.0 Hz, 1H), 8.06 (dt, J = 8.0, 2.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.47 (dd, J = 8.0, 4.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 3.67 (t, J = 5.4 Hz, 2H), 3.55 (t, J = 5.4 Hz, 2H), 3.46 (q, J = 7.0 Hz, 2H), 3.06 (t, J = 5.4 Hz, 2H), 3.02 (t, J = 5.4 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 163.06, 162.69, 148.33, 147.51, 139.18, 135.35, 135.04, 133.95, 129.55, 126.81, 123.88, 42.60, 42.36, 42.19, 39.52, 37.72, 34.15。

[0066] Compound 19: 1 H NMR (600 MHz, DMSO- d 6) δ 9.24 (d, J = 11.4 Hz, 1H), 9.19 (d, J = 2.2 Hz, 1H), 8.87 (d, J = 8.0 Hz, 1H), 8.84 (t, J = 6.0 Hz, 1H), 8.82 (dd, J =5.5, 1.8 Hz, 1H), 8.75 (dt, J = 8.2, 1.8 Hz, 1H), 8.02 (dd, J = 8.2, 5.5 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 3.89 – 3.82 (m, 1H), 3.43 (q, J = 7.0 Hz, 2H), 3.24 (dt, J = 12.9, 3.2 Hz, 2H), 2.93 (dt, J = 12.4, 6.7 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 1.91 – 1.72 (m, 4H). 13 C NMR (151 MHz, DMSO- d 6) δ159.83, 159.59, 141.99, 141.32, 140.83, 137.85, 132.31, 129.63, 127.25,126.50, 44.35, 42.01, 39.52, 34.16, 27.67.

[0067] Compound 20: 1 H NMR (600 MHz, DMSO- d 6) δ 8.87 (d, J = 2.0 Hz, 1H), 8.71 (t, J = 7.2 Hz, 1H), 8.55 (dd, J = 4.8, 2.0 Hz, 1H), 8.05 (dt, J= 8.0, 2.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.47 (dd, J = 8.0, 4.8 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 4.77 (d, J = 4.0 Hz, 1H), 3.84 – 3.74 (m, 1H), 3.73 – 3.62 (m, 1H), 3.44 (dt, J =7.2, 3.6 Hz, 2H), 3.38 – 3.34 (m, 1H), 3.10 – 3.03 (m, 1H), 3.01 – 2.94 (m,1H), 2.82 (t, J = 7.0 Hz, 2H), 1.73 – 1.64 (m, 1H), 1.65 – 1.56 (m, 1H), 1.31 –1.21 (m, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 163.89, 163.28, 148.30, 147.49,139.24, 135.40, 134.95, 133.89, 129.57, 126.73, 123.87, 65.18, 42.87, 39.52,37.99, 34.35, 34.21, 33.43.

[0068] Example 4: Activity study of the series of compounds of the present invention against AC (1) Experimental materials: the series of compounds of this invention and positive control carmoflu, melanoma cells (SK-Mel-28), microplate reader (Bio-Stack), high-speed refrigerated centrifuge (Eppendoff), Rbm14C12 (Merck), lysosomal protein extraction kit (Solarbio), black 96-well plate.

[0069] (2) Experimental Methods: Acrylosomes (AC) are proteins mainly located in lysosomes. This experiment used differential centrifugation, based on the principle of sedimentation rate differences in the centrifugation field caused by density differences between organelles, to achieve stepwise purification of lysosomal proteins through a gradient centrifugation strategy. In this experiment, the inhibitory effect of the target compound and positive control on AC was detected using Rbm14C12 coumarin fluorescent substrate. The single-well inhibition rate of all samples was determined at a concentration of 10 µM, and the experimental results are shown in Table 4.

[0070] Table 4. Inhibition rate of AC by the series of compounds of this invention

[0071] Compounds with inhibition rates greater than 60% were selected, and different concentration gradients were set for compounds with different inhibition rates. Three replicates were used, and nonlinear curve fitting analysis was performed on the inhibition rate at each concentration to calculate the half-maximal inhibitory concentration (IC50). 50 value.

[0072] The black 96-well plate contained three systems: a blank group, a control group, and an experimental group. The three blank groups consisted of 99 μL of reaction buffer added to each well. The three control groups consisted of 94 μL of reaction buffer and 5 μL of lysosomal protein (approximately 29 mg / well) added to each well. The remaining experimental groups consisted of 84 μL of reaction buffer, 5 μL of lysosomal protein (approximately 29 mg / well), and 10 μL of inhibitor (DMSO concentration not exceeding 1% of the reaction volume) added to each well. Carmofluuron was used as a positive control. The plates were pre-incubated at 37 °C for 30 min. 1 μL of fluorescent substrate (final concentration 5 μM) was added to each well, and the plates were incubated at 37 °C for 1 h. 50 μL of methanol was added, followed by 100 μL of stop buffer to terminate the reaction. The plates were then incubated in the dark at 37 °C for another 2 h. Fluorescence intensity was then measured at λex = 355 nm / λem = 460 nm, three times. Calculate the inhibition rate of AC for different concentrations of the compound. Inhibition rate = (fluorescence value of blank well - fluorescence value of experimental well) / (fluorescence value of blank well - fluorescence value of blank well correction group) × 100%. Select compounds with inhibition rates greater than 50%, set up 5 different concentration gradients, 3 replicates, and perform nonlinear curve fitting analysis on the inhibition rate of each concentration. Calculate the half-maximal inhibitory concentration (IC50). 50 Value. Half-maximal inhibitory concentration (IC50) 50 The lower the value, the stronger the inhibitory effect of the compound on AC. The experimental results are shown in Table 5.

[0073] Table 5 IC50 of the compounds of the present invention to AC 50 value

[0074] Experimental results showed that compounds 6 and 10 had significant inhibitory effects on AC, and their effects were significantly better than those of the positive control drug carmoflu.

[0075] Example 5: Study on the effects of the series of compounds of the present invention on cancer cell proliferation (1) Experimental materials: the series of compounds of this invention and positive control carmoflu, trypsin, washing solution PBS, fetal bovine serum (Excell), human malignant melanoma cells SK-Mel-28, mouse skin melanoma cells B16-F10, human colon cancer cells HCT-116, human liver cancer cells HepG2, mouse liver cancer cells Hepa1-6, human breast cancer cells MDA-MB-231, mouse breast cancer cells 4T1 and human breast cancer cells MCF-7 (Shanghai Institute of Microbiology, Chinese Academy of Sciences Cell Bank), microplate reader (thermoscientific) and 96-well plate, DMEM medium (Pronosai), wherein the final concentrations of penicillin and streptomycin were 100 U / mL and 100 U / mL, respectively.

[0076] (2) Experimental method: Cells grown to the logarithmic growth phase were prepared into uniformly dispersed single-cell suspensions, with approximately (2-5) × 10⁶ cells per well. 3 Cells were seeded into 96-well plates and incubated in a 5% CO2, 37°C incubator for 24 h. After cell attachment, 10 μL of each of the six different concentrations of sample was added, with 3-5 replicates for each concentration. Incubation continued for 48 h, followed by incubation of each well with 10 μL of CCK8 for 30-50 min. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. Each experiment was conducted independently in triplicate. Inhibition rate = 1 - [(OD...] 实验组 -OD 空白组 ) / (OD 阴性对照 -OD 空白组 []×100%. Analysis was performed using Graphpad 7.0 software. Linear regression was performed between the logarithm of sample concentration and cell inhibition rate to calculate the half-maximal inhibitory concentration (IC50) of the compound for each cell type. 50 Values. The experimental results are shown in Tables 6 and 7.

[0077] Table 6. IC of AC according to the present invention 50 Compounds with a concentration less than 3 have an effect on cancer cell IC50. 50 Comparison of experimental results

[0078] Experimental results showed that compounds 6 and 10 had significant inhibitory effects on melanoma cells and breast cancer cells, and their effects were significantly better than those of the positive control drug carmoflu. Compounds 5 and 9 had significant inhibitory effects on human colon cancer cells HCT-116, compounds 7 and 8 had significant inhibitory effects on liver cancer cells, and compound 19 had significantly better effects on human breast cancer cells MCF-7 and mouse breast cancer cells 4T1 than those of the positive control drug carmoflu.

[0079] In summary, the above embodiments demonstrate that, according to the present invention, compounds with higher enzyme inhibitory and anticancer activities than the positive control carmoflu have been discovered, wherein compounds 6 or 10 exhibit the best enzyme inhibitory effect and the ability to inhibit the proliferation of various cancer cells, indicating that compounds 6 or 10 can be further developed into drugs targeting AC.

[0080] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any partial changes to the formulation and process therein should be within the scope of protection of the present invention.

Claims

1. A kind N - Phenethylamide compounds or their pharmaceutically acceptable salts, characterized by... The general structural formula of the compound is shown in formula (I) or (II): ， （Ⅰ） （Ⅱ） Wherein, R1 is any one of furanyl, thienyl, pyridinyl, 3-methyl-1H-pyrazolyl, 2-aminothiazolyl, 2-methylthiazolyl, 3-ethyl-3H-indolyl, chlorophenyl, 3-(difluoromethyl)-1-methyl-1H-pyrazolyl, morpholinyl, 4-aminopiperidinyl, 4-aminocyclohexanolyl, and piperazinyl; R2 is any one of pyrimidinyl, pyridinyl, isoxazolyl, phenyl, 4-formylphenyl, 4-chlorophenyl, 4-methylphenyl, and pyrazolyl.

2. As described in claim 1 N - Phenethylamide compounds or their pharmaceutically acceptable salts, characterized by... R1 is , , , , , , , , , , , , , , Any one of them; R2 is , , Any one of them.

3. As described in claim 1 N - Phenethylamide compounds or their pharmaceutically acceptable salts, characterized by... The compound is selected from one of the following structures: 。 4. As described in claim 1 N - Phenethylamide compounds or their pharmaceutically acceptable salts, characterized by... The salt is any one of acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, or methanesulfonate.

5. A method for preparing a compound of formula (I), characterized in that, The synthesis route is as follows: or ， In the formula, R1 and R2 are defined as in claim 1.

6. The preparation method according to claim 5, characterized in that, Includes the following steps: (a) The compound having R1 undergoes a condensation reaction with p-bromophenethylamine to form compound A; (b) Compound A undergoes Suzuki coupling with R2B(OH)2 to produce the compound shown in formula (I); or, (a') p-Bromophenylethylamine undergoes Suzuki coupling with R2B(OH)2 to form compound B; (b') Compound B and phenyl chloroformate undergo a condensation reaction to form compound C; (c') Compound C reacts with a compound having R1 to produce the compound shown in formula (I).

7. A method for preparing a compound of formula (II), characterized in that, The synthesis route is as follows: ， In the formula, R1 and R2 are defined as in claim 1.

8. Use of the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

9. The application according to claim 8, characterized in that, The tumor is melanoma, breast cancer, liver cancer, or colon cancer.

10. A pharmaceutical composition, characterized in that, It comprises the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

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