Application of compound based on 3-phenyl-1,2,4-oxadiazole-5-carboxamide scaffold in preparation of drug for treating tumor diseases

Abstract

Disclosed is an application of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide scaffold in the preparation of a drug for treating tumor diseases. The compound of the 3-phenyl-1,2,4-oxadiazole-5-carboxamide scaffold regulates the level of Cofilin1 palmitoylation modification by inhibiting the activity of PPT2, and finally achieves an anticancer effect.

Description

Application of a compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of drugs for treating tumor diseases Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of drugs for treating tumor diseases. Background Technology

[0002] Studies have shown a close correlation between dysregulation of protein palmitoylation and tumor progression. Research indicates that palmitoyl transferases and depalmitoyl transferases are involved in various aspects of carcinogenesis, cancer cell growth, survival, and treatment resistance. However, the pattern and dynamics of protein palmitoylation in human cancer remain unclear. Cofilin1 is highly expressed in pan-cancer tissues. Our study found that Cofilin1 is a palmitoylated protein, and its palmitoylation level regulates tumor cell migration and invasion. Cofilin1 depalmitoylate is PPT2. Could inhibiting PPT2 activity with small molecule inhibitors regulate Cofilin1 palmitoylation levels, thereby suppressing tumor migration and invasion?

[0003] Therefore, how to obtain a small molecule inhibitor to inhibit the activity of PPT2 and regulate the level of palmitoylation modification is a technical problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating tumor diseases, in order to solve the above-mentioned technical problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of the compound represented by Formula I in the preparation of a drug for treating tumor diseases, as shown below:

[0007] Among them, R 1 Contains a nitrogen-containing heterocyclic hydrocarbon group or a tertiary amine group; R 2 It contains linear alkyl, cycloalkyl, aryl, or ether groups.

[0008] Furthermore, the R 1 Selected from the following structure:

[0009] The R 2 Selected from the following structure:

[0010] Furthermore, the compound of formula I comprises the following structural formula:

[0011] Furthermore, the tumor diseases include cervical cancer, liver cancer, or lung cancer.

[0012] Furthermore, the compound of Formula I inhibits the activity of PPT2 and regulates the palmitoylation modification level of Cofilin1, thereby achieving the effect of inhibiting tumor cells.

[0013] A pharmaceutical composition for treating tumor diseases includes a compound of formula I and a pharmaceutically acceptable excipient, said formula I being shown below:

[0014] Among them, R 1 Contains a nitrogen-containing heterocyclic hydrocarbon group or a tertiary amine group; R 2 It contains linear alkyl, cycloalkyl, aryl, or ether groups.

[0015] Furthermore, the pharmaceutical composition may be formulated as an injection, tablet, capsule, granule, or sustained-release formulation.

[0016] The beneficial effects of this invention are as follows: PPT1 and PPT2 are enzymes with similar functions. Currently, only the commercially available substrate for PPT1, 4-methylcoumarinyl-6-thio-hexadecyl-β-D-glucopyranoside (MU-6S-palm-β-Glc), is available. The inhibitory effects of HCQ and TPPT on PPT1 activity were compared, and the inhibitory effects of HCQ and TPPT on PPT1 activity were calculated by measuring solution fluorescence. The inhibitor drug21 and its analogues in this invention have inhibitory effects on the migration, invasion, and proliferation of tumor cells, and the inhibitor drug21 has a better inhibitory effect on PPT1 than the known PPT1 inhibitor HCQ. Attached Figure Description

[0017] Figures 1 and 2 show the absorbance curves of compounds 1-21 after the addition of CCK8 reagent;

[0018] Figure 3 shows the results of HCQ and TPPT (Drug21) inhibiting the enzyme activity of PPT1;

[0019] Figure 4 shows the results of different concentrations of TPPT (Drug21) inhibiting the proliferation of cervical cancer, liver cancer, and lung cancer cells.

[0020] Figure 5 shows the results of different concentrations of TPPT (Drug21) inhibiting the migration ability of tumor cells.

[0021] Figure 6 shows the results of different concentrations of TPPT (Drug21) inhibiting the invasive ability of cervical cancer, liver cancer, and lung cancer cells. Detailed Implementation

[0022] This invention provides the use of the compound represented by Formula I in the preparation of a drug for treating tumor diseases, as shown below:

[0023] Among them, R 1 Contains a nitrogen-containing heterocyclic hydrocarbon group or a tertiary amine group; R 2 It contains linear alkyl, cycloalkyl, aryl, or ether groups.

[0024] In this invention, the R 1 Preferred from the following structure:

[0025] The R 2 Preferred from the following structure:

[0026] In this invention, the compound of formula I is preferably of the following structural formula:

[0027] Table 1

[0028] In this invention, the preparation method of the compound of formula I includes the following steps:

[0029] (1) Commercially available (E)-4-bromo-N'-hydroxybenzylamidinium (5.00 g, 23.25 mmol) and triethylamine (5.06 ml, 46.50 mmol) were dissolved in 50 mL of acetonitrile, and oxaloyl chloride monoethyl ester (6.32 g, 46.50 mmol) was added dropwise under ice bath. The mixture was stirred at this temperature for 0.5 h, and then heated to 72 °C and refluxed for 7 h. After the reaction was completed, the mixture was filtered to remove the solids in the reaction system. The filtrate was subjected to reduced pressure to remove the solvent. The concentrate was diluted with ethyl acetate and extracted with ethyl acetate. The concentrate was washed with water and saturated brine. The organic phase was dried with anhydrous sodium sulfate and purified by column chromatography to obtain intermediate 1.

[0030] (2) Dissolve intermediate 1 in 50 ml of ethanol, add sodium hydroxide solution at room temperature to adjust the pH to alkaline, hydrolyze at room temperature for 3 hours and remove the solvent under reduced pressure, dissolve in water, slowly add dilute HCl under ice bath to adjust the pH to acidic until a large amount of solid is produced, filter, wash the filter cake with ice water or petroleum ether, and finally dry at 45°C to obtain white solid 2.

[0031] (3) Intermediate 2 (1.5 equiv.), the substituted aromatic or fatty amine (1 equiv.), TCFH (1.1 equiv.), and NMI (2.1 equiv.) were dissolved in 50 ml of tetrahydrofuran and stirred at room temperature for 5-8 h. After the reaction was completed, the solvent was removed under reduced pressure, the concentrate was diluted with ethyl acetate and extracted with ethyl acetate, washed with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, and purified by column chromatography to obtain intermediate 3;

[0032] (4) Intermediate 3 (1 equiv.), the substituted aromatic or aliphatic amine (1.5 equiv.), Pd2(dba)3 (0.1 equiv.), BINAP (0.5 equiv.), and K2CO3 (2.0 equiv.) were dissolved in 50 mL of toluene and stirred at 85 °C for 5–8 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed under reduced pressure, the concentrate was diluted with ethyl acetate and extracted with ethyl acetate, washed with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, and the final product was purified by column chromatography.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Inhibitory effect of the compound on HeLa cells

[0036] The above 20 compounds were dissolved in DMSO to prepare a concentration of 5 mM, which was then added to the culture medium to a concentration of 10 μM. HeLa cells were cultured in 96-well plates using this culture medium and complete culture medium, respectively. CCK8 reagent was added at four time points: 0 h, 24 h, 48 h, and 72 h. The absorbance of the culture medium was measured at 450 nm using a microplate reader, and the results are shown in Figure 1 and Figure 2.

[0037] Example 2

[0038] The inhibitory effects of HCQ and TPPT (Drug21) on PPT1 enzyme activity were compared, and the inhibitory effects of HCQ and TPPT on PPT1 enzyme activity were calculated by measuring the fluorescence of the solution. HCQ and TPPT were prepared at a concentration of 5 mM and added to 96-well plates containing serum, along with the substrate and inhibitor. After incubation, fluorescence was measured using a microplate reader. The enzyme activity was calculated based on the fluorescence intensity emitted after the substrate was digested. The results are shown in Figure 3.

[0039] In cell experiments, the addition of the inhibitor (Drug 21) significantly inhibited the proliferation, migration, and invasion of cervical cancer, liver cancer, and lung cancer cells. As shown in Figures 4, 5, and 6, cell proliferation, scratch assays, and invasion experiments demonstrated that the inhibitor at different concentrations inhibited the proliferation, migration, and invasion of cervical cancer cells, and the inhibitory effect showed a concentration-dependent trend.

[0040] Example 3

[0041] Inhibitory effects of different concentrations of TPPT (Drug21) on cervical cancer cells (HeLa), liver cancer cells (MHCC97-H, Hepg2, Huh7), and lung cancer cells (A549, H1299).

[0042] TPPT was dissolved in DMSO to prepare a concentration of 5 mM, and then added to the culture medium to a concentration of 2.5 μM, 5 μM, and 10 μM. HeLa cells were cultured in 96-well plates using this medium and complete medium, respectively. CCK8 reagent was added at four time points: 0 h, 24 h, 48 h, and 72 h. The absorbance of the culture medium was measured at 450 nm using a microplate reader after 1 h, and the results were plotted as shown in Figure 4.

[0043] Example 4

[0044] Inhibitory effects of different concentrations of TPPT on the migration ability of cervical cancer cells (HeLa).

[0045] TPPT was dissolved in DMSO to prepare a concentration of 5 mM, and then added to the culture medium to a concentration of 2.5 μM, 5 μM, and 10 μM. HeLa cells were cultured in the insert using this medium and complete medium, respectively. The images were taken under a 10x microscope at 0 h and 24 h, and the trace area was plotted as shown in Figure 5.

[0046] Example 5

[0047] Inhibitory effects of different concentrations of TPPT on the invasive ability of cervical cancer cells (HeLa).

[0048] TPPT was dissolved in DMSO to prepare a concentration of 5 mM, and then added to the culture medium to a concentration of 2.5 μM, 5 μM, and 10 μM. HeLa cells were cultured in 24-well plates using this medium and complete medium, respectively. After 96 h, the cells were photographed under a 20x microscope, and the number of migrating cells was counted and plotted as shown in Figure 6.

[0049] Example 6

[0050] Inhibitory effects of compounds 1-21 on HeLa cells

[0051] The above 21 compounds were dissolved in DMSO to prepare a concentration of 5 mM, which was then added to the culture medium to a concentration of 10 μM. HeLa cells were cultured in 96-well plates using this culture medium and complete culture medium, respectively. CCK8 reagent was added at four time points: 0 h, 24 h, 48 h, and 72 h. The absorbance of the culture medium was measured at 450 nm using a microplate reader after 1 h, and the results were plotted.

[0052] The experimental results are shown in Table 2 below.

[0053] Table 2 shows the inhibitory effects of compounds 1-21 on HeLa cells.

[0054] As can be seen from the above embodiments, the present invention provides an application of compounds based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of drugs for treating tumor diseases. The results above show that when R... 1 When various secondary amines (tetrahydropyrrole, 4-methylpiperidine, cyclohexylimine, and dimethylamine) are introduced, most compounds exhibit strong inhibitory effects. When R... 2 When they are alkyl chains (Drug5-6, Drug9-10, Drug14-15, and Drug20), they all have a certain inhibitory effect; R 2 When aliphatic rings were introduced (cycloheptane, cyclopropane, cyclopentane, and cyclohexane), the inhibition rates of Drug1, Drug4, Drug7-8, Drug11, and Drug13 were all reduced, with Drug7-8 and Drug13 showing virtually no inhibitory effect. When the aliphatic ring was changed to an N-containing aliphatic ring (i.e., a tertiary amine), the inhibition rates of compounds Drug2-3 and Drug16-19 were significantly increased, with Drug16 and Drug19 showing inhibition rates of 88.20% and 91.33%, respectively.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a compound of Formula I in the preparation of a drug for treating tumor diseases, characterized in that, Equation I is shown below: Wherein, the R 1 Selected from the following structure: The R 2 Selected from the following structure: The tumor diseases mentioned include cervical cancer, liver cancer, or lung cancer.

2. The application according to claim 1, characterized in that, The compound of formula I comprises the following structural formula:

3. The application according to claim 2, characterized in that, The compound of Formula I inhibits tumor cells by suppressing the activity of PPT2 and regulating the palmitoylation level of Cofilin1.

4. A pharmaceutical composition for treating tumor diseases, characterized in that, It includes a compound of formula I and pharmaceutically acceptable excipients, wherein formula I is shown below: Wherein, the R 1 Selected from the following structure: The R 2 Selected from the following structure:

5. The pharmaceutical composition for treating tumor diseases according to claim 4, characterized in that, The pharmaceutical composition is formulated as an injection, tablet, capsule, granule, or sustained-release formulation.

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