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

Abstract

The present invention provides the use of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating tumor diseases, belonging to the field of biomedicine technology. The compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton provided by the present invention inhibits the activity of PPT2 and regulates the palmitoylation level of cofilin1, ultimately achieving a tumor-suppressing effect.

Description

Technical Field

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

[0002] Research indicates that dysregulated protein palmitoylation is closely linked to tumor progression. Studies have shown that palmitoyl acyltransferases and depalmitoyl acyltransferases are associated with various aspects of carcinogenesis, cancer cell growth, survival, and therapeutic resistance. However, the patterns and dynamics of protein palmitoylation in human cancers remain unclear. Cofilin1 is highly expressed in pan-cancer tissues. Our research has revealed that Cofilin1 is a palmitoylated protein, and its palmitoylation levels regulate tumor cell migration and invasion. The depalmitoylation enzyme for Cofilin1 is PPT2. Is it possible to inhibit the activity of PPT2 through small molecule inhibitors to regulate the palmitoylation level of Cofilin1 and thereby inhibit 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 at present. Summary of the Invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a use of a compound represented by Formula I in the preparation of a drug for treating tumor diseases. Formula I is shown below:

[0007]

[0008] Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

[0009] Furthermore, the R 1 Selected from the following structures:

[0010]

[0011] The R 2 Selected from the following structures:

[0012]

[0013] Furthermore, the compound of formula I comprises the following structural formula:

[0014]

[0015] Furthermore, the tumor disease includes cervical cancer, liver cancer or lung cancer.

[0016] 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.

[0017] A pharmaceutical composition for treating tumor diseases, comprising a compound of formula I and a pharmaceutically acceptable excipient, wherein formula I is as follows:

[0018]

[0019] Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

[0020] Furthermore, the pharmaceutical composition may be in the form of an injection, tablet, capsule, granule or sustained-release formulation.

[0021] The present invention has the beneficial effects of comparing PPT1 and PPT2, enzymes with similar functions. Currently, the only commercially available PPT1 substrate is 4-methylcoumaryl-6-thio-hexadecanoyl-β-D-glucopyranoside, or MU-6S-palm-β-Glc. 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 analogs of the present invention have inhibitory effects on tumor cell migration, invasion, and proliferation, and the inhibitor drug2 exhibits superior inhibitory effects on PPT1 compared to the known PPT1 inhibitor HCQ. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 and Figure 2 is the absorbance curve of compounds 1 to 21 after adding CCK8 reagent;

[0023] Figure 3 The figure shows the results of HCQ and TPPT (Drug21) inhibiting the enzyme activity of PPT1;

[0024] Figure 4 The graph shows the results of the ability of different concentrations of TPPT (Drug21) to inhibit the proliferation of cervical cancer, liver cancer, and lung cancer cells;

[0025] Figure 5The graph shows the results of the ability of different concentrations of TPPT (Drug21) to inhibit tumor cell migration;

[0026] Figure 6 The graph shows the results of different concentrations of TPPT (Drug21) inhibiting the invasion ability of cervical cancer, liver cancer and lung cancer cells. DETAILED DESCRIPTION

[0027] The present invention provides a use of a compound represented by Formula I in the preparation of a drug for treating tumor diseases. Formula I is shown below:

[0028]

[0029] Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

[0030] In the present invention, the R 1 Preferably selected from the following structures:

[0031]

[0032] The R 2 Preferably selected from the following structures:

[0033]

[0034] In the present invention, the compound of formula I is preferably the following structural formula:

[0035] Table 1

[0036]

[0037]

[0038]

[0039] In the present invention, the preparation method of the compound of formula I comprises the following steps:

[0040]

[0041] (1) Commercially available (E)-4-bromo-N'-hydroxybenzamidine (5.00 g, 23.25 mmol) and triethylamine (5.06 ml, 46.50 mmol) were dissolved in 50 mL of acetonitrile, and ethyl oxalyl chloride (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 solid in the reaction system was removed by suction filtration. The solvent was removed from the filtrate under reduced pressure, and the concentrate was diluted with ethyl acetate and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to obtain intermediate 1.

[0042]

[0043] (2) Dissolve the 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, remove the solvent under reduced pressure, add water to dissolve, slowly add dilute HCl in an 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 a white solid 2;

[0044]

[0045] (3) Dissolve intermediate 2 (1.5 equiv.), a substituted aromatic or aliphatic amine (1 equiv.), TCFH (1.1 equiv.), and NMI (2.1 equiv.) in 50 ml of tetrahydrofuran and stir at room temperature for 5-8 hours. After the reaction is complete, remove the solvent under reduced pressure, dilute the concentrate with ethyl acetate, extract with ethyl acetate, wash with water and saturated brine, dry the organic phase over anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 3.

[0046]

[0047] (4) Intermediate 3 (1 equiv.), 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 under nitrogen for 5-8 h. After the reaction was complete, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The concentrate was diluted with ethyl acetate and extracted with ethyl acetate. The mixture was washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to obtain the final product.

[0048] The technical solutions provided by the present invention are 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.

[0049] Example 1

[0050] Inhibitory effect of compounds on Hela cells

[0051] The above 20 compounds were dissolved in DMSO to a concentration of 5 mM and added to the culture medium to a concentration of 10 μM. Hela cells were cultured in 96-well plates with the culture medium and complete culture medium, and the absorbance of the culture medium was measured at 450 nm using a microplate reader at four time points: 0 h, 24 h, 48 h, and 72 h. Figure 1 and Figure 2 .

[0052] Example 2

[0053] Compare the inhibitory effects of HCQ and TPPT (Drug 21) on PPT1 enzyme activity, and then calculate the inhibitory effects of HCQ and TPPT on PPT1 enzyme activity by measuring the fluorescence of the solution. HCQ and TPPT were prepared into a concentration of 5mM and added to a 96-well plate containing serum. Substrate and inhibitor were added. After incubation, fluorescence was measured on a microplate reader. The enzyme activity was calculated based on the fluorescence intensity emitted after the substrate was enzymatically hydrolyzed. The results are shown in Figure 3 .

[0054] In cell experiments, the addition of inhibitors (Drug 21) had a significant inhibitory effect on the proliferation, migration, and invasion of cervical cancer, liver cancer, and lung cancer cells. Figure 4 、 5 ,6. Cell proliferation, scratch and invasion experiments showed that the inhibitors had inhibitory effects on the proliferation, migration and invasion of cervical cancer cells at different concentrations, and the inhibitory effect showed a concentration-dependent trend.

[0055] Example 3

[0056] 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)

[0057] TPPT was dissolved in DMSO to a concentration of 5 mM and 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 with this culture medium and complete culture medium, respectively. The absorbance of the culture medium was measured at 450 nm using a microplate reader at four time points: 0 h, 24 h, 48 h, and 72 h, after adding CCK8 reagent for 1 h. The absorbance was plotted as shown in the figure. Figure 4 shown.

[0058] Example 4

[0059] Inhibitory effect of different concentrations of TPPT on the migration ability of cervical cancer cells (Hela)

[0060] TPPT was dissolved in DMSO to a concentration of 5 mM and added to the culture medium to 2.5 μM, 5 μM, and 10 μM. Hela cells were cultured in the inserts with this culture medium and complete culture medium, respectively. Pictures were taken under a 10x microscope at 0 h and 24 h, and the trace areas were plotted as shown in the figure. Figure 5 shown.

[0061] Example 5

[0062] Inhibitory effect of different concentrations of TPPT on the invasion ability of cervical cancer cells (Hela)

[0063] TPPT was dissolved in DMSO to a concentration of 5 mM and added to the culture medium to 2.5 μM, 5 μM, and 10 μM. Hela cells were cultured in 24-well plates with this culture medium and complete culture medium, respectively. After 96 h, the cells were photographed under a 20x microscope and the number of migrated cells was counted and plotted as shown in the figure. Figure 6 shown.

[0064] Example 6

[0065] Inhibitory effects of compounds 1-21 on Hela cells

[0066] The above 21 compounds were dissolved in DMSO to a concentration of 5 mM and added to the culture medium to a concentration of 10 μM. Hela cells were cultured in a 96-well plate with this culture medium and complete culture medium, respectively. The absorbance of the culture medium was measured at 450 nm using a microplate reader at four time points: 0 h, 24 h, 48 h, and 72 h, after adding CCK8 reagent for 1 h. The absorbance was plotted on a graph.

[0067] The experimental results are shown in Table 2 below.

[0068] Table 2 Inhibitory effect of compounds 1 to 21 on Hela cells

[0069]

[0070]

[0071]

[0072] From the above examples, it can be seen that the present invention provides a compound based on 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton for use in the preparation of drugs for treating tumor diseases. 1 When a variety of secondary amines (tetrahydropyrrole, 4-methylpiperidine, cycloheximide and dimethylamine) were introduced, most compounds showed strong inhibitory effects. 2 When it is an alkyl chain (Drug5-6, Drug9-10, Drug14-15 and Drug20), they all have a certain inhibitory effect; R 2When aliphatic rings (cycloheptane, cyclopropane, cyclopentane and cyclohexane) were introduced, the inhibition rates of Drug1, Drug4, Drug7-8, Drug11 and Drug13 were all reduced, among which Drug7-8 and Drug13 had basically no inhibitory effect; when the aliphatic rings were changed to N-containing aliphatic rings (i.e., tertiary amines), the inhibition rates of compounds Drug2-3 and Drug16-19 were significantly improved, among which the inhibition rates of Drug16 and Drug19 were 88.20% and 91.33%, respectively.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a compound represented by formula I in the preparation of a drug for treating tumor diseases, characterized in that: Formula I is shown below: Among them, the R 1 Selected from the following structures: The R 2 Selected from the following structures: The tumor disease includes cervical cancer, liver cancer or lung cancer.

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

3. The use according to claim 2, characterized in that 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.

4. A pharmaceutical composition for treating tumor diseases, characterized in that: The invention comprises a compound of formula I and a pharmaceutically acceptable excipient, wherein the formula I is as follows: Among them, the R 1 Selected from the following structures: The R 2 Selected from the following structures:

5. The pharmaceutical composition for treating tumor diseases according to claim 4, characterized in that: The pharmaceutical composition is in the form of injection, tablet, capsule, granule or sustained-release preparation.

Images