Application of compound based on 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton in preparation of medicine for treating tumor diseases

Abstract

The invention provides an application of a compound based on a 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton in preparation of a medicine for treating tumor diseases, and belongs to the technical field of biological medicines. The compound based on the 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton provided by the invention regulates and controls the palmitoylation modification level of Cofilin1 by inhibiting the activity of PPT2, and finally achieves the effect of inhibiting cancer.

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] Studies have shown that protein palmitoylation disorders are closely related to tumor progression. Studies have shown that palmitoyl acyltransferases and depalmitoyl acyltransferases are associated with various aspects of carcinogenesis, cancer cell growth, survival, and treatment resistance. However, the pattern and dynamics of protein palmitoylation in human cancers are still unclear. Cofilin1 is highly expressed in pan-cancer tissues. Through our research, we found that Cofilin1 is a palmitoylated protein, and its palmitoylation level regulates the migration and invasion of tumor cells. Cofilin1 depalmitoylation enzyme is PPT2. Can we inhibit the activity of PPT2 by small molecule inhibitors to regulate the palmitoylation level of Cofilin1, thereby inhibiting the migration and invasion of tumors?

[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 an application of a compound represented by formula I in the preparation of a drug for treating tumor diseases, wherein formula I is as follows:

[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 is in the form of an injection, tablet, capsule, granule or sustained-release agent.

[0021] The beneficial effects of the present invention are as follows: PPT1 and PPT2 are enzymes with similar functions. Currently, only the commercialized substrate of PPT1 is 4-methylcoumarinyl-6-thio-hexadecanoyl-β-D-pyranoglucoside, namely: MU-6S-palm-β-Glc. The inhibitory effects of HCQ and TPPT on the enzyme activity of PPT1 are compared, and then the inhibitory effects of HCQ and TPPT on the enzyme activity of PPT1 are calculated by measuring the fluorescence of the solution. The inhibitor drug21 and its analogs in the present invention have an inhibitory effect on the migration, invasion and proliferation of tumor cells, and the inhibitor drug2 has a better inhibitory effect on PPT1 than 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 results of HCQ and TPPT (Drug21) inhibiting the enzyme activity of PPT1;

[0024] Figure 4 The results of the inhibition of proliferation of cervical cancer, liver cancer and lung cancer cells by different concentrations of TPPT (Drug21) are shown in Figure 2.

[0025] Figure 5The results of the inhibition of tumor cell migration by different concentrations of TPPT (Drug21) are shown in Figure 2.

[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 an application of a compound represented by formula I in the preparation of a drug for treating tumor diseases, wherein formula I is as follows:

[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, and the mixture was stirred for 0.5 h at the same temperature, and then the mixture was 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, and the solvent was removed from the filtrate under reduced pressure. The concentrate was diluted with ethyl acetate, extracted with ethyl acetate, 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 generated, 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.), substituted aromatic amine 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 completed, 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 with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 3;

[0046]

[0047] (4) Intermediate 3 (1 equiv.), substituted aromatic amine or aliphatic amine (1.5 equiv.), Pd 2 (dba) 3 (0.1equiv.), BINAP (0.5equiv.) and K 2 CO 3 (2.0 equiv.) was dissolved in 50 ml of toluene and stirred at 85°C for 5-8 h under nitrogen protection. After the reaction was completed, 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, washed with water and saturated brine, and 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] Inhibitory effects 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. The culture medium and complete culture medium were used to culture Hela cells in 96-well plates. The absorbance of the culture medium was measured by a 450 nm microplate reader at four time points: 0 h, 24 h, 48 h, and 72 h, and the results were plotted. Figure 1 and Figure 2 .

[0052] Example 2

[0053] Compare the inhibitory effects of HCQ and TPPT (Drug21) on the enzyme activity of PPT1, and then calculate the inhibitory effects of HCQ and TPPT on the enzyme activity of PPT1 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 by the fluorescence intensity emitted after the substrate was enzymatically hydrolyzed. The results are shown in Figure 3 .

[0054] After adding the inhibitor (Drug 21) to the cell experiment, it has 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. The culture medium and complete culture medium were used to culture Hela cells in 96-well plates, respectively. CCK8 reagent was added at four time points of 0 h, 24 h, 48 h, and 72 h, and the absorbance of the culture solution was measured by a 450 nm microplate reader after 1 h of adding CCK8 reagent, and the graphs were plotted as follows: 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 the culture medium and complete culture medium, respectively. Pictures were taken under a 10x microscope at 0 h and 24 h, and the trace area was 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 the culture medium and complete culture medium, respectively. After 96 h, photos were taken under a 20x microscope, and the number of migrated cells was counted and plotted into a graph, as shown in Figure 2. Figure 6 shown.

[0064] Example 6

[0065] Inhibitory effects of compounds 1 to 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 the 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 graphs were drawn.

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

[0068] Table 2 Inhibition rate 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 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. 1 When various secondary amines (tetrahydropyrrole, 4-methylpiperidine, cycloheximide and dimethylamine) were introduced, most compounds showed strong inhibitory effects.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 (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 principle of the present invention. These improvements and modifications should also be regarded as 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, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

2. The use according to claim 1, characterized in that: The R 1 Selected from the following structures: The R 2 Selected from the following structures:

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

4. The use according to any one of claims 1 to 3, characterized in that: The tumor disease includes cervical cancer, liver cancer or lung cancer.

5. The use according to claim 4, 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.

6. 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, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

7. The pharmaceutical composition for treating tumor diseases according to claim 6, characterized in that: The pharmaceutical composition may be in the form of injection, tablet, capsule, granule or sustained-release preparation.

Images