CN114573548A - Nifurozide heterocyclic benzylidene hydrazide derivative and synthesis method and application thereof - Google Patents
Nifurozide heterocyclic benzylidene hydrazide derivative and synthesis method and application thereof Download PDFInfo
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Abstract
The invention provides nifurozide heterocyclic benzylidene hydrazide derivatives, which have a structural formula shown in a formula I. The derivative can effectively inhibit proliferation of hepatocellular carcinoma cells, induce apoptosis and obviously inhibit liver cellsExpression of PD-L1 protein in a cell carcinoma cell.
Description
Technical Field
The invention belongs to the field of tumor drug research, and particularly relates to nifurozide heterocyclic benzylidene hydrazide derivatives, and a synthesis method and application thereof.
Background
Hepatocellular carcinoma (HCC), the most common primary liver cancer, is also the fourth leading cause of cancer-related death worldwide. The current treatment options mainly comprise surgical resection, liver transplantation, radio frequency ablation, interventional therapy, targeted therapy, chemoradiotherapy and the like. In recent years, with the development of immunology, tumor immunotherapy has become a promising method for tumor therapy. Recent studies have shown that blocking the immune checkpoint pathway PD-1/PD-L1 has been shown to be effective in treating tumors.
Programmed cell death-ligand 1 (PD-L1), a transmembrane protein of 40kDa in size, also known as surface antigen cluster 274, is mainly expressed in T cells, B cells, and,DC. Macrophages, mesenchymal stem cells, bone marrow derived mast cells and tumor cell surfaces. It can bind with Programmed cell death receptor-1 (PD-1), transmit inhibitory signal, and inhibit lymph node CD8+The proliferation of T cells and the accumulation of antigen-specific T cells in lymph nodes significantly inhibit the anti-tumor effects of effector T cells. Moreover, high expression of PD-L1 also inhibited the effect of neighboring macrophages in tumor tissue. In tumor-bearing mice, the blocking of the PD-L1 signaling pathway can significantly activate macrophages, and can still exert significant anti-tumor activity even in the absence of T cells. Therefore, the development of inhibitors of the PD-1/PD-L1 pathway is still very important.
Disclosure of Invention
In view of the technical problems, the invention provides nifurozide heterocyclic benzylidene hydrazide derivatives, which can effectively inhibit proliferation of hepatocellular carcinoma cells, induce apoptosis and remarkably inhibit expression of PD-L1 protein in the hepatocellular carcinoma cells.
The nifurozide heterocyclic benzylidene hydrazide derivative or pharmaceutically acceptable salt thereof provided by the invention has a structural formula shown as a formula I:
wherein X is selected from a sulfur atom, an oxygen atom or a nitrogen atom; r is selected from C1-C5 alkyl, nitro, amino, substituted amino, methoxy, hydroxyl or carboxyl.
Preferably, X is selected from the group consisting of sulfur atoms and R is selected from the group consisting of methoxy groups.
The invention also provides a synthesis method of the derivative, which comprises the following steps:
mixing benzoate or a substitute thereof with a hydrazine hydrate solution with the mass fraction of 40-80%, and reacting for 4-8 hours at the temperature of 25-80 ℃ to obtain a hydrazide compound;
mixing a hydrazide compound and nitro heterocyclic aldehyde, adding the mixture into a mixed solvent of water and ethanol or methanol for dissolving, adjusting the pH value to 5-7, refluxing at 60-90 ℃ for 1-6 h to obtain a crude product, and filtering to obtain a target product.
Preferably, the mass ratio of the benzoate or the substitute thereof to the hydrazine hydrate solution is 1: 1.5-10;
the molar ratio of the hydrazide compound to the nitro heterocyclic aldehyde is 1-2: 1;
the volume ratio of ethanol or methanol to water in the mixed solvent is 5-20: 3-8.
The invention also provides application of the derivative in preparing a PD-L1 expression inhibitor.
Preferably, the derivative can kill cancer cells, inhibit cancer cell proliferation and metastasis, and promote cancer cell apoptosis.
Preferably, the cancer cells are HepG2 cells or a549 cells.
The invention also provides a pharmaceutical composition which comprises the nifurozide heterocyclic benzylidene hydrazide derivative or pharmaceutically acceptable salt thereof.
Preferably, a pharmaceutically acceptable adjuvant or carrier is also included.
Compared with the prior art, the invention has the beneficial effects that:
the nifurozide heterocyclic benzylidene hydrazide derivative provided by the invention can effectively inhibit proliferation of hepatocellular carcinoma cells, induce apoptosis and obviously inhibit expression of PD-L1 protein in the hepatocellular carcinoma cells; meanwhile, the derivative also has a certain proliferation inhibition effect on lung cancer cells. The compound provided by the invention can be used as a PD-L1 inhibitor and has a better anti-tumor application prospect.
Drawings
FIG. 1 shows the killing effect of compounds on hepatocellular carcinoma cells after 12 hours of action;
FIG. 2 is a graph showing the killing effect of a compound on hepatocellular carcinoma cells 24 hours later;
FIG. 3 is the killing effect of a compound on hepatocellular carcinoma cells after 36 hours of action;
FIG. 4 is the effect of compounds on proliferation of hepatocellular carcinoma cells;
FIG. 5 is the effect of compounds on migration of hepatocellular carcinoma cells;
FIG. 6 is a graph showing the effect of compounds on apoptosis 24 hours after they act on hepatocellular carcinoma cells;
FIG. 7 is a graph showing the effect of compounds on apoptosis 48 hours after they act on hepatocellular carcinoma cells;
FIG. 8 is a graph of the effect of compounds on PD-L1 expression 12 hours after treatment on hepatocellular carcinoma cells;
FIG. 9 is a graph of the effect of compounds on PD-L1 expression 24 hours after exposure to hepatocellular carcinoma cells;
FIG. 10 is a graph showing the effect of compounds on PD-L1 expression after 36 hours of exposure to hepatocellular carcinoma cells.
FIG. 11 is the effect of compounds on lung cancer cell proliferation;
FIG. 12 is a graph of the effect of compounds on PD-L1 expression 24 hours after they acted on lung cancer cells.
Detailed Description
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative and not limiting of the scope of the invention.
The nifurozide heterocyclic benzylidene hydrazide derivative provided by the embodiment of the invention has a structural formula shown in a formula I:
wherein X is selected from a sulfur atom, an oxygen atom or a nitrogen atom; r is selected from C1-C5 alkyl, nitro, amino, substituted amino, methoxy, hydroxyl or carboxyl.
The basic parent nucleus of the derivative is the dotted box part in formula I. The invention utilizes substituted benzoyl hydrazine and nitro heterocyclic aldehyde to synthesize the derivative with the structure similar to that of a classic antibacterial drug, namely nifuratel, wherein hetero atoms of the nitro heterocyclic aldehyde can be oxygen, nitrogen or sulfur atoms. Experiments prove that the compound can effectively inhibit the proliferation and migration of tumor cells, promote the apoptosis of the tumor cells and show obvious anti-tumor effect. Importantly, the compound remarkably inhibits the expression of an immunosuppressive molecule PD-L1, and has the potential of improving the anti-tumor immunity of an organism.
The derivative is prepared by the following method:
mixing benzoate or a substitute thereof with a hydrazine hydrate solution with the mass fraction of 40-80%, and reacting for 4-8 hours at the temperature of 25-80 ℃ to obtain a hydrazide compound;
mixing a hydrazide compound and nitro heterocyclic aldehyde, adding the mixture into a mixed solvent of water and ethanol or methanol for dissolving, adjusting the pH value to 5-7, refluxing for 1-5 h at the temperature of 60-90 ℃, obtaining a crude product, and filtering to obtain a target product.
The following description will be given with reference to specific examples.
Example 1
A nifurozide heterocyclic benzylidene hydrazide derivative has a structural formula shown in a formula I:
the derivative is obtained according to the following synthetic route:
the specific synthetic process is as follows:
dissolving 10mmol of p-methoxybenzoate in 25mL of methanol, adding 100mmol of hydrazine hydrate with the mass fraction of 80%, heating the mixed solution to 80 ℃, refluxing for 6h, and removing the solvent under reduced pressure and drying after the p-methoxybenzoate completely reacts to obtain p-methoxybenzoyl hydrazine;
dissolving p-methoxybenzoyl hydrazine and 2-nitrothiophene-4-formaldehyde into a mixed solution of methanol, glacial acetic acid, concentrated sulfuric acid and water according to a molar ratio of 1: 1 (the volume ratio of the four is 20: 8: 7: 8, the mixed solvent of water and methanol can increase the yield of a final reaction product and is beneficial to separation of a target product), heating to 60 ℃, refluxing for 6 hours, after the reaction is finished, filtering the reaction product in a suction manner, and recrystallizing the precipitate with methanol to obtain a yellow target compound, wherein the yield is as follows: 79.4 percent.
Characterization data for this derivative:1HNMR：(400MHz,DMSO-d6)δ12.14(s,1H),8.68(s,1H),8.14(d,J＝4.3Hz,1H),7.91(d,J＝8.4Hz,2H),7.57(d,J＝4.3Hz,1H),7.08(d,J＝8.6Hz,2H),3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ157.87,153.39,151.12,147.48,144.62,133.99,131.03,130.84,130.21,129.88,125.24,114.29,55.94。
example 2
The nifurozide heterocyclic benzylidene hydrazide derivative has a structural formula shown in a formula II:
the specific synthetic process is as follows:
dissolving 10mmol of m-methyl benzoate in 25mL of methanol, adding 50mmol of hydrazine hydrate with the mass fraction of 40%, heating the mixed solution to 25 ℃, refluxing for 4h, and removing the solvent under reduced pressure and drying after the p-methoxybenzoate completely reacts to obtain m-methyl benzoyl hydrazine;
dissolving m-methylbenzoyl hydrazine and 2-nitrothiophene-4-formaldehyde in a mixed solution of methanol, glacial acetic acid, concentrated sulfuric acid and water according to a molar ratio of 2: 1 (the volume ratio of the four is 15: 8: 4: 5), heating to 65 ℃, refluxing for 4 hours, after the reaction is finished, filtering the reaction product, recrystallizing the precipitate with methanol to obtain a yellow target compound, wherein the yield is as follows: 81.2 percent.
Example 3
A nifurozide heterocyclic benzylidene hydrazide derivative has a structure shown as a formula III:
the specific synthetic method of the derivative comprises the following steps:
dissolving 10mmol of o-hydroxybenzoates in 25mL of methanol, adding 15mmol of hydrazine hydrate with the mass fraction of 60%, heating the mixed solution to 55 ℃, refluxing for 5h, and removing the solvent under reduced pressure and drying after all the methoxybenzoates react to obtain o-hydroxybenzoates;
dissolving o-hydroxybenzoyl hydrazine and 5-nitro-2-furfural in a mixed solution of methanol, glacial acetic acid, concentrated sulfuric acid and water according to a molar ratio of 2: 1 (the volume ratio of the four is 2: 1), heating to 90 ℃, refluxing for 1h, after the reaction is finished, filtering the reaction product, recrystallizing the precipitate with methanol to obtain a yellow target compound, wherein the yield is as follows: 80.3 percent.
Example 4
A nifurozide heterocyclic benzylidene hydrazide derivative has a structure shown in a formula IV:
the specific synthetic method of the derivative comprises the following steps:
dissolving 10mmol of m-dimethylaminobenzoate in 25mL of methanol, adding 80mmol of hydrazine hydrate with the mass fraction of 55%, heating the mixed solution to 75 ℃, refluxing for 6h, and removing the solvent under reduced pressure after all the methoxybenzoate reacts to obtain the m-dimethylaminobenzoate;
dissolving m-dimethylaminobenzoate and 5-nitropyrrole-2-formaldehyde in a mixed solution of methanol, glacial acetic acid, concentrated sulfuric acid and water according to a molar ratio of 2: 1 (the volume ratio of the four is 12: 5: 7: 8), heating to 65 ℃, refluxing for 3 hours, after the reaction is finished, filtering the reaction product, recrystallizing the precipitate with methanol to obtain a yellow target compound, wherein the yield is as follows: 78.9 percent.
Hereinafter, the antitumor effect of the nifurozide heterocyclic benzylidene hydrazide derivatives provided by the present invention (the antitumor effect of the compounds provided in each example is substantially the same, and therefore, the compound provided in example 1 is only described as an example below).
Effects on liver cancer cells:
1. killing effect of nifurozide heterocyclic benzylidene hydrazide derivative on hepatocellular carcinoma cells
Spreading the hepatocellular carcinoma HepG2 cells on a cell 96-well plate, at 37 deg.C and 5% CO2After incubation for 16 hours in an incubator, different concentrations were addedAfter culturing the compound (0, 0.625, 1.25, 2.5, 5, 10, 20, 40. mu.g/ml) for 12 hours, 24 hours and 36 hours, the killing ability of the compound against hepatocellular carcinoma cells was examined by CCK8 test.
2. Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on proliferation of hepatocellular carcinoma cells
Spreading the HepG2 cell to 6-well plate at 37 deg.C and 5% CO2After 16 hours incubation in the incubator, different concentrations of compound (0, 0.025, 0.05, 0.1, 0.2, 0.4. mu.g/ml) were added. After further culturing for 24 hours, the culture medium in the corresponding 6-well plate was discarded, replaced with a complete culture medium of 10% fetal calf serum without drug, and placed at 37 ℃ in 5% CO2And (3) continuously culturing in the incubator, when a macroscopic cell clone block mass is observed to be formed and the cell block mass is moderate in size, taking out the 6-hole plate, discarding the culture medium, washing twice with 3ml of PBS (phosphate buffer solution), adding 1ml of 4% tissue cell fixing solution into each hole, removing the tissue cell fixing solution after fixing for 30min, adding crystal violet staining solution into each hole, staining for 30min, then discarding the crystal violet staining solution, flushing the residual crystal violet with ultrapure water, airing the cell plate, photographing and analyzing the influence of the compound on the proliferation of the hepatocellular carcinoma cells.
3. Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on migration of hepatocellular carcinoma cells
The effect of the new compounds on the migration of hepatocellular carcinoma cells was examined using a cell scratch test. Spreading the HepG2 cell to 6-well plate at 37 deg.C and 5% CO2After incubation in the incubator for 16 hours, the cell plates were marked with a "straight" line using a 200. mu.l pipette tip, and then different concentrations of compounds (0, 0.625, 1.25, 2.5, 5, 10. mu.g/ml) were added and incubation was continued for 24 hours and 48 hours, respectively, and the cell migration was photographed by microscopy.
4. Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on hepatocellular carcinoma cell apoptosis
Spreading hepatocellular carcinoma HepG2 cells on 6-well cell plates at 37 deg.C and 5% CO2After incubation in an incubator for 16h, compounds (0, 0.3125, 0.625, 1.25, 2.5, 5 μ g/ml) of different concentrations were added, incubation continued for 24h and 48h,discarding the culture medium, collecting cells, and detecting the influence of the compound on the hepatocellular carcinoma cell apoptosis according to an Annexin V-FITC/PI cell apoptosis flow detection kit.
5. Influence of nifurozide heterocyclic benzylidene hydrazide derivatives on expression of PD-L1 protein in hepatocellular carcinoma cells
Spreading hepatocellular carcinoma HepG2 cells on 6-well cell plate, 37 deg.C, 5% CO2After incubation in an incubator for 16 hours, compounds (0, 0.3125, 0.625, 1.25, 2.5, 5. mu.g/ml) were added at different concentrations and after 12 hours, 24 hours and 36 hours of co-incubation, the effect of the compounds on the expression of PD-L1 protein in hepatocellular carcinoma cells was examined by the Westernblot assay.
6. As a result, the
6.1 killing effect of nifurozide heterocyclic benzylidene hydrazide derivatives on hepatocellular carcinoma cells
The CCK8 test shows that the compound acts on hepatocellular carcinoma cells for 12 hours, 24 hours and 36 hours, and then kills the cells.
The results show that the compound significantly kills hepatocellular carcinoma cells when the drug concentration is 10, 20, 40 μ g/ml after 12 hours of compound action on the cells (fig. 1). When the compound acts on the cells for 24 hours and 36 hours, the compound kills hepatocellular carcinoma cells remarkably when the drug concentration is 5, 10, 20 and 40 mu g/ml (figures 2 and 3). The derivative has a certain killing effect on hepatocellular carcinoma cells.
6.2 Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on proliferation of hepatocellular carcinoma cells
The influence of nifurozide heterocyclic benzylidene hydrazide derivatives with different concentrations (0, 0.025, 0.05, 0.1, 0.2 and 0.4 mu g/ml) on the proliferation of hepatocellular carcinoma cells is detected by a cell cloning experiment.
The results showed that the proliferation of hepatocellular carcinoma cells was significantly inhibited when the concentrations of the compounds were 0.05, 0.1, 0.2, 0.4. mu.g/ml (FIG. 4). The derivative has the function of inhibiting the proliferation of hepatocellular carcinoma cells.
6.3 Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on migration of hepatocellular carcinoma cells
The effect of different concentrations (0, 0.625, 1.25, 2.5, 5, 10. mu.g/ml) of the compound on cell migration after 24 hours and 48 hours of the action on hepatocellular carcinoma cells was examined using a cell scratch test.
The results show that the migration of hepatocellular carcinoma cells is remarkably inhibited when the concentration of the compound is 1.25, 2.5, 5 and 10 mu g/ml after the compound acts on the cells for 12 hours; after 48 hours of cell action, the migration of hepatocellular carcinoma cells was significantly inhibited at concentrations of 0.625, 1.25, 2.5, 5, and 10. mu.g/ml (FIG. 5). The derivative has the function of inhibiting the migration of hepatocellular carcinoma cells.
6.4 Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on hepatocellular carcinoma apoptosis
The influence of different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5. mu.g/ml) of the compounds on apoptosis of hepatocellular carcinoma cells after 24 hours and 48 hours was examined by flow cytometry.
The results show that the compound significantly increased apoptosis of hepatocellular carcinoma cells at concentrations of 1.25, 2.5 and 5 μ g/ml after 24 hours of cell action (fig. 6); after the cell is acted for 48 hours, the apoptosis of the hepatocellular carcinoma cells is remarkably increased when the concentration is 0.3125, 0.625, 1.25, 2.5 and 5 mu g/ml (figure 7). The derivative is shown to have the function of remarkably increasing the apoptosis of hepatocellular carcinoma cells.
6.5 Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on expression of related proteins in hepatocellular carcinoma cells
Westernblot experiments were used to test the effect of compounds at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5. mu.g/ml) on the expression of PD-L1 protein in hepatocellular carcinoma cells after 12 hours, 24 hours and 36 hours of exposure.
The results showed that the compound significantly inhibited the expression of PD-L1 protein at concentrations of 2.5 and 5. mu.g/ml after 12 hours, 24 hours and 36 hours of the action on hepatocellular carcinoma cells (FIGS. 8-10). The compound is shown to be capable of being used as an inhibitor of PD-L1.
Effect on lung cancer cells:
1. effect of nifurozide heterocyclic benzylidene hydrazide derivatives on lung cancer cell proliferation
The influence of nifurozide heterocyclic benzylidene hydrazide derivatives with different concentrations (0, 0.025, 0.05, 0.1, 0.2 and 0.4 mu g/ml) on the proliferation of lung cancer cells is detected by a cell cloning experiment.
The results showed that the proliferation of hepatocellular carcinoma cells was significantly inhibited at concentrations of 0.1, 0.2, 0.4. mu.g/ml compound (FIG. 11). The derivative is shown to have the function of inhibiting the proliferation of lung cancer cells.
2. Effect of nifurozide heterocyclic benzylidene hydrazide derivatives on expression of PD-L1 in lung cancer cells
Westernblot experiments are used to detect the influence of compounds with different concentrations (0, 0.3125, 0.625, 1.25 and 2.5 mu g/ml) on the expression of PD-L1 protein in lung cancer cells after 24 hours of action.
The results show that the compound significantly inhibited the expression of PD-L1 protein at concentrations of 0.625, 1.25 and 2.5. mu.g/ml after 24 hours of action on lung cancer cells (FIG. 12). The compound is shown to be capable of being used as an inhibitor of PD-L1.
In conclusion, the nifurozide heterocyclic benzylidene hydrazide derivative provided by the invention can obviously kill hepatocellular carcinoma cells and lung cancer cells, inhibit proliferation and migration of the cells, promote apoptosis of the cells, obviously inhibit expression of PD-L1 in tumor cells, and can be used as a novel PD-L1 inhibitor.
It should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A nifurozide heterocyclic benzylidene hydrazide derivative or a pharmaceutically acceptable salt thereof has a structural formula shown as a formula I:
wherein X is selected from a sulfur atom, an oxygen atom or a nitrogen atom; r is selected from C1-C5 alkyl, nitro, amino, substituted amino, methoxy, hydroxyl or carboxyl.
2. The nifurozide heterocyclic benzylidene hydrazide derivative or the pharmaceutically acceptable salt thereof according to claim 1, wherein X is selected from a sulfur atom, and R is selected from a methoxy group.
3. A method for synthesizing nifurozide heterocyclic benzylidene hydrazide derivative as claimed in claim 1, comprising the following steps:
mixing benzoate or a substitute thereof with a hydrazine hydrate solution with the mass fraction of 40-80%, and reacting at 25-80 ℃ for 4-8 h to obtain a hydrazide compound;
mixing a hydrazide compound and nitro heterocyclic aldehyde, adding the mixture into a mixed solvent of water and ethanol or methanol for dissolving, adjusting the pH value to 5-7, refluxing for 1-6 h at the temperature of 60-90 ℃ to obtain a crude product, and filtering to obtain a target product.
4. The method of synthesis according to claim 3,
the mass ratio of the benzoate or the substitute thereof to the hydrazine hydrate solution is 1: 1.5-10;
the molar ratio of the hydrazide compound to the nitro heterocyclic aldehyde is 1-2: 1;
the volume ratio of ethanol or methanol to water in the mixed solvent is 5-20: 3-8.
5. Use of nifurozide heterocyclic benzylidene hydrazide derivative as defined in claim 1 in preparing PD-L1 expression inhibitor.
6. The use according to claim 5, wherein said derivative is capable of killing cancer cells, inhibiting cancer cell proliferation and metastasis, and promoting cancer cell apoptosis.
7. The use according to claim 6, wherein the cancer cells are HepG2 cells or A549 cells.
8. A pharmaceutical composition comprising the nifurozide heterocyclic benzylidene hydrazide derivative as claimed in claim 1 or 2, or a pharmaceutically acceptable salt thereof.
9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable adjuvant or carrier.
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| CN116496198A (en) * | 2023-06-26 | 2023-07-28 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 4-hydroxy-2' - (1-benzyl-5-nitropyrrole methylene) -benzoyl hydrazine derivative as well as preparation method and application thereof |
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