CN118908989B - Pyruvate derivative nano-atomizing agent for preventing malignant transformation of lung nodules and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a pyruvate derivative nano-atomization agent for preventing malignant transformation of lung nodules and a preparation method thereof. The invention provides a pyruvate derivative nano-atomizing agent for preventing malignant transformation of pulmonary nodules, which is prepared from pyruvate derivatives obtained by structurally improving sodium pyruvate which is a common anti-pulmonary disease drug. The pyruvate derivative can reduce the damage of lung tissues, change the levels of cytokines and chemokines in tumor microenvironment, reduce factors for promoting tumor growth and increase factors for inhibiting tumor growth so as to inhibit malignant transformation of lung nodules by inhibiting the expression of IL-6, and can obviously improve the transcriptional expression level of epithelial markers and inhibit the transcriptional level of mesenchymal cell markers after being treated by the pyruvate derivative so as to inhibit the epithelial-mesenchymal transformation process and further achieve the aim of inhibiting malignant transformation of lung nodules.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a pyruvate derivative nano-atomization agent for preventing malignant transformation of lung nodules and a preparation method thereof.
Background
Lung nodules refer to solid or sub-solid lesions that are imaging quasi-circular, less than 3cm in diameter, surrounded by air-bearing lung tissue. These lesions appear as elevated density areas of the lung on chest X-rays or chest CT, either isolated or multiple. The formation of lung nodules may be caused by a variety of causes including, but not limited to, chronic inflammatory diseases (e.g., granulomatous polyangiitis, sarcoidosis, rheumatoid arthritis involving the lungs, etc.), infectious agents (e.g., bacterial infections, tubercular infections, fungal infections, etc.), benign tumors (e.g., hamartoma, cellulose tumors, smooth muscle tumors, etc.), malignant tumors (i.e., lung cancer), although most lung nodules are benign, some nodules may be malignant.
Malignant transformation of a pulmonary nodule refers to the occurrence of malignant changes in the pulmonary nodule during growth and development, i.e., the pulmonary nodule changes from benign to malignant, forming a neoplastic-like lesion with invasive growth and metastatic potential. This malignant transformation is typically due to an imbalance in cell proliferation and apoptosis caused by genetic mutations, such that lung epithelial cells are malignant transformed and hyperproliferative to form nodules (epithelial-mesenchymal transformation to form nodules).
The existing medicines for treating the lung nodules are various, and specific selection needs to be determined according to the etiology and the properties of the lung nodules and the specific conditions of patients. Some common classes of drugs for treating pulmonary nodules include anti-inflammatory drugs (for pulmonary nodules caused by inflammatory infections, improving pulmonary conditions by anti-inflammatory sterilization), antifungal drugs (for treating pulmonary nodules caused by fungal infections, eliminating etiology by inhibiting fungal activity), cancer cell inhibiting drugs (for pulmonary nodules caused by tumors, by inhibiting cancer cell growth and spread).
In terms of pulmonary disease, sodium pyruvate reduces the injury and inflammatory response of pulmonary tissue through its antioxidant and anti-inflammatory effects, thereby improving pulmonary function. However, how to effectively utilize the beneficial effect exhibited by sodium pyruvate in the aspect of treating pulmonary diseases and further expand the application range by modifying the same, particularly in the key field of preventing malignant transformation of pulmonary nodules, is an important and urgent problem at present.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a pyruvate derivative for preventing malignant transformation of lung nodules, which can effectively prevent the malignant transformation of lung nodules.
One of the purposes of the invention is realized by adopting the following technical scheme:
A pyruvate derivative for use in preventing malignant transformation of pulmonary nodules, the pyruvate derivative having the structural formula shown in formula I:
。
the second object of the present invention is to provide a method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules, comprising the steps of:
(1) Adding 3-fluoro-4-aldehyde phenylboronic acid and sodium pyruvate into a sodium hydroxide solution, reacting for 0.5-2 hours, and then treating to obtain an intermediate 4A;
(2) Adding 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimido chloride and 3, 4-dibromoaniline into sodium bicarbonate solution, reacting for 1-2 hours at 60-70 ℃ and then treating to obtain an intermediate 1;
(3) Adding the intermediate 1 and N, N' -carbonyl diimidazole into a solvent, reacting for 3-5 hours in a reflux state, and then treating to obtain an intermediate 2;
(4) Adding the intermediate 2 into trifluoroacetic acid and 30% hydrogen peroxide solution, reacting for 5-7 hours at 60-70 ℃ and then treating to obtain an intermediate 3;
(5) Sequentially adding the intermediate 3, 2- (3, 4-dihydroxyphenyl) ethylamine and triethylamine into a solvent, reacting for 0.5-2 h, and then treating to obtain an intermediate 4;
(6) And in the environment with the pH value of 8-9, reacting the intermediate 4 with the intermediate 4A for 3-5 hours, and then treating to obtain the pyruvate derivative.
Further, in the step (1), the molar ratio of the 3-fluoro-4-aldehyde phenylboronic acid to the sodium pyruvate to the sodium hydroxide is 10 (20-25) (40-60).
Further, in the step (2), the molar use ratio of the 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimido chloride, 3, 4-dibromoaniline and sodium bicarbonate is 1 (1.1-1.5) to 0.2-0.4.
Further, in the step (3), the molar ratio of the intermediate 1 to the N, N' -carbonyldiimidazole is 1 (1.4-1.6).
Further, in the step (4), the use amount ratio of the acid and the 30% hydrogen peroxide solution added to the intermediate 2 is 2mmol (15-20 mL) and 11-12 mL.
Further, in the step (5), the molar ratio of the intermediate 3 to the 2- (3, 4-dihydroxyphenyl) ethylamine to the triethylamine is 5 (6-9) to 12-20.
Further, in the step (6), the molar ratio of the intermediate 4 to the intermediate 4A is 1 (1.2-1.8).
The invention further provides a pyruvate derivative nano-atomizing agent for preventing malignant transformation of lung nodules, which comprises 5-8 parts of pyruvate derivative nano-particles and 100 parts of water for injection, wherein the preparation method of the pyruvate derivative nano-particles comprises the steps of preparing Tween 80 or Tween 20 into a solution with the concentration of 0.02-0.05wt% by using water, refrigerating at the temperature of 2-4 ℃ to obtain a water phase solution, adding 8-10 mg/mL of the pyruvate derivative methylene dichloride solution into the water phase solution at the temperature of-15 to-18 ℃, emulsifying for 5-7 min at the speed of 1000-1200 r/min, evaporating the solvent for 3-4 h, centrifuging, collecting nano-particles and drying to obtain the pyruvate derivative nano-atomizing agent.
The fourth object of the invention is to provide a preparation method of a pyruvate derivative nano-atomizing agent for preventing malignant transformation of pulmonary nodules, which is obtained by adding sodium pyruvate derivative nano-particles into water for injection according to the formula.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a pyruvate derivative for preventing malignant transformation of pulmonary nodules, which is prepared by nucleophilic substitution of aldehyde group of 3-fluoro-4-aldehyde phenylboronic acid and ketocarbonyl of sodium pyruvate to obtain an intermediate 4A, amination of 4-amino-N '-hydroxy-1, 2, 5-oxadiazole-3-carboimido chloride to obtain an intermediate 1, 4-dibromoaniline, cyclization of the intermediate 1 under the action of N, N' -carbonyl diimidazole to obtain an intermediate 2, oxidation of amino of the intermediate 2 to nitro by the combined action of hydrogen peroxide and trifluoroacetic acid to obtain an intermediate 3, substitution reaction of the intermediate 3 and 2- (3, 4-dihydroxyphenyl) ethylamine to obtain an intermediate 4, and esterification of group o-diol of the intermediate 4 and group boric acid of the intermediate 4A to obtain the pyruvate derivative for preventing malignant transformation of pulmonary nodules.
The pyruvate derivative can reduce the damage of lung tissues, maintain the normal structure and function of the lung tissues, change the levels of cytokines and chemokines in tumor microenvironment, reduce factors for promoting tumor growth, increase factors for inhibiting tumor growth so as to inhibit malignant transformation of lung nodules, and obviously improve the transcriptional expression level of epithelial markers and inhibit the transcriptional level of mesenchymal cell markers after being treated by the pyruvate derivative so as to inhibit the epithelial cell-mesenchymal transformation (EMT) process, thereby achieving the purpose of inhibiting malignant transformation of lung nodules. In addition, the pyruvate derivative nano-atomization agent has a better inhibition effect on the swelling of lung nodules, and can prevent further deterioration.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of the pyruvate derivatives of the present invention;
FIG. 2 is a schematic representation of the hydrogen spectrum results of the pyruvate derivative intermediate 4A of the present invention;
FIG. 3 is a schematic representation of the mass spectrum results of the pyruvate derivative intermediate 4A of the present invention;
FIG. 4 is a schematic representation of the hydrogen spectrum results of the pyruvate derivative intermediate 1 of the present invention;
FIG. 5 is a schematic representation of the mass spectrum results of the pyruvate derivative intermediate 1 of the present invention;
FIG. 6 is a schematic representation of the hydrogen spectrum results of the pyruvate derivative intermediate 2 of the present invention;
FIG. 7 is a schematic representation of the hydrogen spectrum results of pyruvate derivative intermediate 3 of the present invention;
FIG. 8 is a graphical representation of the hydrogen spectrum results of the pyruvate derivative intermediate 4 of the present invention;
FIG. 9 is a schematic representation of the hydrogen spectrum results of pyruvate derivatives of the present invention;
FIG. 10 is a schematic representation of the mass spectrum results of the pyruvate derivatives of the present invention;
FIG. 11 is a schematic representation of the effect of pyruvate derivatives of the present invention on interleukin-6 (IL-6) in BEAS-2B cells;
FIG. 12 is a graph showing the inhibition of BEAS-2B cell EMT by pyruvate derivatives of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used, unless otherwise specified, are all conventional products obtained from commercial sources.
Example 1
A pyruvate derivative nano-atomizing agent for preventing malignant transformation of lung nodules comprises the following components in parts by weight of 5 parts of pyruvate derivative nano-particles and 100 parts of water for injection.
The preparation method of the pyruvate derivative nano atomizing agent comprises the step of adding pyruvate derivative nano particles into water for injection according to the formula.
The preparation method of the pyruvate derivative nanoparticle comprises the steps of preparing Tween 80 into a solution with the concentration of 0.02wt% by using water, refrigerating at the temperature of 2 ℃ to obtain an aqueous phase solution, adding 8mg/mL of a pyruvate derivative dichloromethane solution into the aqueous phase solution (the volume ratio of dichloromethane to aqueous phase is 1:10) at the temperature of 15 ℃ below zero, emulsifying for 5min at the speed of 1000r/min, evaporating the solvent for 3h, centrifuging, collecting the nanoparticle and drying to obtain the pyruvate derivative nanoparticle.
The preparation method of the pyruvate derivative comprises the following steps, and the reaction scheme is shown in figure 1:
A. Preparation of intermediate 4A
Dissolving sodium hydroxide in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200mL of sodium hydroxide solution with a final concentration of 0.2M, adding 3-fluoro-4-aldehyde phenylboronic acid (10 mmol) and sodium pyruvate (20 mmol) into the solution, stirring the solution at room temperature for reaction for 1h, filtering the reaction solution after the reaction is finished to obtain a filter cake, washing the filter cake with cold ethanol, dissolving the filter cake with ethyl acetate, regulating pH=5 with 1M HCl, separating ethyl acetate phase, and concentrating the solution with anhydrous sodium sulfate to obtain an intermediate 4A;
The 1 H NMR of intermediate 4A is shown in FIG. 2 (C 11H10 BFO,400MHz,DMSO-d 6): delta 7.42-7.32 (M, 2H), 6.99-6.85 (M, 2H), 6.08 (M, 1H), 4.19 (d, 4H), and the mass spectrum of intermediate 4A is shown in FIG. 3: [ M+H ] + M/z:253.06, ESI-MS (M/z): 253.11.
B. preparation of intermediate 1
Adding ethanol (40 mL) into 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimidyl chloride (10 mmol) and 3, 4-dibromoaniline (11 mmol), then adding 20mL of 1M sodium bicarbonate solution, reacting for 2h at 60 ℃, concentrating the reaction solution, dissolving in ethyl acetate (100 mL), washing an organic phase with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, pulping the crude product in a mixed solvent (N-hexane: ethyl acetate=7:3, 1g of crude product: 8mL of mixed solvent), filtering, collecting a filter cake, and drying to obtain an intermediate 1;
1 H NMR of intermediate 1 is shown in FIG. 4 ：（C9H7Br2N5O2,400MHz,DMSO-d6）：δ11.08（s,1H）,8.45（s,1H）,7.28（d,1H）,6.78(d,2H),6.31（s,2H）; mass spectrum of intermediate 1 is shown in FIG. 5 [ M+H ] + M/z 375.90, ESI-MS (M/z): 375.91.
C. Preparation of intermediate 2
Adding intermediate 1 (50 mmol) into 120mL of ethyl acetate, adding N, N' -carbonyl diimidazole (75 mmol) into the mixture, and reacting for 5h at 60 ℃, adding water to quench the reaction, separating liquid, extracting water phase with ethyl acetate, merging organic phases, washing the organic phases with 2M HCl solution and saturated saline in sequence, drying the organic phases with anhydrous ammonium sulfate, and concentrating to obtain intermediate 2;
The 1 H NMR of intermediate 2 is shown in FIG. 6 (C 10H5Br2N5O3, 400MHz, DMSO-d 6): delta 7.88-7.60 (m, 3H), 6.60 (s, 2H).
D. Preparation of intermediate 3
Slowly adding the intermediate 2 (20 mmol) into a mixed solution of 200mL of trifluoroacetic acid and 100mL of 30% hydrogen peroxide at the temperature of 0 ℃, stirring uniformly after the addition, heating to 70 ℃ for reaction for 5h, quenching the reaction solution with water in an ice bath, extracting a water phase with ethyl acetate, drying the ethyl acetate phase with anhydrous sodium sulfate, and concentrating to obtain an intermediate 3;
The 1 H NMR of intermediate 3 is shown in FIG. 7 (C 10H3Br2N5O5, 400MHz, DMSO-d 6): delta 7.88-7.60 (m, 3H).
E. preparation of intermediate 4
Adding intermediate 3 (5 mmol) and 2- (3, 4-dihydroxyphenyl) ethylamine (7.5 mmol) into 50mL tetrahydrofuran, adding triethylamine (15 mmol) into the mixture, reacting for 1H at room temperature, adding ethyl acetate into the reaction solution to dilute the reaction solution after the reaction, adding saturated sodium bicarbonate aqueous solution to obtain a mixed solution, separating the mixed solution, washing the organic phase with water, saturated brine, drying the organic phase with anhydrous sodium sulfate, passing through a column (eluent, dichloromethane: methanol=100:1) to obtain intermediate 4, wherein 1 H NMR of intermediate 4 is shown in FIG. 8 ：（C18H13Br2N5O5,400MHz,DMSO-d6）：δ 9.52（s,2H）,7.88-7.60（m,3H）,6.80-6.60（m,3H）,6.47（s,1H）,3.43（t,2H）,2.97（t,2H）.
F. Preparation of pyruvate derivatives
Adding the intermediate 4 (10 mmol) and the intermediate 4A (10 mmol) into a mixed solution of 100mL of ethanol and water in a ratio of 1:1, regulating the pH to 8.2 by using 1M NaOH, and reacting for 6 hours, concentrating the reaction solution, purifying by a column (eluent, dichloromethane: methanol=80:20), and obtaining a pyruvate derivative;
1 H NMR of the pyruvate derivative is shown in FIG. 9 and mass spectrum of ：（C29H18BBr2FN5NaO8,400MHz,DMSO-d6）：δ 7.60-7.48（m,4H）,7.32（m,1H）,6.99（d,1H）,6.85（d,1H）,6.64-6.52（m,3H）,6.27（m,1H）,6.08（s,1H）,3.43（t,2H）,3.10（d,2H）,2.97（t,2H）; pyruvate derivative is shown in FIG. 10 [ M+H ] + M/z 775.95.11, ESI-MS (M/z): 775.98.
Example 2
A nanometer pyruvate derivative atomizing agent for preventing malignant transformation of lung nodules comprises, by weight, 6 parts of nanometer pyruvate derivative particles and 100 parts of water for injection.
The preparation method of the pyruvate derivative nano atomizing agent comprises the step of adding pyruvate derivative nano particles into water for injection according to the formula.
The preparation method of the pyruvate derivative nanoparticle comprises the steps of preparing Tween 20 into a solution with the concentration of 0.03wt% by using water, refrigerating at the temperature of 3 ℃ to obtain an aqueous phase solution, adding a dichloromethane solution with the concentration of 9mg/mL of the pyruvate derivative into the aqueous phase solution (the volume ratio of dichloromethane to aqueous phase is 1:15) at the temperature of 17 ℃ below zero, emulsifying for 6min at the speed of 1100r/min, evaporating the solvent for 3h, centrifuging, collecting the nanoparticle and drying to obtain the pyruvate derivative nanoparticle.
The preparation method of the pyruvate derivative comprises the following steps:
A. Preparation of intermediate 4A
Sodium hydroxide is dissolved in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200mL of sodium hydroxide solution with a final concentration of 0.3M, 3-fluoro-4-aldehyde phenylboronic acid (10 mmol) and sodium pyruvate (22 mmol) are added to the solution, the solution is stirred at room temperature for reaction for 0.5h, the reaction solution is filtered after the reaction is finished to obtain a filter cake, the filter cake is washed with cold ethanol and then is dissolved with ethyl acetate, pH=6 is regulated by using 1M HCl, the ethyl acetate phase is separated by extraction, and then anhydrous sodium sulfate and concentration are carried out to obtain an intermediate 4A, and the results of 1 HNMR and MS show that the structure is consistent with that of example 1.
B. preparation of intermediate 1
Ethanol (40 mL) was added to 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimidyl chloride (10 mmol) and 3, 4-dibromoaniline (15 mmol), then 20mL of 1.5m sodium bicarbonate solution was added thereto and reacted at 70 ℃ for 1h, the reaction solution was concentrated and dissolved in ethyl acetate (100 mL), the organic phase was washed with saturated brine water, the organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product, the crude product was slurried in a mixed solvent (N-hexane: ethyl acetate=7:3, 1g of crude product: 8mL of mixed solvent) for 1h, the filter cake was collected by filtration and dried to give intermediate 1, and the results of 1 HNMR and MS showed that the structure was consistent with example 1.
C. Preparation of intermediate 2
Intermediate 1 (50 mmol) was added to 100mL of ethyl acetate, then N, N' -carbonyldiimidazole (80 mmol) was added thereto and reacted at 70℃for 3 hours, the reaction was quenched by water, the layers were separated, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, the organic phase was washed with a 2M HCl solution, saturated saline solution, and the organic phase was dried over anhydrous ammonium sulfate and concentrated to give intermediate 2, and 1 HNMR and MS showed that the structure was the same as in example 1.
D. Preparation of intermediate 3
Intermediate 2 (20 mmol) was slowly added to a mixture of 150mL trifluoroacetic acid and 120mL 30% hydrogen peroxide at 0deg.C, after the addition was completed, the mixture was stirred and heated to 65deg.C for 7h, the reaction mixture was quenched with water in an ice bath, the aqueous phase was extracted with ethyl acetate, the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to give intermediate 3, and 1 HNMR and MS showed the structure as in example 1.
E. Preparation of intermediate 4
Intermediate 3 (5 mmol) and 2- (3, 4-dihydroxyphenyl) ethylamine (9 mmol) were added to 50mL of tetrahydrofuran, then triethylamine (20 mmol) was added thereto and reacted at room temperature for 0.5h, ethyl acetate was added to the reaction solution after the completion of the reaction to dilute the reaction solution, then saturated aqueous sodium bicarbonate solution was added to the mixed solution, the mixed solution was separated, and the organic phase was washed with water, saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and passed through a column (eluent, dichloromethane: methanol=100:1) to give intermediate 4, and 1 HNMR and MS showed that the structure was the same as in example 1.
F. Preparation of pyruvate derivatives
Intermediate 4 (10 mmol) and intermediate 4A (12 mmol) were added to a 1:1 mixture of 100mL ethanol and water, pH was adjusted to 9 with 1M NaOH, and the reaction was allowed to react for 5h, and the reaction solution was purified by column (eluent, dichloromethane: methanol=80:20) to give the final product, and 1 HNMR and MS showed the structure consistent with example 1.
Example 3
A pyruvate derivative nano-atomizing agent for preventing malignant transformation of lung nodules comprises the following components in parts by weight of 8 parts of pyruvate derivative nano-particles and 100 parts of water for injection.
The preparation method of the pyruvate derivative nano atomizing agent comprises the step of adding pyruvate derivative nano particles into water for injection according to the formula.
The preparation method of the pyruvate derivative nanoparticle comprises the steps of preparing Tween 80 into a solution with the concentration of 0.05wt% by using water, refrigerating at the temperature of 4 ℃ to obtain an aqueous phase solution, adding 10mg/mL of a pyruvate derivative dichloromethane solution into the aqueous phase solution (the volume ratio of dichloromethane to aqueous phase is 1:20) at the temperature of-18 ℃, emulsifying for 7min at the speed of 1200r/min, evaporating the solvent for 4h, centrifuging, collecting the nanoparticle and drying to obtain the pyruvate derivative nanoparticle.
The preparation method of the pyruvate derivative comprises the following steps:
(1) Preparation of pyruvate derivatives
A. Preparation of intermediate 4A
Sodium hydroxide is dissolved in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200mL of sodium hydroxide solution with a final concentration of 0.25M, 3-fluoro-4-aldehyde phenylboronic acid (10 mmol) and sodium pyruvate (25 mmol) are added into the solution, the solution is stirred at room temperature for 2h to react, the reaction solution is filtered after the reaction is finished to obtain a filter cake, the filter cake is washed with cold ethanol and then is dissolved with ethyl acetate, pH=6 is regulated by 1M HCl, the ethyl acetate phase is separated by extraction, and then anhydrous sodium sulfate and concentration are carried out to obtain an intermediate 4A, and the results of 1 HNMR and MS show that the structure is consistent with that of example 1.
B. preparation of intermediate 1
Ethanol (40 mL) was added to 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimidyl chloride (10 mmol) and 3, 4-dibromoaniline (13 mmol), then 20mL of 2m sodium bicarbonate solution was added thereto and reacted at 60 ℃ for 1.5 hours, the reaction solution was concentrated and dissolved in ethyl acetate (100 mL), the organic phase was washed with saturated brine water, the organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product, the crude product was slurried in a mixed solvent (N-hexane: ethyl acetate=7:3, 1g of crude product: 8mL of mixed solvent), and the filter cake was collected by filtration and dried to give intermediate 1, and 1 HNMR and MS showed that the structure was consistent with example 1.
C. Preparation of intermediate 2
Intermediate 1 (50 mmol) was added to 100mL of ethyl acetate, then N, N' -carbonyldiimidazole (70 mmol) was added thereto and reacted at 65℃for 4h, the reaction was quenched with water, the layers were separated, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, the organic phase was washed with a 2M HCl solution, saturated saline solution, and the organic phase was dried over anhydrous ammonium sulfate and concentrated to give intermediate 2, and 1 HNMR and MS showed that the structure was the same as in example 1.
D. Preparation of intermediate 3
Intermediate 2 (20 mmol) was slowly added to 180mL of a mixed solution of trifluoroacetic acid and 110mL of 30% hydrogen peroxide at 0 ℃, stirred after the addition was completed, heated to 60 ℃ for reaction for 6h, the reaction solution was quenched with water under ice bath, the aqueous phase was extracted with ethyl acetate, the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to give intermediate 3, and 1 HNMR and MS showed that the structure was consistent with example 1.
E. Preparation of intermediate 4
Intermediate 3 (5 mmol) and 2- (3, 4-dihydroxyphenyl) ethylamine (6 mmol) were added to 50mL of tetrahydrofuran, then triethylamine (12 mmol) was added thereto and reacted at room temperature for 2 hours, ethyl acetate was added to the reaction solution after the completion of the reaction to dilute the reaction solution, then saturated aqueous sodium bicarbonate solution was added to the mixed solution, the mixed solution was separated, and the organic phase was washed with water, saturated aqueous sodium chloride and dried over anhydrous sodium sulfate, and after passing through the column (eluent, dichloromethane: methanol=100:1), intermediate 4 was obtained, and 1 HNMR and MS showed that the structure was the same as in example 1.
F. Preparation of pyruvate derivatives
Intermediate 4 (10 mmol) and intermediate 4A (18 mmol) were added to a 1:1 mixture of 100mL ethanol and water, pH was adjusted to 8.5 with 1M NaOH, reacted for 3H, the reaction was concentrated and purified by column (eluent, dichloromethane: methanol=80:20) to give the final product, and 1 H NMR, MS results showed the structure to be identical to example 1.
Test example 1
Effects of pyruvate derivatives of the invention on IL & #x2011;6 levels in human lung epithelial cells BEAS-2B
In order to examine the influence of pyruvate derivatives on lung inflammation, lipopolysaccharide (LPS) is adopted to stimulate human lung epithelial cells BEAS-2B to establish an inflammatory cell model, and the specific steps are as follows:
(1) Human lung epithelial cells BEAS-2B in logarithmic growth phase were digested with pancreatin, centrifuged, resuspended, counted and inoculated into 24-well plates at 2.5X10 5/well;
(2) LPS was added to HG-DMEM medium containing 10% FBS at a concentration of 100ng/mL, and the pyruvate derivative of the present invention was dissolved in DMSO to prepare a 1mM pyruvate derivative solution. Diluting pyruvate derivatives to corresponding concentrations (LPS+2μM pyruvate derivatives, LPS+5μM pyruvate derivatives, LPS+10μM pyruvate derivatives) with culture medium without LPS as blank control group, culturing for 24 hr, washing with PBS once, adding cell extraction buffer solution into each cell, centrifuging at 12000rpm for 15min, and collecting supernatant;
(3) Taking an ELISA plate coated with a capture antibody, washing the plate for 3 times by using PBST (poly-butylene-styrene) and spin-drying, respectively adding 100 mu L of sample supernatant, incubating for 2 hours at room temperature, washing the plate for 3 times and spin-drying, adding 100 mu L of HRP (high-rate polyethylene) labeled antibody working solution into a reaction hole, incubating for 40 minutes at room temperature in a dark place, washing the plate for 3 times and spin-drying;
(4) Adding 100 μL of chromogenic substrate TMB into a reaction hole, developing for 10min at room temperature in the dark, adding 100 μL of 1M sulfuric acid to terminate the reaction, performing dual-wavelength detection by using an enzyme-labeled instrument, measuring the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, subtracting the OD value of 630nm from the OD value of 450nm as a final result, drawing a standard curve by taking the standard concentration as an abscissa (pg/mL) and taking the OD450-OD630 as an ordinate, and calculating the expression level of IL-6 in the sample, wherein the result is shown in FIG. 11.
As can be seen from fig. 11, the LPS group significantly up-regulated the expression of IL-6 in bees-2B compared to the blank group. The pyruvate derivatives of the present invention can inhibit inflammatory response stimulated by LPS, and can achieve the drug effect almost consistent with that of asterone by using lower concentration of administration compared with the positive control group. The pyruvate derivative can reduce the damage and fibrosis of lung tissues by inhibiting the expression of IL-6, maintain the normal structure and function of the lung tissues, change the level of cytokines and chemokines in tumor microenvironment, reduce factors for promoting tumor growth and increase factors for inhibiting tumor growth, thereby inhibiting the malignant transformation of lung nodules.
Test example 2
Effect of pyruvate derivatives of the invention on BEAS-2B cell EMT
Epithelial cell-mesenchymal transition (EMT) refers to a process in which epithelial cells lose their original polarity, intercellular junction and adhesion under specific conditions, and acquire characteristics of mesenchymal cells such as migration ability, invasiveness, and anti-apoptotic ability. EMT plays an important role in malignant transformation, growth and metastasis of lung nodules as a process of epithelial cell transformation into mesenchymal cells. In order to examine the influence of pyruvate derivatives on sarcoidosis, we used TGF-beta to stimulate human lung epithelial cells BEAS-2B to build an EMT model, the inhibition process of pyruvate derivatives on cell EMT of the invention comprises the following specific steps:
(1) Taking human lung epithelial cells BEAS-2B in logarithmic growth phase, using pancreatin to digest, centrifuge, resuspension, count, inoculating into 96-well plate with 3000 pieces/mL, and 100 μl per well;
(2) Starving the cells with a medium containing 0.5% FBS for 12h after cell attachment;
(3) Preparing a TGF-beta solution with concentration of 5ng/mL by using a medium of 0.5% FBS, dissolving a pyruvate derivative by using DMSO, diluting the pyruvate derivative to corresponding concentration by using a medium added with TGF-beta (TGF-beta+2mu M pyruvate derivative, TGF-beta+5mu M pyruvate derivative and TGF-beta+10mu M pyruvate derivative), and taking the medium without TGF-beta as a blank control group;
(4) Finally, the culture medium for starved cells is aspirated, and the diluted compound is placed into the well. After 24h incubation, wash once with PBS, add 200. Mu.L Trizol to lyse the RNA samples;
(5) RNA transcription was performed using PRIMESCRIPT RT MASTER Mix kit, followed by real-time fluorescent quantitative PCR detection using 2 XSYBR GREEN QPCR MASTER Mix kit. The results of inhibition of cell EMT by pyruvate derivatives are shown in figure 12.
As can be seen from fig. 12, the transcriptional expression level of the epithelial marker E-cadherein was reduced in the BEAS-2B cells exposed to TGF- β, the transcriptional expression levels of the mesenchymal cell markers Vimentin, fibronectin and N-cadherein were higher, and the pyruvate derivative treatment significantly increased the transcriptional expression level of the epithelial markers, inhibiting the transcriptional level of the mesenchymal cell markers, demonstrating that the pyruvate derivative of the present invention can inhibit the cell EMT process, and has the ability to inhibit malignant transformation of lung nodules.
Test example 3
Influence of the pyruvate derivative nano-atomizing agent on pulmonary nodules in rats
20 SD rats (340+ -10 g) were intraperitoneally injected with pentobarbital sodium solution (mass fraction: 2%) at a dose of 40mg/kg, which was fixed after anesthesia. The skin of the rat neck was sterilized with medical ethanol, cut along the median line of the neck, and the muscle was blunt-separated to expose the trachea. The non-exposure type trachea cannula is used for contamination, and 250 mu L of silicon dioxide suspension with the mass concentration of 200g/L is injected into a trachea for molding.
The method comprises the steps of taking 16 rats with successful modeling, dividing the rats into two groups, namely an atomization administration group and a model group, wherein the blank group is a healthy rat (340+/-10 g), the administration amount of the atomization administration group is 5mg of pyruvate derivatives/kg and1 time per day, the atomization administration process is as follows, adding a pyruvate derivative nanometer atomizing agent into an aerosol atomizer to treat the rats, specifically, placing a pyruvate derivative nanometer atomizing agent solution into a beaker connected with the atomizer, placing the rats into a fixer, exposing the mouth and nose, placing the fixer on the atomizer, performing atomization administration for 15min, taking out the rats, and completing administration. The blank and model groups were given an equal amount of physiological saline daily for a total of 60 days. After the end of the administration, each group of rats was sacrificed after anesthesia, the lung tissue weight of the rats was weighed, and the lung swelling coefficient and the lung nodule swelling inhibition rate were calculated. Lung swelling coefficient = lung wet weight (mg)/body weight (g), lung nodule swelling inhibition = 1- [ (nebulization dosing group lung swelling system-blank group lung swelling coefficient)/(model group lung swelling coefficient-blank group lung swelling coefficient) ]x100%, specifically as described in table 1.
As can be seen from the data in table 1, the pulmonary nodule swelling inhibition rate of the atomized administration group is 55.41%, which indicates that the pyruvate derivative nano-nebulizer of the invention has a better inhibition effect on pulmonary nodule swelling and can prevent further deterioration.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (10)
1. A pyruvate derivative for preventing malignant transformation of pulmonary nodules, wherein the pyruvate derivative has a structural formula as shown in formula I:
。
2. A method of preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 1, comprising the steps of:
(1) Adding 3-fluoro-4-aldehyde phenylboronic acid and sodium pyruvate into a sodium hydroxide solution, reacting for 0.5-2 hours, and then treating to obtain an intermediate 4A;
(2) Adding 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimido chloride and 3, 4-dibromoaniline into sodium bicarbonate solution, reacting for 1-2 hours at 60-70 ℃ and then treating to obtain an intermediate 1;
(3) Adding the intermediate 1 and N, N' -carbonyl diimidazole into a solvent, reacting for 3-5 hours in a reflux state, and then treating to obtain an intermediate 2;
(4) Adding the intermediate 2 into trifluoroacetic acid and 30% hydrogen peroxide solution, reacting for 5-7 hours at 60-70 ℃ and then treating to obtain an intermediate 3;
(5) Sequentially adding the intermediate 3, 2- (3, 4-dihydroxyphenyl) ethylamine and triethylamine into a solvent, reacting for 0.5-2 h, and then treating to obtain an intermediate 4;
(6) And in the environment with the pH value of 8-9, reacting the intermediate 4 with the intermediate 4A for 3-5 hours, and then treating to obtain the pyruvate derivative.
3. The method for preparing pyruvate derivatives for preventing malignant transformation of pulmonary nodules according to claim 2, wherein the molar ratio of 3-fluoro-4-aldehyde phenylboronic acid, sodium pyruvate and sodium hydroxide in the step (1) is 1 (2-2.5): 4-6.
4. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 2, wherein in the step (2), the molar ratio of the 4-amino-N' -hydroxy-1, 2, 5-oxadiazole-3-carboimidyl chloride, 3, 4-dibromoaniline and sodium bicarbonate is 1 (1.1-1.5): 2-4.
5. The method for producing a pyruvate derivative according to claim 2, wherein the molar ratio of the intermediate 1 to N, N' -carbonyldiimidazole in the step (3) is 1 (1.4 to 1.6).
6. The method for preparing pyruvate derivatives for preventing malignant transformation of pulmonary nodules according to claim 2, wherein in the step (4), trifluoroacetic acid and 30% hydrogen peroxide solution are added to the intermediate 2 in a dosage ratio of 2mmol (15-20 mL) to (11-12 mL).
7. The method for preparing pyruvate derivatives for preventing malignant transformation of pulmonary nodules according to claim 2, wherein the molar ratio of the intermediates 3, 2- (3, 4-dihydroxyphenyl) ethylamine and triethylamine in the step (5) is 5 (6-9): 12-20.
8. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 2, wherein the molar ratio of the intermediate 4 to the intermediate 4A in the step (6) is 1 (1.2-1.8).
9. A pyruvate derivative nano-atomizing agent for preventing malignant transformation of lung nodules is characterized by comprising the following components, by weight, 5-8 parts of pyruvate derivative nano-particles and 100 parts of water for injection, wherein the preparation method of the pyruvate derivative nano-particles comprises the steps of preparing Tween 80 or Tween 20 into a solution with the concentration of 0.02-0.05wt% by using water, refrigerating at the temperature of 2-4 ℃ to obtain an aqueous phase solution, adding 8-10 mg/mL of the pyruvate derivative methylene dichloride solution of claim 1 into the aqueous phase solution at the temperature of-15 to-18 ℃, emulsifying for 5-7 min at the speed of 1000-1200 r/min, evaporating the solvent for 3-4h, centrifuging, collecting nano-particles and drying to obtain the pyruvate derivative nano-atomizing agent.
10. The method for preparing the pyruvate derivative nano-aerosol for preventing malignant transformation of pulmonary nodules according to claim 9, wherein the pyruvate derivative nano-particles are added into water for injection according to the formula.
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