A pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules and a preparation method thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules and a preparation method thereof. The present invention provides a pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules, and the pyruvate derivative obtained after structural improvement of the commonly used anti-pulmonary disease drug sodium pyruvate is prepared as a nano atomizer. The pyruvate derivative of the present invention inhibits the expression of IL-6, reduces the damage of lung tissue, changes the levels of cytokines and chemokines in the tumor microenvironment, reduces factors that promote tumor growth, and increases factors that inhibit tumor growth, thereby inhibiting the malignant transformation of pulmonary nodules; secondly, after treatment with pyruvate derivatives, the transcriptional expression level of epithelial markers is significantly increased, and the transcriptional level of mesenchymal cell markers is inhibited, which can inhibit the epithelial cell-mesenchymal transformation process, and then achieve the purpose of inhibiting the malignant transformation of pulmonary nodules.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules and a preparation method thereof. Background Art

[0002] Pulmonary nodules refer to solid or subsolid lesions that are round in shape, less than 3 cm in diameter, and surrounded by air-containing lung tissue. These lesions appear as increased density in the lung area on chest X-ray or chest CT, and can be isolated or multiple. The formation of pulmonary nodules can be caused by a variety of reasons, including but not limited to: chronic inflammatory diseases (such as granulomatosis with polyangiitis, sarcoidosis, rheumatoid arthritis involving the lungs, etc.), infectious factors (such as bacterial infection, tuberculosis infection, fungal infection, etc.), benign tumors (such as hamartoma, fibroma, leiomyoma, etc.), and malignant tumors (i.e. lung cancer). Although most pulmonary nodules are benign, some nodules may still be malignant.

[0003] Malignant transformation of pulmonary nodules refers to the malignant transformation of pulmonary nodules during their growth and development, that is, the pulmonary nodules change from benign to malignant, forming tumor-like lesions with invasive growth and metastatic potential. This malignant transformation is usually caused by gene mutations that lead to an imbalance between cell proliferation and apoptosis, causing the malignant transformation of pulmonary epithelial cells and excessive proliferation to form nodules (epithelial-mesenchymal transformation to form nodules).

[0004] There are many types of drugs available for treating lung nodules, and the specific choice should be determined based on the cause, nature and specific conditions of the lung nodules. Some common drug categories for treating lung nodules include: anti-inflammatory drugs (for lung nodules caused by inflammatory infections, improving lung conditions by reducing inflammation and killing bacteria), antifungal drugs (for treating lung nodules caused by fungal infections, eliminating the cause by inhibiting fungal activity), and anticancer drugs (for lung nodules caused by tumors, treating by inhibiting the growth and spread of cancer cells).

[0005] In terms of lung diseases, sodium pyruvate reduces lung tissue damage and inflammatory responses through its antioxidant and anti-inflammatory effects, thereby improving lung function. However, how to effectively utilize the beneficial effects of sodium pyruvate in the treatment of lung diseases and further expand its application range by modifying it, especially in the key area of ​​preventing malignant transformation of lung nodules, is an important and urgent problem to be solved. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a pyruvate derivative for preventing the malignant transformation of pulmonary nodules, which can effectively prevent the malignant transformation of pulmonary nodules.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A pyruvate derivative for preventing malignant transformation of pulmonary nodules, wherein the structural formula of the pyruvate derivative is as shown in Formula I:

[0009] .

[0010] 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 following steps:

[0011]

[0012] (1) Add 3-fluoro-4-formaldehyde phenylboronic acid and sodium pyruvate to a sodium hydroxide solution, react for 0.5 to 2 h, and then treat to obtain intermediate 4A;

[0013] (2) Add 4-amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride and 3,4-dibromoaniline to a sodium bicarbonate solution, react at 60-70°C for 1-2h, and then treat to obtain an intermediate 1;

[0014] (3) Adding intermediate 1 and N,N'-carbonyldiimidazole to a solvent, reacting under reflux for 3-5 hours, and then treating to obtain intermediate 2;

[0015] (4) adding intermediate 2 to trifluoroacetic acid and 30% hydrogen peroxide solution, reacting at 60-70°C for 5-7h, and then treating to obtain intermediate 3;

[0016] (5) adding intermediate 3, 2-(3,4-dihydroxyphenyl)ethylamine and triethylamine to a solvent in sequence, reacting for 0.5 to 2 hours and then treating to obtain intermediate 4;

[0017] (6) At pH 8-9, intermediate 4 and intermediate 4A are reacted for 3-5 h and then treated to obtain a pyruvate derivative.

[0018] Furthermore, in step (1), the molar ratio of 3-fluoro-4-formaldehyde phenylboronic acid, sodium pyruvate and sodium hydroxide is 10:(20-25):(40-60).

[0019] Furthermore, in step (2), the molar ratio of 4-amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride, 3,4-dibromoaniline and sodium bicarbonate is 1:(1.1-1.5):(0.2-0.4).

[0020] Furthermore, in step (3), the molar ratio of the intermediate 1 to N,N'-carbonyldiimidazole is 1:(1.4-1.6).

[0021] Furthermore, in step (4), the ratio of the amount of acid and 30% hydrogen peroxide solution added to the intermediate 2 is 2 mmol: (15-20) mL: (11-12) mL.

[0022] Furthermore, in step (5), the molar ratio of the intermediate 3, 2-(3,4-dihydroxyphenyl)ethylamine and triethylamine is 5:(6-9):(12-20).

[0023] Furthermore, in step (6), the molar ratio of intermediate 4 to intermediate 4A is 1:(1.2-1.8).

[0024] The third object of the present invention is to provide a pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules, comprising the following components: 5 to 8 parts of pyruvate derivative nanoparticles and 100 parts of water for injection; the preparation method of the pyruvate derivative nanoparticles comprises: preparing a 0.02 to 0.05 wt % solution of Tween 80 or Tween 20 with water, refrigerating at 2 to 4°C to obtain an aqueous phase solution; adding 8 to 10 mg / mL of a dichloromethane solution of the pyruvate derivative to the aqueous phase solution at -15 to -18°C, emulsifying at 1000 to 1200 r / min for 5 to 7 minutes, evaporating the solvent for 3 to 4 hours, centrifuging, collecting the nanoparticles and drying to obtain the obtained product; the volume ratio of the dichloromethane to the aqueous phase is 1:10-20.

[0025] The fourth object of the present invention is to provide a method for preparing a pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules. According to the above formula, sodium pyruvate derivative nanoparticles are added to water for injection to obtain the atomizer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The pyruvate derivative for preventing malignant transformation of pulmonary nodules provided by the present invention has a specific preparation mechanism as follows: first, the aldehyde group of 3-fluoro-4-formylphenylboronic acid and the ketocarbonyl group of sodium pyruvate are subjected to nucleophilic substitution to obtain intermediate 4A; secondly, 4-amino-N'-hydroxy-1,2,5-oxadiazole-3-carbonyl imide chloride is subjected to amination to introduce 3,4-dibromoaniline to obtain intermediate 1, and then intermediate 1 is subjected to cyclization under the action of N,N'-carbonyldiimidazole to obtain intermediate 2, and then intermediate 2 is subjected to the combined action of hydrogen peroxide and trifluoroacetic acid to oxidize the amino group of intermediate 2 to nitro to obtain intermediate 3, and intermediate 3 and 2-(3,4-dihydroxyphenyl)ethylamine are subjected to substitution reaction under the action of triethylamine to obtain intermediate 4; finally, the group vicinal diol of intermediate 4 is subjected to esterification with the group boronic acid of intermediate 4A to obtain the pyruvate derivative for preventing malignant transformation of pulmonary nodules of the present invention.

[0028] The pyruvate derivatives of the present invention can reduce the damage of lung tissue by inhibiting the expression of IL-6, maintain the normal structure and function of lung tissue, change the levels of cytokines and chemokines in the tumor microenvironment, reduce factors that promote tumor growth, and increase factors that inhibit tumor growth, thereby inhibiting the malignant transformation of lung nodules; secondly, after treatment with pyruvate derivatives, the transcriptional expression level of epithelial markers is significantly increased and the transcriptional level of mesenchymal cell markers is inhibited, thereby inhibiting the epithelial cell-mesenchymal transformation (EMT) process, thereby achieving the purpose of inhibiting the malignant transformation of lung nodules. In addition, the pyruvate derivative nano atomizer of the present invention has a good inhibitory effect on the swelling of lung nodules and can prevent them from further deterioration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the synthesis route of the pyruvate derivative of the present invention;

[0030] Figure 2 It is a schematic diagram of the hydrogen spectrum result of the pyruvate derivative intermediate 4A of the present invention;

[0031] Figure 3 Schematic diagram of mass spectrometry results of pyruvate derivative intermediate 4A of the present invention;

[0032] Figure 4 It is a schematic diagram of the hydrogen spectrum result of the pyruvate derivative intermediate 1 of the present invention;

[0033] Figure 5 Schematic diagram of mass spectrometry results of pyruvate derivative intermediate 1 of the present invention;

[0034] Figure 6 It is a schematic diagram of the hydrogen spectrum result of the pyruvate derivative intermediate 2 of the present invention;

[0035] Figure 7 It is a schematic diagram of the hydrogen spectrum result of the pyruvate derivative intermediate 3 of the present invention;

[0036] Figure 8 It is a schematic diagram of the hydrogen spectrum result of the pyruvate derivative intermediate 4 of the present invention;

[0037] Fig. 9 It is a schematic diagram of the hydrogen spectrum results of the pyruvate derivatives of the present invention;

[0038] Fig.10 Schematic diagram of mass spectrometry results of pyruvate derivatives of the present invention;

[0039] Fig.11 It is a schematic diagram of the effect of the pyruvate derivatives of the present invention on interleukin-6 (IL-6) in BEAS-2B cells;

[0040] Fig.12 Schematic diagram of the inhibition of BEAS-2B cell EMT by the pyruvate derivatives of the present invention. DETAILED DESCRIPTION

[0041] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0042] Example 1

[0043] A pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules comprises the following components in parts by weight: 5 parts of pyruvate derivative nanoparticles and 100 parts of water for injection.

[0044] The preparation method of the pyruvate derivative nano atomizer is as follows: according to the above formula, pyruvate derivative nano particles are added into water for injection to obtain the atomizer.

[0045] The preparation method of the above-mentioned pyruvate derivative nanoparticles is as follows: Tween 80 is prepared into a 0.02wt% solution with water, and refrigerated at 2°C to obtain an aqueous phase solution; at -15°C, an 8mg / mL dichloromethane solution of the pyruvate derivative is added to the aqueous phase solution (the volume ratio of dichloromethane to the aqueous phase is 1:10), emulsified at 1000r / min for 5min, the solvent is evaporated for 3h, centrifuged, the nanoparticles are collected and dried, and the pyruvate derivative nanoparticles are obtained.

[0046] The preparation method of the above pyruvate derivative comprises the following steps, and the reaction flow chart is as follows: Figure 1 As shown:

[0047] A. Preparation of Intermediate 4A

[0048] Sodium hydroxide was dissolved in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200 mL of a sodium hydroxide solution with a final concentration of 0.2 M, and then 3-fluoro-4-formaldehyde phenylboronic acid (10 mmol) and sodium pyruvate (20 mmol) were added thereto, followed by stirring at room temperature for 1 h. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed with cold ethanol, and then dissolved with ethyl acetate. The pH was adjusted to 5 with 1 M HCl, and the ethyl acetate phase was separated, and then anhydrous sodium sulfate was used and concentrated to obtain intermediate 4A.

[0049] Intermediate 4A 1 H NMR Figure 2 As shown: (C11 H 10 BFO, 400MHz, DMSO- d6 ): δ 7.42-7.32 (m, 2H), 6.99-6.85 (m, 2H), 6.08 (m, 1H), 4.19 (d, 4H); the mass spectrum of intermediate 4A is shown in Figure 3 Shown: [M+H] + m / z: 253.06, ESI-MS (m / z): 253.11.

[0050] B. Preparation of Intermediate 1

[0051] 4-Amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (10 mmol) and 3,4-dibromoaniline (11 mmol) were added to ethanol (40 mL), and then 20 mL of 1 M sodium bicarbonate solution was added thereto, and the mixture was reacted at 60°C for 2 h; the reaction solution was concentrated and dissolved in ethyl acetate (100 mL), and the organic phase was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was slurried in a mixed solvent (n-hexane: ethyl acetate = 7:3, 1 g of crude product: 8 mL of mixed solvent) for 1 hour, and the filter cake was collected by filtration and dried to obtain intermediate 1;

[0052] Intermediate 1 1 H NMR Figure 4 As shown: (C9H7Br2N5O2, 400MHz, DMSO- d6 ): δ11.08 (s, 1H), 8.45 (s, 1H), 7.28 (d, 1H), 6.78 (d, 2H), 6.31 (s, 2H); The mass spectrum of intermediate 1 is as follows Figure 5 Shown: [M+H] + m / z: 375.90, ESI-MS (m / z): 375.91.

[0053] C. Preparation of Intermediate 2

[0054] Add intermediate 1 (50 mmol) to 120 mL of ethyl acetate, then add N,N'-carbonyldiimidazole (75 mmol) thereto, and react at 60°C for 5 h; add water to quench the reaction, extract the aqueous phase with ethyl acetate after separation, combine the organic phases, wash the organic phases with 2M HCl solution and saturated brine in sequence, dry the organic phase with anhydrous ammonium sulfate, and concentrate to obtain intermediate 2;

[0055] Intermediate 2 1 H NMR Figure 6 As shown: (C 10 H5Br2N5O3, 400MHz, DMSO-d6 ): δ 7.88-7.60 (m, 3H), 6.60 (s, 2H).

[0056] D. Preparation of Intermediate 3

[0057] The intermediate 2 (20 mmol) was slowly added to a mixed solution of 200 mL trifluoroacetic acid and 100 mL 30% hydrogen peroxide at 0°C. After the addition was completed, the mixture was stirred evenly and heated to 70°C for 5 h. The reaction solution was quenched with water in an ice bath, the aqueous phase was extracted with ethyl acetate, and the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to obtain the intermediate 3.

[0058] Intermediate 3 1 H NMR Figure 7 As shown: (C 10 H3Br2N5O5, 400MHz, DMSO- d6 ): δ 7.88-7.60 (m, 3H).

[0059] E. Preparation of Intermediate 4

[0060] Intermediate 3 (5 mmol) and 2-(3,4-dihydroxyphenyl)ethylamine (7.5 mmol) were added to 50 mL of tetrahydrofuran, and then triethylamine (15 mmol) was added thereto, and the mixture was reacted at room temperature for 1 h. After the reaction was completed, ethyl acetate was added to the reaction solution to dilute the reaction solution, and then a saturated sodium bicarbonate aqueous solution was added to obtain a mixed solution. The mixed solution was separated, and the organic phase was washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After passing through a column (eluent, dichloromethane: methanol = 100: 1), intermediate 4 was obtained. 1 H NMR Figure 8 As shown: (C 18 H 13 Br2N5O5, 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).

[0061] F. Preparation of Pyruvate Derivatives

[0062] Intermediate 4 (10 mmol) and intermediate 4A (10 mmol) were added to a 100 mL mixed solution of ethanol and water (1:1), and the pH was adjusted to 8.2 with 1 M NaOH. The reaction was allowed to proceed for 6 h. The reaction solution was concentrated and purified by column chromatography (eluent, dichloromethane:methanol = 80:20) to obtain a pyruvate derivative.

[0063] Pyruvate derivatives 1H NMR Fig. 9 As shown: (C 29 H 18 BBr2FN5NaO8, 400MHz, DMSO- d6 ): δ7.60-7.48 (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); The mass spectrum of the pyruvate derivative is shown in Fig.10 Shown: [M+H] + m / z: 775.95.11, ESI-MS (m / z): 775.98.

[0064] Example 2

[0065] A pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules comprises the following components in parts by weight: 6 parts of pyruvate derivative nanoparticles and 100 parts of water for injection.

[0066] The preparation method of the pyruvate derivative nano atomizer is as follows: according to the above formula, pyruvate derivative nano particles are added into water for injection to obtain the atomizer.

[0067] The preparation method of the above-mentioned pyruvate derivative nanoparticles is as follows: Tween 20 is prepared into a 0.03wt% solution with water, and refrigerated at 3°C ​​to obtain an aqueous phase solution; at -17°C, a 9mg / mL dichloromethane solution of the pyruvate derivative is added to the aqueous phase solution (the volume ratio of dichloromethane to the aqueous phase is 1:15), emulsified at 1100r / min for 6min, the solvent is evaporated for 3h, centrifuged, the nanoparticles are collected and dried, and the pyruvate derivative nanoparticles are obtained.

[0068] The preparation method of the above pyruvate derivative comprises the following steps:

[0069] A. Preparation of Intermediate 4A

[0070] Sodium hydroxide was dissolved in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200 mL of a sodium hydroxide solution with a final concentration of 0.3 M, and then 3-fluoro-4-formaldehyde phenylboronic acid (10 mmol) and sodium pyruvate (22 mmol) were added thereto, followed by stirring and reacting at room temperature for 0.5 h; after the reaction, the reaction solution was filtered to obtain a filter cake, which was washed with cold ethanol, and then dissolved with ethyl acetate, and the pH was adjusted to 6 with 1 M HCl, and the ethyl acetate phase was separated by extraction, and then anhydrous sodium sulfate was used and concentrated to obtain intermediate 4A; 1The results of HNMR and MS showed that the structure was consistent with Example 1.

[0071] B. Preparation of Intermediate 1

[0072] 4-Amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (10 mmol) and 3,4-dibromoaniline (15 mmol) were added to ethanol (40 mL), and then 20 mL of 1.5 M sodium bicarbonate solution was added thereto, and the mixture was reacted at 70°C for 1 h; the reaction solution was concentrated and dissolved in ethyl acetate (100 mL), and the organic phase was washed with saturated saline water. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was slurried in a mixed solvent (n-hexane: ethyl acetate = 7:3, 1 g of crude product: 8 mL of mixed solvent) for 1 hour, and the filter cake was collected by filtration and dried to obtain intermediate 1; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0073] C. Preparation of Intermediate 2

[0074] Add intermediate 1 (50 mmol) to 100 mL of ethyl acetate, then add N,N'-carbonyldiimidazole (80 mmol) thereto, and react at 70°C for 3 h; add water to quench the reaction, separate the layers, extract the aqueous phase with ethyl acetate, combine the organic phases, wash the organic phases with 2M HCl solution and saturated brine, dry the organic phase with anhydrous ammonium sulfate, and concentrate to obtain intermediate 2; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0075] D. Preparation of Intermediate 3

[0076] The intermediate 2 (20 mmol) was slowly added to a mixed solution of 150 mL trifluoroacetic acid and 120 mL 30% hydrogen peroxide at 0°C. After the addition was completed, the mixture was stirred evenly and heated to 65°C for 7 h. The reaction solution was quenched with water in an ice bath, the aqueous phase was extracted with ethyl acetate, and the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to obtain the intermediate 3. 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0077] E. Preparation of Intermediate 4

[0078] Add intermediate 3 (5 mmol) and 2-(3,4-dihydroxyphenyl)ethylamine (9 mmol) to 50 mL tetrahydrofuran, then add triethylamine (20 mmol) to it, and react at room temperature for 0.5 h; after the reaction, add ethyl acetate to dilute the reaction solution, then add saturated sodium bicarbonate aqueous solution to the mixed solution, separate the mixed solution, wash the organic phase with water, wash with saturated brine, dry with anhydrous sodium sulfate, and pass through a column (eluent, dichloromethane: methanol = 100: 1) to obtain intermediate 4; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0079] F. Preparation of pyruvate derivatives

[0080] Intermediate 4 (10 mmol) and intermediate 4A (12 mmol) were added to 100 mL of a 1:1 mixed solution of ethanol and water, and the pH was adjusted to 9 with 1 M NaOH. The reaction was allowed to react for 5 h. The reaction solution was concentrated and purified by column (eluent, dichloromethane: methanol = 80:20) to obtain the final product. 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0081] Example 3

[0082] A pyruvate derivative nano atomizer for preventing malignant transformation of pulmonary nodules comprises the following components in parts by weight: 8 parts of pyruvate derivative nanoparticles and 100 parts of water for injection.

[0083] The preparation method of the pyruvate derivative nano atomizer is as follows: according to the above formula, pyruvate derivative nano particles are added into water for injection to obtain the atomizer.

[0084] The preparation method of the above-mentioned pyruvate derivative nanoparticles is as follows: Tween 80 is prepared into a 0.05wt% solution with water, and refrigerated at 4°C to obtain an aqueous phase solution; at -18°C, a 10mg / mL dichloromethane solution of the pyruvate derivative is added to the aqueous phase solution (the volume ratio of dichloromethane to the aqueous phase is 1:20), emulsified at 1200r / min for 7min, the solvent is evaporated for 4h, centrifuged, the nanoparticles are collected and dried, and the pyruvate derivative nanoparticles are obtained.

[0085] The preparation method of the above pyruvate derivative comprises the following steps:

[0086] (1) Preparation of pyruvate derivatives

[0087] A. Preparation of Intermediate 4A

[0088] Sodium hydroxide was dissolved in a mixed solution of water and ethanol in a volume ratio of 1:1 to obtain 200 mL of a sodium hydroxide solution with a final concentration of 0.25 M, and then 3-fluoro-4-formaldehyde phenylboronic acid (10 mmol) and sodium pyruvate (25 mmol) were added thereto, followed by stirring and reacting at room temperature for 2 h; after the reaction, the reaction solution was filtered to obtain a filter cake, the filter cake was washed with cold ethanol, and then the filter cake was dissolved with ethyl acetate, the pH was adjusted to 6 with 1 M HCl, the ethyl acetate phase was separated by extraction, and then anhydrous sodium sulfate was used and concentrated to obtain intermediate 4A; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0089] B. Preparation of Intermediate 1

[0090] 4-Amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (10 mmol) and 3,4-dibromoaniline (13 mmol) were added to ethanol (40 mL), and then 20 mL of 2M sodium bicarbonate solution was added thereto, and the mixture was reacted at 60°C for 1.5 h; the reaction solution was concentrated and dissolved in ethyl acetate (100 mL), and the organic phase was washed with saturated saline water. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was slurried in a mixed solvent (n-hexane: ethyl acetate = 7:3, 1 g of crude product: 8 mL of mixed solvent) for 1 hour, and the filter cake was collected by filtration and dried to obtain intermediate 1; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0091] C. Preparation of Intermediate 2

[0092] Add intermediate 1 (50 mmol) to 100 mL of ethyl acetate, then add N,N'-carbonyldiimidazole (70 mmol) thereto, and react at 65°C for 4 h; add water to quench the reaction, separate the layers, extract the aqueous phase with ethyl acetate, combine the organic phases, wash the organic phases with 2M HCl solution and saturated brine, dry the organic phase with anhydrous ammonium sulfate, and concentrate to obtain intermediate 2; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0093] D. Preparation of Intermediate 3

[0094] The intermediate 2 (20 mmol) was slowly added to a mixed solution of 180 mL trifluoroacetic acid and 110 mL 30% hydrogen peroxide at 0°C. After the addition was completed, the mixture was stirred evenly and heated to 60°C for 6 h. The reaction solution was quenched with water in an ice bath, the aqueous phase was extracted with ethyl acetate, and the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to obtain the intermediate 3. 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0095] E. Preparation of Intermediate 4

[0096] Add intermediate 3 (5 mmol) and 2-(3,4-dihydroxyphenyl)ethylamine (6 mmol) to 50 mL tetrahydrofuran, then add triethylamine (12 mmol) to it, and react at room temperature for 2 h; after the reaction, add ethyl acetate to dilute the reaction solution, then add saturated sodium bicarbonate aqueous solution to the mixed solution, separate the mixed solution, wash the organic phase with water, wash with saturated brine, dry with anhydrous sodium sulfate, and pass through a column (eluent, dichloromethane: methanol = 100: 1) to obtain intermediate 4; 1 The results of HNMR and MS showed that the structure was consistent with Example 1.

[0097] F. Preparation of pyruvate derivatives

[0098] Intermediate 4 (10 mmol) and intermediate 4A (18 mmol) were added to 100 mL of a 1:1 mixed solution of ethanol and water, and the pH was adjusted to 8.5 with 1 M NaOH. The reaction was allowed to react for 3 h. The reaction solution was concentrated and purified by column (eluent, dichloromethane: methanol = 80:20) to obtain the final product. 1 The results of H NMR and MS showed that the structure was consistent with that of Example 1.

[0099] Test Example 1

[0100] Effects of pyruvate derivatives of the present invention on IL-6 levels in human lung epithelial cells BEAS-2B

[0101] In order to investigate the effect of pyruvate derivatives on lung inflammation, this test example used lipopolysaccharide (LPS) to stimulate human lung epithelial cells BEAS-2B to establish an inflammatory cell model. The specific steps are as follows:

[0102] (1) Human lung epithelial cells BEAS-2B in the logarithmic growth phase were digested with trypsin, centrifuged, resuspended, and counted. 5 pcs / well were inoculated into 24-well plates;

[0103] (2) Add LPS to a 10% FBS HG-DMEM medium to make the concentration 100 ng / mL, and dissolve the pyruvate derivative of the present invention in DMSO to prepare a 1 mM pyruvate derivative solution. Use the medium added with LPS to dilute the pyruvate derivative to the corresponding concentration (LPS+2μM pyruvate derivative, LPS+5μM pyruvate derivative, LPS+10μM pyruvate derivative); use the medium without LPS as the blank control group, and use "LPS+10μM asterone" as the positive control group; after culturing for 24 hours, wash once with PBS, add cell extraction buffer to each well of cells, centrifuge at 12000rpm for 15 minutes, and take the supernatant for use;

[0104] (3) Take the capture antibody-coated ELISA plate, wash the plate three times with PBST and spin dry; add 100 μL of sample supernatant, incubate at room temperature for 2 h, wash the plate three times and spin dry; add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature in the dark for 40 min, wash the plate three times and spin dry;

[0105] (4) Add 100 μL of chromogenic substrate TMB to the reaction well, develop color at room temperature in the dark for 10 min, and add 100 μL of 1 M sulfuric acid to terminate the reaction; use an ELISA reader to perform dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and use the OD value at 450 nm minus the OD value at 630 nm as the final result; draw a standard curve with the standard concentration as the horizontal axis (pg / mL) and OD450-OD630 as the vertical axis, and calculate the expression level of IL-6 in the sample. The results are as follows: Fig.11 shown.

[0106] Depend on Fig.11 It can be seen that compared with the blank control group, the LPS group significantly upregulated the expression of IL-6 in BEAS-2B. The pyruvate derivatives of the present invention can inhibit the inflammatory response stimulated by LPS; compared with the positive control group, the present invention can achieve almost the same efficacy as asterone by administering at a lower concentration. The pyruvate derivatives of the present invention can reduce the damage and fibrosis of lung tissue by inhibiting the expression of IL-6, maintain the normal structure and function of lung tissue, change the levels of cytokines and chemokines in the tumor microenvironment, reduce factors that promote tumor growth, and increase factors that inhibit tumor growth, thereby inhibiting the malignant transformation of lung nodules.

[0107] Test Example 2

[0108] Effects of the pyruvate derivatives of the present invention on EMT of BEAS-2B cells

[0109] Epithelial-mesenchymal transition (EMT) refers to the process in which epithelial cells lose their original polarity, intercellular connections and adhesion under specific conditions and acquire the characteristics of mesenchymal cells, such as migration ability, invasiveness and anti-apoptosis ability. EMT, as the process of epithelial cells transforming into mesenchymal cells, plays an important role in the malignant transformation, growth and metastasis of pulmonary nodules. In order to investigate the effect of pyruvate derivatives on sarcoidosis, we used TGF-β to stimulate human lung epithelial cells BEAS-2B to establish an EMT model. The specific steps of the inhibition of cell EMT by pyruvate derivatives of the present invention are as follows:

[0110] (1) Human lung epithelial cells BEAS-2B in the logarithmic growth phase were digested with trypsin, centrifuged, resuspended, counted, and inoculated into a 96-well plate at 3000 cells / mL, with 100 µL per well;

[0111] (2) After the cells adhered, starve the cells with medium containing 0.5% FBS for 12 h;

[0112] (3) A TGF-β solution with a concentration of 5 ng / mL was prepared in a culture medium containing 0.5% FBS, and the pyruvate derivative of the present invention was dissolved in DMSO; the pyruvate derivative was diluted to the corresponding concentration (TGF-β+2 μM pyruvate derivative, TGF-β+5 μM pyruvate derivative, TGF-β+10 μM pyruvate derivative) using a culture medium added with TGF-β; a culture medium without TGF-β was used as a blank control group;

[0113] (4) Finally, remove the medium used for starving cells and add the diluted compound into the wells. After 24 hours of incubation, wash once with PBS and add 200 μL Trizol to lyse and collect RNA samples;

[0114] (5) RNA transcription was performed using the PrimeScript RT Master Mix Kit, and then real-time fluorescence quantitative PCR was performed using the 2×SYBRGreen qPCR Master Mix Kit. Fig.12 shown.

[0115] Depend on Fig.12It can be seen that the transcriptional expression level of the epithelial marker E-cadherin in BEAS-2B cells exposed to TGF-β is reduced, and the transcriptional expression levels of the mesenchymal cell markers Vimentin, Fibronectin and N-cadherin are relatively high. After treatment with pyruvate derivatives, the transcriptional expression levels of epithelial markers are significantly increased and the transcriptional expression levels of mesenchymal cell markers are inhibited, indicating that the pyruvate derivatives of the present invention can inhibit the cellular EMT process and have the ability to inhibit the malignant transformation of lung nodules.

[0116] Test Example 3

[0117] Effects of the pyruvate derivative nano-atomizer of the present invention on pulmonary nodules in rats

[0118] Twenty SD rats (340±10g) were taken: sodium pentobarbital solution (mass fraction 2%) was injected intraperitoneally at a dose of 40mg / kg, and they were fixed after anesthesia. The skin of the rat neck was disinfected with medical ethanol, and the neck was cut along the midline of the neck, and the muscles were bluntly separated to expose the trachea. The rats were intubated using non-exposed endotracheal intubation, and 250μL of silica suspension with a mass concentration of 200g / L was injected into the trachea at one time for modeling.

[0119] Sixteen rats with successful modeling were taken and divided into two groups, namely, the aerosol administration group and the model group. The blank group was healthy rats (340±10g). The aerosol administration group was administered with a dosage of 5mg pyruvate derivative / kg, once a day. The aerosol administration process was as follows: the pyruvate derivative nano-aerosol was added to the aerosol nebulizer to treat the rats; specifically, the pyruvate derivative nano-aerosol solution was placed in a beaker connected to the nebulizer, the rat was placed in a fixator, the mouth and nose were exposed, the fixator was placed on the nebulizer, the aerosol administration was performed for 15 minutes, the fixator was removed, the rat was taken out, and the administration was completed. The blank group and the model group were given an equal amount of normal saline every day for a total of 60 days. After the end of the administration, the rats in each group were anesthetized and killed, 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 rate = 1-[(lung swelling system of nebulized drug administration group - lung swelling coefficient of blank group) / (lung swelling coefficient of model group - lung swelling coefficient of blank group)] × 100%, as described in Table 1.

[0120]

[0121] Combined with the data in Table 1, it can be seen that the inhibition rate of lung nodule swelling in the aerosol administration group is 55.41%, which indicates that the pyruvate derivative nano aerosol agent of the present invention has a good inhibitory effect on the swelling of lung nodules and can prevent its further deterioration.

[0122] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A pyruvate derivative for preventing malignant transformation of pulmonary nodules, characterized in that: The structural formula of the pyruvate derivative is shown in Formula I: 。 2. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 1, characterized in that: The following steps are involved: (1) Add 3-fluoro-4-formaldehyde phenylboronic acid and sodium pyruvate to a sodium hydroxide solution, react for 0.5 to 2 h, and then treat to obtain intermediate 4A; (2) Add 4-amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride and 3,4-dibromoaniline to a sodium bicarbonate solution, react at 60-70°C for 1-2h, and then treat to obtain an intermediate 1; (3) Adding intermediate 1 and N,N'-carbonyldiimidazole to a solvent, reacting under reflux for 3-5 hours, and then treating to obtain intermediate 2; (4) adding intermediate 2 to trifluoroacetic acid and 30% hydrogen peroxide solution, reacting at 60-70°C for 5-7h, and then treating to obtain intermediate 3; (5) Adding intermediate 3, 2-(3,4-dihydroxyphenyl)ethylamine and triethylamine to a solvent in sequence, reacting for 0.5 to 2 hours, and then treating to obtain intermediate 4; (6) At pH 8-9, intermediate 4 and intermediate 4A are reacted for 3-5 h and then treated to obtain a pyruvate derivative.

3. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 2, characterized in that: The molar ratio of 3-fluoro-4-formaldehyde phenylboronic acid, sodium pyruvate and sodium hydroxide in 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, characterized in that: The molar ratio of 4-amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride, 3,4-dibromoaniline and sodium bicarbonate in step (2) is 1:(1.1-1.5):(2-4).

5. The method for preparing a pyruvate derivative according to claim 2, characterized in that: The molar ratio of the intermediate 1 to N,N'-carbonyldiimidazole in step (3) is 1:(1.4-1.6).

6. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 2, characterized in that: In step (4), the intermediate 2 is added with trifluoroacetic acid and 30% hydrogen peroxide solution in a ratio of 2 mmol: (15-20) mL: (11-12) mL.

7. The method for preparing a pyruvate derivative for preventing malignant transformation of pulmonary nodules according to claim 2, characterized in that: In step (5), the molar ratio of the intermediate 3, 2-(3,4-dihydroxyphenyl)ethylamine and triethylamine 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, characterized in that: The molar ratio of intermediate 4 to intermediate 4A in step (6) is 1:(1.2-1.8).

9. A pyruvate derivative nano-atomizer for preventing malignant transformation of pulmonary nodules, characterized in that: The invention comprises the following components in parts by weight: 5 to 8 parts of pyruvate derivative nanoparticles and 100 parts of water for injection; the preparation method of the pyruvate derivative nanoparticles comprises: preparing a 0.02 to 0.05 wt % solution of Tween 80 or Tween 20 with water, refrigerating at 2 to 4°C to obtain an aqueous phase solution; adding a 8 to 10 mg / mL dichloromethane solution of the pyruvate derivative according to claim 1 to the aqueous phase solution at -15 to -18°C, emulsifying at 1000 to 1200 r / min for 5 to 7 minutes, evaporating the solvent for 3 to 4 hours, centrifuging, collecting the nanoparticles and drying to obtain the nanoparticles; the volume ratio of the dichloromethane to the aqueous phase is 1:10-20.

10. The method for preparing the pyruvate derivative nano-atomizer for preventing malignant transformation of pulmonary nodules according to claim 9, characterized in that: According to the above formula, the pyruvate derivative nanoparticles are added into water for injection to obtain the nanoparticles.

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