Deoxyvasicinone compounds, preparation method thereof and anti-inflammatory application thereof

Abstract

The present application relates to deoxyvaccinum ketone compound and its preparation method and anti-inflammatory application, belong to the medical technical field.The deoxyvaccinum ketone compound provided in the present application can reduce the expression of inflammatory factors in RAW 264.7 cell inflammation model, and the deoxyvaccinum ketone compound ZZMV-17 can reduce neutrophil infiltration, inflammatory factor cascade release and endothelial injury by antagonizing CXCR2 receptor, so as to achieve the anti-inflammatory effect;In summary, the deoxyvaccinum ketone compound can antagonize CXCR2 receptor, regulate downstream signal pathways such as NF-κB, reduce neutrophil infiltration, reduce the release of inflammatory factors, and has great clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to deoxydaunophyton ketone compounds, their preparation methods, and their anti-inflammatory applications. Background Technology

[0002] Inflammation is a defensive response of the body to infection or tissue damage. Macrophages mediate the immune response by releasing inflammatory factors such as nitric oxide and tumor necrosis factor-α. However, excessive release of inflammatory factors can lead to tissue damage and is a core pathological link in many acute and chronic inflammatory diseases. Therefore, developing highly effective and low-toxicity inhibitors of inflammatory factors is of significant clinical importance.

[0003] Natural products have always been an important source of innovative drug development. Draconis ketones are a class of natural alkaloids derived from medicinal plants. Existing literature has reported that their core structure possesses certain anti-inflammatory activity, capable of inhibiting the release of inflammatory mediators to some extent. This makes them a highly anticipated lead compound skeleton in anti-inflammatory drug development.

[0004] However, the direct application of natural products as drugs often faces numerous challenges: while exerting anti-inflammatory effects, natural products frequently exhibit cytotoxicity, resulting in a narrow therapeutic window and limiting their clinical translational potential. The anti-inflammatory mechanisms of most naturally derived ketone compounds are not well understood, and their specific molecular targets remain unclear, hindering structure-based rational drug optimization. Furthermore, the impact of different substituents on the parent nucleus of natural products on their activity, toxicity, and pharmacokinetic properties lacks systematic research, making it difficult to obtain candidate molecules with superior performance.

[0005] Therefore, it is particularly important to find ways to obtain novel derivatives with stronger activity, lower toxicity, and clear mechanisms of action through structural modification while retaining the anti-inflammatory activity of the alkaloid nucleus, so as to overcome the limitations of natural products themselves. Summary of the Invention

[0006] This invention relates to deoxydaunoside ketone compounds, their preparation methods, and their anti-inflammatory applications, aiming to address the aforementioned technical problems. This invention discovers that deoxydaunoside ketone compounds competitively antagonize the CXCR2 receptor, downregulate the NF-κB signaling pathway, reduce neutrophil infiltration, and decrease the release of inflammatory factors, thus possessing significant clinical application value.

[0007] To achieve the above objectives, the present invention provides the following solution: The first aspect of this invention proposes the following deoxydaunocarpine ketone compounds and their pharmaceutically acceptable salts, selected from: .

[0008] The second aspect of this invention provides a method for preparing the aforementioned deoxydaunophyton ketone compounds and their pharmaceutically acceptable salts, comprising the following steps: (1) Dissolve 2-amino-6-methoxybenzoic acid and 2-pyrrolidone in POCl3 and heat under reflux. After the reaction is completed and cooled, slowly add the reaction solution to ice water with a pipette and adjust the pH to weakly alkaline to obtain compound 3. (2) Compound 3 was added to concentrated sulfuric acid, heated and stirred, and then concentrated nitric acid was added and the mixture was heated and stirred again. After the reaction was completed and cooled, the reaction solution was pipetted into ice water. The pH was roughly adjusted to 8-9 and then finely adjusted to weakly alkaline with saturated sodium carbonate solution to obtain compound 4. (3) Add 4,4'-bipyridine to a DMF solution containing B2(OH)4 and stir at room temperature for 5-8 min. Then add intermediate compound 4 and stir the reaction to obtain compound 5. (4) Dissolve intermediate compound 5, diethyl squaric acid, and DIPEA in MeOH and heat and stir overnight to obtain compound 6; (5) Compound 6 and DIPEA were dissolved in anhydrous ethanol and heated and stirred to obtain target compounds ZZMV-2, ZZMV-4, ZZMV-7, ZZMV-11, ZZMV-12, ZZMV-17 and ZZMV-19.

[0009] Furthermore, in step (5), the benzylamine compound is one of 3,4,5-trifluorobenzylamine, 4-fluorobenzylamine, 4-bromobenzylamine, 4-fluorophenylethylamine, 2,6-difluorobenzylamine, 4-N-butylbenzylamine, and 4-(trifluoromethoxy)benzylamine.

[0010] The third aspect of the present invention provides for the use of deoxydrysticatine ketone compounds and their pharmaceutically acceptable salts in the preparation of any one or more of the following (1) to (5); (1) Preparation of drugs that inhibit cellular inflammatory factors; (2) Preparation of anti-inflammatory drugs; (3) To prepare drugs for treating diseases characterized by cytokine storms; (4) Prepare drugs that regulate inflammatory signaling pathways; (5) Prepare drugs that antagonize CXCR2.

[0011] Furthermore, the deoxydaunophyton ketone compounds regulate the NF-κB signaling pathway by antagonizing the CXCR2 receptor.

[0012] Furthermore, the inflammatory factors include TNF-α and NO.

[0013] Furthermore, the deoxydaunophyton ketone compounds and their pharmaceutically acceptable salts are used as the active ingredients of the drug.

[0014] Furthermore, the drug may also include pharmaceutically acceptable carriers or adjuvants.

[0015] Beneficial effects 1. This invention provides a novel class of deoxy-Daucus ketone compounds and their preparation method. In vitro anti-inflammatory activity screening confirmed that these compounds, especially the preferred compound ZZMV-17, significantly reduced the release levels of lipopolysaccharide-induced inflammatory factors—nitric oxide and tumor necrosis factor-α—in a RAW 264.7 macrophage inflammation model. Compared with known Daucus ketone core compounds in the prior art, the compounds of this invention exhibit superior inhibitory effects on inflammatory factors, demonstrating stronger anti-inflammatory potential.

[0016] 2. This invention further elucidates the anti-inflammatory mechanism of compound ZZMV. Studies have found that it achieves its anti-inflammatory effect by specifically antagonizing CXC chemokine receptor 2, thereby regulating the activation of the downstream NF-κB signaling pathway. The clear elucidation of the molecular target and signaling pathway provides a solid theoretical foundation for the development of this compound as a targeted anti-inflammatory drug, overcoming the shortcomings of the ambiguous mechanisms of traditional natural products.

[0017] 3. The compounds of this invention exhibit extremely low cytotoxicity to normal cells at effective anti-inflammatory concentrations, demonstrating a wider safety and therapeutic window. This indicates that while inheriting the core activity of the natural product, the compounds of this invention effectively mitigate its potential toxicity risks and possess better prospects for clinical translation.

[0018] In summary, the deoxydaunophyton ketone compounds provided by this invention have three major advantages: high activity, low toxicity, and clear mechanism. They can be used to prepare anti-inflammatory drugs, drugs that inhibit the expression of macrophage inflammatory factors, and CXCR2 receptor antagonists, and have important clinical application value and development prospects. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation methods of compounds ZZMV-2, ZZMV-4, ZZMV-7, ZZMV-11, ZZMV-12, ZZMV-17, and ZZMV-19 of the present invention. Figure 2 The toxicity of the compound of this invention to RAW 264.7 cells; compared with the control group, n = 5. Ns indicates no significant difference, ** indicates no significant difference. p <0.001, **** indicates p <0.0001;

[0020] Figure 3The ELISA assay was used to measure the TNF-α release of RAW 264.7 cells. A represents the inhibition rate of the ZZMV compound on LPS-induced NO release from RAW 264.7 cells; B represents the inhibition rate of the ZZMV compound on LPS-induced TNF-α release from RAW 264.7 cells. Compared with the control group, n = 5. **** indicates... p <0.0001. Detailed Implementation

[0021] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0022] The structure of the compound was determined by nuclear magnetic resonance (NMR). The NMR measurements were performed using a Bruker AVANCE-300 / 500 NMR spectrometer, and the solvent was DMSO. d 6 and CDCl3, with TMS as the internal standard.

[0023] Preparation method as follows Figure 1 As shown, the steps are as follows: (1) Dissolve 2-amino-6-methoxybenzoic acid and 2-pyrrolidone in POCl3 and heat under reflux. After the reaction is completed and cooled, slowly add the reaction solution to ice water with a pipette and adjust the pH to weakly alkaline to obtain compound 3. (2) Compound 3 was added to concentrated sulfuric acid, heated and stirred, and then concentrated nitric acid was added and the mixture was heated and stirred again. After the reaction was completed and cooled, the reaction solution was pipetted into ice water. The pH was roughly adjusted to 8-9 and then finely adjusted to weakly alkaline with saturated sodium carbonate solution to obtain compound 4. (3) Add 4,4'-bipyridine to a DMF solution containing B2(OH)4 and stir at room temperature for 5-8 min. Then add intermediate compound 4 and stir the reaction to obtain compound 5. (4) Dissolve intermediate compound 5, diethyl squaric acid, and DIPEA in MeOH and heat and stir overnight to obtain compound 6; (5) Compound 6, DIPEA, was dissolved in anhydrous ethanol with 3,4,5-trifluorobenzylamine, 4-fluorobenzylamine, 4-bromobenzylamine, 4-fluorophenylethylamine, 2,6-difluorobenzylamine, 4-N-butylbenzylamine, and 4-(trifluoromethoxy)benzylamine, respectively, and heated and stirred to obtain target compounds ZZMV-2, ZZMV-4, ZZMV-7, ZZMV-11, ZZMV-12, ZZMV-17, and ZZMV-19.

[0024] Example 1 8-Methoxy-2,3-dihydropyrrolo[2,1- b Quinazoline-9(1) H )-ketone (3) 2-Amino-6-methoxybenzoic acid and 2-pyrrolidone were dissolved in POCl3 and heated under reflux at 110 °C for 3 h. After the reaction was completed and cooled, the reaction solution was slowly added dropwise to ice water with stirring to quench the reaction. Under ice bath conditions, the pH was roughly adjusted to 8-9 with 4M sodium hydroxide solution, and then finely adjusted to a slightly alkaline state with saturated sodium carbonate. The solution was filtered and dried to give compound 3 as a yellowish-brown solid, with a yield of 45%.

[0025] Example 2 8-Methoxy-7-nitro-2,3-dihydropyrrolo[2,1- b Quinazoline-9(1) H )-Ketone (4) Compound 3 was added to concentrated sulfuric acid and heated and stirred at 80°C for 1 hour. Concentrated nitric acid was then added, and heating and stirring continued for another 2 hours. After the reaction was complete and cooled, the reaction mixture was pipetted into ice water while stirring. Under ice bath conditions, the pH was roughly adjusted to 8-9 with 4M sodium hydroxide solution, and then finely adjusted to weakly alkaline with saturated sodium carbonate solution. The mixture was filtered and dried to give compound 4 as a yellow solid, with a yield of 78%.

[0026] Example 3 7-Amino-8-hydroxy-2,3-dihydropyrrolo[2,1- b Quinazoline-9(1) H )-Ketone (5) 4,4'-bipyridine was added to a DMF solution containing B2(OH)4 and stirred at room temperature. When the solution turned purple, compound 4 was added to give compound 5 as a green solid with a yield of 40%.

[0027] Example 4 3-Ethoxy-[4-(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (6) Compound 5, diethyl squaric acid, DIPEA as an acid-binding agent, and anhydrous MeOH as a solvent were stirred overnight at 40°C to obtain compound 6 as a green solid, with a yield of 25%.

[0028] Example 5 3-[(4-fluorobenzyl)amino]-4-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (ZZMV-2) Compound 6 (1 eq), 4-fluorobenzylamine (1 eq), DIPEA (6 eq), and anhydrous ethanol were used as solvents, and the mixture was stirred at 80 °C for 4 h. After the reaction was complete, the mixture was filtered, and the filter cake was washed with hot ethanol to give compound ZZMV-2, a green solid, in 81% yield. ¹H NMR (400 MHz, DMSO-) d6) d 11.68 (s, 1H), 8.92 (s, 1H), 8.15 (t, J = 6.2 Hz, 1H), 7.71(d, J = 8.9 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 7.9 Hz, 2H), 6.53(d, J = 8.9 Hz, 1H), 4.39 (d, J = 6.2 Hz, 2H), 3.49 (t, J = 7.3 Hz, 2H), 2.50(t, J = 7.9 Hz, 2H), 1.64 (p, J = 7.6 Hz, 2H).13C NMR (100 MHz, DMSO- d 6) d 184.29, 180.70, 169.24, 164.46, 163.57, 158.91, 147.72, 144.94, 143.54,128.51, 128.22, 126.97, 125.89, 125.85, 125.81, 123.09, 122.99, 116.40,106.46, 46.78, 46.06, 31.55, 21.81. HR-ESI-MS m / z 471.1282 [M+H]+ , (calcdfor C23H18F3N4O4, 471.1280).

[0029] Example 6 3-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [[Quinazolin-7-yl)amino]-[4-(4-trifluoromethylbenzyl)amino]cyclobut-3-ene-1,2-dione (ZZMV-4) Using compounds 6 and 3,4,5-trifluorobenzylamine as starting materials, compound ZZMV-4 was synthesized using the same method as ZZMV-2, yielding a yellow solid in 85% yield. ¹H NMR (400 MHz, DMSO-) d 6) d 11.38 (s, 1H), 8.59 (s, 1H), 7.78(t, J = 6.2 Hz, 1H), 7.41 (d, J= 8.9 Hz, 1H), 6.64 – 6.56 (m, 2H), 6.44 –6.35 (m, 2H), 6.25 (d, J = 8.9 Hz, 1H), 3.97 (d, J = 6.0 Hz, 2H), 3.20 (t, J = 7.3 Hz, 2H), 2.21 (t, J = 7.9 Hz, 2H), 1.34 (p, J = 7.6 Hz, 2H). 13C NMR (100 MHz, DMSO- d 6) d 183.7, 179.9, 168.5, 163.9, 162.8, 162.4, 160.0, 158.4,158.3, 147.1, 144.3, 134.3, 129.5, 129.4, 126.4, 122.4, 115.8, 115.2, 115.0,105.9, 45.5, 31.0. HR-ESI-MS m / z 421.1314 [M+H]+ , (calcd for C22H18FN4O4,421.1312).

[0030] Example 7 3-[(4-bromobenzyl)amino]-4-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (ZZMV-7) Using compounds 6 and 4-bromobenzylamine as starting materials, compound ZZMV-7 was synthesized using the same method as ZZMV-2, yielding a white solid in 86% yield. ¹H NMR (400 MHz, DMSO-) d 6) d 11.56 (s, 1H), 8.79 (s, 1H), 7.96 (t, J =6.3 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 6.97 – 6.90 (m, 2H), 6.69 (d, J = 8.2Hz, 2H), 6.43 (d, J = 8.9 Hz, 1H), 4.13 (d, J = 6.2 Hz, 2H), 3.38 (t, J= 7.3Hz, 2H), 2.39 (t, J = 7.9 Hz, 2H), 1.52 (p, J = 7.7 Hz, 2H). 13C NMR (100MHz, DMSO- d 6) d 184.5, 180.8, 169.4, 164.7, 163.7, 159.2, 148.0, 145.2,138.4, 132.1, 130.3, 130.4, 127.3, 123.2, 121.1, 116.6, 106.7, 49.0, 46.8,46.3, 31.8, 19.5. HR-ESI-MS m / z 481.0504 [M+H]+ , (calcd for C22H18BrN4O4,481.0511).

[0031] Example 8 3-[(4-Butylbenzyl)amino]-4-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (ZZMV-11) Using compounds 6 and 4-N-butylbenzylamine as starting materials, compound ZZMV-11 was synthesized using the same method as ZZMV-2, yielding a yellow solid in 89% yield. ¹H NMR (400 MHz, DMSO-) d 6) d 12.24 (s, 1H), 9.43 (s, 1H), 8.60(t, J = 6.1 Hz, 1H), 8.27 (d, J = 8.9 Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 7.22(d, J = 7.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.05(t, J = 7.1 Hz, 2H), 3.06 (t, J = 7.7 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.19(p, J = 7.6 Hz, 2H), 1.54 (p,J = 7.5 Hz, 2H), 1.29 (q, J = 7.6 Hz, 2H), 0.89(t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO- d 6) d 183.8, 179.8, 168.5, 163.9,162.6, 158.3, 147.0, 144.3, 141.4, 135.2, 128.2, 128.2, 127.3, 127.3, 126.4,122.4, 115.8, 105.8, 46.6, 45.4, 34.1, 32.8, 30.9, 21.3, 18.6, 13.3. HR-ESI-MS m / z 459.2035 [M+H]+ , (calcd for C26H27N4O4, 459.2032).

[0032] Example 9 3-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [[Quinazolin-7-yl)amino]-4-[(4-(trifluoromethoxy)benzyl)amino]cyclobut-3-ene-1,2-dione (ZZMV-12) Using compounds 6 and 4-(trifluoromethoxy)benzylamine as starting materials, compound ZZMV-12 was synthesized using the same method as ZZMV-2, yielding a yellow solid with a yield of 74%. ¹H NMR (400 MHz, DMSO-) d 6) d 12.24 (s, 1H), 9.46 (s, 1H), 8.63 (t, J = 6.2 Hz, 1H), 8.26 (d, J = 8.9 Hz, 1H), 7.58 – 7.49 (m, 2H), 7.45 – 7.38 (m, 2H), 7.10 (d, J = 8.8 Hz, 1H), 4.87 (d, J = 6.1 Hz, 2H), 4.09– 4.01 (t, 2H), 3.06 (t, J = 7.9 Hz, 2H), 2.25 – 2.13 (m, 2H). 13C NMR (100MHz, DMSO- d 6) d184.6, 180.9, 169.4, 164.8, 163.7, 159.2, 148.2, 148.0, 145.2, 138.5, 130.1, 127.3, 123.3, 121.9, 121.8, 119.3, 116.7, 106.8, 46.8, 46.3, 31.8, 19.5 . HR-ESI-MS m / z 487.1229 [M+H]+ , (calcd for C23H18F3N4O5,487.1229).

[0033] Example 10 3-[(4-fluorophenylethyl)amino]-4-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (ZZMV-17) Using compounds 6 and 4-fluorophenylethylamine as starting materials, compound ZZMV-17 was synthesized using the same method as ZZMV-2, yielding a yellow solid with a yield of 93%. ¹H NMR (400 MHz, DMSO-) d 6) d 12.23 (s, 1H), 9.39 (s, 1H), 8.29 (t, J = 6.1 Hz, 1H), 8.23 ​​(d, J = 8.8 Hz, 1H), 7.36 – 7.27 (m, 2H), 7.20 – 7.10(m, 2H), 7.07 (d, J = 8.8 Hz, 1H), 4.04 (t, J = 7.3 Hz, 2H), 3.85 (q, J = 6.7Hz, 2H), 3.04 (t, J = 7.9 Hz, 2H), 2.89 (t, J = 7.0 Hz, 2H), 2.24 – 2.12 (m,2H).13C NMR (101 MHz, DMSO- d 6) d184.1, 180.2, 169.3, 164.3, 162.9, 162.2,159.8, 158.7, 147.5, 144.6, 134.6, 130.7, 126.8,122.9, 116.2, 115.3, 115.1,106.3, 45.9, 44.9, 36.0, 31.4, 19.1. HR-ESI-MS m / z 435.1473 [M+H]+ , (calcdfor C23H20FN4O4, 435.1469).

[0034] Example 11 3-[(2,6-difluorobenzyl)amino]-4-[(8-hydroxy-9-oxo-1,2,3,9-tetrahydropyrrolo[2,1- b [Quinazolin-7-yl)amino]cyclobut-3-ene-1,2-dione (ZZMV-19) Using compounds 6 and 2,6-difluorobenzylamine as starting materials, compound ZZMV-19 was synthesized using the same method as ZZMV-2, yielding a yellow solid with a yield of 79%. ¹H NMR (400 MHz, DMSO-) d 6) d 11.41 (s, 1H), 8.56 (s, 1H), 7.71(t, J = 5.9 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 6.67 (tt, J = 8.3, 6.6 Hz, 1H), 6.37 (t, J = 7.9 Hz, 2H), 6.27 (d, J = 8.9 Hz, 1H), 4.14 (d, J = 5.8 Hz, 2H), 3.22 (t, J = 7.3 Hz, 2H), 2.23 (t, J = 7.8 Hz, 2H), 1.36 (p, J = 7.6 Hz,2H). 13C NMR (100 MHz, DMSO- d 6) d183.3, 180.1, 168.4, 163.9, 162.5, 161.6,159.1, 159.1, 158.4, 147.1, 144.3, 130.7, 126.4, 122.4, 115.8, 112.9, 111.6,111.4, 105.9, 45.5, 34.8, 18.6. HR-ESI-MS m / z 461.1039 [M+Na]+ , (calcd forC22H16F2N4O4Na, 461.1037).

[0035] Example 12: Evaluation of the toxicity of deoxydaunocarpine ketone compounds to RAW 264.7 cells RAW 264.7 cells in the logarithmic growth phase were selected and cultured at a rate of 4 × 10⁻⁶ cells / year. 4 The cells were seeded at a density of 1 / 2 well in a 96-well plate and incubated overnight at 37°C with 5% CO2. For each compound group, 100 μL of the corresponding deoxydaunocarbazone compound (final concentration 10 μM) was added. The blank group contained only blank medium, and the control group contained an equal volume of medium containing DMSO. The plates were incubated for another 24 h. Under dark conditions, 20 μL of MTT solution (5 mg / mL) was added to each well. After 4 h of reaction, the medium was discarded, and 150 μL of DMSO solution was added. The 96-well plate was then placed on a horizontal shaker and shaken for 5-10 min to completely dissolve the crystals. Finally, the absorbance at 490 nm was measured using a microplate reader. The cell viability was calculated based on the OD value. The results are shown in Table 1.

[0036] Table 1. Toxicity evaluation of seven deoxydaunocarpine ketone compounds on RAW 264.7 cells.

[0037] Experimental results showed that cells from all seven groups of deoxydaunocarpine ketone compounds survived. Among them, the cell survival rate of the groups of compounds ZZMV-2, ZZMV-4, ZZMV-7, and ZZMV-17 was greater than 70%, and the cell survival rate of the groups of ZZMV-2, ZZMV-4, and ZZMV-17 was as high as 90% or more, with low cytotoxicity.

[0038] Example 13 Effects of deoxydaunoside ketones on NO release from RAW 264.7 cells RAW 264.7 cells at 10 per well 5Cells were seeded at a density of [insert density here] into 96-well plates and incubated overnight at 37°C. The supernatant was discarded, and the medium was replaced with 100 μL of DMEM. Each compound group was treated with 50 μL of the corresponding deoxy-duckbill ketone compound at a final concentration of 10 μM, while the Control group was treated with 50 μL of medium containing DMSO. After 1 h of incubation, the Control group was treated with 50 μL of DMEM, and the other groups were treated with 50 μL of LPS (lipopolysaccharide) at a final concentration of 10 μg / mL. After 24 h of incubation, the supernatant was collected. The NO level in the cell supernatant was determined using the Griess method according to the NO detection kit instructions. The results are shown in Table 2.

[0039] Table 2. Effects of deoxydaunoside ketones on LPS-induced NO release in RAW 264.7 cells.

[0040] Experimental results showed that all seven deoxydaunoside ketone compounds could inhibit NO production to varying degrees, with compounds ZZMV-2, ZZMV-4, ZZMV-12, and ZZMV-17 showing particularly strong inhibitory effects on NO production.

[0041] Example 14 Effects of deoxydaunoside ketones on TNF-α release in RAW 264.7 cells RAW 264.7 with 2×10 4 Cells were seeded at a density of cells / well into 96-well plates and incubated overnight at 37°C. The compound group received 50 μL / well (final concentration 10 μM) of deoxydaunocarbazone ketone compound, the control group received 50 μL of medium containing DMSO (final concentration 10 μM), and incubated at 37°C for 1 h. The control group received 50 μL of medium, and the remaining groups received 50 μL of LPS (final concentration 10 ng / mL), and incubated for another 2 h. The supernatant was collected and used to detect the expression level of the inflammatory cytokine TNF-α (tumor necrosis factor-α) in the cell supernatant according to the instructions of a commercial ELISA kit. The experimental results are shown in Table 3.

[0042] Table 3. Effects of deoxydaunoside ketones on LPS-induced TNF-α release in RAW 264.7 cells.

[0043] Experimental results showed that all seven deoxydaunoside ketone compounds could reduce the expression level of TNF-α after LPS stimulation to varying degrees, especially compounds ZZMV-4 and ZZMV-17, which could significantly reduce the expression level of TNF-α after LPS stimulation.

[0044] ZZMV-17 was selected from the above-mentioned deoxydaunophyton ketone compounds for further experiments. Other deoxydaunophyton ketone compounds have the same or similar effects, which will not be repeated here.

[0045] Example 15: Antagonistic activity of ZZMV-17 against CXCR2 1× stimulation buffer was prepared according to the LANCE Ultra cAMP kit (Revvity) instructions, and the positive compound SB 265610 and the test samples were diluted. HEK-293T cells at 80% confluence and stably expressing CXCR2 were then trypsinized, counted, and seeded at a density of 1000 cells / well in 384-well plates (5 μL per well). Next, 1 μL of the diluted 10× compound was added to each well, and the plates were incubated at 37°C for 10 minutes. Then, 4 μL of 1× stimulation buffer containing 1.5 μM Forskolin and 0.5 nM IL-8 agonist was added to induce cAMP production for 30 minutes. Finally, fluorescently labeled Eu-cAMP and cAMP antibody were diluted to working concentrations, and 4 μL were added to each well. After centrifugation, the plates were incubated at room temperature for 1 hour, and data were read using a microplate reader at 665 nm and 620 nm wavelengths. The results are shown in Table 4.

[0046] Table 4. Antagonistic activity of deoxydaunocarpine ketones against CXCR2

[0047] The results showed that compounds with electron-withdrawing groups exhibited relatively good antagonistic activity against CXCR2, with ZZMV-17 showing the highest antagonistic activity against CXCR2, with an inhibition rate of 33.35% at 10 μM.

[0048] In summary, this invention investigated the anti-inflammatory efficacy of deoxydaunoside ketone compounds using an LPS-induced RAW 264.7 cell in vitro inflammation model. In vitro results showed that the seven deoxydaunoside ketone compounds exhibited low cytotoxicity, with ZZMV-2, ZZMV-4, ZZMV-7, and ZZMV-17 showing particularly low cytotoxicity. All seven compounds inhibited NO production, with ZZMV-2, ZZMV-4, ZZMV-12, and ZZMV-17 showing the best inhibitory effects. Furthermore, all seven compounds reduced TNF-α expression levels after LPS stimulation, with compounds ZZMV-4 and ZZMV-17 significantly reducing TNF-α expression levels after LPS stimulation. Meanwhile, ZZMV-4 and ZZMV-17 may exert potential anti-inflammatory effects by competitively binding to the CXCR2 receptor site, blocking the cAMP signaling pathway mediated by the endogenous ligand CXCL8, and regulating downstream inflammatory signal transduction.

[0049] Therefore, deoxydaunoside ketones possess excellent anti-inflammatory effects. Using deoxydaunoside ketones or their pharmaceutically acceptable salts as active substances, alone or in combination with other pharmacologically active compounds and / or extracts, they can be formulated into various dosage forms of anti-inflammatory drugs according to conventional pharmaceutical preparation methods. Alternatively, they can be combined with other anti-inflammatory drugs to create compound preparations. These formulations aim to reduce adverse drug reactions while maintaining efficacy, providing a safe, effective, and economical solution for the prevention and treatment of inflammation.

[0050] The deoxyduckbilline ketone compounds can be used to prepare drugs against cytoinflammatory factors, drugs that inhibit the expression of inflammatory factors in the RAW 264.7 cell model, and drugs that antagonize the CXCR2 receptor. The dosage forms of these drugs include, but are not limited to, tablets, powders, pills, granules, capsules, solutions, suspensions, or injections. The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims.

Claims

1. The following deoxydaunophyton ketone compounds and their pharmaceutically acceptable salts are selected from: 。 2. A method for preparing the deoxydrysticatine ketone compound as described in claim 1 and its pharmaceutically acceptable salt, characterized in that, The steps are as follows: (1) Dissolve 2-amino-6-methoxybenzoic acid and 2-pyrrolidone in POCl3 and heat under reflux. After the reaction is completed and cooled, slowly add the reaction solution to ice water with a pipette and adjust the pH to weakly alkaline to obtain compound 3. (2) Compound 3 was added to concentrated sulfuric acid, heated and stirred, and then concentrated nitric acid was added and the mixture was heated and stirred again. After the reaction was completed and cooled, the reaction solution was pipetted into ice water. The pH was roughly adjusted to 8-9 and then finely adjusted to weakly alkaline with saturated sodium carbonate solution to obtain compound 4. (3) Add 4,4'-bipyridine to a DMF solution containing B2(OH)4 and stir at room temperature for 5-8 min. Then add intermediate compound 4 and stir the reaction to obtain compound 5. (4) Dissolve intermediate compound 5, diethyl squaric acid, and DIPEA in MeOH and heat and stir overnight to obtain compound 6; (5) Compound 6 and DIPEA were dissolved in anhydrous ethanol and heated and stirred to obtain target compounds ZZMV-2, ZZMV-4, ZZMV-7, ZZMV-11, ZZMV-12, ZZMV-17 and ZZMV-19.

3. The method for preparing the deoxydrysticatine ketone compound and its pharmaceutically acceptable salt according to claim 2, characterized in that, In step (5), the benzylamine compound is one of 3,4,5-trifluorobenzylamine, 4-fluorobenzylamine, 4-bromobenzylamine, 4-fluorophenylethylamine, 2,6-difluorobenzylamine, 4-N-butylbenzylamine, and 4-(trifluoromethoxy)benzylamine.

4. The use of the deoxydaunocarpine ketone compound as described in claim 1 and its pharmaceutically acceptable salt in the preparation of any one or more of the following (1) to (5); (1) Preparation of drugs that inhibit cellular inflammatory factors; (2) Preparation of anti-inflammatory drugs; (3) To prepare drugs for treating diseases characterized by cytokine storms; (4) Prepare drugs that regulate inflammatory signaling pathways; (5) Prepare drugs that antagonize CXCR2.

5. The application according to claim 4, characterized in that, The deoxyduckbill ketone compounds regulate the NF-κB signaling pathway by antagonizing the CXCR2 receptor.

6. The application according to claim 4, characterized in that, The inflammatory factors include TNF-α and NO.

7. The application according to claim 4, characterized in that, The deoxyduckbill ketone compounds and their pharmaceutically acceptable salts are used as the active ingredients of the drug.

8. The application according to claim 4, characterized in that, The drug may also include pharmaceutically acceptable carriers or adjuvants.

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