A pyridinone compound, and a preparation method and application thereof

Abstract

The application provides a pyridinone compound and a preparation method and application thereof, and belongs to the technical field of compound synthesis.The pyridinone compound is (E)-5-[(hydroxyl methylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione, and the molecular formula is C6H4N2O4.The pyridinone compound of the application simultaneously contains a hydroxyl group, an imino group and multiple carbonyl functional groups in the structure, forms a unique conjugated system and hydrogen bond network, and has the potential to be used as a bioactive precursor or a functional material.The pyridinone compound with a purity greater than 95% and a yield greater than 94% can be obtained by using the preparation method of the application, the compound shows good performance in dyeing applications, not only expands the application range of the pyridinone compound, but also provides a new way for realizing high-value utilization of fermentation broth resources, and has the advantages of environmental protection and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of compound synthesis technology, and relates to a pyridone compound, its preparation method and application. Background Technology

[0002] Pyridones are a class of heterocyclic molecules with broad biological activities, playing an important role in medicinal chemistry and natural product chemistry. Studies have shown that these compounds generally exhibit antibacterial, anti-inflammatory, and antitumor pharmacological activities, and have significant applications in drug synthesis and functional materials. For example, 4-pyridone derivatives can coordinate with metal ions such as iron and aluminum to treat iron overload diseases; 6-amino-2-pyridone can be used as a dye intermediate in the synthesis of azo dyes.

[0003] Existing pyridone compounds often exhibit limited or singular substitution modes, resulting in narrow bioactivity profiles, less than ideal physicochemical properties (such as solubility and stability), or difficulties in efficient preparation using environmentally friendly processes. Specifically, traditional chemical synthesis methods for these compounds typically involve lengthy routes, low overall yields, and often rely on hazardous reagents or generate difficult-to-manage waste in key steps, contradicting the principles of green chemistry and sustainable production.

[0004] Therefore, the development of pyridone compounds with good staining properties remains a key research focus in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a pyridone compound, its preparation method, and its applications. This invention isolates a pyridone compound from the fermentation broth of *Gynostemma pentaphyllum*, which can be used as a precursor in the synthesis of bioactive substances, and as a raw material or intermediate in the development and preparation of dyes.

[0006] To achieve this objective, the present invention adopts the following technical solution: On one hand, the present invention provides a pyridone compound, the structure of which is shown in Formula I; the pyridone compound of the present invention is named (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione, and has the molecular formula C6H4N2O4.

[0007] Formula I.

[0008] This invention isolates a novel pyridone compound—(E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione—from the fermentation broth of Indigoidine. This compound simultaneously contains hydroxyl, imino, and multiple carbonyl functional groups in its structure, forming a unique conjugated system and hydrogen bond network, possessing the potential as a bioactive precursor or functional material. Experiments show that this compound exhibits excellent performance in staining applications, not only expanding the application range of pyridone compounds but also providing a new approach for the high-value utilization of fermentation broth resources, combining environmental and economic benefits.

[0009] On the other hand, the present invention provides a method for preparing the pyridone compounds as described above, the method comprising the following steps: (1) The fermentation broth of the engineered bacteria was subjected to solid-liquid separation to obtain insoluble matter; (2) Add N,N-dimethylformamide to the insoluble matter, heat and extract, and then perform solid-liquid separation to obtain the supernatant; (3) The supernatant was evaporated to dryness to obtain a solid; (4) Add dichloromethane to the solid for extraction, separate the solid and liquid, and evaporate to dryness to obtain (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione.

[0010] The pyridone compounds of the present invention can be synthesized using engineered bacteria IN10-06 with accession number CCTCC NO: M 2024514. Escherichia coli IN10-06 (derived from patent application number 202410484458.2) and engineered bacteria IN11 of CCTCC NO: M 2024243 (Escherichia coli) IN11 (derived from patent application number 202410284862.5) and the engineered bacteria CG07 of CCTCC NO: M2024075 ( Corynebacterium glutemicun It was found in the fermentation broth of the strain CG07 (derived from patent application number 202410135824.3), and pure product was obtained through separation and purification.

[0011] In this invention, the fermentation broth is subjected to solid-liquid separation to obtain insoluble matter, N,N-dimethylformamide is added and heated for extraction, and after solid-liquid separation, a supernatant is obtained, which is evaporated to dryness to obtain a solid, and then dichloromethane is added for extraction. After solid-liquid separation, the solid is evaporated to dryness to obtain pyridinone compounds. Using the above preparation method, pyridinone compounds with a purity greater than 95% and a yield greater than 94% can be obtained.

[0012] Preferably, the heating and extraction temperature in step (2) is 60-80℃ (e.g., 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 76℃ or 80℃, etc.), and the extraction time is 1-2h (e.g., 1h, 1.25h, 1.5h, 1.7h or 2h, etc.).

[0013] Preferably, the amount of N,N-dimethylformamide added in step (2) is 2 to 5 times the amount of insoluble substance (e.g., 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times or 5 times, etc.).

[0014] The present invention employs the above-mentioned heating temperature and holding time, which allows the target compound to dissolve fully and improves the extraction efficiency; furthermore, the method of the present invention, by using the above-mentioned amount of N,N-dimethylformamide, can simultaneously improve the purity and yield of the product.

[0015] Preferably, the temperature of the rotary evaporation in step (3) is 60~80℃ (e.g., 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 76℃ or 80℃, etc.), and the vacuum degree is -0.08~-0.1MPa (e.g., -0.1MPa, -0.095MPa, -0.09MPa, -0.085MPa or -0.08MPa, etc.).

[0016] The present invention employs the above-mentioned evaporation conditions, which can prevent compound decomposition and ensure product stability.

[0017] Preferably, the amount of dichloromethane added in step (4) is 5 to 10 times the solid mass (e.g., 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times or 10 times, etc.).

[0018] Preferably, the extraction temperature in step (4) is 25~35℃ (e.g., 25℃, 27℃, 30℃, 32℃ or 35℃, etc.), and the extraction time is 0.5~1h (e.g., 0.5h, 0.6h, 0.7h, 0.8h or 1h, etc.).

[0019] This invention employs dichloromethane extraction under the above conditions to better remove impurities, and utilizes the differences in solubility of substances in different solvents for purification to obtain pyridone compounds with higher purity, greater than 95%.

[0020] Preferably, the solid-liquid separation in steps (1), (2) and (4) is performed by centrifugation or filtration.

[0021] Preferably, the centrifugation conditions are 5000~10000g (e.g. 5000g, 6250g, 7500g, 8750g or 10000g, etc.) and the centrifugation time is 10~20min (e.g. 10min, 12.5min, 15min, 17min or 20min, etc.).

[0022] The present invention employs the above-mentioned solid-liquid separation conditions, which can efficiently separate solids and liquids and improve operational efficiency.

[0023] Preferably, the evaporation temperature in step (4) is 40~60℃ (e.g., 40℃, 45℃, 50℃, 55℃, or 60℃), and the vacuum degree is -0.08~-0.1MPa (e.g., -0.1MPa, -0.095MPa, -0.09MPa, -0.085MPa, or -0.08MPa). These evaporation conditions can prevent compound decomposition and ensure product stability.

[0024] On the other hand, the present invention provides the application of pyridone compounds as described above in dyes.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The pyridone compounds of this invention simultaneously contain hydroxyl, imino, and multiple carbonyl functional groups in their structure, forming a unique conjugated system and hydrogen bond network, possessing the potential to serve as bioactive precursors or functional materials. The preparation method of this invention can yield the pyridone compounds with a purity greater than 95% and a yield greater than 94%. These compounds exhibit excellent performance in staining applications, not only expanding the application range of pyridone compounds but also providing a new approach for the high-value utilization of fermentation broth resources, combining environmental and economic benefits. Attached Figure Description

[0026] Figure 1A The liquid chromatogram of the pure product (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione) in Example 1 is shown below. Figure 1B The UV absorption spectrum of the pure (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione) in Example 1; Figure 2A The mass spectrum (M / Z Na) of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione) prepared in Example 1 is shown. + ); Figure 2BThe mass spectrum (M / ZH) of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione prepared in Example 1. - ); Figure 3 The 1H NMR spectrum of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione prepared in Example 1; Figure 4 The carbon NMR spectrum of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione prepared in Example 1; Figure 5 The HSQC diagram of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione prepared in Example 1; Figure 6 Images showing the solubility of the insoluble substance obtained in Example 1 in different solvents; Figure 7 Images showing the solubility of the brownish-brown crude solid obtained in Example 1 in different solvents; Figure 8 Fabric pieces of cotton fabric dyed with the pyridone compound prepared in Example 1 and other pyridone compounds. Detailed Implementation

[0027] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0029] In the following embodiments, the engineered bacteria IN10-06 with accession number CCTCC NO: M 2024514 ( Escherichia coli The engineered bacteria IN11 (IN10-06), CCTCC NO:M 2024243 Escherichia coli IN11) and CCTCC NO: M 2024075 engineered bacteria CG07 ( Corynebacterium glutemicun The fermentation broth of CG07 was provided by Nanjing Hegu Life Biotechnology Co., Ltd. IN10-06, IN11, and CG07 are all deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0030] Preparation of fermentation broth for engineered strain IN10-06 with accession number CCTCC NO: M 2024514: The activated recombinant strain seed fermentation broth was inoculated into a basic fermentation medium for the first stage of fermentation culture, with the pH maintained at 7.0 and the temperature controlled at 27℃ for 24 hours to obtain the first stage fermentation broth; the pH of the first stage fermentation broth was maintained at 7.5 and the temperature controlled at 25℃, and a synthase inducer was added for the second stage fermentation culture for 28 hours to obtain the second stage fermentation broth; the pH of the second stage fermentation broth was maintained at 8.5 and the temperature controlled at 40℃, and a synthase inducer and lactose were added for the third stage fermentation culture for 30 hours to obtain the third stage fermentation broth.

[0031] Preparation of fermentation broth of engineered strain IN11 (Escherichia coli IN11) with preservation number CCTCC NO:M 2024243: The activated recombinant strain seed fermentation broth was inoculated into a basic fermentation medium for the first stage of fermentation culture, with the pH maintained at 6.8, the temperature controlled at 25℃, and the culture time at 24h to obtain the first stage fermentation broth; the pH of the first stage fermentation broth was maintained at 7.0, the temperature controlled at 30℃, and a synthase inducer was added for the second stage fermentation culture, with the culture time at 24h to obtain the second stage fermentation broth; the pH of the second stage fermentation broth was maintained at 8.0, the temperature controlled at 35℃, and a synthase inducer and lactose were added for the third stage fermentation culture, with the culture time at 24h to obtain the third stage fermentation broth.

[0032] Preparation of fermentation broth of engineered strain CG07 (Corynebacterium glutemicunCG07) with preservation number CCTCC NO:M 2024075: The activated recombinant strain seed fermentation broth was inoculated into a basic fermentation medium for the first stage of fermentation culture, with the pH maintained at 7.2 and the temperature controlled at 23℃ for 24 h to obtain the first stage fermentation broth; the pH of the first stage fermentation broth was maintained at 7.2 and the temperature controlled at 28℃, and a synthase inducer was added for the second stage fermentation culture for 24 h to obtain the second stage fermentation broth; the pH of the second stage fermentation broth was maintained at 7.5 and the temperature controlled at 30℃, and a synthase inducer and lactose were added for the third stage fermentation culture for 24 h to obtain the third stage fermentation broth.

[0033] Preparation of 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione: It was prepared using the method described in Example 1 of patent CN2025118424834, as follows: 1) The activated engineered bacteria IN10-06 (Escherichia coli IN10-06) was transferred to an Erlenmeyer flask containing basal fermentation medium at an inoculation volume of 1% of the fermentation medium volume. Kanamycin was added to the flask and cultured under the following conditions: temperature 35℃, rotation speed 200 rpm, time 9 h, until the OD600 of the bacterial solution was 3.5 to obtain the seed culture. 2) Prepare 50L of basic fermentation medium and place it in a 100L fermenter for autoclaving. After sterilization, wait for the basic fermentation medium to cool to 25℃, then use ammonia water to adjust its pH to 7.0 and add 0.5g / L of arabinose to the fermenter. Inoculate the seed liquid obtained in step (1) into the fermenter and start fermentation. The temperature is about 25℃, the initial aeration ratio is 0.8vvm, the initial rotation speed is 200rpm, and the dissolved oxygen rotation speed is set to 35%. When the rotation speed reaches 900rpm, the dissolved oxygen rotation speed is decoupled, and the dissolved oxygen is controlled at 35% by increasing the aeration ratio (1.0vvm). The pH is controlled at 7.0 throughout the fermentation process. When the dissolved oxygen rebounds rapidly to above 50%, feed medium is added for feeding. The initial feeding rate is 3g / L / h, and the residual sugar is controlled to be less than 5g / L throughout the fermentation process. Feeding is stopped after 72h. Fermentation is ended when the pH rebounds to 7.5 or the dissolved oxygen rebounds to 70%, and the fermentation broth is obtained.

[0034] 3) Take 5L of fermentation broth, heat it to 70℃ and keep it warm for 2h, centrifuge at 5000g for 10min to remove insoluble matter, concentrate it by rotary evaporation at 80℃ for 30X to obtain concentrated liquid, add 2X isopropanol to the concentrated liquid, cool it to 8℃ and stir for 2h, after crystallization is complete, centrifuge at 5000g to obtain crude crystals; add the obtained crude crystals to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 8℃, stir for 2h and then separate the solid and liquid, and dry the solid to obtain the product.

[0035] The structure was confirmed to be 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. 5-Amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione.

[0036] Liquid chromatography detection methods: Mobile phase: Gradient elution of methanol and 0.1% acetic acid in water, with the gradient as follows: Flow rate: 0.6 ml / L, wavelength: 400 nm and full-band scanning, column temperature: 35 ℃, injection volume: 10 μL, run time: 20 min.

[0037] Sample preparation solvent: water Chromatographic column: Aglient Eclipse plus C18 4.6 250,5um.

[0038] Example 1: Preparation of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione Take 10 L of fermentation broth of engineered bacteria IN10-06 with preservation number CCTCC NO: M 2024514, centrifuge at 8000 g for 15 min at 4℃, discard the supernatant, collect the wet bacterial insoluble matter, and weigh it to be 520 g.

[0039] Add 1.56 L (approximately 3 times the mass of the insoluble matter) of N,N-dimethylformamide (DMF) to the above insoluble matter, and heat and stir at 70°C for 1.5 h to extract. After extraction, centrifuge at 8000 g for 15 min while hot to obtain the supernatant (extract).

[0040] The obtained supernatant was subjected to rotary evaporation in a water bath at 70°C and a vacuum of -0.09 MPa until it was evaporated to dryness and DMF was removed, yielding a brownish-brown crude solid.

[0041] Add 150 mL (approximately 6 times the solid mass) of dichloromethane to the above crude solid product and extract with stirring at 30°C for 0.75 h. After extraction, filter and collect the filtrate. Evaporate the filtrate to dryness under a 50°C water bath and a vacuum of -0.09 MPa to obtain a pale yellow solid powder, which is the target compound (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione. The weight was 18.2 g, and the purity was 96.1% according to HPLC analysis (e.g., ...). Figure 1A As shown in the figure, the yield was estimated to be 95.8% based on the fermentation broth volume and the theoretical yield of the target substance. Figure 1B The product showed a maximum absorption wavelength of 422 nm, confirming that the target compound (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione is a pale yellow solid powder.

[0042] Compound identification Hydrogen spectrum (H NMR) 1 H NMR (e.g.) Figure 3 (as shown) Solvent: CDCl3; Hydrogen spectrum description: Two sets of proton signals were observed. The δ8.37 ppm (1H, s) signal was assigned to a methine proton with an imine group (N=CH-) on the side chain. The δ6.50 ppm (1H, br s) signal was assigned to a proton with an enol structure on the ring (C5-H). The other active hydrogen (-OH) in the molecule was not observed in CDCl3, possibly due to hydrogen exchange or a broad peak shape.

[0043] The peak assignments are shown in Table 1.

[0044] Table 1 Carbon spectrum (CMR) 13 C NMR (such as C NMR) Figure 4 (as shown) Solvent: CDCl3; Carbon spectrum description: The carbon spectrum shows signals in multiple low-field regions, with carbonyl carbon signals (C2, C4, C6) appearing in the δ 170-175 ppm region. The signal at δ ~ 152 ppm is assigned to the side-chain imine carbon (N=CH-). The signal at δ ~ 102 ppm is assigned to the methine carbon (C5-H) with an enol structure on the ring. This carbon is a quaternary carbon in the original structure, but becomes a protonated carbon after tautomerism, and is directly linked to the proton at δ 6.50 ppm in the proton spectrum.

[0045] The peak assignments are shown in Table 2.

[0046] Table 2 Figure 5 The HSQC spectra of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione prepared in Example 1 are shown in Table 3.

[0047] Table 3 HSQC Description: HSQC spectra showed two distinct groups 1 H- 13 C direct correlation signal: δH 8.37 ppm / δC~152 ppm: confirms that the proton is a methine proton (N=CH-) attached to the imine carbon. δH 6.50 ppm / δC~102 ppm: confirms that the proton is attached to a sp... 2 On the hybridized methine carbon, there is a C5-H corresponding to the tautomerism formed on the ring. The protons (such as OH) on the remaining carbonyl carbons and heteroatoms show no relevant signal in the HSQC, consistent with expectations.

[0048] The mass spectra in Figure 2 show: mass spectrum 167 (M / ZH)- ) and 191 (M / Z Na + The molecular weight can be determined to be 168 (M / Z), and the structure can be determined to be (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione by combining the characterization of proton and carbon spectra.

[0049] Example 2: Preparation of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione Take 10 L of the fermentation broth of engineered bacteria IN11, centrifuge at 5000 g for 20 min at 4℃, and collect 500 g of wet insoluble bacterial matter.

[0050] Add 1.0 L (twice the mass of the insoluble matter) of DMF to the insoluble matter and heat and stir at 60°C for 2 h. After extraction, centrifuge at 5000 g for 20 min while hot and collect the supernatant.

[0051] The supernatant was evaporated to dryness in a water bath at 60°C and a vacuum of -0.08 MPa to obtain a solid.

[0052] Dichloromethane (5 times the mass of the solid) was added to the solid, and the mixture was stirred and extracted at 25°C for 1 h. The mixture was filtered, and the filtrate was rotary evaporated to dryness under a water bath at 40°C and a vacuum of -0.08 MPa to obtain a pale yellow solid. The solid weighed 17.5 g, had an HPLC purity of 95.3%, and a yield of 94.6%.

[0053] Example 3: Preparation of (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione Take 10 L of fermentation broth from engineered bacteria CG07, centrifuge at 10000 g for 10 min at 4℃, and collect the wet insoluble matter of the bacteria, which weighs 540 g.

[0054] Add 2.7 L (5 times the mass of the insoluble matter) of DMF to the insoluble matter and heat and stir at 80°C for 1 h. After extraction, centrifuge at 10000 g for 10 min while hot and collect the supernatant.

[0055] The supernatant was evaporated to dryness in an 80°C water bath under a vacuum of -0.1 MPa to obtain a solid.

[0056] Dichloromethane (10 times the mass of the solid) was added to the solid, and the mixture was stirred and extracted at 35°C for 0.5 h. The mixture was filtered, and the filtrate was rotary evaporated to dryness under a water bath at 60°C and a vacuum of -0.1 MPa to obtain a pale yellow solid. The solid weighed 18.6 g, had an HPLC purity of 96.5%, and a yield of 96.2%.

[0057] Comparative Example 1 10 L of fermentation broth from engineered strain IN10-06 was centrifuged under the conditions of Example 1 to obtain 520 g of wet cell insoluble matter. First, 1.56 L (3 times the mass of the insoluble matter) of dichloromethane was added to the insoluble matter, and the mixture was stirred and extracted at 30°C for 1.5 h. After extraction, centrifugation was performed, and almost no signal of the target compound was detected in the supernatant.

[0058] Comparative Example 2 10 L of fermentation broth from engineered strain IN10-06 was centrifuged under the conditions of Example 1 to obtain 520 g of wet cell insoluble matter. 1.56 L (3 times the mass of the insoluble matter) of a methanol-water mixture (methanol:water = 4:1, v / v) was added to the insoluble matter, and the mixture was heated and stirred at 70 °C for 1.5 h for extraction. Since the product is intracellularly secreted, after extraction with the methanol-water mixture (methanol:water = 4:1, v / v), centrifugation revealed almost no detectable signal of the target compound in the supernatant.

[0059] Comparative Example 3 10 L of fermentation broth from engineered strain IN10-06 was centrifuged under the conditions of Example 1 to obtain 520 g of wet insoluble bacterial cell matter. 1.56 L (approximately 3 times the mass of the insoluble matter) of DMF was added to the insoluble matter, and extraction was carried out at 70°C for 0.5 h. The supernatant was obtained by centrifugation. The supernatant was rotary evaporated to dryness under a vacuum of -0.1 MPa in an 80°C water bath to obtain a solid. Ethyl acetate (10 times the mass of the solid) was added to the solid, and extraction was carried out with stirring at 35°C for 0.5 h. The solid was filtered, and the filtrate was rotary evaporated to dryness under a vacuum of -0.1 MPa in a 60°C water bath to obtain a solid. The solid weighed 14.8 g, and HPLC analysis showed a purity of only 15.7%, with a dark color and many impurities.

[0060] Comparative Example 4 10 L of fermentation broth from engineered strain IN10-06 was centrifuged under the conditions of Example 1 to obtain 520 g of wet cell insoluble matter. 4.16 L (8 times the mass of the insoluble matter) of DMF was added to the above insoluble matter, and the mixture was heated and stirred at 70°C for 1.5 h for extraction. Subsequent steps were the same as in Example 1. A pale yellow solid of 15.4 g was finally obtained, with a purity of 82.3% and a yield of 81.1%.

[0061] Comparative Example 5 10 L of fermentation broth from engineered strain IN10-06 was centrifuged under the conditions of Example 1 to obtain 520 g of wet cell insoluble matter. 1.56 L (approximately 3 times the mass of the insoluble matter) of DMF was added to the above insoluble matter, and the mixture was heated and stirred at 40°C for 1.5 h for extraction. Subsequent steps were the same as in Example 1. A pale yellow solid of 9.2 g with a purity of 95.5% was finally obtained, but the yield was only 48.4%.

[0062] The products of Examples 2-3 were also analyzed by 1H NMR, 1C NMR, HPLC, and mass spectrometry, which confirmed the correctness of the structure.

[0063] Results analysis: Examples 1-3 strictly followed the preparation method described in this invention, employing a process route starting from fermentation insolubles and involving heating extraction with a specific ratio of DMF, rotary evaporation, and selective extraction with dichloromethane. Results showed that this method is simple and efficient, yielding products with purity exceeding 95% and yields exceeding 94%, and exhibiting good product color. Different strains treated with this method showed highly similar product purity and yield, confirming the method's good universality across different strains. It also fully validated the rationality and superiority of the process parameters determined in this application (such as 2-5 times the DMF dosage, extraction temperature of 60-80℃, and 5-10 times the dichloromethane dosage), enabling the achievement of high purity and high yield.

[0064] In Comparative Examples 1 and 2, the mixed solvents of dichloromethane and methanol-water failed to disrupt cell interactions and dissolve the target product, confirming the necessity of using the highly polar aprotic solvent DMF. In Comparative Example 3, ethyl acetate, instead of dichloromethane, failed to achieve selective purification. This confirms the necessity of using dichloromethane, a solvent that precisely utilizes differences in solubility (dissolving products but not impurities), to obtain samples with high purity. In Comparative Example 4, while adding more than 5 times the amount of DMF aided dissolution, it also led to the simultaneous extraction of a large amount of intracellular impurities (such as pigments, lipids, and protein fragments). Excessive solvent not only increased the energy consumption and time of subsequent rotary evaporation (leading to excessive product degradation due to prolonged heating), but also placed an excessive burden on dichloromethane for impurity removal, making it difficult to obtain samples with high purity. In Comparative Example 5, when the extraction temperature was below 60℃, the dissolution rate and solubility of the target compound in DMF decreased significantly, resulting in a large amount of product remaining in the cell residue and failing to be extracted, severely impacting the yield. This confirms that an extraction temperature of 60-80℃ is crucial for ensuring a high yield. This confirms that the various process steps and parameter settings described in this invention can guarantee the acquisition of high-purity, high-yield products.

[0065] Experimental Example 1: The insoluble matter obtained by centrifugation of the fermentation broth in Example 1 was subjected to solubility experiments using different solvents. The experimental results are shown below. Figure 6 Since the product is an intracellular product, based on the solvent solubility state and experimental results, it was found that only DMF and DMSO can dissolve the insoluble matter. However, because DMSO has a high boiling point and is difficult to evaporate to dryness, DMF was used for extraction.

[0066] Example 2: The extract was evaporated to dryness to remove DMF, yielding a brownish-red crude solid. Dissolution experiments were conducted using different solvents. The experimental results are shown below. Figure 7After extracting the product from the cell using DMF, the crude product was extracted with different solvents. It was found that dichloromethane could dissolve the product but not the impurities. Therefore, dichloromethane was used for a second extraction.

[0067] Application Example 1 Take 1.5 g each of the pyridone compound (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione and 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione prepared in Example 1, and disperse them evenly in 500 mL of distilled water. Adjust the pH to 5 with acetic acid, and simultaneously place cotton fabric samples in the water for dyeing. Heat to 130 °C and maintain the temperature for 30 min. After cooling, rinse the samples with water and perform color testing. The liquor ratio is 1:50.

[0068] Color depth (K / S): Measured using a colorimeter. The colorimeter measures the reflectance R of the dyed textile, and then R is used according to the Kubelka-Munk formula: K / S = (1-R). 2 / 2R is used to calculate color depth (K / S), where R is reflectance. Average color depth (K / S) is calculated from average reflectance.

[0069] Color fastness to washing with soap: Tested in accordance with GB / T 3921-2008 "Textiles - Tests for color fastness to washing with soap".

[0070] Color fastness to wet and dry rubbing: Tested in accordance with GB / T 3920-2008 "Textiles - Tests for color fastness to rubbing".

[0071] Color fastness to light: Tested in accordance with GB / T 8427-2019 "Textiles - Tests for color fastness to artificial light: Xenon arc".

[0072] The test results are shown in Table 4.

[0073] Table 4 The compounds prepared by this invention have better color depth (K / S) and various color fastness properties.

[0074] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A pyridone compound, characterized in that, The chemical name of the pyridone compound is (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione, with the molecular formula C6H4N2O4 and the structure shown in Formula I: Equation I.

2. The method for preparing pyridone compounds according to claim 1, characterized in that, The preparation method includes the following steps: (1) The fermentation broth of the engineered bacteria was subjected to solid-liquid separation to obtain insoluble matter; (2) Add N,N-dimethylformamide to the insoluble matter, heat and extract, and then perform solid-liquid separation to obtain the supernatant; (3) The supernatant was evaporated to dryness to obtain a solid; (4) Add dichloromethane to the solid for extraction, separate the solid and liquid, and evaporate to dryness to obtain (E)-5-[(hydroxymethylene)amino]-1,2,3,6-tetrahydropyridine-2,3,6-trione.

3. The preparation method according to claim 2, characterized in that, The engineered bacteria mentioned in step (1) are engineered bacteria IN10-06 with accession number CCTCC NO: M 2024514, engineered bacteria IN11 with accession number CCTCC NO: M 2024243, or engineered bacteria CG07 with accession number CCTCC NO: M2024075.

4. The preparation method according to claim 2, characterized in that, The heating and extraction temperature in step (2) is 60-80℃, and the extraction time is 1-2 hours. The amount of N,N-dimethylformamide added in step (2) is 2-5 times the amount of the insoluble substance.

5. The preparation method according to claim 2, characterized in that, The temperature of the rotary evaporation in step (3) is 60~80℃ and the vacuum degree is -0.08~-0.1MPa.

6. The preparation method according to claim 2, characterized in that, The amount of dichloromethane added in step (4) is 5 to 10 times the mass of the solid.

7. The preparation method according to claim 2, characterized in that, The extraction temperature in step (4) is 25~35℃ and the extraction time is 0.5~1h.

8. The preparation method according to claim 2, characterized in that, In steps (1), (2), and (4), the solid-liquid separation is performed by centrifugation or filtration. The centrifugation conditions are 5000~10000g and centrifugation time is 10~20min.

9. The preparation method according to claim 2, characterized in that, The temperature for evaporation in step (4) is 40~60℃ and the vacuum degree is -0.08~-0.1MPa.

10. The use of the pyridone compounds according to claim 1 in dyes.

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