A method for preparing pyridazine compounds

Abstract

The application belongs to the field of green manufacturing, and discloses a preparation method of a pyridazine compound, comprising the following steps: (1) adding an enzyme catalyst, a bio-based furan and a co-substrate into a buffer solution to react, and removing cells after the reaction; (2) adding hydrogen peroxide or an oxidase and a titanium-silicon molecular sieve to react, and removing the titanium-silicon molecular sieve after the reaction; (3) slowly adding hydrazine under stirring at 0-60 DEG C to obtain the pyridazine compound after the reaction; the enzyme catalyst is at least one of the following: an alcohol dehydrogenase / glucose dehydrogenase double enzyme system, an alcohol dehydrogenase / formate dehydrogenase double enzyme system and a recombinant bacterium co-expressing one or both of the above two double enzyme systems. The application has the advantages of high selectivity, mild reaction conditions, environmental friendliness, fast reaction speed and no need for intermediate separation and purification.

Description

Technical Field

[0001] This invention belongs to the field of green manufacturing, specifically relating to a method for catalytically converting furan compounds from biomass into pyridazine compounds through one-pot biocatalytic reduction, chemobiocatalytic oxidation, and spontaneous cycloaddition reactions. Background Technology

[0002] Pyridazines are a class of six-membered aromatic heterocyclic compounds with two adjacent nitrogen atoms. Pyridazine derivatives (such as azathanthrones and pyridazinamides) exist in nature but are relatively rare. Nevertheless, the pyridazine skeleton has attracted considerable attention in the pharmaceutical and agrochemical fields due to the diverse biological activities of its derivatives, including anticancer, anti-HIV, anti-Alzheimer's, antithrombotic, antibacterial, and herbicidal activities. For example, the pyridazine-containing inhibitors declavatinib (targeting tyrosine kinase 2) and ensartinib (targeting anaplastic lymphoma kinase / tyrosine kinase) were approved for clinical use by the U.S. Food and Drug Administration (FDA) in 2022 and 2024, respectively. Furthermore, the pyridazine skeleton, due to its proximity to sp... 2 The presence of -N bonds gives pyridazine units excellent optoelectronic properties. For example, they can serve as excellent electron-attracting units for developing electron-transfer materials with high electron mobility, as well as electron-push-pull molecular systems exhibiting separated and balanced electron / hole transfer capabilities. This makes pyridazine units valuable in the synthesis of organic optoelectronic materials, such as organic field-effect transistors, organic photovoltaic devices, and organic light-emitting diodes. Furthermore, the pyridazine framework possesses high-energy N-N bonds and a planar conjugated framework structure, making it highly promising for the synthesis of high-energy compounds.

[0003] Significant progress has been made in the synthesis of pyridazine compounds in recent decades, but most of these synthesis currently uses non-renewable fossil resources as starting materials (Russ. Chem. Rev., 2020, 89, 393). For example, Chang et al. reported the synthesis of substituted pyridazines from γ-acetylenes via dioxane-mediated [4+2] cyclocondensation and dehydrogenation aromatization (Tetrahedron, 2015, 71, 6840). Herdewijn's team developed a two-step process for the synthesis of 6-substituted-4-hydroxy-3-methoxycarbonylpyridazines, in which the substituted α-diazo-β-keto-δ-hydroxybutane is oxidized with 2-iodooxybenzoic acid (IBX) and then reacted with phosphine via a diazo-Wittig reaction (J. Org. Chem., 2013, 78, 7845). Levin's team revealed a three-step chemical route to prepare pyridazines from pyridine via skeleton editing (Science, 2025, 389, 295).

[0004] In recent years, the synthesis of chemicals, fuels, and materials from renewable biomass and its derivatives has attracted much attention. Bio-based furans are furan compounds obtained by dehydrating various pentoses and hexoses. As early as 2001, Lichtenthaler et al. pioneered the synthesis of bio-based 3,6-pyridazinediethanol (3,6-PDM) from 5-hydroxymethylfurfural (HMF) and its derivatives (Green Chem., 2001, 3, 201). 3,6-PDM is a potential bio-based monomer that can be used to manufacture bioplastics, organic optoelectronic materials, and energy materials. This process involves chemical reduction, acylation protection, oxidative ring-opening, cycloaddition, and deprotection reactions, with a 3,6-PDM yield of approximately 46%. Furthermore, each reaction step requires the separation and purification of intermediates, consuming significant amounts of solvent and time, and generating substantial waste. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a novel, green and efficient synthetic method for synthesizing pyridazine compounds using bio-based furans as substrates.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing a pyridazine compound includes the following steps:

[0008] (1) Add the enzyme catalyst, bio-based furan, and co-substrate to the buffer solution for reaction, and remove the cells after the reaction;

[0009] (2) Add hydrogen peroxide or oxidase and titanium silicon molecular sieve to react. After the reaction is completed, remove the titanium silicon molecular sieve.

[0010] (3) Hydrazine compounds were slowly added at 0-60 °C with stirring, and the reaction yielded pyridazine compounds;

[0011] The enzyme catalyst is at least one of the following: an alcohol dehydrogenase / glucose dehydrogenase dual enzyme system, an alcohol dehydrogenase / formate dehydrogenase dual enzyme system, or a recombinant bacterium expressing one or both of the above dual enzyme systems.

[0012] Preferably, the pyridazine compound is shown in general formula I, and the bio-based furan is shown in general formula II:

[0013] I, II

[0014] R- represents a hydrogen atom group, hydroxymethyl, methyl, ethyl, acetoxymethyl, methoxymethyl, or ethoxymethyl.

[0015] Preferably, the alcohol dehydrogenase is one or more of RbADH, EcYjgB, EcYahK, and the EcYjgB mutant S199D / S200R.

[0016] Preferably, the host cell of the recombinant bacteria is Escherichia coli.

[0017] Preferably, the co-substrate in step (1) is glucose or sodium formate, and its amount is 1.2-4.0 times the molar amount of bio-based furan.

[0018] Preferably, in step (1), when the co-substrate is glucose, calcium carbonate or NaOH needs to be added to maintain the pH at 7.0±1.0.

[0019] Preferably, the buffer solution in step (1) is a phosphate buffer solution with a concentration of 50-200 mmol / L and a pH of 6.0-8.0.

[0020] Preferably, the titanium-silicon molecular sieve in step (2) is TS-1, and the dosage is 5-20 g / L.

[0021] Preferably, the oxidase in step (2) is glucose oxidase or formate oxidase, and the amount used is 0.01-1 g / L; the amount of hydrogen peroxide is 1.1-2.0 times the initial molar amount of bio-based furan; and the amount of hydrazine in step (3) is 1.1-2.0 times the initial molar amount of bio-based furan.

[0022] Preferably, the reaction conditions for step (1) are 30±10 ℃ and 150±50 rpm for 2±1 h; the reaction conditions for step (2) are 40±20 ℃ and 150±50 rpm for 1-12 h; and the reaction conditions for step (3) are 4±4 ℃ and 250±50 rpm for 5±3 min.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) This invention discloses a green route for selectively synthesizing pyridazine compounds using bio-based furans as raw materials, which not only avoids dependence on petroleum-based resources, but also achieves the goal of carbon neutrality.

[0025] (2) This invention uses water as a solvent and utilizes non-toxic enzyme catalysts and titanium-silicon molecular sieve TS-1 as catalysts. It eliminates the need for intermediate separation and purification, and the reaction process is simple, easy to control, and environmentally friendly.

[0026] (3) The process has high selectivity, requires no protection and deprotection steps, and has high atom economy. Attached Figure Description

[0027] Figure 1This is a schematic diagram of the synthesis of pyridazine compounds.

[0028] Figure 2 The liquid chromatogram is shown for the sample analysis obtained in Example 1.

[0029] Figure 3 3,6-pyridazine dimethyl alcohol was prepared as described in Example 2. 1 H NMR spectrum.

[0030] Figure 4 3,6-pyridazine dimethyl alcohol was prepared as described in Example 2. 13 C NMR spectrum. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0032] Titanium silicate molecular sieve TS-1 (specific surface area: 350-450 m²) 2 (g, pore size: 0.56-0.58 nm) was purchased from Maclean Biotechnology Co., Ltd.; glucose oxidase (GOx, >180 U / mg) was purchased from Shanghai Aladdin Biotechnology Co., Ltd.

[0033] Alcohol dehydrogenases RbADH, EcYjgB, EcYahK, glucose dehydrogenase (GDH), and recombinant E. coli co-expressing alcohol dehydrogenase / GDH have been disclosed in the literature "Wu, Q.; Zong, M.-H.; Li, N. One-Pot Chemobiocatalytic Production of 2,5-Bis(hydroxymethyl)furan and Its Diester from Biomass in Aqueous Media. ACS Catal. 2023, 13, 9404-9414." The EcYjgB mutant S199D / S200R, formate dehydrogenase (FDH), and recombinant E. coli co-expressing this mutant / FDH have been disclosed in Chinese invention patent CN120290502A. Formate oxidase has been disclosed in “Tieves, F.; Willot, SJ-P.; van Schie, MMCH; Rauch, MCR; Younes, SHH; Zhang, W.; Dong, J.; Gomez deSantos, P.; Robbins, JM; Bommarius, B.; et al. Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2O2-Dependent BiocatalyticOxidation Reactions. Angew. Chem. Int. Ed. 2019, 58, 7873-7877.”

[0034] Example 1

[0035] Add 5-hydroxymethylfurfural (HMF, final concentration 50 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 200 mM glucose, and 15 g / L calcium carbonate to 2 mL phosphate buffer (200 mM, pH 7.0). After reacting at 30 °C and 150 rpm for 2 h, centrifuge at 8000 rpm for 5 min to remove cells and residual calcium carbonate. Add glucose oxidase GOx (final concentration 1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L). Continue reacting at 30 °C and 150 rpm for another 2 h. Cool to 4 °C, and slowly add 100 mM hydrazine under stirring (250 rpm). After reacting for 5 min, analyze by HPLC. Figure 2The yield of 3,6-pyridazine dimethyl alcohol was 86%.

[0036] Example 2

[0037] In 50 mL of phosphate buffer (200 mM, pH 7.0), HMF (final concentration 250 mM), 50 g / L recombinant E. coli-EcYjgB-GDH (wet weight), and 375 mM glucose were added. The reaction was carried out at 25 °C and 250 rpm, with 10 M sodium hydroxide solution added during the reaction to maintain pH 7.0. After 3 h of reaction, the cells were removed, and 500 mM hydrogen peroxide and TS-1 titanium silicate molecular sieve (20 g / L) were added. The reaction was continued at 50 °C and 250 rpm for another 2 h. After cooling to 4 °C, 500 mM hydrazine was slowly added under stirring (250 rpm). After 30 min of reaction, the pH of the reaction solution was adjusted to 11.0, and sodium chloride was added to make it supersaturated. The solution was extracted with tetrahydrofuran (50 mL × 10). The organic phases were combined, dried overnight with anhydrous sodium sulfate, and the organic solvent was removed to obtain 3,6-pyridazine diethanol with a separation yield of 70%.

[0038] Example 3

[0039] In 2 mL of phosphate buffer (200 mM, pH 6.0), 5-methylfurfural (final concentration 50 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 200 mM glucose, and 15 g / L calcium carbonate were added. After reacting at 30 °C and 150 rpm for 2 h, cells and residual calcium carbonate were removed. Glucose oxidase GOx (final concentration 1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L) were added, and the reaction was continued at 30 °C and 150 rpm for another 2 h. After cooling to 4 °C, 100 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 6-methyl-3-pyridazine methanol was obtained with a yield of 81%.

[0040] Example 4

[0041] In 2 mL of phosphate buffer (200 mM, pH 7.0), furfural (final concentration 20 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 25 mM glucose, and 5 g / L calcium carbonate were added. After reacting at 30 °C and 150 rpm for 1 h, cells and residual calcium carbonate were removed. Glucose oxidase GOx (final concentration 1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L) were added, and the reaction was continued at 30 °C and 150 rpm for another 2 h. After cooling to approximately 0 °C, 25 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 3-pyridazine methanol was obtained with a yield of 58%.

[0042] Example 5

[0043] In 2 mL of phosphate buffer (200 mM, pH 7.0), 5-methoxymethylfurfural (final concentration 50 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 110 mM glucose, and 15 g / L calcium carbonate were added. After reacting at 30 °C and 150 rpm for 3 h, cells and residual calcium carbonate were removed. Glucose oxidase GOx (final concentration 0.1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L) were added, and the reaction was continued at 30 °C and 150 rpm for 12 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 6-methoxymethyl-3-pyridazine methanol was obtained with a yield of 69%.

[0044] Example 6

[0045] In 2 mL of phosphate buffer (200 mM, pH 7.0), 5-ethoxymethylfurfural (final concentration 50 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 110 mM glucose, and 15 g / L calcium carbonate were added. After reacting at 30 °C and 150 rpm for 3 h, cells and residual calcium carbonate were removed. Glucose oxidase GOx (final concentration 0.1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L) were added. The reaction was continued at 30 °C and 150 rpm for 12 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 6-ethoxymethyl-3-pyridazine methanol was obtained with a yield of 54%.

[0046] Example 7

[0047] In 2 mL of phosphate buffer (200 mM, pH 7.0), 5-ethoxymethylfurfural (final concentration 50 mM), 10 g / L recombinant E. coli-EcYjgB-GDH (wet weight), 110 mM glucose, and 15 g / L calcium carbonate were added. After reacting at 30 °C and 150 rpm for 3 h, cells and residual calcium carbonate were removed. Glucose oxidase GOx (final concentration 0.1 g / L) and titanium silicate molecular sieve TS-1 (20 g / L) were added. The reaction was continued at 60 °C and 150 rpm for 4 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 6-ethoxymethyl-3-pyridazine methanol was obtained with a yield of 78%.

[0048] Example 8

[0049] In 2 mL of phosphate buffer (200 mM, pH 7.0), 5-hydroxymethylfurfural (HMF, final concentration 50 mM), NADPH (0.2 mM), EcYjgB (0.3 g / L), GDH (0.5 g / L), glucose oxidase GOx (1 g / L), glucose (150 mM), calcium carbonate (15 g / L), and titanium silicate molecular sieve TS-1 (12.5 g / L) were added. The mixture was reacted at 30 °C and 150 rpm for 3 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 3,6-pyridazine dimethylethanol was obtained with a yield of 81%.

[0050] Example 9

[0051] 5-Hydroxymethylfurfural (HMF, final concentration 50 mM), EcYjgB mutant S199D / S200R (0.3 g / L), formate dehydrogenase (0.5 g / L), NADH (0.2 mM), formate oxidase (1 g / L), sodium formate (150 mM), and titanium silicate molecular sieve TS-1 (12.5 g / L) were added to 2 mL of phosphate buffer (200 mM, pH 7.0). The mixture was reacted at 30 °C and 150 rpm for 5 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 3,6-pyridazine dimethylethanol was obtained in 80% yield.

[0052] Example 10

[0053] 5-Hydroxymethylfurfural (HMF, final concentration 50 mM), recombinant E. coli-EcYjgB S199D / S200R-FDH (10 g / L), and sodium formate (150 mM) were added to 2 mL of phosphate buffer (200 mM, pH 7.0). The mixture was reacted at 30 °C and 150 rpm for 4 h. After removing the cells, formate oxidase (1 g / L) and titanium silicate molecular sieve TS-1 (12.5 g / L) were added. The mixture was reacted at 30 °C and 150 rpm for 5 h. After cooling to 4 °C, 55 mM hydrazine was slowly added under stirring (250 rpm). After reacting for 5 min, 3,6-pyridazine dimethylethanol was obtained with a yield of 83%.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a pyridazine compound, characterized in that, Includes the following steps: (1) Add the enzyme catalyst, bio-based furan, and co-substrate to the buffer solution for reaction, and remove the cells after the reaction; (2) Add hydrogen peroxide or oxidase and titanium silicon molecular sieve to react. After the reaction is completed, remove the titanium silicon molecular sieve. (3) Hydrazine compounds were slowly added at 0-60 °C with stirring, and the reaction yielded pyridazine compounds; The enzyme catalyst is at least one of the following: an alcohol dehydrogenase / glucose dehydrogenase dual enzyme system, an alcohol dehydrogenase / formate dehydrogenase dual enzyme system, or a recombinant bacterium expressing one or both of the above dual enzyme systems.

2. The preparation method according to claim 1, characterized in that, The pyridazine compounds are shown in general formula I, and the bio-based furans are shown in general formula II: I、 Ⅱ R- represents a hydrogen atom group, hydroxymethyl, methyl, ethyl, acetoxymethyl, methoxymethyl, or ethoxymethyl.

3. The preparation method according to claim 1, characterized in that, The alcohol dehydrogenase is one or more of RbADH, EcYjgB, EcYahK, and the EcYjgB mutant S199D / S200R.

4. The preparation method according to claim 1, characterized in that, The host cell of the recombinant bacteria is Escherichia coli.

5. The preparation method according to any one of claims 1 to 4, characterized in that, In step (1), the co-substrate is glucose or sodium formate, and the amount used is 1.2-4.0 times the molar amount of bio-based furan.

6. The preparation method according to claim 5, characterized in that, In step (1), when the co-substrate is glucose, calcium carbonate or NaOH needs to be added to maintain the pH at 7.0±1.

0.

7. The preparation method according to claim 5, characterized in that, The buffer solution in step (1) is a phosphate buffer with a concentration of 50-200 mmol / L and a pH of 6.0-8.

0.

8. The preparation method according to any one of claims 1 to 4, characterized in that, The titanium-silicon molecular sieve used in step (2) is TS-1, and the dosage is 5-20 g / L.

9. The preparation method according to any one of claims 1 to 4, characterized in that, The oxidase in step (2) is glucose oxidase or formate oxidase, and the dosage is 0.01-1 g / L; the amount of hydrogen peroxide is 1.1-2.0 times the initial molar amount of bio-based furan; the amount of hydrazine in step (3) is 1.1-2.0 times the initial molar amount of bio-based furan.

10. The preparation method according to claim 9, characterized in that, The reaction conditions for step (1) are 30±10 ℃ and 150±50 rpm for 2±1 h; the reaction conditions for step (2) are 40±20 ℃ and 150±50 rpm for 1-12 h; the reaction conditions for step (3) are 4±4 ℃ and 250±50 rpm for 5±3 min.

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