A process for the preparation of pyrazolotriazines

Abstract

The present application relates to a kind of preparation method of pyrazole triazine, using Pseudomonas mosselii 923 strain fermentation, to prepare to obtain pyrazole triazine.Pseudomonas mosselii 923 strain fermentation conditions are as follows: in culture medium, under the condition of 28~32 ℃, 200~250rpm is fermented 30~40h, pyrazole triazine is contained in fermentation liquor, pyrazole triazine in fermentation liquor is extracted using ethyl acetate, again, the organic phase of ethyl acetate is rotary evaporated, after drying, obtain pyrazole triazine crude extract;Pyrazole triazine crude extract is dissolved with methanol and added C18 reverse phase silica gel, is made into methanol-silica gel homogenate, after being packed glass chromatography column, using mobile phase is eluted, purple phase rotary evaporation is dried, is purified by HPLC, obtain pyrazole triazine pure product.The present application overcomes the shortcomings of complex steps, harsh conditions, low yield of chemical synthesis method, the biological activity of pyrazole triazine prepared by the method of the present application provides scientific clues and research ideas for the prevention and treatment of agricultural and medical diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for preparing pyrazolium triazine. Background Technology

[0002] Pyrazolo[4,3-e][1,2,4]triazine is a purple, polar heterocyclic molecule containing multiple nitrogen atoms. It belongs to the pyrazolo[4,3-e][1,2,4]triazine family of natural products, which also includes two other compounds, nostocine A and fluviol A. Pyrazolo[4,3-e]triazine was first isolated in 1972 by Japanese scientists Lindner, HJ, and Schaden, G, from *Pseudomonas fluorescens* var. *pseudoiodinum* (Lindner, HJ, Schaden, G. *Pyrazolo[4,3-e]triazin, ein neues heterocyclisches System aus Pseudomonas fluorescens var. *pseudoiodinum* [J]. Chemische Berichte. 1972, 105(6): 1949-1955.), and later it was discovered that the bacteria *Nostoc spongiaeforme* can also secrete pyrazolo[4,3-e][1,2,4]triazines (Mojzych, M. Cytotoxic activity of some pyrazolo[4,3-e][1,2,4]triazines against human cancer cell lines[J]. J Chem Soc Pak. 2011, 33(1): 123.). Compounds in this family are distinguished from other natural products by their bright colors, and the number of nitrogen atoms often exceeds that of carbon atoms. However, research on their synthesis has been relatively limited.

[0003] To date, research on the biological functions of pseudoiodinine has mainly focused on its derivatives, which have anticancer, antiviral and antitumor activities. Psydiodinine itself also has antiviral and antitumor activities and can be used as a drug against human sarcoma. It is also predicted to be able to treat atherosclerosis (Dembitsky, VM, Gloriozova, TA, Poroiko, VV Pharmacological and predicted activities of natural azocompounds[J]. Natural products and bioprospecting.2017,7(1):151-169.). Since pyrazole triazines are purine analogues, their structures have been modified to enable them to inhibit the activity of various enzymes. Utilizing the visible spectral properties of these derivatives, they can be used for chemotherapy of cancer and viruses, and have great application value in the fields of biological research and pharmacological research (Mojzych, M., Rykowski, A., Wierzchowski, J. Pyrazolo[4,3-e][1,2,4]triazines: Purine analogues with electronic absorption in the visible region[J].Molecules.2005,10(10):1298-1306.).

[0004] The core structure of pseudoiodinine, consisting of a six-membered azine ring and a five-membered azole ring, contains multiple nitrogen atoms, resulting in a very high energy barrier. This leads to complex chemical synthesis methods with demanding conditions. The reaction process requires the use of the toxic gas diazomethane, which is prone to explosion and is quite dangerous. Moreover, the yield of its chemical synthesis is relatively low (Kelly, TR, Elliott, EL, Lebedev, R. et al. Synthesis of the pyrazolo[4,3-e][1,2,4]triazine family of natural products: nostocine A, fluviol A, and pseudoiodinine[J]. Journal of the American Chemical Society. 2006, 128(17): 5646-5647.). Therefore, no in-depth research on the biosynthesis of pseudoiodinine has been conducted since then.

[0005] There are currently no literature reports on the production of pseudoiodinine using microbial fermentation. Summary of the Invention

[0006] Given the complex and demanding chemical synthesis methods of pyrazole triazine in existing technologies, this invention provides a method for preparing pyrazole triazine. This invention utilizes microbial fermentation, which will contribute to the efficient production and application of pyrazole triazine.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] This invention provides a method for preparing pyrazolium triazine, comprising the following steps: fermentation using Pseudomonas mosselii strain 923 to prepare pyrazolium triazine.

[0009] In this invention, the structure of the pyrazole triazine is as follows:

[0010]

[0011] In this invention, *Pseudomonas mosselii* 923 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number M 2018252, deposited on May 7, 2018, at the Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University). It is disclosed in patent CN108998389A.

[0012] In one embodiment of the present invention, the fermentation conditions of Pseudomonas mosselii strain 923 are as follows: fermentation in a culture medium at 28-32°C and 200-250 rpm for 30-40 h, and the fermentation broth contains pyrazolium triazine.

[0013] In one embodiment of the present invention, the fermentation conditions of Pseudomonas mosselii strain 923 are as follows: fermentation in a culture medium at 30°C and 220 rpm for 36 h, and the fermentation broth contains pyrazolium triazine.

[0014] In one embodiment of the present invention, the fermentation conditions of Pseudomonas mosselii strain 923 are as follows: using TSB medium.

[0015] In one embodiment of the present invention, pyrazolium is extracted from the fermentation broth after fermentation with Pseudomonas mosselii strain 923 to obtain pyrazolium.

[0016] In one embodiment of the present invention, the extraction method is as follows: pyrazole triazine is extracted from the fermentation broth using ethyl acetate, and then the organic phase of ethyl acetate is rotary evaporated and dried to obtain crude pyrazole triazine extract.

[0017] In one embodiment of the present invention, the amount of ethyl acetate used is equal to the volume of the fermentation broth. In this invention, pyrazolium triazine in the fermentation broth can be dissolved by an equal volume of ethyl acetate.

[0018] In one embodiment of the present invention, the rotary evaporation condition is 35°C.

[0019] In one embodiment of the present invention, the extraction method further includes the following steps: the crude extract of pyrazole triazine is dissolved in methanol and C18 reversed-phase silica gel is added to prepare a methanol-silica gel homogenate, which is then packed into a glass chromatography column and eluted with a mobile phase. The purple phase (absorption peak at 500 nm) is dried by rotary evaporation and purified by HPLC to obtain pure pyrazole triazine.

[0020] In one embodiment of the present invention, the mobile phase is a mixture of methanol and water (v / v) = 3:7-9:1.

[0021] In one embodiment of the present invention, the rotary evaporation condition is 35°C.

[0022] Compared with existing technologies, this invention provides a biological fermentation method for preparing pyrazolium triazine. This method overcomes the shortcomings of chemical synthesis methods, such as complex steps, harsh conditions, and low yield. The bioactivity of pyrazolium triazine prepared by this method provides scientific clues and research ideas for the prevention and treatment of diseases in agriculture and medicine. Attached Figure Description

[0023] Figure 1 Isolation and purification of pyrazole triazine: A: Determination of antibacterial activity of different extraction phases; B: TCL thin-layer chromatography detection; C: Effect on PXO99 A Determination of antibacterial activity; D: High-performance liquid chromatogram of the antibacterial compound; E: Ultraviolet absorption peak of the antibacterial compound. From Figure 1 As can be seen, the main antibacterial substance produced by P. mosselii 923 is present in the ethyl acetate phase, which contains a characteristic ultraviolet absorption peak at 500 nm.

[0024] Figure 2 LC-MS spectrum of pseudoiodinine.

[0025] Figure 3 : Pyrazole triazine 1 H-NMR spectrum.

[0026] Figure 4 : Pyrazole triazine 1 C-NMR spectrum.

[0027] Figure 5 HMBC spectrum of pseudoiodinine.

[0028] Figure 6 HSQC spectrum of pseudoiodinine.

[0029] Figure 7 Chemical structural formula of pyrazole triazine.

[0030] Figure 8 Crystal structure diagram of pyrazole triazine. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0032] The culture medium formulations used in the following examples are as follows:

[0033] (1) TSB medium: used for culturing Pseudomonas (28℃), the formula is as follows: 17g pancreatic digest of casein, 3g soybean digest, 2.5g glucose, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, dissolved in pure water, diluted to 1000mL, pH adjusted to 7.3±0.2, autoclaved at 121℃ for 15min. For TSB solid medium, 15g agar powder needs to be added.

[0034] (2) Beef extract peptone medium NA (g / L): 3g beef extract, 5g polypeptone, 10g sucrose, 1g yeast extract, 15g agar powder, add water to dissolve, finally bring the volume to 1000mL, adjust the pH to 7.0-7.2, dispense and autoclave (121℃, 20min).

[0035] Example 1

[0036] Preparation of crude extract from fermentation broth of Pseudomonas mosselii 923

[0037] In this embodiment, *Pseudomonas mosselii* 923 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number M 2018252, deposited on May 7, 2018, at the Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University). It is disclosed in patent CN108998389A.

[0038] Pseudomonas mosselii 923 (hereinafter referred to as strain 923 or P. mosselii 923) was activated by streaking on TSB solid medium after being removed from the -80℃ freezer and cultured overnight at 30℃. After single colonies grew, a single colony was picked and inoculated into 4 mL of TSB liquid medium and cultured at 30℃ and 220 rpm for 12 h to obtain the fermentation seed culture of 923. The seed culture was transferred to a 250 mL Erlenmeyer flask containing 50 mL of TSB liquid medium at a volume ratio of 1:100 and cultured at 30℃ and 220 rpm with shaking for 36 h. At room temperature, the fermentation broth of P. mosselii 923 was extracted three times consecutively with equal volumes of ethyl acetate, petroleum ether, and n-butanol, respectively, at a 1:1 volume ratio. After thorough mixing and standing for 15 min, the mixture separated into organic and aqueous phases. The organic phases were then combined and dried by rotary evaporation at 35°C to obtain the crude extract. 500 μL of methanol (100X concentrate) was used to dissolve the crude extract for antibacterial activity testing. The aqueous phase was freeze-dried to a powder state and dissolved in 1 mL of methanol (100X concentrate). Plate antibacterial assays were used to determine the components containing the antibacterial active compounds, with methanol and the corresponding extractants serving as negative controls. The results showed that the main pyrazolite produced by P. mosselii 923 was concentrated in the ethyl acetate phase (…). Figure 1 A).

[0039] The active components were then subjected to large-scale batch fermentation, totaling 150L. After thorough extraction with ethyl acetate, the organic phases were combined to obtain a crude extract, which was used for subsequent compound separation and purification experiments.

[0040] Example 2: Reversed-phase silica gel column chromatography separation of crude extract from pyrazole triazine.

[0041] (1) Sample mixing: The crude extract obtained by the above ethyl acetate phase extraction was dissolved in methanol, and an appropriate amount of C18 reversed-phase silica gel was added and stirred evenly. The mixture was then dried by rotary evaporation at 35°C.

[0042] (2) Column packing: Measure about 30.0g of C18 reversed silica gel powder in a clean small beaker, add 2 times the volume of methanol, and stir continuously with a glass rod until the mixture is homogeneous to make a methanol-silica gel slurry.

[0043] Beforehand, firmly press the bottom of the glass chromatography column with cotton to prevent silica gel leakage. Then, slowly pour the methanol-silica gel homogenate into the column in one go, stirring gently with a thin iron wire while pouring. Be careful not to generate air bubbles during this process. Stop packing the column when the methanol-silica gel homogenate is about 9-10 cm from the top of the column opening.

[0044] (3) Equilibration: Use 30% methanol to equilibrate the silica gel column. Fix the glass chromatography column on the support and keep it vertical overnight to allow the column to settle completely. Do not vibrate it during this period. To prevent methanol from evaporating, the end of the chromatography column can be sealed with sealing film.

[0045] (4) Sample loading: Slowly add the powder prepared in step (1) into the tube opening, making sure the column surface is neat so as to facilitate subsequent gradient separation.

[0046] (5) Elution: Elution was performed sequentially using a mobile phase of methanol:water (v / v) = 1:1, with the flow rate controlled at around 1 mL / min.

[0047] (6) Collecting components: Based on the different colors of different compounds in the chromatography column, use 250mL conical flasks to collect the components eluted from each gradient.

[0048] (7) TLC test: Each collected sample was subjected to TCL thin-layer chromatography for spot testing. Samples were appropriately combined based on the migration positions of different components. The collected samples were rotary dried at 35°C and then stored at -80°C for subsequent antibacterial and HPLC detection. Results are as follows: Figure 1 As shown in BCD, the antibacterial substance is component 2-2, which contains a characteristic ultraviolet absorption peak at 500 nm.

[0049] Example 3: Mass spectrometry and nuclear magnetic resonance analysis of pyrazole triazine

[0050] The most active fraction obtained by reversed-phase silica column chromatography was dissolved in methanol and centrifuged at 12,000 rpm at 4°C for 20 min. Further purification and large-scale preparation were then performed using a preparative liquid chromatography-mass spectrometry (LC-MS) C18 (20.0*250 mm, 5 μm) system. Mobile phase: methanol / water; flow rate: 2 mL / min; detection wavelengths: 210 nm, 250 nm, 295 nm, 500 nm; injection volume: 80-100 μL. Molecular weight was determined using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS).

[0051] The prepared pure compound was dissolved in deuterated methanol, and its carbon, proton, and two-dimensional NMR spectra were analyzed using an AVANCE NEO 600MHz nuclear magnetic resonance analyzer. The spectra were then interpreted. The results are as follows: Figures 2-6 As shown.

[0052] Example 4: Crystal structure analysis of pyrazole triazine

[0053] To further determine the chemical structure of this antibacterial substance, X-ray crystal diffraction experiments were performed on pseudoiodinine, and its crystal structure was analyzed based on the measured data. The results are shown in Table 1 and... Figure 7 , Figure 8 As shown.

[0054] The structure of pyrazole triazine is shown below:

[0055]

[0056] The structural information of pyrazole triazine is as follows:

[0057] Mp 120–123℃(lit.112℃).1H NMR(400MHz,CD3OD,δ):4.27(s,3H),4.41(s,3H),8.95(s,1H).13C NMR(125MHz,CD3OD,δ):43.0,57.6,139.0,142.6,146.0,160.7.IR(NaCl,thin film)(cm-1):2988,2924,2851,1586,1537.UV(CH2Cl2)λmax(logε):230(3.6),255(3.5),295(3.2),533 (2.6).UV(EtOH)λmax(logε):220(3.7),254(3.6),295sh(3.4),520(2.6).HRMS-ESI(m / z):[M+H]+calcd for C6H8N5O,166.0729; found,166.0737.

[0058] Table 1. Crystal structure analysis data of pyrazole triazine (pseudoiodinine)

[0059]

[0060]

[0061] Example 5: Different isolated components against PXO99, the pathogen of rice bacterial blight. A Antibacterial activity assay

[0062] PXO99 A Inoculated into NA liquid medium and incubated overnight at 28°C and 180 rpm in a shaker. 200 μL of bacterial suspension was then mixed thoroughly with the NA medium. After the plates solidified, 7 mm diameter Oxford cups were evenly affixed to the NA plates, each containing 50 μL of a different liquid composition. Each treatment was repeated in triplicate. The plates were incubated at 28°C for 24 hours, and the presence of inhibition zones was observed. Results are as follows: Figure 1 As shown. This illustrates the effect of pyrazole triazine on rice bacterial blight pathogen PXO99. A It has antibacterial activity.

[0063] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A process for the preparation of a pyrazolotriazine characterized in that, Using pseudomonas moorei Pseudomonas mosselii 923 strains to ferment to produce pyrazolotriazine; Pseudomonas moorei Pseudomonas mosselii 923, which was preserved in China Center for Type Culture Collection (CCTCC) on May 7, 2018, and the preservation number is CCTCC No: M 2018252.

2. The method for preparing pyrazolium triazine according to claim 1, characterized in that, Pseudomonas moorei Pseudomonas mosselii 923The conditions for fermentation of the strain are: fermentation at 28-32 °C, 200-250 rpm in the culture medium for 30-40 h, and the fermentation broth contains pyrazolotriazine.

3. The method for preparing pyrazolium triazine according to claim 2, characterized in that, pseudomonas moorei Pseudomonas mosselii 923The conditions for the fermentation of the strain were 30°C, 220 rpm for 36 h in a culture medium containing pyrazolotriazine in the fermentation broth.

4. The method for preparing pyrazolium triazine according to claim 2, characterized in that, pseudomonas moorei Pseudomonas mosselii 923The conditions for fermentation of the strain were: TSB medium was used.

5. The method for preparing a pyrazole triazine according to claim 1, characterized in that, Using pseudomonas moorei Pseudomonas mosselii 923After fermentation of the strain, the pyrazolotriazine is extracted from the resulting fermentation broth to obtain the pyrazolotriazine.

6. The method for preparing pyrazolium triazine according to claim 5, characterized in that, The extraction method is: using ethyl acetate to extract pyrazole triazine in fermentation liquor, then rotary evaporation of ethyl acetate organic phase, drying to obtain pyrazole triazine crude extract.

7. The method for preparing a pyrazole triazine according to claim 6, characterized in that, The amount of ethyl acetate is equal to the volume of fermentation liquor.

8. The method for preparing pyrazolium triazine according to claim 6, characterized in that, The rotary evaporation condition is 35℃.

9. The method for preparing a pyrazole triazine according to claim 6, characterized in that, The extraction method further comprises the following steps: pyrazole triazine crude extract is dissolved in methanol and added with C18 reverse phase silica gel to prepare methanol-silica gel homogenate, then the homogenate is loaded into a glass chromatography column, elution is carried out using mobile phase, the purple phase is rotary evaporated and dried, and pyrazole triazine pure product is obtained through HPLC purification.

10. The method for preparing pyrazolium triazine according to claim 9, characterized in that, The rotary evaporation condition is 35℃.

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