A process for the preparation of artemiseen and desoxyartemisin B from artemisin B

Abstract

The application discloses a method for preparing artemisitene (ARE) and deoxyarteannuin B (DB) from arteannuin B (ART-B), wherein ART-B is used as a reaction substrate, a Fenton oxidation reaction system is constructed by adding ferrous salt and hydrogen peroxide in a water-methanol mixed solvent system, and a UVA light is used to assist in a catalytic oxidation reaction, so that the ARE and DB products are generated. The application realizes specific directional conversion of ART-B to ARE and DB by using a light-Fenton synergistic oxidation system for the first time, confirms that ART-B is a natural synthesis precursor of ARE, and proves an oxidation conversion path of ART-B to ARE, and the whole reaction system has the advantages of mild conditions, clear conversion path, controllable product selectivity and the like, and provides a new technical method and a key theoretical basis for artificial synthesis of artemisitene and analogues thereof, deep analysis of a biosynthesis path of artemisinin compounds, and innovative application of the Fenton reaction in the field of directional structure conversion of natural products.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and organic synthesis technology, specifically relating to a method for preparing artemisitene (ARE) and deoxyarteannuin B (DB) from artemisinin B (ART-B). Background Technology

[0002] Artemisia annua L. is a traditional Chinese medicine for treating malaria. Its active ingredient, artemisinin (ART), is a sesquiterpene lactone compound with a unique peroxide bridge structure and is currently a core effective drug for the clinical treatment of malignant malaria. Artemisitene (ARE) is highly homologous to artemisinin in structure, differing only in the double bond structure at the C11-C13 positions. It retains the key peroxide bridge core structure that enables artemisinin's activity. In addition to its antimalarial potential, ARE has also been shown to possess good anti-inflammatory and antitumor pharmacological activities, making it extremely valuable for medicinal development.

[0003] Existing research on natural product transformation has clarified the synthetic pathways of some artemisinin derivatives: dihydroartemisinic acid (DHAA) can be converted to artemisinin (ART) via photooxidation, and artemisinic acid (AA) can be converted to artemisinin B (ART-B) via photooxidation. However, the oxidation conversion mechanism and synthetic pathway of ART-B are not yet clear, the biosynthetic precursors of ART are not well traced, and there is a lack of efficient and mature chemical synthesis processes, which greatly limits its large-scale preparation, in-depth analysis of pharmacological mechanisms, and development and application of new drugs.

[0004] The Fenton reaction can be achieved via Fe... 2+ Catalytic decomposition of hydrogen peroxide generates highly oxidizing hydroxyl radicals (·OH), enabling efficient oxidative modification of organic matter. The photo-Fenton system, constructed based on the principle of photocoupled synergistic catalysis, can further enhance catalytic oxidation efficiency and is currently widely used in the degradation of environmental pollutants. However, this highly efficient oxidation technology has not yet been applied to the directional structural transformation of artemisinin-type sesquiterpene natural products and the research on precursor traceability.

[0005] Therefore, developing a new, efficient, and mild conversion process for the directional preparation of ARE using ART-B as a raw material is of great research significance and application prospect for filling the research gap in the chemical conversion process and biosynthetic pathway of artemisinin derivatives. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a method for preparing ARE and DB from ART-B, which utilizes a photo-Fenton synergistic oxidation system to achieve the specific directional conversion of ART-B into ARE and DB.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing artemisinin and deoxyartemisinin B from artemisinin B includes the following steps:

[0009] (1) Using artemisinin B as a substrate, ferrous salt and hydrogen peroxide were added to a water-methanol mixed solvent, the pH of the system was adjusted to acidic by acid, and the mixture was stirred to obtain Fenton reaction solution.

[0010] (2) The Fenton reaction solution was irradiated with a UVA ultraviolet light source to carry out the Fenton oxidation reaction. After the reaction was completed, a crude product containing artemisinin and deoxyartemisinin B was obtained.

[0011] (3) The crude product was separated and purified by preparative high performance liquid chromatography, the target peak fraction was collected and concentrated and dried to obtain artemisinin and deoxyartemisinin B, respectively.

[0012] Preferably, in step (1), the volume ratio of water to methanol in the water-methanol mixed solvent is 2:1-4:1, more preferably 3:1.

[0013] Preferably, in step (1), the ferrous salt is FeSO4·7H2O with a molar concentration of 0.08-0.12 mM.

[0014] Preferably, in step (1), the molar concentration of hydrogen peroxide is 50-200 mM, more preferably 80-120 mM.

[0015] Preferably, in step (1), dilute sulfuric acid is used to adjust the pH of the system to 2.5-3.5.

[0016] Preferably, in step (2), the wavelength of the UVA ultraviolet light source is 350-400 nm and the reaction time is 15-60 min.

[0017] Preferably, in step (3), the conditions for the preparative high performance liquid chromatography are as follows: a C18 column is used; acetonitrile-water is used as the mobile phase at a volume ratio of 70:30, and isocratic elution is employed; the detection wavelength is 209 nm; the column temperature is 25-35℃; and the flow rate is 1-3 mL / min.

[0018] More preferably, the C18 chromatographic column has dimensions of 250 mm × 10.0 mm and 5 μm.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention is the first to utilize a photo-Fenton synergistic oxidation system to achieve the specific and directional conversion of ART-B to ARE and DB, confirming that ART-B is a natural synthetic precursor of ARE and elucidating the oxidative conversion pathway of ART-B to ARE. The entire reaction system has outstanding advantages such as mild conditions, high product selectivity, clear conversion pathway, and stable and controllable system. It provides a brand-new technical method and key theoretical basis for the artificial synthesis of artemisinin and its analogues, the in-depth analysis of the biosynthetic pathway of artemisinin-like compounds, and the innovative application of the Fenton reaction in the field of directional structural conversion of natural products. Attached Figure Description

[0021] Figure 1 Here is a structural diagram of ART, ARE, and DB;

[0022] Figure 2 This is a chromatogram obtained by HPLC-DAD detection;

[0023] Figure 3 Chromatograms and mass spectra detected by GC-MS;

[0024] Figure 4 HPLC chromatograms at different H2O2 concentrations;

[0025] Figure 5 HPLC chromatograms for different solvent systems;

[0026] Figure 6 These are HPLC chromatograms under different light exposure times. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Preparation and identification of artemisinin and deoxyartemisinin B

[0029] 1. Artemisinin and deoxyartemisinin B

[0030] (1) Using artemisinin B as a substrate, 0.1 mM FeSO4·7H2O and 100 mM hydrogen peroxide were added to 1 mL of water-methanol (3:1) mixed solvent. The pH of the system was adjusted to 3.0 with 1 μL of dilute sulfuric acid (6 M). The mixture was stirred and mixed to obtain the Fenton reaction solution.

[0031] (2) The Fenton reaction solution was irradiated with a long-wave ultraviolet (UVA) light source (365 nm) for 60 min to carry out the Fenton oxidation reaction. After the reaction was completed, a crude product containing artemisinin and deoxyartemisinin B was obtained.

[0032] (3) The crude product was separated and purified by preparative high-performance liquid chromatography (HPLC). The chromatographic peak fractions corresponding to artemisinin and deoxyartemisinin B were collected, the collected fractions were concentrated, and then dried under vacuum to obtain artemisinin (ARE) and deoxyartemisinin B (DB), respectively. GC-MS quantitative analysis showed that the ARE formation rate was 4.8% and the DB formation rate was 12.5%.

[0033] The conditions for the preparative high-performance liquid chromatography are as follows:

[0034] Column: YMC-Pack ODS-A C18 (250 mm × 10.0 mm, 5 μm);

[0035] Mobile phase: Acetonitrile-water mixed at a volume ratio of 70:30, eluted using isocratic elution;

[0036] Flow rate: 2 mL / min; column temperature: 30℃; detection wavelength: 209 nm; injection volume: 50 μL.

[0037] 2. Analysis and identification

[0038] The reaction product was extracted three times with ethyl acetate, filtered through a 0.22 μm organic filter membrane, dried under nitrogen, redissolved in methanol, and detected by GC-MS and HPLC-DAD.

[0039] The HPLC-DAD detection conditions were as follows: YMC-Pack ODS-A C18 column, acetonitrile (A)-water (B) gradient elution: 0-2 min: A = 30%; 2-10 min: A linearly increases to 70%; 10-25 min: A = 70%; 25-35 min: A linearly increases to 100%; 35-45 min: A = 100%; 45-45.1 min: A decreases to 30%; 45.1-50 min: A = 30%, detection wavelength 209 nm. A comparison with standards was performed. Figure 2 As shown, ART-B was subjected to Fenton reaction treatment under UVA irradiation, and compared with ARE and DB standards. Peaks 1 and 2 were generated, and their retention times were consistent with those of ARE and DB standards, thus confirming them as ARE and DB standards.

[0040] The GC-MS detection conditions were as follows: HP-5MS column, programmed temperature rise: initial temperature 60℃, hold for 1 min; rise to 170℃ at 12℃ / min, hold for 12 min; then rise to 300℃ at 10℃ / min, run for 3 min; ion source: EI source, 70 eV; extracted ion currents m / z 191.0 and m / z 232.1, retention time and mass spectrometry fragmentation were compared with the standard.

[0041] like Figure 3 As shown in Figure A, by comparing the GC-MS chromatograms of Fenton group ART-B under UVA irradiation and Fenton group ART-B without UVA irradiation (mass-to-charge ratio m / z 191.0), and comparing them with the ARE standard, the results showed that Fenton group ART-B under UVA irradiation showed a new peak at a retention time of 25.3 min, which was consistent with the retention time of the ARE standard peak. However, no such peak was generated in Fenton group ART-B without UVA irradiation, confirming that the generation of ARE is UV-dependent.

[0042] Figure 3 Figure B shows the fragment ion peaks of Fenton group ART-B and ARE standard under UVA irradiation, with a retention time of 25.3 min. The results show that the fragment ions of the two are consistent, indicating that the peak produced by Fenton group ART-B under UVA irradiation is ARE.

[0043] Figure 3 In the middle section, by comparing the GC-MS chromatograms of Fenton group ART-B under UVA irradiation and Fenton group ART-B without UVA irradiation (mass-to-charge ratio m / z 232.1), and comparing them with the DB standard, the results showed that Fenton group ART-B under UVA irradiation showed a new peak at a retention time of 21.7 min, which was consistent with the retention time of the DB standard peak.

[0044] Figure 3 The image shows the fragment ion peaks of Fenton group ART-B and DB standard under UVA irradiation, with a retention time of 21.7 min. The results show that the fragment ion similarity between the two is almost identical, indicating that the peak produced by Fenton group ART-B under UVA irradiation is DB.

[0045] Example 2: Optimization of reaction conditions

[0046] (1) Optimization of H2O2 concentration

[0047] To investigate the effect of H2O2 concentration on ART-B conversion, this invention uses H2O2 concentrations of 50 mM, 100 mM, 200 mM, and 400 mM for the reaction, and the peak areas of ART-B and its products are detected by HPLC-DAD.

[0048] The results are as follows Figure 4 As shown, when the concentration of H2O2 is 50 mM, due to insufficient ·OH, the oxidation ability is weak, the ART-B peak area is high, the conversion is incomplete, and the product conversion rate and yield are very low. When the concentration of H2O2 is 400 mM, it will lead to excessive degradation of ART-B and the product. When the concentration of H2O2 is 100 mM, the ·OH concentration is moderate, and it can be seen that the ARE and DB peak areas are the largest, and the substrate is retained to a greater extent, indicating that the ART-B conversion is sufficient and the degradation is minimal.

[0049] (2) Solvent optimization

[0050] This invention investigated the effects of pure water Fenton systems and water-methanol (3:1) Fenton systems on ART-B conversion, and the results are as follows: Figure 5 As shown, in the pure water system, ·OH has high stability, fast diffusion, and extremely strong oxidizing power, causing rapid degradation of ART-B and its products. The liquid phase is almost at the baseline, which is not conducive to the conversion of ART-B. The water-methanol (3:1) system can inhibit excessive oxidation, reduce the degradation of ART-B caused by the strong oxidizing power of ·OH, and the liquid phase shows a significant increase in the generation of new peaks, thus improving the directional conversion efficiency.

[0051] (3) Optimization of illumination time

[0052] This invention investigated the effect of UVA illumination time on ART-B conversion, setting illumination times of 5 min, 15 min, 30 min, 60 min, and 120 min, respectively. The results are as follows: Figure 6 As shown, in the initial stage of the reaction at 5 min, little ·OH was generated, and ART-B was almost not converted; while at 120 min, the liquid phase baseline was almost flat, indicating that the reaction time was too long and both the substrate and product were significantly degraded; compared with 15 min and 30 min, the peak areas of ARE and DB were the largest at 60 min, and the conversion reached the peak value, indicating that 60 min irradiation was the optimal for ART-B conversion in the Fenton reaction.

Claims

1. A method for preparing artemisinin and deoxyartemisinin B from artemisinin B, characterized in that, Includes the following steps: (1) Using artemisinin B as a substrate, ferrous salt and hydrogen peroxide were added to a water-methanol mixed solvent, the pH of the system was adjusted to acidic by acid, and the mixture was stirred to obtain Fenton reaction solution. (2) The Fenton reaction solution was irradiated with a UVA ultraviolet light source to carry out the Fenton oxidation reaction. After the reaction was completed, a crude product containing artemisinin and deoxyartemisinin B was obtained. (3) The crude product was separated and purified by preparative high performance liquid chromatography, the target peak fraction was collected and concentrated and dried to obtain artemisinin and deoxyartemisinin B, respectively.

2. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (1), the volume ratio of water to methanol in the water-methanol mixed solvent is 2:1-4:

1.

3. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (1), the ferrous salt is FeSO4·7H2O with a molar concentration of 0.08-0.12 mM.

4. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (1), the molar concentration of hydrogen peroxide is 50-200 mM, preferably 80-120 mM.

5. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (1), dilute sulfuric acid is used to adjust the pH of the system to 2.5-3.

5.

6. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (2), the wavelength of the UVA ultraviolet light source is 350-400 nm and the reaction time is 15-60 min.

7. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 1, characterized in that, In step (3), the conditions for the preparative high performance liquid chromatography are as follows: a C18 column is used; acetonitrile-water is used as the mobile phase at a volume ratio of 70:30, and isocratic elution is employed; the detection wavelength is 209 nm; the column temperature is 25-35℃; and the flow rate is 1-3 mL / min.

8. The method for preparing artemisinin and deoxyartemisinin B from artemisinin B according to claim 7, characterized in that, The C18 column has dimensions of 250 mm × 10.0 mm and a diameter of 5 μm.

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