A method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine

Abstract

The application relates to the fields of biological medicine and microbial conversion technology, and discloses a method for preparing an antitumor m-phenylenediamine derivative by using fungal bioconversion of m-phenylenediamine. The method uses cheap potato glucose liquid culture medium from agricultural and sideline products as a fermentation matrix, externally adds m-phenylenediamine into the fungal culture medium as a skeleton substrate for directional conversion, uses the metabolic enzyme system of abundant oxidases, reductases and hydroxylases of the fungus to perform accurate structure modification, does not need gene modification, performs bioconversion under normal temperature and pressure conditions, does not depend on high temperature, high pressure and noble metal catalysts, and can obtain a novel m-phenylenediamine derivative with significant antitumor cytotoxic activity, the IC 50 value of which is much lower than that of the raw material m-phenylenediamine IC 50 , and the m-phenylenediamine derivative can be used as an antitumor lead compound for subsequent drug research and development, and fills the domestic and foreign technical blank of preparing an antitumor derivative by using fungal bioconversion of m-phenylenediamine.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbial transformation technology, specifically a method for preparing antitumor m-phenylenediamine derivatives by utilizing fungal biotransformation of m-phenylenediamine. Background Technology

[0002] Malignant tumors pose a serious threat to human health. Existing clinical chemotherapy drugs generally suffer from significant toxic side effects, easy development of drug resistance, and environmentally unfriendly synthesis processes. Therefore, developing green, efficient, and low-toxicity novel anti-tumor lead compounds has become an urgent direction in drug research and development. Microbial biotransformation technology utilizes the metabolic enzyme systems such as oxidases, reductases, and hydroxylases contained in fungi and other microorganisms to precisely modify the structure of exogenous substrates under ambient temperature and pressure conditions. It has significant advantages such as high reaction selectivity, mild conditions, environmental friendliness, and few byproducts, and has gradually become an important technological route for the green preparation of small molecule derivatives. m-Phenylenediamine (CAS No. 108-45-2), as an readily available aromatic diamine chemical raw material, is inexpensive and readily available, but its inhibitory activity against tumor cell proliferation is extremely low (IC50). 50 >100 μM) lacks the necessary pharmacodynamic groups, making it unsuitable for direct use as an antitumor active substance. Chemical modification of its benzene ring typically requires multiple organic reactions, consuming large amounts of highly toxic organic solvents such as dichloromethane and DMF. Furthermore, it relies on precious metal catalysts and high-temperature, high-pressure conditions, resulting in numerous byproducts, difficult separation and purification, high costs for waste treatment, and significant equipment investment, making large-scale production extremely challenging. On the other hand, conventional microbial synthesis methods heavily depend on the inherent secondary metabolic capabilities of the strain, making it difficult to directionally synthesize derivatives with an exogenous m-phenylenediamine skeleton. Directed synthesis through genetic modification requires complex molecular biology operations, is time-consuming and costly, and the activity of the obtained product is unpredictable. To date, no publicly available technical schemes exist for the biotransformation of m-phenylenediamine by fungi to prepare antitumor derivatives, either domestically or internationally. To address the aforementioned problems, this invention provides a green, mild, and low-cost preparation process. It uses inexpensive potato glucose liquid culture medium derived from agricultural byproducts as the fermentation substrate, and leverages the abundant biological enzyme system of fungi to perform targeted biotransformation of m-phenylenediamine. Standardized shaker culture parameters are then used to stably produce a novel m-phenylenediamine derivative with potent antitumor cytotoxic activity. This effectively overcomes a series of technical challenges, including severe pollution from chemical synthesis, insufficient raw material activity, poor directionality of traditional microbial synthesis, and high preparation costs. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing antitumor m-phenylenediamine derivatives by utilizing fungal biotransformation of m-phenylenediamine. This method is green and mild, low-cost, easy to operate, and can directionally transform the m-phenylenediamine skeleton to obtain derivatives with high antitumor activity. Furthermore, the product can be stably produced through standardized culture. This method solves the problems of heavy pollution, high cost, and difficulty in scaling up chemical synthesis routes, low activity of raw materials that cannot be directly used for pharmaceutical purposes, and the inability of conventional microbial synthesis to directionally prepare specific exogenous skeleton derivatives.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine, comprising the following steps: S1. Preparation of potato glucose liquid culture medium: Cut fresh potatoes into small cubes, add distilled water and boil, then filter. Add glucose in a set ratio to the filtrate, stir, dilute with distilled water to volume and dispense into Erlenmeyer flasks. Sterilize under high pressure steam and cool to room temperature for later use. S2, Substrate addition: After the potato glucose liquid culture medium prepared in step S1 has cooled to room temperature, add the substrate m-phenylenediamine to the culture medium so that the m-phenylenediamine reaches the set final concentration in the culture medium, and shake thoroughly to mix. S3. Inoculation: Inoculate the mycelium of *Epipremnum aureum* into the potato glucose liquid culture medium containing m-phenylenediamine added in step S2. S4. Constant temperature shaking culture: Place the shake flasks inoculated in step S3 into a constant temperature shaking shaker and continue shaking fermentation under the set conditions to complete the biotransformation of the substrate and obtain a fermentation broth containing the transformation product. S5. Post-processing of fermentation broth: Collect all fermentation broth, add an equal volume of organic solvent ethyl acetate for extraction, repeat the extraction 3 times, combine the organic phases, and evaporate the organic phases under reduced pressure at a set temperature to obtain crude product. S6. Crude product separation and purification: The crude product obtained in step S5 is initially separated by silica gel column chromatography, the target fraction is collected, and then further purified by semi-preparative high performance liquid chromatography to obtain a high-purity target m-phenylenediamine derivative. S7. Structural identification: The target m-phenylenediamine derivative obtained in step S6 was identified using spectroscopic techniques such as proton nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry. S8. Activity Verification: The MTT / SRB assay was used to detect the inhibitory activity of the target m-phenylenediamine derivative purified in S6 on the cell proliferation of L-02, K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 cell lines, and the IC50 was calculated. 50 The value was set up, and a positive control group of doxorubicin was also set up.

[0005] Preferably, the preparation process of the potato glucose liquid culture medium in S1 is as follows: S1.1 Take 200 g of fresh potatoes, cut them into 1-2 cm³ cubes, add 500 mL of distilled water, boil for 10 minutes, filter with 4-6 layers of sterile gauze, add 20 g of glucose to the filtrate, and stir at 100-200 rpm to dissolve it completely. S1.2. Dilute to 1 L with distilled water, dispense 100 mL into 500 mL pre-sterilized conical flasks, autoclave at 121 °C for 20 min, and cool to room temperature before use.

[0006] Preferably, in S2: the substrate m-phenylenediamine is dissolved in dimethyl sulfoxide to prepare a 0.2 g / mL stock solution, which is then filtered through a 0.22-micron filter membrane for sterilization, and 100 μL is added to the culture medium to bring the final concentration to 0.2 g / L.

[0007] Preferably, the constant temperature shaker biotransformation culture conditions in S4 are set as follows: at a temperature of 28°C and a shaking speed of 180 r / min, the fermentation is carried out continuously for 6 days.

[0008] Preferably, in step S5, the organic phase is subjected to reduced pressure rotary evaporation at 30°C to remove the solvent.

[0009] Preferably, the crude product separation conditions in S6 are as follows: preliminary separation is carried out under silica gel column chromatography with a 300-400 mesh mesh and an eluent system of dichloromethane:methanol = 30:1.

[0010] Preferably, the crude product purification in S6 involves: after preliminary separation, the target fraction is collected and further purified by semi-preparative high-performance liquid chromatography under reversed-phase C18, 10×250 mm, 5 μm, 3 mL / min, 254 nm, methanol:water = 55:45 conditions.

[0011] Preferably, in step S7, the structure identification involves performing a structure identification on the target m-phenylenediamine derivative obtained in step S6, and its chemical structure is as follows:

[0012] In the formula, the product structure is methyl acetate [4-formyl-1-[3-(3-formyl-4-hydroxymethyl-2-methyl-1H-pyrrolo-1-yl)phenyl]-5-methyl-1H-pyrrolo-3-yl] ester.

[0013] Preferably, the m-phenylenediamine derivative exhibits an IC50 inhibitory effect on K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 tumor cell lines. 50 The values ​​reached 0.07-0.51 μM.

[0014] Preferably, the m-phenylenediamine derivative is used in the field of preparing drugs for the prevention and treatment of malignant tumors.

[0015] Compared with the prior art, the present invention provides a method for preparing antitumor m-phenylenediamine derivatives by utilizing fungal biotransformation of m-phenylenediamine, which has the following beneficial effects: 1. This invention utilizes the exogenous addition of m-phenylenediamine to fungal culture medium as a scaffold substrate for targeted transformation, and leverages the abundant metabolic enzyme systems of fungi, such as oxidases, reductases, and hydroxylases, to precisely modify its structure. This achieves the beneficial effect of obtaining novel m-phenylenediamine derivatives that fungi cannot synthesize independently and that possess significant antitumor cytotoxic activity without genetic modification. Its IC50 value is [not specified in the original text]. 50 The value is much lower than that of the raw material m-phenylenediamine IC. 50 With a concentration of >100 μM, it can serve as an anti-tumor lead compound for subsequent drug development, filling the technological gap in the domestic and international production of anti-tumor derivatives through fungal biotransformation of m-phenylenediamine.

[0016] 2. This invention uses inexpensive potato glucose liquid culture medium derived from agricultural by-products as the fermentation substrate, and carries out biotransformation under normal temperature and pressure conditions. It does not rely on high temperature and pressure or precious metal catalysts, and does not use DMF, a highly toxic organic solvent. It achieves the green and environmentally friendly benefits of mild reaction conditions, low energy consumption, minimal emissions of waste, significantly reduced environmental burden, and low production safety risks, effectively replacing the high-pollution and high-cost chemical synthesis route.

[0017] 3. By adopting conventional shaker culture and a simple product separation process, combined with standardized shaker culture parameters, this invention achieves the beneficial effects of simple operation, few by-products, low separation and purification difficulty, low fermentation raw material cost, and stable and reproducible production of the target product, making it easy to scale up production and promote application. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the reaction principle of the present invention; Figure 2 The NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 3 The NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 4 The COSY NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention; Figure 5 The HSQC NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 6 The HMBC NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention; Figure 7 This is a high-resolution mass spectrum of the m-phenylenediamine derivative from Example 1 of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, 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.

[0020] Please see Figures 1-7 A method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine includes the following steps: S1. Preparation of potato-glucose liquid culture medium: Take 200 g of fresh potatoes, cut them into small pieces (1-2 cm³), add 500 mL of distilled water, boil for 10 minutes, and then filter through 4-6 layers of sterile gauze to remove solid residue. Add 20 g of glucose to the filtrate and stir until fully dissolved. Make up to 1 L with distilled water, dispense 100 mL of the solution into 500 mL pre-sterilized Erlenmeyer flasks, autoclave at 121℃ for 20 min, and cool to room temperature before use. S2, Substrate Addition: After the potato-glucose liquid culture medium prepared in step S1 has cooled to room temperature (25-30℃), add the substrate m-phenylenediamine to the culture medium, dissolving the substrate m-phenylenediamine in dimethyl sulfoxide (DMSO) to prepare a 0.2 g / mL stock solution. After sterilization by filtration through a 0.22-micron filter membrane, take 100 μL and add it to the culture medium to bring the final concentration to 0.2 g / L. Shake thoroughly to mix. The raw materials in this step are simple. Potatoes and glucose are both bulk agricultural by-products, which are easy to purchase and inexpensive. The cost of preparing the culture medium is much lower than that of artificially synthesized inorganic salt culture medium, making it suitable for industrial-scale batch fermentation production. S3, Inoculation with fungal strain: Inoculate *Lactobacillus verrucae* Myrothecium verrucaria The mycelium of HPU-ZMN (GenBank no.PV082666) was inoculated into potato glucose liquid medium containing m-phenylenediamine in step S2. S4. Constant temperature shaking culture: Place the shake flasks inoculated in step S3 in a constant temperature shaking shaker and continue shaking fermentation for 6 days at a temperature of 28℃ and a shaking speed of 150-220 r / min to complete the biotransformation of the substrate and obtain a fermentation broth containing the transformation product. S5. Post-processing of fermentation broth: Collect all fermentation broth, add an equal volume of ethyl acetate (an organic solvent) for extraction, repeat the extraction three times, and combine the organic phases. Remove the solvent from the organic phase by rotary evaporation under reduced pressure at 30°C to obtain the crude product; S6. Crude product separation and purification: The crude product obtained in step S5 was initially separated by silica gel column chromatography (300-400 mesh, eluent system: dichloromethane:methanol = 30:1). The target fraction was collected and then further purified by semi-preparative high performance liquid chromatography (reversed phase C18, 10×250 mm, 5 μm, 3 mL / min, 254 nm, methanol:water = 55:45) to obtain high-purity target m-phenylenediamine derivative. S7. Structural identification: Hydrogen nuclear magnetic resonance spectroscopy was used (H1N1 NMR spectroscopy). 1 H NMR, carbon nuclear magnetic resonance (NMR) 13 The target m-phenylenediamine derivative obtained in step S6 was structurally identified using spectroscopic techniques such as C NMR and high-resolution mass spectrometry (HR-ESI-MS), confirming the chemical structural formula (I) of the m-phenylenediamine derivative as follows:

[0021] In formula (I), the product structure is methyl acetate [4-formyl-1-[3-(3-formyl-4-hydroxymethyl-2-methyl-1H-pyrrolo-1-yl)phenyl]-5-methyl-1H-pyrrolo-3-yl], which was confirmed by high-resolution mass spectrometry and nuclear magnetic resonance, as shown in the following results. Figure 2-7 .

[0022] Figure 2 The NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 3 The NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 7 The high-resolution mass spectrum of the m-phenylenediamine derivative in Example 1 of this invention is shown in Table 1. Table 1 shows the NMR 1H and NMR 1C spectra of the m-phenylenediamine derivative of this invention. Figure 2-3 As shown in Tables 7 and 1. High-resolution mass spectrometry (HRESIMS) in... m / z [M+H] is given at position 395.1598. + Peak (C) 22 H 23 Based on the N2O5 (395.1601) and the NMR 1H and NMR 1C spectra, the molecular formula of this compound is inferred to be C. 22 H 22 N₂O₅, with an unsaturation degree of 13. 1D NMR data (NMR H and NMR C spectra) show that this compound contains 3 methyl groups, 2 methylene groups, and 6... sp 2 Methyl group, 9 quaternary carbons and 2 formyl carbons (see Table 1).

[0023] Table 1. NMR data of the compounds [ 13 C (150 MHz), 1 H (600 MHz), DMSO-d 6.] 1 138.8, C - 2 123.3, CH 7.61, t (1.9) 3 138.5, C - 4 125.5, CH 7.56, d (1.9) 5 130.7, CH 7.72, t (8.0) 6 125.6, CH 7.57, d (1.9) 2′ 139.7, C - 3′ 120.3, C - 4′ 118.9, C - 5′ 123.2, CH 7.15, s 6′ <![CDATA[10.9, CH3]]> 2.48, s 7′ 186.0, CH 9.97, s 8′ <![CDATA[58.3 CH2]]> 5.17, s 9′ 170.2, C - 10′ <![CDATA[20.7, CH3]]> 2.02, s 2′′ 139.3, C - 3′′ 120.4, C - 4′′ 126.3, C - 5′′ 120.9, CH 6.97, s 6′′ <![CDATA[10.6, CH3]]> 2.48, s 7′′ 186.4, CH 9.96, s 8′′ <![CDATA[56.2 CH2]]> 4.60, d (4.3) Figure 4 The COSY NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention; Figure 5 The HSQC NMR spectrum of the m-phenylenediamine derivative of Example 1 of this invention is shown below. Figure 6 This is the HMBC NMR spectrum of the m-phenylenediamine derivative from Example 1 of the present invention. Figure 4-6 As shown, by analyzing and comparing the structure of m-phenylenediamine, and combining the 2D NMR data (COSY, HSQC and HMBC NMR spectra), the structural formula is as follows, which confirms the correctness of the planar structure.

[0024]

[0025] S8. Activity Verification: The MTT or SRB assay was used to detect the inhibitory activity of the target m-phenylenediamine derivative purified in step S6 on the cell proliferation of K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 cell lines, and the IC50 was calculated. 50 The value was set up, and a positive control group of doxorubicin was also set up.

[0026] By providing the aforementioned green, mild, and safe fungal biotransformation preparation process to replace the highly polluting and costly chemical synthesis route, the green preparation of m-phenylenediamine derivatives can be achieved, ultimately yielding novel m-phenylenediamine derivatives with high tumor cytotoxic activity and improving their IC50 value. 50 The value is significantly lower than that of the raw material m-phenylenediamine, which can be used as an anti-tumor lead compound for subsequent drug development; by using low-cost potato glucose liquid culture medium derived from agricultural by-products as the fermentation substrate, the cost of fermentation raw materials is greatly reduced, and the stable and reproducible production of the target product is achieved through standardized shaker culture parameters.

[0027] The cytotoxic activity of the m-phenylenediamine derivatives was verified, as shown in Table 2. Table 2. Cell toxicity data of m-phenylenediamine (IC50) 50 μM) K562 0.073 NCI-H446 0.26 NCI-H446 / EP 0.26 MDA-MB-231 0.51 The advantages are: This invention uses potato glucose liquid culture medium as the fermentation substrate and exogenously added m-phenylenediamine as the substrate to generate m-phenylenediamine derivatives through fungal biotransformation. The prepared m-phenylenediamine derivatives show high IC50 values ​​for K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 tumor cell lines. 50 The value reached 0.07-0.51 μM, compared to the raw material m-phenylenediamine (IC50). 50The antitumor activity was increased by more than 200 times (>100 μM), showing outstanding drug development potential. Furthermore, the entire biotransformation process can be completed using only a conventional constant temperature shaker (low energy consumption due to 28℃ low-temperature culture). The operation process is simple, easy to standardize and scale up. No highly corrosive or toxic organic reagents are used throughout the process (only ethyl acetate is used in post-processing), resulting in a significant reduction in waste emissions. There are no dangerous high-temperature or high-pressure reactions, and the production safety is high.

[0028] The use of m-phenylenediamine derivatives or pharmaceutically acceptable salts, solvates or prodrugs as shown in formula (I) above in the preparation of drugs for the prevention and / or treatment of malignant tumors.

[0029] The preparation method of the present invention is applied to the following Example 1, including: Example 1 (Standard conversion process of this invention) (1) Preparation of culture medium: Take 200 g of fresh potatoes, cut them into pieces, add 500 mL of distilled water and boil for 10 min, filter with 6 layers of sterile gauze, add 20 g of glucose to the filtrate, make up to 1000 mL, dispense into 500 mL Erlenmeyer flasks that have been sterilized at high temperature (each flask contains 100 mL of liquid), autoclave at 121℃ for 20 min, and cool to room temperature.

[0030] (2) Substrate addition: Under aseptic conditions, add m-phenylenediamine to each culture medium to a final concentration of 0.2 g / L and shake thoroughly to mix.

[0031] (3) Inoculation: Myrothecium verrucaria An appropriate amount of HPU-ZMN mycelium was inoculated into the above-mentioned culture medium.

[0032] (4) Biotransformation culture: Place the inoculated shake flask in a constant temperature shaker at 28℃ and 180 r / min for 6 days.

[0033] (5) Post-processing and purification: 1 L of fermentation broth was collected and extracted three times with an equal volume of ethyl acetate. The organic phases were combined and evaporated under reduced pressure at 30 °C to obtain 0.6 g of crude product. The crude product was separated by silica gel column chromatography (300-400 mesh, eluent system: dichloromethane: methanol = 30:1) and then purified by semi-preparative HPLC (reversed-phase C18, 10×250 mm, 5 μm, 3 mL / min, 254 nm, methanol: water = 55:45) to obtain 7.1 mg of pure m-phenylenediamine derivative, with a yield of 1.0%.

[0034] (6) Structural identification: The product structure was identified by ¹H NMR, ¹³C NMR and HR-MS as methyl acetate [4-formyl-1-[3-(3-formyl-4-hydroxymethyl-2-methyl-1H-pyrrole-1-yl)phenyl]-5-methyl-1H-pyrrole-3-yl] ester.

[0035] (7) Activity assay: The cytotoxic activity (IC50) of the obtained derivatives against K562, NCI-H446, NCI-H446 / EP and MDA-MB-231 tumor cell lines was detected by MTT or SRB assay. 50 The value reached 0.07-0.51 μM, while the IC50 of the raw material m-phenylenediamine under the same conditions was... 50 > 100 μM.

[0036] Example 2 (Comparative Experiment of Different Fungal Strains) Selected strains Clonostachys rogersoniana , Penicillium citrinum , Eucasphaeria capensis and Fusarium verticillioides Transformation was performed under the same conditions, but no fungi were obtained. Myrothecium verrucaria The transformation of m-phenylenediamine derivatives by HPU-ZMN indicates that this transformation reaction can only occur in specific strains.

[0037] Example 3 (m-phenylenediamine derivatives capable of undergoing the same transformation) Comparative experiments with different m-phenylenediamine derivatives used 4-chloro-1,3-phenylenediamine, 2,4-diaminotoluene, 4-bromo-1,3-phenylenediamine, 4-fluoro-1,3-diaminobenzene, methyl 3,5-diaminobenzoate, and 3,5-diaminotrifluorotoluene as substrates instead of m-phenylenediamine. The remaining operations were exactly the same as in Example 1. HPLC analysis showed that significant conversion product peaks with UV absorption were detected in each reaction system.

[0038] The above results indicate that changing the substrate structure can correspondingly alter the structure of the conversion product, and this conversion reaction exhibits good substrate adaptability to a variety of m-phenylenediamine derivatives.

[0039] Comparative Example 1 (Changing the Culture Medium Components) When 1.0% peptone and 1.0% corn steep liquor powder were added to the original culture medium, and the rest of the operation was exactly the same as in Example 1, the fermentation product changed and no target conversion product was generated. The results indicate that potatoes should be the only natural culture medium component besides glucose, and adding other natural culture media is detrimental to the conversion.

[0040] Comparative Example 2 (blank control without the addition of m-phenylenediamine) Except for the absence of the m-phenylenediamine substrate, the operation was exactly the same as in Example 1. The results showed that no target m-phenylenediamine derivative was detected in the fermentation product without the addition of substrate, indicating that the target product did indeed originate from the biotransformation of m-phenylenediamine, rather than from the metabolism of the fungus itself.

[0041] In summary, based on structural identification by ¹H NMR, ¹³C NMR, and HR-MS, activity testing by MTT / SRB method, comparative experiments with different fungal strains, adaptability experiments with different substrates, comparative experiments with culture medium components, and a blank control experiment without substrate addition, the target product obtained in this invention has been confirmed as methyl acetate [4-formyl-1-[3-(3-formyl-4-hydroxymethyl-2-methyl-1H-pyrrolo-1-yl)phenyl]-5-methyl-1H-pyrrolo-3-yl], which exhibits significant inhibitory activity (IC50) against the proliferation of K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 tumor cell lines. 50 = 0.07-0.51 μM), far superior to the raw material m-phenylenediamine (IC50). 50 > 100 μM), and this transformation reaction is strain-specific, occurring only when... Myrothecium verrucaria The HPU-ZMN culture medium exhibits good adaptability to various m-phenylenediamine derivative substrates. It also confirms that potato is the only natural component in potato-glucose liquid culture medium besides glucose. The addition of nitrogen sources such as peptone and corn steep liquor is not conducive to the formation of the target product. Without the addition of substrate, the fungus itself does not produce the target product, thus confirming that the target product originates from the directed fungal biotransformation of exogenous m-phenylenediamine rather than the fungal metabolite itself.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine, characterized in that, Includes the following steps: S1. Preparation of potato glucose liquid culture medium: Cut fresh potatoes into small cubes, add distilled water and boil, then filter. Add glucose in a set ratio to the filtrate, stir, dilute with distilled water to volume and dispense into Erlenmeyer flasks. Sterilize under high pressure steam and cool to room temperature for later use. S2, Substrate addition: After the potato glucose liquid culture medium prepared in step S1 has cooled to room temperature, add the substrate m-phenylenediamine to the culture medium so that the m-phenylenediamine reaches the set final concentration in the culture medium, and shake thoroughly to mix. S3. Inoculation: Inoculate the mycelium of *Epipremnum aureum* into the potato glucose liquid culture medium containing m-phenylenediamine added in step S2. S4. Constant temperature shaking culture: Place the shake flasks inoculated in step S3 into a constant temperature shaking shaker and continue shaking fermentation under the set conditions to complete the biotransformation of the substrate and obtain a fermentation broth containing the transformation product. S5. Post-processing of fermentation broth: Collect all fermentation broth, add an equal volume of organic solvent ethyl acetate for extraction, repeat the extraction 3 times, combine the organic phases, and evaporate the organic phases under reduced pressure at a set temperature to obtain crude product. S6. Crude product separation and purification: The crude product obtained in step S5 is initially separated by silica gel column chromatography, the target fraction is collected, and then further purified by semi-preparative high performance liquid chromatography to obtain a high-purity target m-phenylenediamine derivative. S7. Structural identification: The target m-phenylenediamine derivative obtained in step S6 was identified by spectroscopic techniques including proton nuclear magnetic resonance (NMR), carbon nuclear magnetic resonance (NMR), and high-resolution mass spectrometry. S8. Activity Verification: The MTT / SRB assay was used to detect the inhibitory activity of the target m-phenylenediamine derivative purified in S6 on the cell proliferation of K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 cell lines, and the IC50 was calculated. 50 The value was set up, and a positive control group of doxorubicin was also set up.

2. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, The preparation process of the potato glucose liquid culture medium in S1 is as follows: S1.1 Take 200 g of fresh potatoes, cut them into 1-2 cm³ cubes, add 500 mL of distilled water, boil for 10 minutes, filter with 4-6 layers of sterile gauze, add 20 g of glucose to the filtrate, and stir at 100-200 rpm to dissolve it completely. S1.

2. Dilute to 1 L with distilled water, dispense 100 mL into 500 mL pre-sterilized conical flasks, autoclave at 121℃ for 20 min, and cool to room temperature before use.

3. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, In S2: the substrate m-phenylenediamine is dissolved in dimethyl sulfoxide to prepare a 0.2 g / mL stock solution, which is then filtered through a 0.22-micron filter membrane for sterilization. 100 μL of the stock solution is then added to the culture medium to bring the final concentration to 0.2 g / L.

4. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, The biotransformation culture conditions in the constant temperature shaker in S4 are set as follows: fermentation is carried out continuously for 6 days at a temperature of 28℃ and a shaking speed of 150-220 r / min.

5. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, In step S5, the organic phase is subjected to reduced pressure rotary evaporation at 30°C to remove the solvent.

6. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, The crude product separation conditions in S6 are as follows: preliminary separation is carried out under silica gel column chromatography with a 300-400 mesh mesh and an eluent system of dichloromethane:methanol = 30:

1.

7. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 6, characterized in that, Purification of the crude product in S6: After preliminary separation, the target fraction is collected and further purified by semi-preparative high performance liquid chromatography under reversed-phase C18, 10×250 mm, 5 μm, 3 mL / min, 254 nm, methanol:water = 55:45 conditions.

8. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, S7: Structural identification of the target m-phenylenediamine derivative obtained in step S6. Its chemical structure is as follows: In the formula, the product structure is methyl acetate [4-formyl-1-[3-(3-formyl-4-hydroxymethyl-2-methyl-1H-pyrrolo-1-yl)phenyl]-5-methyl-1H-pyrrolo-3-yl] ester.

9. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, The m-phenylenediamine derivative showed IC50 values ​​against K562, NCI-H446, NCI-H446 / EP, and MDA-MB-231 tumor cell lines. 50 The values ​​reached 0.07-0.51 μM.

10. The method for preparing antitumor m-phenylenediamine derivatives by fungal biotransformation of m-phenylenediamine according to claim 1, characterized in that, The m-phenylenediamine derivative is used in the preparation of drugs for the prevention and treatment of malignant tumors.

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