(S)-(+)-5-(3, 4-dihydroxybutyl) butyl picolinate and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to a novel compound (S)-(+)-5-(3, 4-dihydroxybutyl) butyl picolinate extracted and separated from a secondary metabolite of salvia plebeia endophytic fungus Fusarium oxysporum LZC03 and application of the novel compound (S)-(+)-5-(3, 4-dihydroxybutyl) butyl picolinate, and relates to application of the (S)-(+)-5-(3, 4-dihydroxybutyl) butyl picolinate. The invention relates to an application of 2, 4-dihydroxybutyl) butyl picolinate in preparation of antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to butyl (S)-(+)-5-(3,4-dihydroxybutyl)picolinate and applications thereof. Background Art

[0002] Salvia plebaia R. Brown, also known as toad grass, snow primrose, and frog grass, is a biennial herb in the genus Salvia of the family Lamiaceae. The whole plant is used as a medicinal herb, known for its heat-clearing, detoxifying, cough-relieving, and asthma-relieving properties. It primarily contains flavonoids, polyphenols, polysaccharides, triterpenes, and volatile oils. Recent studies have revealed the presence of a variety of pharmacologically active chemicals in plebaia.

[0003] Research has shown that endophytic fungi are abundant in plants, producing a diverse array of secondary metabolites, including terpenes, alkaloids, flavonoids, and steroids, with diverse biological activities such as anti-tumor, antibacterial, and antioxidant activities. Some endophytic fungi can also synthesize active ingredients similar to those in their host plants, offering significant potential for pharmaceutical development. Isolating and studying the metabolites of endophytic fungi in medicinal plants not only helps shed light on the mechanisms of plant-microbial symbiosis but also provides a new avenue for the discovery of new drug leads.

[0004] Currently, there are no reports on butyl (S)-(+)-5-(3,4-dihydroxybutyl)picolinate and its activity. Summary of the Invention

[0005] The present invention provides (S)-(+)-butyl 5-(3,4-dihydroxybutyl)picolinate and its uses. (S)-(+)-butyl 5-(3,4-dihydroxybutyl)picolinate and its uses are isolated from the fermentation broth of the endophytic fungus Fusarium Oxysporum LZCO3. This metabolite has antitumor and antibacterial effects.

[0006] The technical solutions to the above-mentioned technical problems to be solved by the present invention are as follows:

[0007] (S)-(+)-butyl 5-(3,4-dihydroxybutyl)picolinate, the structural formula of which is shown in Formula 3c:

[0008]

[0009] The compound is isolated from the secondary metabolite of the endophytic fungus Fusarium Oxysporum LZC03 isolated from the roots of the plant Litchi grass and is the first discovered by the present invention. Through biological activity assays, it was found that it has anti-tumor and antibacterial activities.

[0010] Furthermore, the strain name of the endophytic fungus is Fusarium Oxysporum LZC03, the strain preservation number is CGMCC No.41251, the preservation unit is the General Microbiology Center of the China Culture Collection Administration, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the preservation date is May 31, 2024.

[0011] The present invention provides a method for preparing the above-mentioned secondary metabolites, comprising the following steps:

[0012] (1) The endophytic fungus Fusarium Oxysporum LZC03 was inoculated into PDB culture medium and cultured continuously at 28°C and 150 rpm in a shaking incubator for 7 days; the fermentation seed liquid was inoculated into a sterilized rice-containing culture medium, wherein the rice-containing culture medium includes rice and water, and the ratio of rice to water is 100 g:120 mL. During inoculation, 100 mL of fermentation seed liquid was poured into each bottle of rice culture medium (each bottle containing 100 g of rice and 120 mL of water), and the culture was allowed to stand at room temperature for 50 days;

[0013] (2) After the fermentation of the strain is completed, the fermentation product is added to methanol and soaked and ultrasonicated for 30 minutes, filtered, and the filtrate is taken, and the above steps are repeated. The filtrate is combined, filtered, and rotary evaporated to obtain an extract; then the extract is dispersed in water, extracted with n-butanol, and concentrated to obtain an n-butanol layer extract;

[0014] (3) The n-butanol layer extract was separated by silica gel column chromatography, and the fraction Fr.2 was eluted with a volume ratio of CH2Cl2:CH3OH=100:2; Fr.2 was separated by an octadecylsilane bonded silica gel packing ODS medium-high pressure column, and eluted with a volume ratio of CH3OH:H2O=45:55 to obtain Fr.2-2; Fr.2-2 was separated by a Sephadex LH-20 dextran gel column, and eluted with methanol as the mobile phase to obtain Fr.2-2-2(CH3OH); Fr.2-2-2 was purified by semi-preparative HPLC, the semi-preparative conditions included: CH3CN and H2O volume ratio of 40:60, Agela semi-preparative chromatography column XB-C18-L 5μm, flow rate 3mL / min, wavelength 210nm, to obtain compound 3c (retention time 18.00min).

[0015] The present invention provides application of a new compound (S)-(+)-5-(3,4-dihydroxybutyl) butyl picolinate in the preparation of anti-tumor products.

[0016] The present invention provides application of a novel compound (S)-(+)-5-(3,4-dihydroxybutyl) butyl picolinate in the preparation of antibacterial products.

[0017] The above products include but are not limited to one or more of medicines, foods, and health products.

[0018] The present invention provides the use of the above-mentioned Fusarium oxysporum in preparing a new compound (S)-(+)-5-(3,4-dihydroxybutyl)picolinate.

[0019] The novel compound (S)-(+)-5-(3,4-dihydroxybutyl) picolinate can be prepared by using the above-mentioned Fusarium oxysporum. The compound has anti-tumor and anti-bacterial effects.

[0020] The beneficial effects of adopting the above scheme include: the new compound (S)-(+)-5-(3,4-dihydroxybutyl)picolinate extracted and isolated from the secondary metabolites of the endophytic fungus Fusarium Oxysporum LZC03 isolated from the rhizomes of the plant Litchi grass has anti-tumor and antibacterial activities and can be used to research and develop new anti-tumor or antibacterial products, such as: food, medicine, health products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the phylogenetic tree of strain Fusarium Oxysporum LZC03;

[0022] Figure 2 For compound 3c 1 H NMR spectrum

[0023] Figure 3 For compound 3c 13 C NMR spectrum;

[0024] Figure 4 is the HRESI-MS spectrum of compound 3c;

[0025] Figure 5 is the HSQC spectrum of compound 3c;

[0026] Figure 6 is the HMBC spectrum of compound 3c. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0028] Potato dextrose agar (PDA) medium (catalog number: 02-023) was purchased from Beijing Aoboxing Biotechnology Co., Ltd. and prepared according to the following proportions: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water.

[0029] PDB medium was prepared using the following proportions: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, and 20% potato extract in water. All percentages are expressed by weight relative to water. Mannitol (Cat. No. 01-063), peptone (Cat. No. 01-001), yeast extract (Cat. No. 01-014), and glucose (Cat. No. 01-076) were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0030] IRPMI-1640 culture medium (Cat. No. R2405), fetal bovine serum (Cat. No. F8687), and penicillin-streptomycin solution (Cat. No. TMS-AB2) were purchased from Merck.

[0031] 75% ethanol was purchased from Dezhou Chengze Disinfection Technology Co., Ltd.; sodium hypochlorite (NaClO) was purchased from Tianjin Tianda Chemical Reagent Factory; methanol (CH3OH) (batch number: 20211121) and dichloromethane (CH2Cl2) (batch number: 20211103) were both purchased from Tianjin Tianli Chemical Reagent Co., Ltd.; acetonitrile (CH3CN) (batch number: R142278) was purchased from Beijing Dima Technology Co., Ltd.; column chromatography silica gel was purchased from Qingdao Ocean Chemical Plant Branch; thin layer chromatography silica gel plate (model: GF254) was purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; Sephadex LH-20 dextran gel column was purchased from Hefei Bomei Biological Company; thiazolyl blue (MTT) (batch number: A4586) was purchased from Tianjin Alpha Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) (product number: D6370) was purchased from Beijing Biotopped Co., Ltd.; PBS buffer (product number: P917808) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0032] Test cells: Human leukemia cell line (HL-60) (Cat. No.: CL-0110), human colon cancer cell line (HCT-116) (Cat. No.: CL-0096) and prostate cancer cell line (PC-3) (Cat. No.: CL-0185) were purchased from Wuhan Prosai Life Science Technology Co., Ltd.

[0033] In the present invention, unless otherwise specified, all methods are conventional in the art. The materials, reagents, methods, and instruments used, unless otherwise specified, are conventional in the art. Those skilled in the art can obtain them through commercial channels or prepare the solutions used in the experiments using conventional methods in the art.

[0034] The following is an introduction through specific embodiments.

[0035] Example 1

[0036] Fresh samples of lychee grass collected from Huangshan, Anhui Province, were washed and surface-disinfected using a 75% ethanol and 5% NaClO solution in a clean bench. The samples were then rinsed four times with sterile water. The roots of the lychee grass that had passed surface disinfection were cut into 2 mm slices using a sterile knife and inoculated onto PDA culture plates. The plates were then incubated at 28°C for 3-7 days, during which time the presence of bacterial colonies around the root tissue was observed.

[0037] Strain purification: Observe the status of the colony. When it grows well, pick the tip of the hyphae and transfer it to a PDA medium plate. Culture it in a constant temperature incubator at 28°C for 3-5 days for isolation and purification. Repeat the above steps until a single colony is obtained, which is named LZC03.

[0038] Morphological characteristics of the strain: The front of the colony on the PDA culture medium plate appears dark white, and the back appears reddish-brown flocculent colonies.

[0039] Identification of strains: ITS sequencing analysis of the strains was performed by Shanghai Bioengineering Technology Service Co., Ltd. The sequences were Blast-matched in the NCBI database. Strain sequences with similar homology to the strain sequences were downloaded and aligned using MEGA 11.0 software. The ITS gene phylogenetic tree was constructed using the neighbor-joining method (1000 bootstrap replicates). The results are shown in the figure. Figure 1 Analysis of the phylogenetic tree showed that the strain was in the same branch as Fusarium oxysporum (Accession No. OQ818157.1) and Fusarium oxysporum (Accession No. PP380145.1). Combined with morphological analysis, it was identified as Fusarium oxysporum ( Figure 1 The strain marked with a red dot) was named Fusarium oxysporum LZC03.

[0040] On May 31, 2024, it was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The strain name is Fusarium Oxysporum LZC03, and the strain deposit number is CGMCC No. 41251.

[0041] Example 2

[0042] Fusarium Oxysporum LZC03 was inoculated into 500mL Erlenmeyer flasks containing 200mL of PDB medium and cultured continuously at 28°C, 150rpm, in a shaker for 7 days to prepare a fermentation seed solution using the aforementioned method. Eighty 500mL Erlenmeyer flasks were each filled with 100g of rice and 120mL of water. These flasks were autoclaved at 121°C for 30 minutes to obtain sterilized rice culture medium. The rice culture medium was then removed and cooled to room temperature. 100mL of fermentation seed solution was then added to each flask and incubated at room temperature (20-25°C) for 50 days. After fermentation is completed, add 150 mL of methanol to each bottle and soak it and ultrasonicate for 30 minutes. Then filter it with 4 layers of sterile medical gauze, squeeze out the filtrate, collect the filtrate, put the residue into a conical culture flask and continue to add 150 mL of methanol to soak. Repeat this ultrasonication for a total of 3 times. Combine the extracts and filter them. Evaporate them at 75°C and 80 r / min to dryness to obtain 500 g of extract.

[0043] 500 g of the extract was dispersed in water and extracted three times with n-butanol. The extract was concentrated under reduced pressure at 90°C and 80 rpm to obtain 98.0 g of the extract (i.e., the n-butanol layer extract). The n-butanol layer extract (98.0 g) was mixed with 147 g of silica gel (100-200 mesh). The column silica gel consisted of 588 g of 200-300 mesh silica gel and 65 g of 80-100 mesh silica gel. The retention volume was 2 L. The extract was separated by silica gel column chromatography using a volume ratio of CH2Cl2:CH3OH = 100:2 to obtain Fraction Fr.2 (20.2 g).

[0044] Fr.2 was separated by octadecylsilane bonded silica gel packing ODS medium-high pressure column (20-35 μm, Tianjin Bona Aijieer Technology Co., Ltd.), and the fraction Fr.2-2 (4.9 g) was obtained with a volume ratio of CH3OH:H2O=45:55.

[0045] Fr.2-2 was separated by Sephadex LH-20 column chromatography with methanol as the mobile phase for elution, and fractions were collected with a collection volume of 10 mL to obtain fraction Fr.2-2-2 (CH3OH, 0.9 g).

[0046] Fr.2-2-2 (0.9 g) was purified by semi-preparative HPLC to give compound 3c (9 mg, retention time 18.00 min); the preparation conditions included: CH3CN-H2O (volume ratio 40:60), Agela semi-preparative column XB-C18-L 5μm, flow rate 3mL / min, wavelength 210nm.

[0047] Example 3

[0048] Compound 3c prepared by the method of Example 2 is a white amorphous powder (methanol). 1 H NMR and 13 C NMR data are shown in Table 1. This compound was discovered for the first time by the present invention.

[0049] Table 1 Compound 3c 1 H NMR and 13 C NMR data

[0050]

[0051] Figures 2 to 6 is compound 3c of the present invention 1 H NMR, 13 The structure of the compound was confirmed by combining the C NMR, HRESI-MS, HSQC, and HMBC spectra with the relevant analytical data. The mass spectrometry of compound 3c was performed by Dalian Mengdi Technology Co., Ltd. The mass-to-charge ratio measured by HRESI-MS was m / z 290.1396 [M+Na] + , its molecular formula is speculated to be C 14 H 21 NO4, unsaturation is 5.

[0052] 1 H NMR (600 MHz, DMSO-d6) spectrum showed that H 8.56 (1H, s), 7.96 (1H, d, J = 7.90 Hz), 7.81 (1H, d, J = 7.90 Hz) at the signal confirmed the presence of a 3, 6 substituted pyridine ring. H There is a methoxy hydrogen signal at 4.28 (2H, t, J = 6.6 Hz); δ H There is a hydrogen signal of oxymethylene at 3.38 (1H, dd, J = 9.38, 5.29 Hz); there are hydrogen signals of hydroxymethyl at 3.31 (1H, dd, J = 5.60, 10.50 Hz) and 3.24 (1H, dd, J = 5.60, 10.50 Hz); H There are two methylene hydrogen signals at 2.81 (1H, m), 2.69 (1H, m) and 1.76 (1H, m), 1.54 (1H, m); δ H 0.92 (3H, t, J = 7.20 Hz) There is a methyl hydrogen signal.

[0053] 13 C NMR (150 MHz, DMSO-d6) spectroscopy produces 14 carbon signals, including C There is an ester carbonyl carbon signal at 164.7, δ CThere are pyridine ring carbon signals at 149.8, 145.3, 142.0, 136.8 and 124.5, δ C There is a secondary alcohol carbon signal at 70.1, δ C There is a hydroxymethyl carbon signal at 65.7; δ C There is a methylene carbon signal connected to oxygen at 64.5; C There are methylene carbon signals at 34.5, 28.2, 30.1, and 18.6; δ C There is a methyl carbon signal at 13.5.

[0054] All carbon and hydrogen signals were assigned using the HSQC spectral data, as shown in Table 1.

[0055] The structure of compound 3c was confirmed by the multiplicities and correlations shown in the HMBC spectrum. In the HMBC spectrum, 3.31 (H-10) and 3.24 (H-10) correlated with 70.1 (C-9) and 34.5 (C-8); 3.38 (H-9) correlated with 65.7 (C-10) and 34.5 (C-8); and 1.76 (H-8) and 1.54 (H-8) correlated with 70.1 (C-9), 28.2 (C-7), and 142.0 (C-4). Furthermore, 2.81 (H-7) and 2.69 (H-7) correlated with 142.0 (C-4) and 34.5 (C-8), confirming the structure of pentane-1,2-diol. The correlations of 8.56 (H-3) with 145.3 (C-2), 142.0 (C-4), 136.8 (C-5) and 28.2 (C-7); the correlations of 7.81 (H-5) with 149.8 (C-3) and 145.3 (C-2); and 7.96 (H-6) with 142.0 (C-4) confirmed the presence of a 3,6-substituted pyridine ring structure. 4.28 (H-11) correlated with 164.7 (C-1); 7.81 (H-5), 7.96 (H-6) correlated with 164.7 (C-1); 2.69 (H-7), 2.81 (H-7) correlated with 149.8 (C-3), 124.5 (C-6), 136.8 (C-5) and 8.56 (H-3), 7.81 (H-5) correlated with 28.2 (C-7), confirming the connection between pentane-1,2-diol and butyl acetate of the pyridine ring.

[0056] For compounds containing only a single chiral center, the direction of its optical rotation can usually be used as an important basis for inferring its absolute configuration when compared with known analogs. The optical rotation of compound 3c is (CH3OH, c=0.003g / mL). Since compound 3c has only one chiral center and is an analog of the compound (S)-(+)-methyl fusarinolate ((S)-(+)-fusaric acid methyl ester) in the Synthesis of Substituted Pyridines via Regiocontrolled[4+2]Cycloadditions of Oximinosulfonates published by Adam et al. in The Journal of Organic Chemistry, with the same optical rotation sign, compound 3c and compound (S)-(+)-methyl fusarinolate have the same absolute configuration and are named (S)-(+)-5-(3,4-dihydroxybutyl)picolinic acid butyl ester.

[0057] Example 3

[0058] Cell preparation: Human leukemia cells (HL-60), human colon cancer cells (HCT-116), and prostate cancer cells (PC-3) were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Penicillin (100 U / ml) and streptomycin (100 μg / mL) were added to the medium and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 2-3 days, and cells in the exponential growth phase were used for experiments.

[0059] Drug preparation: Compound 3c was dissolved in DMSO (final DMSO concentration <0.1%) and diluted to the desired concentration using prepared culture medium before the experiment.

[0060] Preparation of MTT solution: Weigh 50 mg of MTT powder and dissolve it in 10 mL of PBS to prepare a 5 mg / mL solution. Stir in the dark, filter sterilize with a 0.22 μM filter membrane, aliquot, and store in the dark.

[0061] Take the test cells at (2-3)×10 4 Cells were seeded at a density of 100 μL per well in a 96-well culture plate at 1 μg / mL. After 24 hours of attachment, the supernatant was removed and 100 μL of drug-containing culture medium was added to each well. The final concentration of compound 3c was 41, 31, 21, 11, and 1 μg / mL. A blank control group was treated with an equal volume of culture medium and DMSO (i.e., cells in a single well were not treated and cultured as usual). The positive control was hydroxycamptothecin. Three replicates were set for each treatment. Culture was continued at 37°C for 24 hours. 10 μL of MTT solution was added to each well and incubated at 37°C for 4 hours. The supernatant was discarded and 150 μL of DMSO was added to each well. The cells were shaken at room temperature for 10 minutes. The absorbance of each well was measured at 490 nm using a microplate reader.

[0062] The tumor cell growth inhibition rate (%) was calculated using the following formula: 1-(absorbance value of drug-added well / absorbance value of control well)×100%.

[0063] Experimental results

[0064] The experimental results are shown in Table 2. Compound 3c has certain cytotoxic activity against human colon cancer cells (HCT-116). Its IC 50 The value was 14.93 μg / mL.

[0065] Table 2 Inhibitory effect of compound 3c on tumor cell proliferation (IC 50 , μg / mL)

[0066]

[0067] “-” in the table means that no activity was detected within the experimental concentration range.

[0068] Example 4

[0069] Antibacterial activity test

[0070] The tested bacterial strains were: Staphylococcus aureus (CMCC(B)26003), Bacillus subtilis (CMCC(B)63534), Escherichia coli (CMCC(B)44102) and Pseudomonas aeruginosa (CMCC(B)10104).

[0071] Compound 3c was prepared into a 2 mg / mL stock solution with DMSO (DMSO concentration ≤ 0.5%). The bacterial strains used in the activity test were activated in a 37°C constant temperature incubator for 2-3 h. Afterwards, a small amount of the activated strains was picked up and placed in sterile water, shaken well, and prepared to a concentration of 1×10 8 CFU / mL bacterial suspension. Add 188 μL of the corresponding culture medium and 2 μL of the stock solution of the test compound to the first well of a 96-well plate. Perform a 2-fold serial dilution in the 96-well plate. Finally, add 10 μL of the test bacterial suspension to each well. The final concentrations of the test compound added to the 96-well plate are 200, 100, 50, 25, 12.5, 6.25, and 3.12 μg / mL, respectively. Streptomycin is used as a positive control, 0.5% DMSO as a negative control, and empty culture medium as a blank control. Each experiment is repeated three times. After culturing in a 37°C incubator for 3 days, observe the turbidity of the bacterial suspension in the 96-well plate. The minimum concentration at which the culture solution becomes clear is the minimum inhibitory concentration (MIC) of the compound.

[0072] 10 μL of the growth-inhibited bacteria was aspirated from each well and spread onto nutrient agar medium and cultured in a 37°C constant temperature incubator for 24-48 hours. Each experiment was repeated 3 times, and the lowest concentration at which the bacteria were completely killed was defined as the minimum bactericidal concentration (MBC).

[0073] Experimental results

[0074] The MIC and MBC values ​​of compound 3c and the positive control drug streptomycin against four standard bacterial strains are shown in Table 3. The results showed that compound 3c had a certain inhibitory effect on the four bacterial strains, among which the inhibitory effect on Staphylococcus aureus was better than that of the positive drug streptomycin (MIC 50 μg / mL), with a MIC value of 25 μg / mL.

[0075] Table 3 MIC and MBC values ​​of compound 3c against four tested bacterial strains (μg / mL)

[0076]

[0077] “-” in the table means that the corresponding MIC and MBC values ​​were not detected within the experimental concentration range.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. (S)-(+)-butyl 5-(3,4-dihydroxybutyl)picolinate, characterized in that The structural formula of the compound is shown in Formula 3c:

2. Application of butyl-(+)-5-(3,4-dihydroxybutyl)picolinate in the preparation of antibacterial drugs.

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