Diketopiperazine alkaloids and their use

Abstract

The application belongs to the technical field of microbial preparation of compounds. In view of this, the application provides a diketopiperazine alkaloid, a chemical structural formula of which is shown in the description. The diketopiperazine alkaloid provided by the application shows excellent inhibitory activity on IAV virus and OC43 virus.

Description

Technical Field

[0001] This invention belongs to the field of microbial compound preparation technology. Background Technology

[0002] Oseltamivir-resistant influenza virus, a typical drug-resistant virus, was first reported in Europe around 2007. In the following years, this resistant strain dominated seasonal influenza outbreaks in many parts of the world. According to the World Health Organization, by 2050, drug-resistant viruses could cause more than 10 million deaths annually. Faced with the increasingly severe situation of antiviral drug resistance, developing new drugs that can effectively inhibit highly pathogenic and drug-resistant viruses has become an urgent medical task.

[0003] Active metabolites derived from fungi in natural products are potential lead compounds for many antibiotics, anticancer drugs, and pesticides, possessing significant research value and economic worth. However, with the deepening research on fungal secondary metabolites, the success rate of finding novel and highly active secondary metabolites from common habitats has greatly decreased. In recent years, researchers have gradually turned their attention to fungi in extreme habitats. These fungi live in extreme environments, mainly including volcanoes, deep seas, deserts, and plant roots. To survive under extreme conditions, fungi in extreme habitats often produce unique secondary metabolites. Existing studies have shown that some fungi in extreme habitats exhibit excellent biological activity. Summary of the Invention

[0004] In view of this, the present invention provides a class of diketopiperazine alkaloids, the chemical structural formula of which is as follows: .

[0005] The diketopiperazine alkaloids provided by this invention exhibit excellent inhibitory activity against IAV virus and OC43 virus. Attached Figure Description

[0006] Figure 1 This is the structural diagram of the compound Reduced gliotoxin.

[0007] Figure 2 This is the ESIMS diagram of reduced gliotoxin.

[0008] Figure 3 It is reduced gliotoxin. 1 H NMR spectrum.

[0009] Figure 4 It is reduced gliotoxin. 13 C NMR spectrum. Detailed Implementation

[0010] Example

[0011] 1. Activation of microbial strains

[0012] 1. Pre-set the constant temperature water bath to ensure that the water temperature is maintained at a constant 37 ℃;

[0013] 2. Remove the Aspergillus fumigatus CGMCC 3.11434 glycerol tube from the -80 ℃ ultra-low temperature freezer and place it in a prepared constant temperature water bath. Let it stand for about 5 minutes to ensure that the contents of the glycerol tube are completely thawed;

[0014] 3. Perform subsequent aseptic procedures on a clean bench. Using a pre-sterilized metal inoculation loop, accurately inoculate the strain onto the surface of modified Martin solid medium using the streak plate method;

[0015] 4. Place the modified Martin solid medium containing the inoculated strain into an incubator for incubation;

[0016] 5. Take out the cultured strain and inoculate it into modified Martin liquid medium. Incubate at 180 rpm and 28 ℃ for 2 days. The resulting culture medium can be used as the stock solution for subsequent experiments.

[0017] 2. Liquid fermentation and extraction of the strain

[0018] Mycelia were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of modified Martin liquid medium and cultured at 28 °C and 180 r / min for 24 h to obtain the mother liquor. The mother liquor was transferred to the remaining liquid medium for co-fermentation, totaling 70 L. After 15 days of culture at 28 °C and 180 r / min, the mixture was centrifuged at 3500 r / min for 15 min. The fermentation supernatant was extracted with ethyl acetate at a volume ratio of 1:1, and the extraction was repeated three times until the ethyl acetate layer was nearly colorless. The upper ethyl acetate extract was collected, and the ethyl acetate phase was distilled under reduced pressure at 40 °C to obtain the crude extract. The bacterial precipitate was lysed by soaking in 80% ethanol for 2 h, followed by sonication for 5 min, centrifugation at 3500 r / min for 15 min, and collection of the supernatant. The supernatant was also distilled under reduced pressure at 42 °C to obtain the crude bacterial extract.

[0019] 3. Solid-state fermentation and extraction of bacterial strains

[0020] Place 160 g of rice and 240 mL of distilled water in a 1000 mL Erlenmeyer flask and sterilize at high temperature. Add 2 to 3 drops of stock solution containing *Aspergillus fumigatus* CGMCC 3.11434 to each culture medium. Prepare a total of 30 kg of rice solid culture medium using this method. After completing the above steps, place the rice culture medium in a 30°C light incubator for 20 days.

[0021] First, a portion of ethyl acetate was poured into a rice solid culture medium containing *Aspergillus fumigatus* CGMCC 3.11434 to prevent fungal spore splashing. Then, the mixture was transferred to a 5-liter beaker, and ethyl acetate was added until the solid culture medium was completely covered. The mixture was soaked for 24 hours, then sonicated for 30 minutes. The mixture was then poured out, stirred, and ethyl acetate was added again to completely cover the solid culture medium. This process was repeated three times. After this step, 95% ethanol was added until the solid culture medium was completely covered. The mixture was sonicated for 30 minutes, poured out, stirred, and 95% ethanol was added again. This process was repeated three times. The liquids extracted with ethyl acetate and 95% ethanol were collected separately and then distilled under reduced pressure at 40 °C to obtain a crude extract (480.6 g) for further processing.

[0022] 4. Isolation and purification of secondary metabolites

[0023] The crude extracts obtained from liquid (modified Martin) fermentation and solid (rice) fermentation were mixed with an equal weight of 80-100 mesh silica gel, moistened with a small amount of methanol, and thoroughly dried. The mixture was then packed into a 200-300 mesh silica gel column using a dry-packing method. Gradient elution was performed using a normal-phase atmospheric pressure column chromatography system with petroleum ether-ethyl acetate (100:0-0:100) and dichloromethane-methanol (100:0-0:100). The eluents were concentrated under reduced pressure and dried to obtain the fractions eluted from each gradient, which were then labeled. The fractions were subsequently dissolved with a small amount of reagent and detected by TLC (thin-layer chromatography). The TLC results were observed under UV light at 254 nm. Similar or identical fractions were combined to obtain six distinct components (Fr.A–Fr.F).

[0024] Fr.C is moderately polar and its components are dark brown solids. Gradient elution was performed using normal-phase atmospheric pressure column chromatography, resulting in 10 fractions (Fr.C.1-Fr.C.10). Fr.C.1 and Fr.C.2 were yellow oily substances; however, they exhibited significant tailing after thin-layer chromatography and were therefore not separated. Fr.C.3 was separated using silica gel column chromatography and dextran gel column chromatography (LH-20).

[0025] 5. Structural identification of compounds

[0026] By carefully comparing the nuclear magnetic resonance spectral data of the compounds ( 1 H and 13 The structure of the compound was determined by combining C10 NMR and ESI-MS data. Figure 1 As shown.

[0027] The compound is a yellow solid, soluble in methanol, with the molecular formula C2. 13 H16 N2O4S2. 1 1H-NMR showed three olefin carbon signals at δ 5.98 (1H, m), 5.93 (1H, m) and δ 5.70 (1H, d, J = 9.6 Hz); one oxygen-bound methylene at δ 4.34 (1H, d, J = 11.6 Hz) and 4.08 (1H, d, J = 11.6 Hz); and one nitrogen-bound methine. 13 C-NMR showed that the compound had 13 carbon signals, including 2 amide carbon signals, δ 132.4, 131.2, 124.7 and 121.5 were 2 groups of alkene carbon signals, and δ 41.3 was an N-methyl carbon signal.

[0028] The NMR data of the compound are as follows: 1 H-NMR (MeOD, 400 MHz): δ 5.98 (1H, m, H-9), 5.93 (1H, m, H-8), δ 5.70 (1H, d, J = 9.6 Hz, H-7), 5.07 (1H, d, J = 14.4 Hz, H-6), 4.68 (1H, d, J = 13.6 Hz, H-5a), 4.34 (1H, d, J = 11.6 Hz, H-12a), 4.08 (1H, d, J = 11.6 Hz, H-12b), 3.35 (1H, overlapped, H-10a), 3.06 (1H, d, J =16.4 Hz, H-12b), 3.13 (3H, s, H-11). 13 C-NMR (MeOD, 100 MHz) δ: 170.9 (C-4), 170.0 (C-1), 132.4 (C-9a), 131.2 (C-7), 124.7 (C-8), 121.5 (C-9), 80.1 (C-3), 74.5 (C-10a), 74.0 (C-5a), 71.3 (C-6), 62.2 (C-12), 41.3 (C-10), 29.2 (C-11).

[0029] After reviewing relevant literature, the physicochemical properties and proton and carbon spectra of the compound were found to be largely consistent with the data reported in the literature. Therefore, the compound was identified as a diketopiperazine alkaloid (Reduced gliotoxin).

[0030] 6. Screening of compounds against IAV virus

[0031] Test principle: Using 293T-GLUC cells as the virus host, the activity of the sample inhibiting the luciferase activity of the reporter gene carried by the virus was measured.

[0032] Test materials and methods:

[0033] 1. Virus strain: Infection of MDCK cells yielded an IAV (influenza A virus, WSN strain) titer of 10. 7 , stored at -80℃.

[0034] 2. Sample preparation: Dissolve the sample in DMSO to prepare a suitable initial concentration (the final concentration of the sample in this experiment was 10 μM), and take a single concentration point for testing.

[0035] 3. Test Method: 293T-GLUC cells were seeded in 96-well culture plates and incubated at 37°C with 5% CO2 for 24 hours. Drug was added pre-incubated for 2 hours, and then the viral protovirus was diluted and inoculated with the virus at an MOI of 0.3. After 24 hours of incubation, the luciferase activity in infected cells was measured, and the inhibition rate of each sample was calculated.

[0036] Table 1: Anti-IAV Activity of Compounds

[0037]

[0038] 7. Screening of compounds against OC43 virus

[0039] Test principle: Using BHK21 cells as the virus host, the activity of the sample inhibiting the luciferase activity of the reporter gene carried by the virus is measured.

[0040] Test materials and methods:

[0041] 1. Virus strain: HCoV-OC43 (GenBank accession number AY391777.1)

[0042] 2. Sample preparation: Dissolve the sample in DMSO to prepare a suitable concentration gradient for testing.

[0043] 3. Test Method: BHK21 cells were seeded in 24-well culture plates and incubated at 37 °C for 24 hours with 5% CO2. After 24 hours, the supernatant was discarded and replaced with 2% medium containing OC43 virus (MOI = 0.01). A diluted compound (1 μL / well) was added as a negative control (1 μL DMSO) and a positive control (1 μL remdesivir, final concentration 10 μM). The plates were then incubated at 37 °C for 48 hours. After 48 hours of incubation, the supernatant was discarded, and 50 μL of 1*cell lysis solution was added to each well. The cells were lysed at 37 °C for 30 minutes. The luciferase activity in the infected cells was then measured, and the inhibition rate of each sample was calculated.

[0044] Table 2: Anti-OC43 Virus Activity of Compounds

[0045]

Claims

1. Diketopiperazine alkaloids, with the following chemical structural formula: .

2. The application of the diketopiperazine alkaloids as described in claim 1 in the treatment of IAV virus.

3. The application of the diketopiperazine alkaloids as described in claim 1 in the treatment of OC43 virus.

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