o-Pyrrolidone C, its preparation method and application

Abstract

This invention relates to similanpyrone C, its preparation method, and its applications, particularly its application in inhibiting acetylcholinesterase. The similanpyrone C is shown in formula (I) below:

Description

Technical Field

[0001] This invention relates to the field of marine biological natural products, specifically to the preparation of o-pyrrolidone C from the marine fungus Aspergillus sp. WXF1904 (deposited on March 24, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC No. 62320) and its application in inhibiting acetylcholinesterase. Pyrrolidone C is a novel bromoisocoumarin, and the first naturally occurring bromoisocoumarin compound discovered to date. Background Technology

[0002] As is well known, natural products are an important source of innovative drugs. Marine microbial resources are abundant and unique. The special environment of high pressure, high salinity, and low light enables them to produce natural products with novel structures and unique activities, distinct from terrestrial microorganisms. Isocumarins are natural products containing lactone rings, and are quite abundant in microorganisms and higher plants. Compounds with isocumarin skeletons have been shown to have physiological and biological activities such as antibacterial, anti-inflammatory, anticancer, protease inhibitory, and herbicidal effects. At the same time, isocumarin compounds are also important skeletons of bioactive substances.

[0003] The activity studies of related isocoumarin compounds provide an example. For instance, a novel dimeric isocoumarin was isolated from the terrestrial Streptomyces p. BCC24731, which exhibited cytotoxic effects on KB and NCI-H187 cell lines, with an IC50 value of [missing information]. 50The values ​​were 24.2 and 8.31 μg / mL, respectively (Boonlarppradab C.; Suriyachadkun C.; Suphothina S.; Tobwor P. Bireticulol, a bioactive isocoumarin dimer from Streptomycess p. BCC24731. J. Antibiot. 2011, 64, 267-70). Bone formation refers to a series of complex events related to osteoblast activity. Isocumarin A isolated from the spreading rhizomes of Polygonum cuspidatum can promote the proliferation and mineralization of non-transformed osteoblast lines by activating the PI3K-Akt / Erk cascade-activated BMP / RUNX2 signaling pathway, thereby promoting fracture and osteoporosis healing (Liu M.Z.; Zhou DC; Liu Q.; Xie FL; Xiang DX; Tang GYLuo SLOsteogenesis activity of isocumarin A through the activation of the PI3K-Akt / Erk cascade-activated BMP / RUNX2 signaling pathway. Eur. J. Pharmacol. 2019, 858, 172480). The isocoumarin analogs 7-hydroxyoospolactone and parapholactone, isolated from the marine-derived fungus Paraphoma sp. CUGBMF180003, showed antibacterial activity against Staphylococcus aureus, with MIC values ​​of 12.5 μg / mL (Xu X.;Li J.;Zhang K.;Wei S.;Lin R.;Polyak SW;Yang N.;Song F. New Isocoumarin Analogues from the Marine-Derived Fungus Paraphoma sp. CUGBMF180003. Mar. Drugs 2021, 19, 313).A rare, naturally occurring isocoumarin derivative, penicifurans A, was obtained from a secondary metabolite of the sponge-derived Penicillium sp. fungus. J. Nat. Prod. 2013, 76, 571-579. Novel amide-amido isocoumarin derivatives S1 and S2 are two effective compounds that exhibit potent cytotoxic and apoptosis-inducing effects on human breast cancer MCF-7 and MDA-MB-231 cells (Das V.; Kaishap PP; Duarah G.; Chikkaputtaiah C.; Deka Boruah HP; Pal M. Cytotoxic and apoptosis-inducing effects of novel 8-amido isocoumarin derivatives against breast cancer cells. Naunyn Schmiedebergs Arch. Pharmacol. 2021, 394, 1437-1449).

[0004] Acetylcholine (ACh) is a neurotransmitter involved in cognition, autonomic nervous system, and neuromuscular function. Acetylcholinesterase (AChE) is a key enzyme in biological neurotransmission. At cholinergic synapses, this enzyme degrades acetylcholine, terminating the neurotransmitter's excitatory effect on the postsynaptic membrane, ensuring the normal transmission of nerve signals within the body, and is crucial for maintaining the normal function of the central and peripheral nervous systems (Li S.; Li AJ; Zhao J.; Santillo MF; Xia M. Acetylcholinesterase Inhibition Assays for High-Throughput Screening. Methods Mol. Biol. 2022, 2474, 47-58). Acetylcholinesterase inhibitors (AChEIs) extend and enhance the effects of acetylcholine by inducing its accumulation at the synapse. Cholinesterase inhibitors are typically produced chemically or by plants and microorganisms and are used to treat neurodegenerative diseases such as Alzheimer's disease (Su J., Liu H.; Guo K.; Chen L.; Yang M.; Chen Q. Research Advances and Detection Methodologies for Microbe-Derived Acetylcholinesterase Inhibitors: A Systemic Review. Molecules 2017, 22, 176). Therefore, finding highly efficient, low-toxicity, highly selective cholinesterase inhibitors with novel structures and mechanisms of action is of great significance in the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0005] This invention relates to the extraction of o-pyrrolidone C, an analogue of formula (I), from the marine fungus Aspergillus sp. WXF1904, its preparation method, and its application in inhibiting acetylcholinesterase.

[0006] In a first aspect, the present invention provides compounds of the following general formula (A):

[0007]

[0008] Wherein, R1 = H or CH3, and R2 = H or COCH3 or CH2-Ph.

[0009] In a second aspect, the present invention provides a compound represented by formula (I):

[0010]

[0011] Thirdly, the present invention provides a method for preparing the compound shown in formula (I).

[0012]

[0013] The method includes the following steps:

[0014] (1) The marine fungal strain Aspergillus sp. WXF1904 was fermented and cultured.

[0015] (2) The fermentation culture was extracted with ethyl acetate, and the extract was recovered by distillation and concentrated. The concentrate was dissolved in 95% methanol solution, placed in a separatory funnel and extracted three times with petroleum ether. The methanol fraction was collected and the methanol was recovered by distillation. The concentrate obtained was the crude extract, which was a black extract.

[0016] (3) The crude extract was separated by medium-pressure silica gel column and eluted by gradient elution using a mixed organic solvent system of petroleum ether-ethyl acetate-methanol.

[0017] (4) The fraction obtained in step (3) was subjected to column chromatography on a semi-preparative ODS column and eluted isocratically with 72% methanol-water solution to obtain the compound shown in formula (I).

[0018] Preferably, the fermentation culture conditions for the marine fungal strain Aspergillus sp. WXF1904 are as follows:

[0019] (1) Culture of liquid seeds: Mature seeds grown on plates were inoculated into liquid seed culture medium and cultured in a shaker at 25°C at 180 rpm for 2 days.

[0020] (2) Fermentation: Transfer the appropriate amount of liquid seed obtained in step (1) to a solid fermentation medium, place it in a 25℃ incubator and let it stand for 30 days, harvest the culture and use it for the separation and purification of secondary metabolites.

[0021] The liquid seed culture medium is formulated as follows: 15g malt extract, 28g NaBr, 1L tap water, pH 7.4-7.8; the solid fermentation culture medium is formulated as follows: 200g rice, 5.6g NaBr, 200mL tap water, all placed in a 1000mL Erlenmeyer flask.

[0022] Fourthly, the present invention provides a method for preparing a compound represented by the following general formula (A),

[0023]

[0024] Wherein, R1 = H or CH3, and R2 = H or COCH3 or CH2-Ph.

[0025] The method includes the preparation method of the compound shown in formula (I) as described in the third aspect above. The substituents represented by R1 and R2 in general formula (A) are all conventional substituents. Based on obtaining the compound shown in formula (I) above, those skilled in the art can obtain all compounds shown in general formula (A) through conventional experiments.

[0026] Fifthly, the present invention provides a marine fungal strain, Aspergillus sp. WXF1904, for preparing the compound shown in formula (I);

[0027]

[0028] Preferably, the fermentation culture conditions for the marine fungal strain Aspergillus sp. WXF1904 are as follows:

[0029] (1) Culture of liquid seeds: Mature seeds grown on plates were inoculated into liquid seed culture medium and cultured in a shaker at 25°C at 180 rpm for 2 days.

[0030] (2) Fermentation: Transfer the appropriate amount of liquid seed obtained in step (1) to a solid fermentation medium, place it in a 25℃ incubator and let it stand for 30 days, harvest the culture and use it for the separation and purification of secondary metabolites.

[0031] The liquid seed culture medium is formulated as follows: 15g malt extract, 28g NaBr, 1L tap water, pH 7.4-7.8; the solid fermentation culture medium is formulated as follows: 200g rice, 5.6g NaBr, 200mL tap water, all placed in a 1000mL Erlenmeyer flask.

[0032] In a sixth aspect, the present invention provides the use of compounds of the following general formula (A) in the inhibition of acetylcholinesterase:

[0033]

[0034]

[0035] Wherein, R1 = H or CH3, and R2 = H or COCH3 or CH2-Ph.

[0036] Preferably, the compound is a compound represented by formula (I):

[0037]

[0038] This invention involves fermenting the marine fungal strain Aspergillus sp. WXF1904, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 24, 2022 (accession number GDMCC No. 62320), on a solid rice fermentation medium. The fermentation culture was then extracted, separated, and purified to obtain the compound of formula (I). Structural analysis revealed it to be a novel compound containing a bromine-containing isocomonasine structure, named similanpyrone C, with the specific structure shown in formula (I). Evaluation of its inhibitory activity against acetylcholinesterase showed that the compound of formula (I) inhibits acetylcholinesterase. Therefore, a second objective of this invention is to provide the application of the compound of formula (I) in the inhibition of acetylcholinesterase.

[0039] This invention provides candidate compounds for the development of new active substances that inhibit cholinesterase, and is of great significance for the development and utilization of cholinesterase inhibitory compound resources from marine microorganisms in China. Attached Figure Description

[0040] Figure 1 The diagram illustrates the compound of formula (I). 1 H NMR spectral information.

[0041] Figure 2 The diagram illustrates the compound of formula (I). 13 C NMR spectral information.

[0042] Figure 3 The HSQC information for compound (I) is illustrated.

[0043] Figure 4 The HMBC information for compound (I) is illustrated.

[0044] Figure 5 The HRESIMS information for compound (I) is illustrated. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0046] Example 1: Preparation and structural identification of compounds of formula (I)

[0047] (1) Preparation

[0048] 1. Microbial materials:

[0049] The marine fungal strain Aspergillus sp. WXF1904, deposited on March 24, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCCNo.62320, was isolated from a starfish sample collected from the Shishen Seamount area in the northwestern sub-basin of the South China Sea. After morphological identification and ITS strain identification, the strain was determined to belong to the genus Aspergillus sp. and named Aspergillus sp. WXF1904.

[0050] 2. Microbial culture conditions:

[0051] Culture medium:

[0052] Liquid seed culture medium: 15g malt extract, 28g NaBr, 1L tap water, pH 7.4-7.8. Solid fermentation culture medium: 200g rice, 5.6g NaBr, 200mL tap water (into a 1000mL Erlenmeyer flask).

[0053] Mature seeds grown on plates were inoculated into liquid seed culture medium and cultured in a shaker at 25°C at 180 rpm for 2 days. Then, they were transferred to solid fermentation medium and cultured statically in an incubator at 25°C for 30 days. The culture was then harvested for the separation and purification of secondary metabolites.

[0054] 3. Extraction and separation:

[0055] Ethyl acetate was added to the above fermentation culture for extraction, and the extract was recovered by distillation. The resulting concentrate was dissolved in 95% methanol solution and extracted three times with petroleum ether to obtain the petroleum ether extract fraction and the methanol fraction. The methanol fraction was removed by vacuum distillation using a rotary evaporator to obtain 143 g of brownish-black extract. The brownish-black extract was uniformly mixed with 150 g of silica gel H (100-200 mesh). Separation was performed by medium-pressure silica gel (200-300 mesh) column chromatography, with gradient elution using a mixed organic solvent system of petroleum ether-ethyl acetate-methanol. After TLC analysis, similar fractions were combined to obtain 5 fractions (Frs. A-E). Frs. A was eluted by semi-preparative HPLC with 72% methanol-water isocratic elution (3.0 mL·min). -1 ), yielding the compound shown in formula (I) (2.56 mg, t R =12min)

[0056] (2) Structural identification

[0057] The chemical structure of the compound of formula (I) was determined by mass spectrometry (MS), nuclear magnetic resonance (NMR) and other spectroscopic techniques.

[0058] Compound of formula (I): brown solid; UV spectrum (solvent: methanol) is λ max (logε): 337 (0.27), 250 (1.56), 242 (1.45), 207 (1.98) nm; Infrared spectrum (potassium bromide tablet) is v max 2953, 2922, 2850, 1683, 1647, 1558, 1541, 1508, 1456, 1338, 470, 426, 428cm -1 HRESIMS showed a quasi-molecular ion peak at m / z 282.9616 [MH]. - (calc.for C 11 H8BrO4, 282.9611). 1 H NMR (500MHz, DMSO-d6) and 13 The C NMR (176MHz, DMSO-d6) data are shown in Table 1. 13 C NMR ( Figure 2 ) and HSQC spectrum ( Figure 3 The results show the presence of one lactone carbonyl group, seven quaternary carbons, one aromatic methine, and two methyl signals. 1 H NMR spectroscopy ( Figure 1 The HMBC spectrum shows that, in addition to the hydrogen-bonded hydroxyl proton signal, there are two methyl hydrogen signals and one isolated benzene ring hydrogen signal. Figure 4 The correlations shown were H-4 with C-3 and C-8a, Me-3 with C-3 and C-4, Me-7 with C-7 and C-8, and OH-8 with C-7, C-8, and C-8a. The monobromine isotope signatures on the mass spectrometer indicated that the compound was a 5-bromine analog, combined with... 1 H and 13 The structure of this isocoumarin compound was determined by the chemical shift value of C and the correlation between HSQC and HMBC. Literature review revealed that the compound of formula (I) is a new compound, named o-pyrone C, or similanpyrone C in English. It is a novel bromo-containing isocoumarin and the first naturally occurring bromo-containing isocoumarin compound discovered to date.

[0059] Based on the above physicochemical data analysis, the specific structure of the compound of formula (I) is shown below:

[0060]

[0061] Table 1. Compounds of Formula (I) in DMSO 1 H(500MHz), 13 176MHz NMR and HMBC data

[0062]

[0063] Example 2: Experimental data on the inhibition of acetylcholinesterase activity by the compound of formula (I)

[0064] The acetylcholinesterase inhibitory activity of the compound of formula (I) was tested using the Ellman assay, and it was found that the compound of formula (I) exhibits acetylcholinesterase inhibitory activity with an inhibition rate of 28.7%. Therefore, this invention provides an active compound with a novel chemical structure that inhibits cholinesterase, and it is expected to become a lead compound for cholinesterase inhibitors. This is of great significance for the development of highly efficient, low-toxicity, highly selective cholinesterase inhibitors with novel structures and mechanisms of action.

Claims

1. A compound represented by formula (I): ###0001### 2. A preparation method of the compound represented by formula (I), comprising the following steps: (1) fermenting and culturing a marine fungus strain Aspergillus sp. WXF1904; (2) extracting the fermentation culture with ethyl acetate, distilling and recovering the extract, and concentrating the extract; dissolving the concentrated extract with 95% methanol solution, placing the solution in a separatory funnel, and extracting with petroleum ether three times; collecting the methanol part and distilling and recovering the methanol to obtain a black extract; (3) separating the crude extract with a medium-pressure silica gel column, and gradient eluting with a mixed organic solvent system of petroleum ether-ethyl acetate-methanol; (4) column chromatographing the fraction obtained in step (3) in a semi-preparative ODS column, and isocratic eluting with a 72% methanol-water solution to obtain the compound represented by formula (I).

3. The preparation method according to claim 2, wherein the fermenting and culturing conditions of the marine fungus strain Aspergillus sp. WXF1904 are as follows: (1) liquid seed culture: inoculating mature seeds grown on a plate into a liquid seed culture medium, and culturing in a 25°C shaking bed at 180 rpm for 2 days; (2) fermentation: transferring an appropriate amount of the liquid seed obtained in step (1) into a solid fermentation culture medium, and culturing in a 25°C incubator for 30 days; harvesting the culture for separation and purification of secondary metabolites; the formula of the liquid seed culture medium is: malt extract 15 g, NaBr 28 g, tap water 1 L, pH 7.4-7.8; and the formula of the solid fermentation culture medium is: rice 200 g, NaBr 5.6 g, tap water 200 mL, and filled into a 1000 mL triangular flask.

4. Use of the compound represented by formula (I) in preparation of acetylcholinesterase inhibitors. ​

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