CN114478458A - Novel isocoumarin, its preparation method and application - Google Patents

Abstract

The invention relates to isocoumarin, a preparation method and application thereof, in particular to application in inhibiting acetylcholinesterase, wherein the isocoumarin is shown as the following formula (I):

Description

Novel isocoumarin, its preparation method and application

Technical Field

The invention relates to the field of marine organism natural products, in particular to a novel isocoumarin prepared from marine fungus XFW1904 and application thereof in inhibiting acetylcholinesterase.

Background

Marine-derived organisms have great potential to produce new skeletal compounds with diverse activities. Isocoumarins are structurally related to another natural lactone called coumarin, but, as their name suggests, have an inverted lactone ring. In other words, it is an isomer with coumarin. In general, isocoumarins differ in the groups attached to the side chains of the basic structure, such as OH, OMe, OAc, COOH and sugar residues. Isocoumarin has the IUPAC name 1H-isochroman-1-one, and also has other names, such as 1H-2-benzopyran-1-one, 3, 4-benzo-2-pyrone, and isocoumarin. A large number of natural sources of isocoumarins have been discovered and isolated from a variety of natural sources. For example, isocoumarins and 3, 4-dihydroisocoumarins are found not only in fungi, lichens, mosses, bacteria, molds and some higher plants, but also in sponges, insect pheromones and venom species. In particular, fungi such as Penicillium, Ceratodon, Fusarium, Aspergillus, Cladosporium, and Oomyces are rich in isocoumarin. Similarly, higher plants, such as Apiaceae, Leguminosae, Compositae, Asteraceae, Saxifragaceae, Lycopodiaceae, hydrangeaceae, Compositae, and Myricaceae contain isocoumarin metabolites. Marine sponges, such as viscose sponges, sammenol sponges and dendritic illicin sponges, are also sources of various isocoumarin metabolites, which are highly cytotoxic.

Isocoumarins, which are ubiquitous in many natural products, are exemplified. For example, "Bergenin" is a polyphenol derivative isolated from different plants, such as plants of the genus bergenia, plants of the genus Artemisia, plants of the genus Malus, etc. (Barry, R.D. Isooularins. development site 1950.chem.Rev.1964, 64, 229-; Patel, D.K.; Patel, K.; Kumar, R.J.; Gadewar, M.; Tahlyani, V.Pharmacological and Analytical Aspects of Bergenin: A circumcise report. Asian Pac.J.Trop.Dis 2012, 2, 163-) -260). Two Isocoumarins bacilosacins A and B (Azumi, M.; Ogawa, K.; Fujita, T.; Takeshita, M.; Yoshida, R.; Furuma, T.; Igarashi, Y.Bacilosacins A and B, Novel Bioactive Isocoumarins With U.nuous Heterocyclic nucleic acid From the Marine group Bacillus subtilis strain tetrahedron 2008, 64, 6420-. Other natural sources of isocoumarins include paramhaeosphaerins A, chaetocerins A, and paramhaeosphaerins C. The two compounds are respectively separated From two epiphytes Paraphaeospora quatrisita and Chaetomium chinensis in two Sonolan Desert plants, namely cactus and ephedra (Wijerane, E.M.K.; Paranagama, P.A.; Gunataka, A.A.L. five Isocoumarins From Sonorana Desert Plant-Associated Fungal Strains Paraphaeospora quatrisata and Chaetomium chinensis 8446). Isocoumarins are also chemical components of the genus Vatica (Vatica belongs to the Dipterocarpaceae family of the major angiosperm group) and possess a variety of pharmacological properties, including H-resistanceIV, antitumor, antibacterial and anti-inflammatory activity (Chen, G.; Mo, Z.; Song, X.; Yi, W.; Han, C.Advances in the students on Chemical Constitutes and pharmaceutical Effect of viral genus Ziran Kexueban 2008, 21, 180-183. chem.Abstract.No. 150: 274651). Three isocoumarin metabolites, dichlordioportin, Diaportinol, Diaportinic Acid, were isolated from a fungus Penicillium nalgiovense (Larsen, T.O.; Breinholt, J.Dichlorodioportin, Diaportinol, and Diaportinic Acid: Three Novel isocoumarins from Penicillium nalgiovense.J.Nat.prod.1999, 62, 1182-. The phenol derivative is separated from the isocoumarins and 6-methyl-isocoumarins. Also, the C-3 side chain on the isocoumarin ring is a critical structure in all these cases. An unusual tetrahydroxy isocoumarin was isolated from a coral-derived P.pestalotiopsis species in south China sea (Zhang, L., Said, G., Haider, W., Shao, C.L., Chen, G.Y., Wei, M.Y.A. new polyhydroxyisocoumarin from the same-derivative fungus. Isocoumarin reticulol and 6-O-methyl-reticulol are separated from an endophytic fungus Biscogniauxia capnode. Reticulol has moderate antioxidant activity (IC) against PPH free radicals₅₀A value of 58 μ g/mL) (srithan, t., Savitri Kumar, n., Jayasinghe, l., Araya, h., Fujimoto, y., Isocoumarins and dihydroisocoumarins from the endo-hydrodynamic funcus biscogmogiauya capnodides from the from of Averrhoa carbola. nat. pro. commu.2019, 14, 4519378X 19851969). Isocoumarin derivatives (3S, S) -4, 5-dihydroxy-mellein and R- (-) -mellein-8-O-. beta. -d-glucopyranoside (Zhang, L.H., Li, S.G., Wu, H.H., Chen, G., Li, L., Bai, J., Pei, Y.H., 3, 4-Dihydroisocoumarin derivative from the marine-derived lance paraoxonium sporulatum YK-03.phytochem. Lett.2017, 20, 200-Bufonic 203.) were isolated from marine-derived fungi Paraconiothrium sporulatum YK-03.

Alzheimer's Disease (AD), also known as senile dementia, is a degenerative neuropathy of the central nervous system occurring in middle and old aged people, and is the most common type of clinical senile dementia. Due to the lack of specific clinical measures, AD has become the fourth cause of death in the elderly after cardiovascular and cerebrovascular diseases, malignancies, and stroke (Wang XP, Ding HL. Alzheimer's disease: epidemiology, genetics, and beyond. neurosci Bull.2008, 24: 105-. The theory of cholinergic impairment is widely accepted in the study of the pathogenesis of AD. AChE developed based on the theory of cholinergic impairment reduces the rate of hydrolysis of acetylcholine by inhibiting the activity of acetylcholinesterase (AChE), thereby increasing its content in the body of a patient. Of the five drugs approved by the U.S. Food and Drug Administration (FDA) for clinical treatment of AD, four are AChEI drugs, including tacrine (tacrine), donepezil (donepezil), rivastigmine (rivastigmine), galantamine (galantamine), and galantamine (galantamine), for treatment of patients with mild and moderate AD (folk, linkejiang, enlightening. progress of anti-alzheimer cholinergic drug research, proceedings of the western academy of medicine, 2009, 7: 117-. However, most of these drugs have short half-lives and have more serious side effects of the peripheral cholinergic system. Therefore, the search for a cholinesterase inhibitor with high efficiency, high selectivity, small side effect and novel structure and action mechanism has important significance in the prevention and treatment of the Alzheimer disease.

Disclosure of Invention

The invention relates to a novel isocoumarin extracted from marine fungus XFW1904 and shown in the following formula (I), an analogue thereof, a preparation method thereof and application thereof in inhibiting acetylcholinesterase.

In a first aspect, the present invention provides a compound of the general formula (a):

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

In a second aspect, the present invention provides a compound of formula (I):

in a third aspect, the present invention provides a process for the preparation of a compound of formula (I),

the method comprises the following steps:

(1) fermenting and culturing a marine fungus strain XFW 1904;

(2) extracting fermentation culture with ethyl acetate, distilling, recovering, concentrating, dissolving the concentrated solution in 95% methanol solution, extracting with petroleum ether for three times to obtain petroleum ether extract part and methanol part, and distilling and recovering the methanol part to obtain crude extract;

(3) separating the crude extract by using a silica gel column, and performing gradient elution by using a petroleum ether-ethyl acetate-methanol system:

(4) and (4) subjecting the fraction obtained in the step (3) to ODS semi-preparative column HPLC, and eluting with 70% acetonitrile-water isocratic to obtain the compound shown in the formula (I).

Preferably, the fermentation culture conditions of the marine fungus strain XFW1904 are as follows:

(1) inoculating the flat seeds into a liquid seed culture medium, and performing shake culture at 25 ℃ and 180rpm for 2 d:

(2) transferring the culture obtained in the step (1) into a solid fermentation culture medium, standing and culturing at 25 ℃, and harvesting the culture after 30 days to separate and purify a secondary metabolite;

the liquid seed culture medium comprises the following components: 15g of malt extract, 16g of sea salt and 1L of tap water, wherein the pH value is 7.4-7.8; the solid fermentation medium comprises the following components: 200g rice, 200mL water, 3.2g crude sea salt, packed in 1000mL triangular flask.

In a fourth aspect, the present invention provides a process for the preparation of a compound of the formula (A),

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

The method comprises a preparation method of the compound shown in the formula (I) in the third aspect, R¹And R²The substituents are conventional substituents, and on the basis of obtaining the compound shown in the formula (I), a person skilled in the art can obtain all the compounds shown in the general formula (A) through routine tests.

In a fifth aspect, the present invention provides a marine fungal strain XFW1904 useful for the preparation of a compound of formula (I);

preferably, the fermentation culture conditions of the marine fungus strain XFW1904 are as follows:

(1) inoculating the flat seeds into a liquid seed culture medium, and performing shake cultivation for 2d at 25 ℃ and 180 rpm;

(2) transferring the culture obtained in the step (1) into a solid fermentation culture medium, standing and culturing at 25 ℃, and harvesting the culture after 30 days to separate and purify a secondary metabolite;

preferably, the composition of the liquid seed culture medium is: 15g of malt extract, 16g of sea salt and 1L of tap water, wherein the pH value is 7.4-7.8; the solid fermentation culture medium comprises the following components: the solid fermentation medium comprises the following components: 200g rice, 200mL water, 3.2g crude sea salt, packed in 1000mL triangular flask.

In a sixth aspect, the present invention provides the use of a compound of formula (a) below for inhibiting acetylcholinesterase:

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

Preferably, the compound is a compound represented by the following formula (I):

in a seventh aspect, the present invention provides the use of a compound of formula (a) below for inhibiting acetylcholinesterase:

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

Preferably, the compound is a compound represented by the following formula (I):

the compound of the formula (I) is prepared by carrying out large-scale fermentation on a solid rice fermentation medium by using a marine fungus strain XFW1904, and extracting, separating and purifying the fermentation culture. Through structure analysis, the coumarin is determined to be a novel compound with an isocoumarin structure, and the specific structure is shown as a formula (I). The compounds of formula (I) were found to have an acetylcholinesterase-inhibiting effect by evaluating their acetylcholinesterase-inhibiting activity. It is therefore a second object of the present invention to provide the use of a compound of formula (I) for inhibiting acetylcholinesterase.

The invention provides an alternative compound for developing a novel active substance for inhibiting cholinesterase, and has important significance for developing the utilization of cholinesterase inhibiting compound resources derived from Chinese marine microorganisms.

Drawings

Figure 1 illustrates the UV information of the compound of formula (I).

FIG. 2 shows schematically the IR information for the compound of formula (I).

Detailed Description

The technical solution of the present invention is further described below with reference to the following embodiments and the accompanying drawings. These examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

Example 1: preparation and structural characterization of Compounds of formula (I)

(1) Preparation of

1. The microbial material is as follows:

the fungus strain XFW1904 is isolated from a starfish sample collected from the sea area of Shishen Haishan of northwest Hai of south China sea.

2. And (3) microorganism culture conditions:

culture medium:

liquid seed culture medium: malt extract 15g, agar 18g, sea salt 16g, tap water 1L, pH 7.4-7.8

Solid fermentation medium: 200g rice, 3.2g crude sea salt, 200mL water (in 1000mL triangular flask)

Inoculating the flat seeds into a liquid seed culture medium, performing shake culture at 25 ℃ and 180rpm for 2d, transferring the flat seeds into a solid fermentation culture medium, performing static culture at 25 ℃, harvesting the culture after 30 days, and performing secondary metabolite separation and purification.

3. Extraction and separation:

extracting with ethyl acetate, distilling, recovering, dissolving the concentrated solution in 95% methanol solution, and extracting with petroleum ether for three times to obtain petroleum ether extract part and methanol part. Petroleum ether and methanol were removed separately by a rotary evaporator to give 145.5g of oily extract at the petroleum ether extraction site and 46.7g of brownish black extract at the methanol site. And uniformly mixing the brownish black extract with 50g of silica gel H (100-200 meshes). Separating by medium-pressure silica gel (200-300 meshes) column chromatography, and performing gradient elution by using a petroleum ether-ethyl acetate-methanol elution system. Similar fractions were pooled after TLC detection analysis to give 10 fractions (Frs. A. J). Frs.A was subjected to semi-preparative HPLC, and eluted at 70% acetonitrile-water isocratic (3.0 mL. min)^-1) To give Compound 1(10.2mg, t)_R＝7.5min)

(2) Structural identification

The chemical structure of the compound of formula (I) was determined by Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR), UV, IR.

A compound of formula (I): grey powder, ESI-MS [ M ]]^-m/z: 239.0124 formula C₁₁H₉ClO₄。TR：3388.93、1674.21、1651.07、1435.05、1199.72、1139.93、842.89、802.39、723.31、667.37、547.78；UV(MeOH)：λ_max(logε)＝249.40(3.279)nm；¹H NMR(500MHz，CDCl₃) And¹³C NMR(500MHz，CDCl₃) See table 1.

From the analysis of the physicochemical data above, the specific structure of the compound of formula (I) is shown below:

TABLE 1 preparation of compounds of formula (I) in DMSO¹H(500MHz)、¹³C NMR (125MHz) and HMBC data

Example 2: experimental data for inhibition of acetylcholinesterase Activity of Compounds of formula (I)

The acetylcholinesterase inhibitory activity of the compound of formula (I) was tested using the Ellman method, and the compound of formula (I) was found to have acetylcholinesterase inhibitory activity with an inhibition rate of 19.9%. Therefore, the cholinesterase inhibitor provided by the invention has a novel chemical structure, is expected to become a lead compound of the cholinesterase inhibitor, and has important significance for developing the cholinesterase inhibitor which is efficient, high in selectivity, small in side effect and novel in structure and action mechanism.

Claims (11)

1. A compound represented by the general formula (A):

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

2. A compound of formula (I):

3. a process for the preparation of a compound of formula (I),

the method comprises the following steps:

(1) fermenting and culturing a marine fungus strain XFW 1904;

(2) extracting fermentation culture with ethyl acetate, distilling, recovering, concentrating, dissolving the concentrated solution in 95% methanol solution, extracting with petroleum ether for three times to obtain petroleum ether extract part and methanol part, and distilling and recovering the methanol part to obtain crude extract as brownish black extract;

(3) separating the crude extract with medium pressure silica gel column, and performing gradient elution with petroleum ether-ethyl acetate-methanol system;

(4) and (4) carrying out column chromatography on the fraction obtained in the step (3) by using a semi-preparative ODS chromatographic column, and eluting by using acetonitrile-water isocratic elution to obtain the compound shown in the formula (I).

4. The production method according to claim 3, wherein the fermentation culture conditions of the marine fungus strain XFW1904 are as follows:

(1) inoculating the flat seeds into a liquid seed culture medium, and performing shake cultivation for 2d at 25 ℃ and 180 rpm;

(2) transferring the culture obtained in the step (1) into a solid fermentation culture medium, standing and culturing at 25 ℃, and harvesting the culture after 30 days to separate and purify a secondary metabolite;

the liquid seed culture medium comprises the following components: 15g of malt extract, 16g of sea salt and 1L of tap water, wherein the pH value is 7.4-7.8; the solid fermentation medium comprises the following components: 200g of rice, 3.2g of crude sea salt and 200mL of water are filled in a 1000mL triangular flask.

5. A process for producing a compound represented by the general formula (A),

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph，

The method comprises the steps of the method of claim 3 and the steps of the method of claim 4.

6. The process for the fermentative culture of a marine fungal strain XFW1904 according to claim 3, wherein said process comprises the steps of:

(1) inoculating the flat seeds into a liquid seed culture medium, and performing shake cultivation for 2d at 25 ℃ and 180 rpm;

(2) transferring the culture obtained in the step (1) into a solid fermentation culture medium, standing and culturing at 25 ℃, harvesting the culture after 30 days, separating and purifying secondary metabolites,

wherein the liquid seed culture medium comprises the following components: 15g of malt extract, 16g of sea salt and 1L of tap water, wherein the pH value is 7.4-7.8; the solid fermentation medium comprises the following components: 200g of rice, 3.2g of crude sea salt and 200mL of water are filled in a 1000mL triangular flask.

7. Use of a compound of formula (a) for inhibiting acetylcholinesterase:

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

8. Use according to claim 7, wherein the enzyme to be inhibited is acetylcholinesterase.

9. The use according to claim 7 or 8, which is a compound represented by the following formula (I):

10. use of a compound of formula (a) below for inhibiting acetylcholinesterase:

wherein R is₁H or CH₃，R₂H or COCH₃Or CH₂-Ph。

11. The use according to claim 10, wherein the compound is of formula (I):

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