Isocoumarin dimer, preparation method thereof and application of isocoumarin dimer in resisting Alzheimer's disease

Abstract

The invention discloses an isocoumarin dimer, a preparation method thereof and application of the isocoumarin dimer in resisting Alzheimer's disease. The structural formulas of the compounds strepisocomarins A and B are as shown in a formula (I) in the specification. The isocoumarins dimers strepisocomarins A and B which are separated from a fermentation extract of streptomyces AHMU XC001 derived from dendrobium huoshanense and have a neuroprotective effect are both new compounds, and experimental results show that the isocoumarins dimers strepisocomarins A and B can be used for remarkably inhibiting the cell viability attenuation of a human neuroblastoma cell line SH-SY5Y induced by A beta 1-42, so that the activity of the human neuroblastoma cell line SH-SY5Y induced by the human neuroblastoma cell line SH-SY5Y induced by the human neuroblastoma cell line SH-SY5Y induced by the human neuroblastoma cell line SH-SY5Y induced by the human neuroblastoma cell line SH-SY5Y induced by the human neuroblastoma cell line the toxicity of the A beta 1-42 on a human neuroblastoma cell line SH-SY5Y is relieved; both of the two have a good neuroprotective effect, are expected to be developed into an anti-Alzheimer's disease medicine, and have important significance on development of traditional Chinese medicine symbiont resources.

Description

Technical Field

[0001] The invention belongs to the technical field of natural products, and particularly relates to a preparation method and application of isocoumarin compounds strepisocoumarins A and B. Background Art

[0002] Alzheimer's disease (AD), also known as primary senile dementia, is a degenerative disease of the central nervous system characterized by progressive cognitive impairment and memory loss. Its incidence increases with age, making it a highly prevalent disease second only to cardiovascular and cerebrovascular diseases and cancer.

[0003] β-amyloid protein (Aβ) is a polypeptide produced by the proteolytic hydrolysis of amyloid precursor protein by β- and γ-secretases. It contains 39 to 43 amino acid residues and is the main component of senile plaques in AD. In the normal brain, Aβ is a soluble neuronal metabolite that regulates several key physiological functions, exerting neuromodulatory effects on synaptic plasticity, memory, and neurotransmitter release. An imbalance between Aβ production and clearance is one of the early initiating factors of Alzheimer's disease. The most common Aβ isoforms are Aβ1-40 and Aβ1-42. Aβ1-42 is more toxic and more prone to aggregation, forming the core of Aβ deposits and triggering neurotoxic effects. The aggregation of Aβ in brain neurons is a major hallmark and cause of neurodegenerative diseases such as AD. Currently, there are no effective drugs that can halt or reverse the progression of Alzheimer's disease.

[0004] Isocoumarin, also known as isoflavandin, scientifically known as 1-hydro-2-benzopyran-1-one, belongs to the benzopyrone class of compounds. Isocoumarin is an isomer of coumarin with an inverted lactone ring. Hydrogenation saturates it to form dihydroisocoumarin. Isocoumarins and their derivatives are numerous and widely distributed in nature, produced by insects, plants, marine organisms, and microorganisms. Isocoumarin compounds also possess a wide range of biological and pharmacological activities, including antibacterial, anti-inflammatory, anti-tumor, neuroprotective, and herbicidal activities. For example, Yin Li et al. found that isocoumarins can effectively inhibit the cleavage of acute phase reaction protein (APP) by γ-secretase, reducing the generation of Aβ. Its inhibitory activity does not affect the cleavage of Notch1 protein. It is a specific inhibitor targeting γ-secretase cleavage of APP protein ("Yin Li, Zou Yuan, Han Wei, Gong Dezheng, Ma Xiaojian. Inhibitory effect of isocoumarins on Aβ protein in Alzheimer's disease and its mechanism. Chinese Journal of Hospital Pharmacy, 2012, 32(05): 329-332"). In addition, isocoumarins and their derivatives can be used as basic building blocks for the chemical synthesis of many natural products. Therefore, this family of compounds has received extensive research and attention.

[0005] Based on this, we collected rhizosphere soil samples from Dendrobium huoshanense in the Dabie Mountains, from which we isolated and screened a Streptomyces strain AHMU XC001. Utilizing the high stereoselectivity and catalytic ability of the enzymes in the biosynthesis process of this strain, we obtained the natural isocoumarin compounds 6,8-dihydroxy-3-methylisocoumarin and 6,8-dihydroxy-3-hydroxymethylisocoumarin from the fermentation products, and also obtained their polymers, strepisocoumarins A and B. Both strepisocoumarins A and B are new compounds with innovative structures, production strains, production processes and biological activities. Summary of the Invention

[0006] The first object of the present invention is to provide isocoumarins dimers, strepisocoumarins A and B, having neuroprotective effects.

[0007] The structural formulas of the isocoumarin compounds strepisocoumarins A and B are shown in formula (I).

[0008]

[0009] Compound 1 is strepisocoumarins A, and compound 2 is strepisocoumarins B;

[0010] Formula I.

[0011] A second object of the present invention is to provide a method for preparing isocoumarin dimers, strepisocoumarins A and B, wherein the isocoumarins dimers, strepisocoumarins A and B, are isolated from the fermentation product of Streptomyces AHMU XC001.

[0012] The specific steps are as follows:

[0013] (a) preparing a fermentation culture of Streptomyces AHMU XC001, centrifuging the fermentation supernatant and mycelium, extracting the mycelium with acetone, extracting the supernatant with ethyl acetate, and concentrating the extract under reduced pressure to obtain a crude extract of the mycelium and supernatant, which was then combined;

[0014] (b) The crude extract obtained in step (a) was mixed with silica gel and eluted by normal phase silica gel column chromatography using dichloromethane and methanol as eluents in ratios of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100 v / v to obtain the corresponding nine components, Fr.A1–Fr.A9;

[0015] A5 and A6, i.e., fractions eluted with dichloromethane and methanol at a ratio of 92:8 and 90:10, were combined and dichloromethane and methanol were used as eluents. The elution was performed at a ratio of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100 v / v to obtain the corresponding 9 components Fr.B1–Fr.B9.

[0016] The fractions A1, A2, and A3 (i.e., fractions eluted with dichloromethane and methanol in ratios of 100:0, 98:2, and 96:4) were combined, and petroleum ether and ethyl acetate were used as eluents in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.C1–Fr.C11.

[0017] Separate A4, i.e., the fraction eluted with dichloromethane and methanol in a ratio of 94:6, using petroleum ether and ethyl acetate as eluents in a ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.D1–Fr.D11.

[0018] Separate A7, the fraction eluted with dichloromethane and methanol in a ratio of 80:20, using ethyl acetate and methanol as eluents at a ratio of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, and 50:50 v / v to obtain 11 components, Fr.E1–Fr.E11.

[0019] A8, i.e., the fraction eluted with 50:50 dichloromethane and methanol, was separated using an eluent consisting of petroleum ether and ethyl acetate, then transitioning to ethyl acetate and methanol, with elutions in the following v / v ratios: P:E = 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, E:M = 95:5, and 90:10 to obtain the corresponding eight components, Fr.F1–Fr.F8.

[0020] Separation B4, i.e., the fraction eluted with dichloromethane and methanol (94:6 v / v), was eluted with petroleum ether and ethyl acetate in the ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, namely Fr.G1–Fr.G11;

[0021] Fractions B5 and B6 (i.e., fractions eluted with dichloromethane and methanol at a ratio of 92:8 and 90:10 v / v) were combined, and petroleum ether and ethyl acetate were used as eluents at a ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.H1–Fr.H11.

[0022] Separation B3, i.e., the fraction eluted with 96:4 v / v dichloromethane and methanol, used dichloromethane and methanol as eluents, and eluted in the ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90:10 v / v to obtain the corresponding Fr.I 1–Fr.I 11, a total of 11 components;

[0023] Component Fr.G6 contains target compounds 1, 2, and 3, component Fr.G7 contains target compounds 1, 2, and 4, component Fr.G8 contains target compound 2, component Fr.H6 contains target compounds 1, 3, and 4, component Fr.H7 contains target compounds 1 and 4, component Fr.H8 contains target compounds 1 and 2, components Fr.I3 and Fr.I4 contain target compounds 1 and 4. The above components are prepared by semi-preparative liquid chromatography to obtain target compounds 1, 2, 3, and 4, respectively.

[0024] The structural formulas of compounds 1, 2, 3, and 4 are shown below:

[0025]

[0026] The conditions of the semi-preparative liquid chromatography method are as follows: Cypress Basic C18 column 10×250 mm; mobile phase: ddH2O+0.1% acetic acid (phase A), acetonitrile (phase B); UV monitoring wavelength: 254 nm; flow rate: 3 mL / min; elution program: 0–15 min, 20%–35% phase B; 15–15.1 min, 35%–70%, phase B; 15.1–20 min, 70%–100%, phase B; 20–20.1 min, 100%, phase B; 20.1–25 min, 100%, phase B; 25–25.1 min, 100%–20%, phase B; 25.1-–30 min, 20%, phase B;

[0027] The target compound 1 was obtained with a retention time of 19.2 minutes; compound 2 with a retention time of 18.1 minutes; compound 3 with a retention time of 20.8 minutes; and compound 4 with a retention time of 17.1 minutes.

[0028] The third object of the present invention is to provide the use of two isocoumarin dimers, strepisocoumarins A and B, in the preparation of anti-Alzheimer's disease drugs.

[0029] The present invention also provides an anti-Alzheimer's disease drug containing strepisocoumarins A and / or B as an effective ingredient.

[0030] The present invention isolates neuroprotective isocoumarin dimers, strepisocoumarins A and B, from a fermentation extract of Streptomyces AHMU XC001 derived from Huoshan Dendrobium. Both are new compounds. Experimental results show that the isocoumarin dimers, strepisocoumarins A and B, can significantly inhibit Aβ1-42-induced cell viability attenuation in the human neuroblastoma cell line SH-SY5Y and alleviate the toxicity of Aβ1-42 on the human neuroblastoma cell line SH-SY5Y. Both have good neuroprotective effects and are expected to be developed into anti-Alzheimer's disease drugs, which is of great significance for the development of traditional Chinese medicine symbiotic resources.

[0031] Streptomyces sp. AHMU XC001 was published in the literature on April 27, 2025. ttps: / / www.ncbi.nlm.nih.gov / sra / ? term=PRJNA1255850 , whose accession number is PRJNA1255850, which is also held by the applicant and will remain available to the public for 20 years from the date of application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the isocoumarin dimer strepisocoumarins A(1) 1 H NMR (400 MHz) spectrum, solvent: DMSO-d6;

[0033] Figure 2 It is the isocoumarin dimer strepisocoumarins A(1) 13 C NMR (100 MHz) spectrum, solvent: DMSO-d6;

[0034] Figure 3 It is the isocoumarin dimer strepisocoumarins B(2) 1 H NMR (400 MHz) spectrum, solvent: DMSO-d6;

[0035] Figure 4 It is the isocoumarin dimer strepisocoumarins B(2) 13 C NMR (100 MHz) spectrum, solvent: DMSO-d6;

[0036] Figure 5 6,8-dihydroxy-3-methylisocoumarin (3) 1H NMR (400 MHz) spectrum, solvent: DMSO-d6;

[0037] Figure 6 6,8-dihydroxy-3-methylisocoumarin (3) 13 C NMR (100 MHz) spectrum, solvent: DMSO-d6;

[0038] Figure 7 6,8-dihydroxy-3-hydroxymethylisocoumarin (4) 1 H NMR (400 MHz) spectrum, solvent: DMSO-d6;

[0039] Figure 8 6,8-dihydroxy-3-hydroxymethylisocoumarin (4) 13 C NMR (100 MHz) spectrum, solvent: DMSO-d6;

[0040] Figure 9 Effects of compounds 1–4, vitamin E, and isocoumarins on the viability of the human neuroblastoma cell line SH-SY5Y and SH-SY5Y cells treated with Aβ1-42 (5 μmol / L). VE refers to vitamin E; ISM refers to isocoumarins; SPC-A refers to compound 1; SPC-B refers to compound 2; SPC-C refers to compound 3; and SPC-D refers to compound 4. (A) Effects of compound 3 at concentrations of 0, 5, 10, 20, and 40 μmol / L on the viability of SH-SY5Y and SH-SY5Y cells treated with Aβ1-42 (5 μmol / L); (B) Effects of compounds 1–4, vitamin E, and isocoumarins on the viability of the human neuroblastoma cell line SH-SY5Y; (C) Effects of compounds 1–4, vitamin E, and isocoumarins on the viability of SH-SY5Y cells treated with Aβ1-42 (5 μmol / L).

[0041] Figure 10 The chemical structures of strepisocoumarin A (1), strepisocoumarin B (2), 6,8-dihydroxy-3-methylisocoumarin (3), 6,8-dihydroxy-3-hydroxymethylisocoumarin (4), coumarin, and isocoumarin. DETAILED DESCRIPTION

[0042] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0043] Example 1 Isolation and Structural Identification of Isocoumarin Dimers Strepisocoumarins A and B

[0044] 1. Preparation of fermentation culture of Streptomyces AHMU XC001

[0045] (1) Preparation of seed culture medium and fermentation medium: Both the seed culture medium and the fermentation medium were AM2ab medium, with the following formula: soluble starch: 5 g / L; glucose: 20 g / L; soybean powder: 5 g / L; peptone: 2 g / L; yeast powder: 2 g / L; KH2PO4: 0.5 g / L; MgSO4·7H2O: 0.5 g / L; NaCl: 4 g / L, calcium carbonate: 2 g / L, solvent was ddH2O, and the pH was adjusted to 7.2–7.4. The seed culture medium was evenly divided into 250 mL conical flasks, 50 mL per bottle, and sterilized at 115°C for 30 min before use. The culture medium for scaled-up fermentation was divided into 1 L conical flasks, 200 mL per bottle, and sterilized at 115°C for 30 min before use.

[0046] (2) Seed culture: Activated spores of Streptomyces AHMU XC001 were inoculated into a 250 mL conical flask containing 50 mL of AM2ab medium and cultured on a shaker at 28°C and 200 rpm for 36 h to obtain a seed culture solution.

[0047] (3) Large-scale fermentation culture: 50 mL of the seed culture in the above-mentioned Erlenmeyer flask was transferred to a 1-L Erlenmeyer flask containing 200 mL of fermentation medium. The culture was shaken at 28°C and 200 rpm for 7 days to obtain a fermentation culture of Streptomyces AHMU XC001.

[0048] 2. Isolation of isocoumarin dimers strepisocoumarins A and B from the fermentation culture of Streptomyces AHMU XC001

[0049] (1) Extraction of fermentation culture

[0050] The supernatant and mycelium of the fermentation culture were centrifuged; the mycelium was extracted 3–5 times with an equal volume of acetone, and the supernatant was extracted 3–5 times with an equal volume of ethyl acetate, and the extracts were concentrated under reduced pressure to obtain a crude extract of the mycelium and supernatant;

[0051] (2) Separation of isocoumarin dimers strepisocoumarins A and B

[0052] The crude extracts of the mycelium and supernatant were combined and initially purified using normal-phase silica gel column chromatography. Nine fractions, Fr.A1–Fr.A9, were obtained using dichloromethane and methanol as eluents at v / v ratios of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100.

[0053] After combining A5 and A6 (i.e., the fractions eluted with dichloromethane and methanol at a ratio of 92:8 and 90:10), dichloromethane and methanol were used as eluents and eluted at a ratio of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100 v / v to obtain the corresponding 9 components Fr.B1–Fr.B9.

[0054] After combining A1, A2 and A3 (i.e., the fractions eluted with dichloromethane and methanol at 100:0, 98:2, and 96:4), petroleum ether and ethyl acetate were used as eluents and eluted at ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.C1–Fr.C11.

[0055] Component A4 (i.e., the fraction eluted with dichloromethane and methanol in a ratio of 94:6) was separated, and petroleum ether and ethyl acetate were used as eluents. The elution was performed in the ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, namely Fr.D1–Fr.D11.

[0056] Component A7 (i.e., the fraction eluted with dichloromethane and methanol in a ratio of 80:20) was separated using ethyl acetate and methanol as eluents. The elution was performed in a ratio of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, and 50:50 v / v to obtain 11 components, namely Fr.E1–Fr.E11.

[0057] Component A8 (i.e., the fraction eluted with dichloromethane and methanol in a ratio of 50:50) was separated, and an eluent consisting of petroleum ether and ethyl acetate, then transitioning to ethyl acetate and methanol, was used. Elution was performed according to the ratios of P:E = 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, E:M = 95:5, and 90:10 v / v to obtain the corresponding 8 components Fr.F1–Fr.F8.

[0058] The B4 component (i.e., the fraction eluted with 94:6 v / v of dichloromethane and methanol) was separated using petroleum ether and ethyl acetate as eluents. The elution was performed in the ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, namely Fr.G1–Fr.G11.

[0059] After combining B5 and B6 (i.e., the fractions eluted with dichloromethane and methanol at 92:8 and 90:10 v / v), petroleum ether and ethyl acetate were used as eluents, and elution was carried out at ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, namely Fr.H1–Fr.H11.

[0060] The B3 component (i.e., the fraction eluted with 96:4 v / v of dichloromethane and methanol) was separated by using dichloromethane and methanol as eluents, and eluted in the ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90:10 v / v to obtain the corresponding Fr.I 1–Fr.I 11, a total of 11 components.

[0061] HPLC detection revealed that components Fr.D3, Fr.D4, and Fr.D5 contained target compound 3, component Fr.G6 contained target compounds 1, 2, and 3, component Fr.G7 contained target compounds 1, 2, and 4, component Fr.G8 contained target compound 2, component Fr.H6 contained target compounds 1, 3, and 4, component Fr.H7 contained target compounds 1 and 4, component Fr.H8 contained target compounds 1 and 2, and components Fr.I3 and Fr.I4 contained target compounds 1 and 4. Semi-preparative liquid chromatography was used to prepare the above components in sequence. The corresponding samples were collected according to the peak retention time and peak shape, and the samples were spin-dried and weighed. The specific preparation method is shown below. Target compounds 1 (11 mg, retention time 19.2 minutes), 2 (16 mg, retention time 18.1 minutes), 3 (127 mg, retention time 20.8 minutes), and 4 (113 mg, retention time 17.1 minutes) were obtained respectively.

[0062] The conditions for analytical HPLC were as follows: instrument model was Waters E2695+2998 UV detector; Agilent 4.6×150mm reversed-phase C18 column; mobile phase: ddH2O+0.1% acetic acid (phase A), acetonitrile+0.1% acetic acid (phase B); UV monitoring wavelength: 254nm; flow rate: 1mL / min; elution program was 0–20min, 10%–80% phase B; 20–20.1min, 80%–100% phase B; 20.1–25min, 100% phase B; 25–25.1min, 100%–10% phase B; 25.1–30min, 10% phase B.

[0063] The conditions for semi-preparative HPLC were as follows: Cypress Basic C18 column (10×250 mm); mobile phase: ddH2O + 0.1% acetic acid (phase A), acetonitrile (phase B); UV monitoring wavelength: 254 nm; flow rate: 3 mL / min; elution program: 0–15 min, 20%–35% phase B; 15–15.1 min, 35%–70%, phase B; 15.1–20 min, 70%–100%, phase B; 20–20.1 min, 100%, phase B; 20.1–25 min, 100%, phase B; 25–25.1 min, 100%–20%, phase B; 25.1-–30 min, 20%, phase B.

[0064] The invention relates to a preparation process of isocoumarin dimers strepisocoumarins A and B (i.e. compounds 1 and 2), 6,8-dihydroxy-3-methylisocoumarin (compound 3), and 6,8-dihydroxy-3-hydroxymethylisocoumarin (compound 4).

[0065] 3. Structural elucidation and spectral data of isocoumarin dimers strepisocoumarins A and B

[0066] The four target compounds obtained were subjected to mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) tests, and the test results were analyzed to obtain the following physicochemical and spectral data. After structural analysis, they were identified and named as strepisocoumarins A and B (i.e., compounds 1 and 2), 6,8-dihydroxy-3-methylisocoumarin (compound 3) and 6,8-dihydroxy-3-hydroxymethylisocoumarin (compound 4), all of which are isocoumarin compounds.

[0067] The structural analysis process is described as follows:

[0068] Compound 1 is a yellow amorphous powder. Its molecular formula is C according to HRESIMS. 20 H14 O9(m / z 399.0697[M+H] + , calculated value 399.0716), indicating that there are 14 unsaturations. 1 H. 13 C and HSQC data showed that there were six ketone carbonyl signals (δ C 162.2, 162.3, 165.0, 165.2, 165.6, 165.9), four methylene carbons (δ C 101.1, 101.5, 101.9, 102.4), a methyl group (δ C 18.9) and a hydroxymethyl group (δ C 59.4), indicating that 1 is a heterodimer. HMBC showed that H-9 correlated with C-4, H-4 correlated with C-5 and C-8a, and H-7 correlated with C-5 / C-8a, proving that one of the monomer units of compound 1 is compound 4. H-9' correlated with C-4', H-4' correlated with C-5' and C-8a', and H-7' correlated with C-5' and C-8a', proving that compound 3 is another monomer in compound 1. 1 The absence of H-5 and H-5' signals in H NMR and the unsaturation of compound 1 confirmed that compound 1 was a C-5 / C-5' cross-coupled heterodimer of compound 4 and compound 3, and it was named strepisocoumarin A ( Figure 10 ),That 1 H and 13 C NMR spectral data are shown in Table 1.

[0069] Compound 2 is a yellow amorphous powder. According to HRESIMS, it is speculated that it has the same molecular formula C as compound 1. 20 H 14 O9(m / z 399.0697[M+H] + , calculated value 399.0716). 1 H. 13 C and HSQC data analysis showed that it has almost the same chemical groups as 1, including six keto carbonyl signals (δ C 160.9,162.2,164.2,164.3,165.6,166.1), four methylene carbons (δ C 101.2,101.6,102.0,104.2), a methyl group (δ C 18.9) and a hydroxymethyl group (δ C59.3), thus confirming that compound 2 is also a heterodimer. The 1D NMR data of compounds 1 and 2 are very similar. Combined with the HMBC correlation, it was determined that compound 2 is also formed by the dimerization of two monomers of compounds 3 and 4, but the difference is that H-5' is correlated with C-7' and C-8a'. Therefore, compound 2 is a C-5 / C-7' cross-coupled heterodimer of compounds 4 and 3, and it is named strepisocoumarin B ( Figure 10 ),That 1 H and 13 C NMR spectral data are shown in Table 1.

[0070] 6,8-Dihydroxy-3-methylisocoumarin (3): white amorphous powder; 1 H and 13 C NMR spectrum data are shown in Table 2; (+) HR-ESI-MS m / z 193.0490 [M+H] + (calcd.for C 10 H9O4 + ,193.0501), which was identified as the known compound 6,8-dihydroxy-3-methylisocoumarin by consulting the literature.

[0071] 6,8-Dihydroxy-3-hydroxymethylisocoumarin (4): white amorphous powder; 1 H and 13 C NMR spectrum data are shown in Table 2; (+) HR-ESI-MS m / z 209.0442 [M+H] + (calcd.for C 10 H9O5 + ,209.0450), which was identified as the known compound 6,8-dihydroxy-3-hydroxymethylisocoumarin by consulting the literature.

[0072] Table 1. Strepisocoumarins A and B 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data

[0073]

[0074] Table 2. 6,8-dihydroxy-3-methylisocoumarin (3) and 6,8-dihydroxy-3-hydroxymethylisocoumarin (4) 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data

[0075]

[0076]

[0077] The target compounds isolated by the above method, strepisocoumarins dimer strepisocoumarins A (compound 1 - code name SPC-A) and B (compound 2 - code name SPC-B), 6,8-dihydroxy-3-methylisocoumarin (compound 3 - code name SPC-C), and 6,8-dihydroxy-3-hydroxymethylisocoumarin (compound 4 - code name SPC-D) have structures as shown in formula (I) and Figure 10 shown.

[0078] Example 2 Effects of Isocoumarin Dimers Strepisocoumarins A and B on the Viability of Human Neuroblastoma Cell Line SH-SY5Y and SH-SY5Y Cells Treated with Aβ1-42 (5 μmol / L)

[0079] Activity test cell line: human neuroblastoma cell line SH-SY5Y.

[0080] Cell Culture and Grouping: SH-SY5Y cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO2, 37°C incubator. Cells were passaged when the confluence reached 80%–90%. First, the dose of compound 3 was explored at concentrations of 0, 5, 10, 20, and 40 μmol / L. A control group A and an Aβ1-42 (5 μmol / L) experimental group A were set up. Both groups received compound 3 at different concentrations. Aβ1-42 was not added to the control group A. Next, compounds 1, 2, 3, or 4 were added at 5 μmol / L. A control group B and an Aβ1-42 (5 μmol / L) experimental group B were set up. Both groups received compound 1, 2, 3, or 4 at 5 μmol / L. Aβ1-42 was not added to the control group B.

[0081] MTT assay for cell viability: SH-SY5Y cells were plated at 1×10 4The cells were plated at a concentration of 100 μL / well in a 96-well plate. To determine the effects of compounds 1-4, isocoumarins, and vitamin E (both positive controls) on the viability of Aβ1-42-treated SH-SY5Y cells, experimental groups A and B were pretreated with culture medium containing Aβ1-42 (5 μmol / L) for 2 hours to establish an AD cell model. The culture medium containing the compounds was then replaced and cultured for an additional 24 hours. 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well and incubated for a further 4 hours. The supernatant was then removed by carefully aspirating the supernatant from the 96-well plate using a pipette. 100 μL of DMSO was added to the 96-well plate and incubated at 37°C for 15 minutes. Control groups A and B did not receive Aβ1-42, and all other treatments were the same as those for experimental groups A and B. A complete DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin was used as a blank control. Treatment conditions, including time and temperature, were consistent with those of the experimental and control groups. Optical density (OD) values ​​were measured at a wavelength of 490 nm using a microplate reader, and cell viability was calculated for each group. Cell viability (%) = [OD (490) experimental group - OD (490) blank group] / [OD (490) control group - OD (490) blank group] × 100%.

[0082] The results showed that when the human neuroblastoma cell line SH-SY5Y was treated with compound 3 at concentrations of 5, 10, 20 and 40 μmol / L, the cell viability was not affected compared with the control, indicating that compound 3 had no cytotoxicity at the four tested concentrations ( Figure 9 A); At the same time, when the AD cell model constructed by Aβ1-42 (5μmol / L) was treated with compound 3 at concentrations of 5, 10, 20 and 40μmol / L, the cell viability of the experimental group was significantly restored compared with the control group ( Figure 9 A), indicating that compound 3 can reverse the decrease in cell viability induced by Aβ1-42. Therefore, the compound concentration of 5 μmol / L was selected for subsequent activity testing of other compounds.

[0083] When SH-SY5Y cells were treated with 5 μmol / L of compounds 1, 2, 3, 4, isocoumarin (ISM) and vitamin E (VE), their viability was not affected ( Figure 9 B); When the AD cell model constructed by Aβ1-42 (5 μmol / L) was treated with compounds 1, 2, 3, 4, isocoumarin and vitamin E at a concentration of 5 μmol / L, the cell viability of SH-SY5Y in the experimental group with the addition of compounds was significantly restored compared with the control group without the addition of compounds ( Figure 9C), indicating that compounds 1, 2, 3, and 4 can significantly inhibit the reduction of cell viability induced by Aβ1-42, and their effects are superior to those of the positive controls isocoumarin and vitamin E. Therefore, compounds 1, 2, 3, and 4 can mitigate the toxicity of Aβ1-42 on the human neuroblastoma cell line SH-SY5Y; they all have good neuroprotective effects and are expected to be developed as anti-Alzheimer's disease drugs.

Claims

1. Compounds strepisocoumarins A and B, whose structural formula is shown in formula (I): Compound 1 is strepisocoumarins A, and compound 2 is strepisocoumarins B.

2. A method for preparing strepisocoumarins A and B, characterized in that: The strepisocoumarins A and B are separated from the fermentation product of Streptomyces AHMU XC001.

3. The preparation method according to claim 2, characterized in that The specific steps are as follows: (a) preparing a fermentation culture of Streptomyces AHMU XC001, centrifuging the fermentation supernatant and the mycelium, extracting the mycelium with acetone, extracting the supernatant with ethyl acetate, and concentrating the extract under reduced pressure to obtain a crude extract of the mycelium and the supernatant, and combining the crude extracts; (b) The crude extract obtained in step (a) was mixed with silica gel and eluted by normal phase silica gel column chromatography using dichloromethane and methanol as eluents in ratios of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100 v / v to obtain the corresponding nine components, Fr.A1–Fr.A9; A5 and A6, i.e., fractions eluted with dichloromethane and methanol at a ratio of 92:8 and 90:10, were combined and dichloromethane and methanol were used as eluents. The elution was performed at a ratio of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 50:50, and 0:100 v / v to obtain the corresponding 9 components Fr.B1–Fr.B9. The fractions A1, A2, and A3 (i.e., fractions eluted with dichloromethane and methanol in ratios of 100:0, 98:2, and 96:4) were combined, and petroleum ether and ethyl acetate were used as eluents in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.C1–Fr.C11. Separate A4, i.e., the fraction eluted with dichloromethane and methanol in a ratio of 94:6, using petroleum ether and ethyl acetate as eluents in a ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.D1–Fr.D11. Separate A7, the fraction eluted with dichloromethane and methanol in a ratio of 80:20, using ethyl acetate and methanol as eluents at a ratio of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, and 50:50 v / v to obtain 11 components, Fr.E1–Fr.E11. A8, i.e., the fraction eluted with 50:50 dichloromethane and methanol, was separated using an eluent consisting of petroleum ether and ethyl acetate, then transitioning to ethyl acetate and methanol, with elutions in the following v / v ratios: P:E = 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, E:M = 95:5, and 90:10 to obtain the corresponding eight components, Fr.F1–Fr.F8. Separation B4, i.e., the fraction eluted with dichloromethane and methanol (94:6 v / v), was eluted with petroleum ether and ethyl acetate in the ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, namely Fr.G1–Fr.G11; Fractions B5 and B6 (i.e., fractions eluted with dichloromethane and methanol at a ratio of 92:8 and 90:10 v / v) were combined, and petroleum ether and ethyl acetate were used as eluents at a ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 v / v to obtain 11 components, Fr.H1–Fr.H11. Separation B3, i.e., the fraction eluted with 96:4 v / v dichloromethane and methanol, used dichloromethane and methanol as eluents, and eluted in the ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90:10 v / v to obtain the corresponding Fr.I 1–Fr.I 11, a total of 11 components; Component Fr.G6 contains target compounds 1, 2, and 3, component Fr.G7 contains target compounds 1, 2, and 4, component Fr.G8 contains target compound 2, component Fr.H6 contains target compounds 1, 3, and 4, component Fr.H7 contains target compounds 1 and 4, component Fr.H8 contains target compounds 1 and 2, and components Fr.I3 and Fr.I4 contain target compounds 1 and 4. The above components are prepared by semi-preparative liquid chromatography to obtain target compounds 1-4 respectively; The structural formula of the compound 1-4 is shown below:

4. The preparation method according to claim 3, characterized in that The semi-preparative liquid chromatography conditions are as follows: Cypress Basic C18 column 10×250 mm; mobile phase: ddH2O + 0.1% acetic acid (phase A), acetonitrile (phase B); UV monitoring wavelength: 254 nm; flow rate: 3 mL / min; elution program: 0–15 min, 20%–35% phase B; 15–15.1 min, 35%–70%, phase B; 15.1–20 min, 70%–100%, phase B; 20–20.1 min, 100%, phase B; 20.1–25 min, 100%, phase B; 25–25.1 min, 100%–20%, phase B; 25.1–30 min, 20%, phase B; The target compound 1 was obtained with a retention time of 19.2 minutes; compound 2 with a retention time of 18.1 minutes; compound 3 with a retention time of 20.8 minutes; and compound 4 with a retention time of 17.1 minutes.

5. Use of the strepisocoumarins A and / or B according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-Alzheimer's disease drug.

6. An anti-Alzheimer's drug, characterized in that: Contains the strepisocoumarins A and / or B according to claim 1, or a pharmaceutically acceptable salt thereof as an active ingredient.

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