A compound with antiepileptic activity, its preparation method and uses

Abstract

This invention discloses a compound with antiepileptic activity, its preparation method, and its uses. The compound's structural formula is shown in Figure I. The preparation method involves fermenting actinomycetes to obtain pyranone ferments, then extracting the ferments with ethyl acetate to obtain a crude extract. This crude extract is then purified by normal-phase silica gel column chromatography, reversed-phase medium-pressure column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography. This pyranone compound has potential applications as an antiepileptic inhibitor. Its advantage lies in its antiepileptic activity, suggesting its potential for developing drugs for the prevention and treatment of epilepsy.

Description

Technical Field

[0001] This invention relates to an α-pyranone compound, and more particularly to a compound with anti-epileptic activity extracted from marine actinomycetes, its preparation method, and its uses. Background Technology

[0002] Epilepsy is a serious neurological disorder affecting approximately 50 million people worldwide. It is typically characterized by recurrent, unpredictable seizures and can lead to complications such as anxiety, depression, cognitive decline, and increased mortality. According to the World Health Organization, epilepsy significantly impacts patients' quality of life. Currently, antiepileptic drugs (AEDs) are generally used to relieve symptoms, but only about 30 drugs are approved for clinical use. Although these drugs effectively control seizures in most patients, drug resistance develops in about 30% of cases. Early antiepileptic drugs only relieved 15-20% of drug-resistant cases and often caused adverse reactions such as dizziness, fatigue, and cognitive impairment, which are among the main reasons for current AED treatment failures. The pathological mechanism of epilepsy is complex, generally believed to be caused by an imbalance in the brain (including the hippocampus, neocortex, corticothalamus, and basal ganglia networks) resulting from the inhibition of excitatory γ-aminobutyric acid (GABA)-mediated neurotransmission and the activation of inhibitory glutamatergic neurotransmission.

[0003] Natural products are considered an important source of drug lead compounds due to their chemical diversity. Marine actinomycetes, with their unique living environment and metabolic pathways, have become a vital resource for discovering novel drug compounds, playing a crucial role in the development of microbial drugs. Actinomycetes are considered one of the most important and widely used medicinal microbial groups to date. It is known that approximately 50% of natural products originate from actinomycetes, and about 70% of natural antibiotics are also produced by them. Given the complex pathological mechanisms of epilepsy and the limitations of existing treatments, the search for new naturally sourced antiepileptic drugs has significant scientific and clinical implications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a compound with anti-epileptic activity, its preparation method and uses.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] 1. A compound with antiepileptic activity, wherein the compound is a pyranone compound and its structural formula is shown in (I):

[0007]

[0008] 2. The preparation method of the above-mentioned compound with anti-epileptic activity includes the following steps:

[0009] (1) Fermentation production

[0010] Marine actinomycete H-1 with preservation number CGMCC No:24623 was streaked on a plate of Gao's No.1 solid medium and incubated upside down in an incubator at 28℃ for 5 days. After that, a single colony was picked and inoculated into liquid medium A. The culture was then placed on a shaker at 28℃ and 220 rpm / min for 16 days and the fermentation broth was collected.

[0011] (2) Extraction of extract

[0012] Add an equal volume of ethyl acetate to the fermentation broth obtained in step (1), extract three times, and then evaporate the ethyl acetate extract by rotary evaporation to obtain a crude extract paste.

[0013] (3) Isolation, purification and preparation of compounds

[0014] The crude extract obtained in step (2) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 3:7 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then mixed with 100-200 mesh reversed-phase silica gel and subjected to reversed-phase medium-pressure column chromatography. Linear gradient elution was performed using a 30-100% methanol-water solution as the eluent for 120 min. The eluent fractions were collected, arranged in descending order of polarity, and combined to obtain 10 fractions. The 8th fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 23:77 mixture of acetonitrile and water as the mobile phase to obtain compound I, the structure of which is shown in (I).

[0015]

[0016]

[0017] Further, the preparation method of Gao's No. 1 solid culture medium in step (1) is as follows: Dissolve 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.01g of FeSO4·7H2O and 20g of agar in 1000mL of distilled water, adjust the pH to 7.4-7.6, autoclave at 121℃ for 20min, cool to 60℃ and then pour into plates.

[0018] Furthermore, the preparation method of liquid culture medium A in step (1) is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt are dissolved in 1000mL of water to prepare the culture medium.

[0019] Furthermore, the flow rate for the semi-preparative reversed-phase high-performance liquid chromatography compound separation preparation described in step (3) is 2.0 mL / min.

[0020] The uses of the above-mentioned α-pyranone compounds in the preparation of antiepileptic drugs.

[0021] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses a compound with anti-epileptic activity, its preparation method and uses. The fermentation product is obtained by microbial fermentation culture, and then the fermentation product is extracted by soaking in ethyl acetate to obtain a crude extract. The crude extract is then purified by medium-pressure normal phase column chromatography, medium-pressure reverse phase column chromatography and semi-preparative high performance liquid chromatography. The compound has significant anti-epileptic activity and can be used in the development of drugs to inhibit epilepsy-related diseases.

[0022] The aforementioned marine actinomycete (Streptomyces sp.), strain H-1, classified as Streptomycess p., with accession number CGMCC No. 24623, was deposited on March 31, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0023] Figure 1 The nuclear magnetic resonance HR-ESI-MS spectrum of compound I of this invention;

[0024] Figure 2 The nuclear magnetic resonance UV spectrum of compound I of this invention;

[0025] Figure 3 The proton NMR spectrum of compound I of this invention;

[0026] Figure 4 The carbon NMR spectrum of compound I of this invention;

[0027] Figure 5 This is the DEPT-135 nuclear magnetic resonance spectrum of compound I of the present invention;

[0028] Figure 6 The COSY NMR spectrum of compound I of this invention;

[0029] Figure 7 The HSQC nuclear magnetic resonance spectrum of compound I of this invention;

[0030] Figure 8 The nuclear magnetic resonance HMBC spectrum of compound I of this invention;

[0031] Figure 9Compounds 1-3 (5 μg / mL) were shown to improve behavioral deficits caused by epilepsy, including (A) representative movement trajectories under dark conditions (n=20), and (BG) total distance, movement time, average speed, frequency of height movement, cumulative duration of movement, and relative total number of center points after 15 minutes of stimulation. (Mean ± SD; Student's t-test statistical analysis: *p<0.05, **p<0.01, ***p<0.001). Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0033] Example 1

[0034] An α-pyranone compound with antiepileptic activity, the structural formula of which is shown in (I).

[0035]

[0036] Example 2

[0037] The method for preparing pyranone compounds as shown in Example 1 specifically includes the following steps:

[0038] 1. Fermentation production

[0039] Marine actinomycete H-1, with accession number CGMCC No: 24623, was streaked onto Gao's No. 1 solid medium (prepared as follows: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g agar, 1000mL distilled water; pH adjusted to 7.4-7.6; autoclaved at 121℃ for 20min; cooled to 60℃ and then poured into plates) for 5 days. Single colonies were then inoculated into liquid medium A. Liquid medium A consisted of: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate, and 30g sea salt dissolved in 1000mL water. Subsequently, it was placed on a shaker at 28°C and 220 rpm / min and cultured for 11 days. During this period, the fermentation broth was collected for further analysis.

[0040] 2. Extraction of extract

[0041] An equal volume of ethyl acetate was added to the fermentation product obtained in step 1, and the extraction was repeated three times. The ethyl acetate extract was then evaporated under vacuum to obtain a crude extract.

[0042] 3. Isolation and preparation of compounds

[0043] The crude extract obtained in step 2 was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography (using a Silica Flash Column 330g). A gradient elution was performed using a 3:7 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography (using a SEPAFLASH SW040 column). A linear gradient elution was performed using a 30-100% methanol-water solution as the eluent for 120 min. The eluent fractions were collected, arranged in descending order of polarity, and combined to obtain 10 fractions. The 8th fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using a 23:77 mixture of acetonitrile and water as the mobile phase at a flow rate of 2.0 mL / min to obtain the compound, the structure of which is shown in (I).

[0044]

[0045] Example 3

[0046] The α-pyranone compound I prepared in Example 2 above is a white powder. Figure 1 The image shows the HR-ESI-MS spectrum of the compound of this invention, with the quasi-molecular ion peak at m / z 211.1332 [M+H]. + (The calculated molecular weight is 211.1334, C) 12 H 18 O3), its molecular formula was determined to be C 12 H 18 O3 indicates that its degree of unsaturation is 5. For example... Figure 2 As shown, the ultraviolet spectrum exhibits ultraviolet absorption at 223 nm, which is a characteristic absorption peak of the pyranone skeleton. Figure 3 As shown, based on compound I 1 H NMR data showed that in δ H There is an alkene proton group at 6.98 (1H, s), δ H 1.19 (3H,s,J=7.6), δ H 1.97 (3H, s), δ H Three methyl groups are present at 0.90 (3H, d, J = 6.8), and at δ H 2.49 (1H, m), δ H 2.39 (1H, dd, J = 13.7, 6.3), δ H 3.32 (1H, dd, J = 11.4, 6.5), δ H Four methylene signals are shown at 3.46 (1H, m). For example... Figure 4 As shown, combined with 13 C NMR ( Figure 4 DEPT Figure 5 ) and HSQC ( Figure 7 The NMR data showed a total of 12 carbon signals, including one carbonyl carbon (δ¹⁸). C 165.3), three methyl carbons (δ) C 16.7, δ C 15.0, δ C 11.7), three methylene carbons (δ C 24.3, δ C 33.1、δ C 66.1), one methylene carbon (δ C 35.7) and four sp 2 Hybrid carbon (δ C 110.2, δ C 124.2, δ C 146.5, δ C 160.7). These data further support the existence of an additional cycle and three unsaturations in the structure. (e.g.) Figure 8 As shown, by utilizing the HMBC correlations from H-3 to C-1, C-5, H2-6 to C-5, C-4, H3-8 to C-3, C-4, and C-5, and the correlation from H2-9 to C-2, the core structure was identified as 4-methyl-α-pyranone. Furthermore, as... Figure 6 As shown, the COSY correlations of H-10 / H2-9 and H-10 / H2-11 further confirmed this structure. Specifically, based on the key HMBC correlation from H2-6 to C-5 and the COSY correlation from H2-6 / H3-7 to C-5, it was revealed that the methyl acetate fragment should be attached to C-5. The planar structure of compound I was identified as a pyranone derivative with the methyl acetate fragment. The absolute configuration of compound I was determined by quantum chemical calculations. Analysis of the optical rotation values, comparing the experimental data (+45.3) with the calculated data (+40.6), indicated that the absolute configuration at C-10 of compound I is S. Compound I was confirmed as a novel α-pyranone derivative.

[0047] Table 1. Compound I 1 H and 13 C10 NMR data (CDCl3-d6)

[0048]

[0049] Note 1: s - singlet, d - doublet, m - multiplet.

[0050] Note 2: 1H was obtained by 600MHz NMR; 13 C was obtained by 150MHz NMR.

[0051] Example 4

[0052] 1. Antiepileptic activity of pyranone compounds

[0053] (1) Experimental Samples

[0054] Preparation of the test sample solution: Pentylenetetrazole (PTZ) was used as an epilepsy inducer to establish a zebrafish inflammation model. The test sample was the purified compound I isolated in Example 3 above. An appropriate amount of sample was accurately weighed and prepared into a solution of the required concentration using DMSO for testing antiepileptic activity. The zebrafish used in this experiment were housed in a flow-through tank, with the water temperature maintained at 28±0.5℃ and a photoperiod of 14 hours light: 10 hours darkness. The zebrafish were fed newly hatched brine shrimp.

[0055] (2) Experimental methods

[0056] Zebrafish embryos 120 hours post-fertilization were placed in six-well plates, with 20 embryos per well. Zebrafish larvae were randomly assigned to different groups: control group, pentylenetetrazol (PTZ) group, compound 1 treatment group (5 mg / L), and positive rescue group (CBZ) (50 μM carbamazepine). Each experimental condition was performed in triplicate. On day 6 post-fertilization (dpf), 10 mM pentylenetetrazol was introduced into all groups except the control group, followed by incubation for 30 minutes. Then, 20 larvae from each group were transferred to individual wells of a 48-well plate, each well containing 1 mL of E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.4, 0.003% (w / v) propylthiouracil (PTU). After acclimatization in the dark for 10 minutes, behavioral parameters were recorded for 15 minutes using a DanioVision observation chamber. The EthoVision XT 10.0 software was used to evaluate motion behavior parameters, including total distance traveled, duration of motion, average speed, high frequency of motion, cumulative activity time, and curvature.

[0057] (3) Experimental Results

[0058] Compound I exhibited more significant anti-epileptic activity in a PTZ-treated zebrafish model, characterized by a marked reduction in behavioral disturbances associated with PTZ-induced epilepsy.

[0059] like Figure 9As shown, compounds 1-3 at a concentration of 5 μg / mL significantly reduced behavioral disturbances in PTZ-induced (5 μg / mL) epilepsy, including total distance, movement time, average speed, frequency of high-altitude movement, cumulative duration of movement, and relative total center point. Figure 9 (AG). Among these compounds, 1 showed more significant antiepileptic activity in a PTZ-treated zebrafish model, characterized by a significant reduction in behavioral disorders associated with PTZ-induced epilepsy.

[0060] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A compound with anti-epileptic activity, characterized in that... This compound is an α-pyranone compound, and its structural formula is shown in (Ⅰ); 2. A method for preparing the compound with antiepileptic activity according to claim 1, characterized in that... Includes the following steps: (1) Fermentation production Marine actinomycete H-1 with preservation number CGMCC No:24623 was streaked on a plate of Gao's No.1 solid medium and incubated upside down in an incubator at 28℃ for 5 days. After that, a single colony was picked and inoculated into liquid medium A. The culture was then placed on a shaker at 28℃ and 220 rpm / min for 16 days and the fermentation broth was collected. (2) Extraction of extract Add an equal volume of ethyl acetate to the fermentation broth obtained in step (1), extract three times, and then evaporate the ethyl acetate extract by rotary evaporation to obtain a crude extract paste. (3) Isolation, purification and preparation of compounds The crude extract obtained in step (2) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 3:7 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then mixed with 100-200 mesh reversed-phase silica gel and subjected to reversed-phase medium-pressure column chromatography. Linear gradient elution was performed using a 30-100% methanol-water solution as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 10 fractions. The 8th fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 23:77 mixture of acetonitrile and water as the mobile phase to obtain compound I, the structure of which is shown in (I).

3. The method for preparing a compound with antiepileptic activity according to claim 2, characterized in that... The preparation method of Gao's No. 1 solid culture medium in step (1) is as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.01g of FeSO4·7H2O, 20g of agar, and 1000mL of distilled water are added to adjust the pH to 7.4-7.

6.

4. The method for preparing a compound with antiepileptic activity according to claim 2, characterized in that... The preparation method of liquid culture medium A in step (1) is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt are dissolved in 1000mL of water.

5. The method for preparing a compound with antiepileptic activity according to claim 2, characterized in that: In step (3), the methanol volume percentage in the reversed-phase medium-pressure column chromatography linear gradient elution ranges from 30% to 100%, and the elution time is 120 min.

6. The method for preparing a compound with antiepileptic activity according to claim 2, characterized in that: The flow rate for the semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) preparation of compounds in step (3) is 2.0 mL / min.

7. Use of the compound of claim 1 having antiepileptic activity in the preparation of an antiepileptic drug.

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