WO2022246810A1 - 具有抗新冠病毒活性的聚酮类化合物的制备及其应用 - Google Patents
具有抗新冠病毒活性的聚酮类化合物的制备及其应用 Download PDFInfo
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- the invention belongs to the field of compound synthesis, and in particular relates to the preparation and application of polyketide compounds.
- Endophytes in medicinal plants are characterized by diversity, and each endophyte has abundant secondary metabolites.
- Many biologically active alkaloids, polypeptides, and polyketides have been discovered from the secondary metabolites of endophytes.
- These secondary metabolites not only have the same or similar biological activities as the host plant, such as antibacterial, anti-inflammatory, anti-tumor, anti-hyperglycemic, anti-parasitic, etc., but also have many new biological activities. Therefore, the secondary metabolites of medicinal plant endophytes are a huge resource pool of potential medicinal substances and have good development prospects.
- the present invention aims to provide a polyketide compound and its active application.
- the present invention is a compound with a novel structure that has inhibitory activity against SARS-CoV-2 virus isolated from a solid fermentation product of a plant-derived endophyte and an application thereof. Specifically, a solid-state fermentation of the endophytic fungus Alternaria sp. was carried out, and a polyketide monomer compound with a novel structure was obtained from the fermentation product using a series of separation and purification methods. Using techniques such as mass spectrometry and nuclear magnetic resonance, the structure of the compound was determined, and the structure is:
- the present invention claims protection for the preparation method of the above-mentioned polyketide compound.
- Extraction and separation of metabolites Use an equal volume of methanol to ultrasonically extract the solid fermentation product of the plant endophytic fungus Alternaria sp., filter through 8 layers of gauze, separate the extract from mycelium and rice, and concentrate the extract to dryness.
- step (1) fermentation conditions the bacterial strain Alternaria sp. was subjected to shaking culture with Fungi No. 4 culture medium, the culture condition was 28°C, 180r/min, and then the fermentation broth and mycelium of the shaking culture were inoculated in the container Static fermentation culture was carried out in conical flasks with rice solid medium for 40 days, and the culture temperature was 28°C.
- the fungus No. 4 culture medium is composed as follows in terms of mass percentage: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH 2 PO 4 , 0.03% MgSO 4 ⁇ 7H 2 O , 0.1% corn steep liquor, deionized water preparation.
- the rice solid culture medium is as follows: add 80 g of rice to every 110 ml of deionized water, and put it into an Erlenmeyer flask; each bottle of rice culture medium is inoculated with 20 ml of seed solution.
- the present invention makes full use of plant endophyte resources, studies plant endophytes, finds new polyketide active compounds, and conducts anti-new coronavirus activity experiments on them, so as to obtain FDA-approved anti-SARS-CoV -2
- the host-directed drug clofazimine for viral activity was used as a control group to explore the antiviral activity of the compound.
- the experimental results show that the SI index of clofazimine is 1.5, and the SI index of the isolated compound of the present invention is 3, the inhibition rate of the virus is higher than that of the control drug, and it has the activity of inhibiting the new coronavirus, and is looking for new lead compounds for drug development .
- Fig. 2 is the carbon 13 nuclear magnetic resonance spectrogram of embodiment 1.
- Fig. 3 is embodiment 1 HSQC spectrogram.
- Fig. 4 is embodiment 1 HMBC spectrogram.
- Alternaria sp. is a known bacterial species in the art.
- Fermentation of strains the bacterial strain Alternaria sp. was cultured by shaking with fungus No. 4 medium, the culture condition was 28° C., 180 r/min; the composition of the fungus No. 4 medium: 2% mannitol, 2% glucose, 0.5 % yeast extract, 1% peptone, 0.05% KH 2 PO 4 , 0.03% MgSO 4 ⁇ 7H 2 O, 0.1% corn steep liquor, and deionized water.
- the fermented liquid of shaking culture is inoculated in the Erlenmeyer flask that rice solid medium (rice solid medium formula: 80g rice, 110ml deionized water) is housed together with mycelia, every bottle of rice medium is inoculated with 20ml seed liquid, The static fermentation culture was carried out for 40 days, and the culture temperature was 28°C.
- rice solid medium rice solid medium formula: 80g rice, 110ml deionized water
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Abstract
一种具有抗新冠病毒活性的聚酮类化合物的制备方法及其应用，通过对链格孢属内生真菌Alternaria sp.进行固体发酵，使用一系列分离纯化方法从发酵产物中得到了一个聚酮类单体化合物，其结构如式(Ⅰ)所示，该化合物具有抑制新冠病毒的活性。
Description
本发明属于化合物合成领域，具体涉及聚酮类化合物的制备及应用。
药用植物内生菌具有多样性特征，每个内生菌又有丰富的次级代谢产物，目前已从内生菌次级代谢产物中发现了许多具有生物活性的生物碱、多肽、聚酮类、萜类等物质，这些次级代谢物不但具有与宿主植物相同或相似的生物活性，例如抗菌、抗炎、抗肿瘤、抗高血糖、抗寄生虫等，还具有许多新的生物活性。因此药用植物内生菌的次级代谢产物是庞大的潜在药用物质资源库，具有良好的开发前景。
发明内容
为弥补现有技术空白，本发明旨在提供一种聚酮类化合物及其活性应用。本发明是从植物来源的内生菌的固体发酵产物中分离得到一种对SARS-CoV-2病毒具有抑制活性的结构新颖的化合物及其应用。具体的说是对链格孢属内生真菌Alternaria sp.进行固体发酵，使用一系列分离纯化方法从发酵产物中得到了一个结构新颖的聚酮类单体化合物。采用了质谱、核磁共振等技术，确定了该化合物的结构，结构为：
结构如式(Ⅰ)所示：
本发明同时请求保护上述聚酮化合物的制备方法。
(1)菌种发酵：将链格孢属内生真菌Alternaria sp.用大米和纯净水进行固体发酵。
(2)代谢产物的提取分离：使用等体积甲醇对植物内生真菌Alternaria sp. 固体发酵产物进行超声提取，经8层纱布过滤，将提取液与菌丝体及大米分离，提取液浓缩至干，提取物用中压柱色谱，以300-400目硅胶为固定相，以氯仿/甲醇为流动相进行梯度洗脱，以体积计，梯度依次为氯仿：甲醇＝100:0、100:1、100：2、100：3、100：5、100：10、0：100，洗脱剂流速为30ml/min，收集洗脱液(每500ml收集一次)，将洗脱液悬干，每五份洗脱液合并，得到组分1-11；将组分4过硅胶柱，以200-300目硅胶为固定相，流动相为石油醚/乙酸乙酯，以体积计，梯度依次为石油醚：乙酸乙酯＝10：1、10：3、10：5、10：6、10：7、10：9，洗脱剂流速为30ml/min，收集洗脱液(每500ml收集一次)，同每五份洗脱液合并，得到组分4-1至4-16；将组分4-11上Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇1：1，洗脱剂流速为0.3ml/min，收集洗脱液(每10ml收集一次)，每四份洗脱液合并，得组分4-11-1至4-11-6；将组分4-11-3经高效液相色谱法，48％甲醇水制备，得到单体化合物，保留时间11min。
(3)结构鉴定：以氘代二甲基亚砜为溶剂，使用Bruker Avance Ⅱ 500M核磁共振仪测定分离得到的化合物样品的核磁图谱并利用质谱推断分子式，最终对产物结构进行表征。
进一步的，所述步骤(1)发酵条件：将菌株Alternaria sp.采用真菌4号培养基进行震荡培养，培养条件28℃，180r/min，再将震荡培养的发酵液连同菌丝体接种于装有大米固体培养基的锥形瓶中静止发酵培养40天，培养温度为28℃。
进一步的，所述真菌4号培养基以质量百分数计组成如下：2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨，0.05％KH
2PO
4，0.03％MgSO
4·7H
2O，0.1％玉米浆，去离子水配制。
进一步的，所述大米固体养基为：每110ml去离子水加入80g大米，装入锥形瓶中；每瓶大米培养基接种20ml种子液。
本发明另一个目的是请求保护上述聚酮类化合物的应用，该化合物对SARS-CoV-2病毒具有抑制活性，可用于制备抑制SARS-CoV-2病毒药物。
有益效果：本发明充分利用植物内生菌资源，对植物内生菌进行研究，寻找新的聚酮类活性化合物，并对其进行抗新冠病毒活性实验，以经FDA批准的具有抗SARS-CoV-2病毒活性的宿主导向药物氯法齐明作为对照组，来探究化合物 的抗病毒活性。实验结果表明，氯法齐明的SI指数为1.5，本发明分离所得化合物SI指数为3，对病毒的抑制率高于对照药物，具有抑制新型冠状病毒的活性，为药物开发寻找新的先导化合物。
图1为实施例1氢核磁共振谱图。
图2为实施例1碳13核磁共振谱图。
图3为实施例1 HSQC谱图。
图4为实施例1 HMBC谱图。
下面通过具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。链格孢属内生真菌Alternaria sp.为本领域已知菌种，为了举例说明，下述实施例中采集自西藏红景天Rhodiola tibetica叶片部位的链格孢属内生真菌Alternaria sp.
实施例1
(1)菌种发酵：将菌株Alternaria sp.采用真菌4号培养基进行震荡培养，培养条件28℃，180r/min；所述真菌4号培养基组成：2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨，0.05％KH
2PO
4，0.03％MgSO
4·7H
2O，0.1％玉米浆，去离子水配制。
再将震荡培养的发酵液连同菌丝体接种于装有大米固体培养基(大米固体培养基配方：80g大米，110ml去离子水)的锥形瓶中，每瓶大米培养基接种20ml种子液，静止发酵培养40天，培养温度为28℃。
(2)代谢产物的提取分离：使用等体积甲醇对植物内生真菌Alternaria sp.固体发酵产物进行超声提取，经8层纱布过滤，将提取液与菌丝体及大米分离，提取液浓缩至干，提取物用中压柱色谱，以300-400目硅胶为固定相，以氯仿/甲醇为流动相进行梯度洗脱，以体积计，梯度依次为氯仿：甲醇＝100:0、100:1、100：2、100：3、100：5、100：10、0：100，洗脱剂流速为30ml/min，收集洗脱液(每500ml收集一次)；将洗脱液悬干，每五份洗脱液合并，得到组分1-11； 将组份4过硅胶柱，以200-300目硅胶为固定相，流动相为石油醚/乙酸乙酯，以体积计，梯度依次为石油醚：乙酸乙酯＝10：1、10：3、10：5、10：6、10：7、10：9，洗脱剂流速为30ml/min，收集洗脱液(每500ml收集一次)，同每五份洗脱液合并，得到组分4-1至4-16；将组分4-11上Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇1：1，洗脱剂流速为0.3ml/min，收集洗脱液(每10ml收集一次)，每四份洗脱液合并，得组分4-11-1至4-11-6；将组分4-11-3经高效液相色谱法，48％甲醇水制备，得到单体化合物，保留时间11min。
(3)结构鉴定：以氘代二甲基亚砜为溶剂，使用Bruker Avance Ⅱ 500M核磁共振仪测定分离得到的化合物样品的核磁图谱并利用质谱推断分子式，最终对产物结构进行表征。
其光谱数据如下：
1H NMR(500Hz，DMSO-d
6)δ：9.42(1H,brs,3-OH),6.68(1H,d,J＝8.0Hz,H-4),7.04(1H,t,J＝8.0Hz,H-5),6.65(1H,d,J＝8.0Hz,H-6),2.57(1H,t,J＝8.0Hz,H-2’),12.1(1H,brs,3-OH’)。
13C NMR(125Hz,DMSO-d
6)δ：142.9(C-1),122.6(C-2),156.4(C-3),113.6(C-4),129.2(C-5),120.1(C-6),64.9(C-7),57.7(C-8),27.9(C-1’),35.9(C-2’),174.5(C-3’)。
其HMBC相关如下：
(2)将化合物溶解，调节浓度为10mg/mL，起始梯度为30倍稀释，后3倍倍比稀释，连续7个梯度。在96孔细胞板中进行培养，病毒对照组孔加入DMEM完全培养基100μl/孔，细胞对照组孔加入DMEM完全培养基150μl/孔，待测样品孔中加入142.5μl/孔，其余孔加入培养基100μl/孔。待测样品孔中加入待测样品7.5μl。对待测样品孔中液体轻柔的反复吹吸6-8次，然后转移50μl液体至对应的下列孔，之后所有孔都3倍倍比稀释。用DMEM完全培养基将SARS-CoV-2 假病毒(spike-D614G)稀释至1.3×10
4TCID50/mL，于每孔加50μl。将上述96孔板置于细胞培养箱中(37℃，5％CO
2)孵育1小时，待孵育30min后，开始消化Huh7细胞，将细胞浓度稀释至2×10
5cells/mL。孵育结束后，每孔加入100μl细胞，使每孔细胞为2×10
4cells，放入37℃，5％CO
2细胞培养箱中培养24小时，培养完毕后，吸弃150μl上清，加入100μl Bright-GloTM荧光素酶检测试剂，室温避光反应2min后，反复吹打，转移150μl液体至白板中。使用PerkinElmer EnSight多功能成像酶标仪读取发光值(RLU)。利用发光值计算出药物EC
50值。CC
50实验过程同上，只是每孔中不加入假病毒，以此来看药物对细胞活性的影响。
(3)重复上述方法，以经FDA批准的具有抗SARS-CoV-2病毒活性的宿主导向药物氯法齐明作为对照组，以此来对比判断化合物对SARS-CoV-2假病毒的抑制活性。
表1活性实验结果：
| AverageEC 50(ug/ml) | CC 50(ug/ml) | SI(SI＝CC 50/average(EC 50) | |
| 氯法齐明 | 4 | 6 | 1.5 |
| 该化合物 | 52 | 157 | 3.0 |
注：SI>3药物可用
实验结果表明，该化合物对SARS-CoV-2病毒有抑制活性，尤其是对病毒的抑制率比氯法齐明对病毒的抑制率高，可用于制备抑制SARS-CoV-2病毒药物。
以上所述，仅为本发明创造较佳的具体实施方式，但本发明创造的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明创造披露的技术范围内，根据本发明创造的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明创造的保护范围之内。
Claims (7)
- 一种具有抗新冠病毒活性的聚酮类化合物，其特征在于，具有式(Ⅰ)所示结构：
- 如权利要求1所述的聚酮类化合物的制备方法，其特征在于，包括如下步骤：(1)菌种发酵：将链格孢属内生真菌Alternaria sp.用大米和纯净水进行固体发酵；(2)代谢产物的提取分离：使用等体积甲醇对植物内生真菌Alternaria sp.固体发酵产物进行超声提取，经8层纱布过滤，将提取液与菌丝体及大米分离，提取液浓缩至干，提取物用中压柱色谱，以300-400目硅胶为固定相，以氯仿/甲醇为流动相进行梯度洗脱，以体积计，梯度依次为氯仿：甲醇＝100:0、100:1、100：2、100：3、100：5、100：10、0：100，洗脱剂流速为30ml/min，每500ml收集一次洗脱液，将洗脱液悬干，每五份洗脱液合并，得到组分1-11；将组分4过硅胶柱，以200-300目硅胶为固定相，流动相为石油醚/乙酸乙酯，以体积计，梯度依次为石油醚：乙酸乙酯＝10：1、10：3、10：5、10：6、10：7、10：9，洗脱剂流速为30ml/min，每500ml收集一次洗脱液，同每五份洗脱液合并，得到组分4-1至4-16；将组分4-11上Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇1：1，洗脱剂流速为0.3ml/min，每10ml收集一次洗脱液，每四份洗脱液合并，得组分4-11-1至4-11-6；将组分4-11-3经高效液相色谱法，48％甲醇水制备，得到单体化合物，保留时间11min。
- 如权利要求2所述的聚酮类化合物的制备方法，其特征在于，还包括结构鉴定步骤(3)：以氘代二甲基亚砜为溶剂，使用Bruker Avance Ⅱ 500M核磁共振仪测定分离得到的化合物样品的核磁图谱并利用质谱推断分子式，最终对产物结构进行表征。
- 如权利要求2所述的聚酮类化合物的制备方法，其特征在于，所述步骤(1)中发酵条件：将菌株Alternaria sp.采用真菌4号培养基进行震荡培养，培养条件28℃，180r/min，再将震荡培养的发酵液连同菌丝体接种于装有大米固体培养基的锥形瓶中静止发酵培养40天，培养温度为28℃。
- 如权利要求4所述的聚酮类化合物的制备方法，其特征在于，所述真菌4号培养基以质量百分数计组成如下：2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨，0.05％KH 2PO 4，0.03％MgSO 4·7H 2O，0.1％玉米浆，去离子水配制。
- 如权利要求4所述的聚酮类化合物的制备方法，其特征在于，所述大米固体培养基为：每110ml去离子水加入80g大米，装入锥形瓶中；每瓶大米培养基接种20ml种子液。
- 如权利要求1所述的聚酮类化合物在制备抑制SARS-CoV-2病毒药物上的应用。
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