Isoquinoline alkaloid compound and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an isoquinoline alkaloid compound and a preparation method and application thereof. The isoquinoline alkaloid compound provided by the present invention has a structure shown in Formula I, Formula II, Formula III or Formula IV. The isoquinoline alkaloid compound provided by the present invention has good affinity with PCSK9, can inhibit the binding of PCSK9 to low-density lipoprotein receptors, and has potential application value in treating cardiovascular diseases by reducing low-density lipoprotein. The present invention provides a preparation method for the isoquinoline alkaloid compound described in the above technical scheme. The present invention uses fennel as a raw material to obtain the isoquinoline alkaloid compound from fennel. The raw material price is cheap and the preparation cost is low, which is convenient for large-scale promotion and use after drug research and development.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an isoquinoline alkaloid compound and a preparation method and application thereof. Background Art

[0002] According to the "China Cardiovascular Health and Disease Report 2021", cardiovascular disease (CVD) is the "number one killer" of residents, and 2 out of every 5 deaths are caused by CVD. Atherosclerotic cardiovascular disease accounts for more than 95% of all causes of CVD. It is currently believed that the main cause is excessive LDL-C (Low Density Lipoprotein-Cholesterol). Current treatment methods mainly focus on reducing the concentration of LDL-C and reducing the risk of cardiovascular disease.

[0003] Berberine is an alkaloid with good biological activity found in many plants. It has a variety of biological effects, such as antioxidant, anti-inflammatory, antihypertensive, and hypolipidemic activities. Its lipid-lowering effect has aroused great interest among scholars, and more and more evidence has shown that berberine can reduce blood lipid levels such as total cholesterol, triglycerides, and LDL-C. There are many mechanisms by which berberine can improve cholesterol metabolism; in the way of upregulating the expression of low-density lipoprotein receptor (LDL-R), berberine can increase the level of LDL-R by inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9), targeting the degradation of low-density lipoprotein, and lowering plasma cholesterol levels.

[0004] PCSK9 was first discovered in 2003. It can bind to LDL-R, interfere with its recycling, reduce the liver's ability to clear LDL-C from the blood, lead to LDL-C accumulation, and increase the risk of cardiovascular disease. In 2007, the Amgen team in the United States first analyzed the crystal structure of PCSK9 and confirmed that it binds tightly to LDL-R to play a role. Therefore, the current goal is to develop PCSK9 antibodies to increase the level of LDL-R on the surface of stem cells, thereby increasing the clearance of LDL-C and achieving the goal of lowering LDL-C levels. Although there are currently three PCSK9 inhibitors (including two monoclonal antibodies), there is still a blank in the approval of small molecule PCSK9 inhibitors. Summary of the invention

[0005] The purpose of the present invention is to provide an isoquinoline alkaloid compound and a preparation method and application thereof. The isoquinoline alkaloid compound provided by the present invention has good binding ability with PCSK9 and has certain potential to become a lead compound for inhibiting PCSK9 protein.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an isoquinoline alkaloid compound having a structure shown in Formula I, Formula II, Formula III or Formula IV:

[0008]

[0009] The present invention provides a method for preparing the isoquinoline alkaloid compound described in the above technical scheme, comprising the following steps:

[0010] Extracting fennel with an organic solvent, concentrating the obtained extract to obtain an extract;

[0011] The extract is dissolved in water, and the pH is adjusted to acidic to obtain an acidic solution; the acidic solution is subjected to a first extraction with petroleum ether to obtain a petroleum ether phase and a first aqueous phase; the first aqueous phase is subjected to a second extraction with ethyl acetate to obtain an ethyl acetate phase and a second aqueous phase; the second aqueous phase is subjected to a pH adjustment to alkaline to obtain an alkaline solution, and the alkaline solution is subjected to a third extraction with dichloromethane to obtain a dichloromethane phase; the dichloromethane is concentrated to obtain the total alkaloids of anise alkaloids;

[0012] The total alkaloids of anise alkaloids and silica gel are mixed, and the obtained mixture is subjected to a first silica gel column chromatography separation, wherein the first silica gel column chromatography separation includes a first gradient elution and a second gradient elution, wherein the first gradient elution is carried out in a descending volume ratio, using a petroleum ether-acetone system with a volume ratio of petroleum ether to acetone of 60:1 to 1:1, and the second gradient elution is carried out in a descending volume ratio, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 10:1 to 0:1; the same fractions are combined to obtain five fractions, which are named Fr.Ⅰ, Fr.Ⅱ, Fr.Ⅲ, Fr.Ⅳ, and Fr.Ⅴ respectively;

[0013] Fr.Ⅳ was subjected to a second silica gel column chromatography separation, wherein the second silica gel column chromatography separation was performed in a gradient elution using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1, and the same fractions were combined to obtain five sub-fractions, which were named Fr.ⅣA, Fr.ⅣB, Fr.ⅣC, Fr.ⅣD, and Fr.ⅣE respectively;

[0014] Fr.IVB is subjected to a first gel column separation and a first high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in Formula I; the mobile phase used in the first gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the first gel column separation is 1:1; the mobile phase used in the first high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the first high performance liquid chromatography separation is 10:90-100:0, and the water content is not 0;

[0015] Fr.IVD is subjected to a second gel column separation and a second high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula II; the mobile phase used in the second gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the second gel column separation is 1:1; the mobile phase used in the second high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the second high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0;

[0016] Fr.V is subjected to a third silica gel column chromatography separation, wherein the third silica gel column chromatography separation is performed in a gradient elution according to a volume ratio from large to small, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1, and the same fractions are combined to obtain five sub-fractions, which are named Fr.VA, Fr.VB, Fr.VC, Fr.VD, and Fr.VE respectively;

[0017] Fr. VC is subjected to a third gel column separation and a third high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula III; the mobile phase used in the third gel column separation is methanol, and the mobile phase used in the third high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the third high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0;

[0018] Fr.IVE is subjected to a fourth gel column separation and a fourth high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound with a structure shown in formula IV; the mobile phase used in the fourth gel column separation is a dichloromethane-methanol system, the volume ratio of dichloromethane and methanol in the mobile phase used in the third gel column separation is 1:1, the mobile phase used in the fourth high performance liquid chromatography separation is an acetonitrile-water system, the volume ratio of acetonitrile and water in the mobile phase used in the fourth high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0.

[0019] Preferably, the fennel is the whole fennel plant; the water content of the whole fennel plant is ≤5%; and the organic solvent is a low-carbon alcohol solvent.

[0020] Preferably, the extraction is multiple extractions, the extraction is carried out under hot reflux conditions, the hot reflux temperature is 45-60°C, the single extraction time is 7-8h, and the solid-liquid ratio of the whole fennel herb to the organic solvent during the single extraction is 1kg:6-7L.

[0021] Preferably, the pH value of the acidic solution is 2-3; the pH value of the alkaline solution is 9-10.

[0022] Preferably, the number of extractions of the first extraction, the second extraction and the third extraction is independently 3 to 4 times; the volume ratio of the organic phase to the aqueous phase when the first extraction, the second extraction and the third extraction are performed once is independently 1:1.5 to 2.

[0023] Preferably, the volume ratio of acetonitrile to water in the mobile phase used in the first HPLC separation is 30:70 to 60:40;

[0024] The volume ratio of acetonitrile to water in the mobile phase used in the second high performance liquid chromatography separation is 25:75 to 60:40;

[0025] The volume ratio of acetonitrile to water in the mobile phase used in the third high performance liquid chromatography separation is 20:80 to 60:40;

[0026] The volume ratio of acetonitrile to water in the mobile phase used in the fourth high performance liquid chromatography separation is 20:80 to 60:40.

[0027] The invention provides an isoquinoline alkaloid pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises the isoquinoline alkaloid compound described in the above technical scheme or a pharmaceutically acceptable salt, hydrate or solvent compound of the isoquinoline alkaloid compound described in the above technical scheme or an isoquinoline alkaloid compound obtained by the preparation method described in the above technical scheme.

[0028] The present invention provides the use of the isoquinoline alkaloid compound described in the above technical scheme, or the pharmaceutically acceptable salt, hydrate or solvate of the isoquinoline alkaloid compound described in the above technical scheme, or the isoquinoline alkaloid compound obtained by the preparation method described in the above technical scheme, or the isoquinoline alkaloid pharmaceutical composition described in the above technical scheme in the preparation of drugs for treating cardiovascular diseases or the preparation of PCSK9 protein inhibitors.

[0029] Preferably, the cardiovascular disease is a cardiovascular disease caused by increased low-density lipoprotein caused by PCSK9.

[0030] The present invention provides an isoquinoline alkaloid compound having a structure shown in Formula I, Formula II, Formula III or Formula IV. The isoquinoline alkaloid compound provided by the present invention and its pharmaceutically acceptable salt, hydrate or solvent compound have good binding ability with PCSK9, can inhibit the binding of PCSK9 with low-density lipoprotein receptor, and has a certain potential to become a lead compound for inhibiting PCSK9 protein. It has potential application value in treating cardiovascular diseases by reducing low-density lipoprotein.

[0031] The present invention provides a method for preparing the isoquinoline alkaloid compound described in the above technical solution. The present invention uses fennel as a raw material to obtain the isoquinoline alkaloid compound from fennel, the raw material price is cheap, the preparation cost is low, and it is convenient for large-scale promotion and use after drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the interaction diagram between the compounds of Formula I to Formula IV and the positive control PF-06446846 and PCSK9;

[0033] Figure 2 This is the docking result of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV with the PCSK9 protein. DETAILED DESCRIPTION

[0034] The present invention provides an isoquinoline alkaloid compound having a structure shown in Formula I, Formula II, Formula III or Formula IV:

[0035]

[0036] The present invention provides a method for preparing the isoquinoline alkaloid compound described in the above technical scheme, comprising the following steps:

[0037] Extracting fennel with an organic solvent, concentrating the obtained extract to obtain an extract;

[0038] The extract is dissolved in water, and the pH is adjusted to acidic to obtain an acidic solution; the acidic solution is subjected to a first extraction with petroleum ether to obtain a petroleum ether phase and a first aqueous phase; the first aqueous phase is subjected to a second extraction with ethyl acetate to obtain an ethyl acetate phase and a second aqueous phase; the second aqueous phase is subjected to a pH adjustment to alkaline to obtain an alkaline solution, and the alkaline solution is subjected to a third extraction with dichloromethane to obtain a dichloromethane phase; the dichloromethane is concentrated to obtain the total alkaloids of anise alkaloids;

[0039] The total alkaloids of anise alkaloids and silica gel are mixed, and the obtained mixture is subjected to a first silica gel column chromatography separation, wherein the first silica gel column chromatography separation includes a first gradient elution and a second gradient elution, wherein the first gradient elution is carried out in a descending volume ratio, using a petroleum ether-acetone system with a volume ratio of petroleum ether to acetone of 60:1 to 1:1, and the second gradient elution is carried out in a descending volume ratio, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 10:1 to 0:1; the same fractions are combined to obtain five fractions, which are named Fr.Ⅰ, Fr.Ⅱ, Fr.Ⅲ, Fr.Ⅳ, and Fr.Ⅴ respectively;

[0040] Fr.Ⅳ was subjected to a second silica gel column chromatography separation, wherein the second silica gel column chromatography separation was performed in a gradient elution using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1, and the same fractions were combined to obtain five sub-fractions, which were named Fr.ⅣA, Fr.ⅣB, Fr.ⅣC, Fr.ⅣD, and Fr.ⅣE respectively;

[0041] Fr.IVB is subjected to a first gel column separation and a first high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in Formula I; the mobile phase used in the first gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the first gel column separation is 1:1; the mobile phase used in the first high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the first high performance liquid chromatography separation is 10:90-100:0, and the water content is not 0;

[0042] Fr.IVD is subjected to a second gel column separation and a second high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula II; the mobile phase used in the second gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the second gel column separation is 1:1; the mobile phase used in the second high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the second high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0;

[0043] Fr.V is subjected to a third silica gel column chromatography separation, wherein the third silica gel column chromatography separation is performed in a gradient elution according to a volume ratio from large to small, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1, and the same fractions are combined to obtain five sub-fractions, which are named Fr.VA, Fr.VB, Fr.VC, Fr.VD, and Fr.VE respectively;

[0044] Fr. VC is subjected to a third gel column separation and a third high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula III; the mobile phase used in the third gel column separation is methanol, and the mobile phase used in the third high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the third high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0;

[0045] Fr.IVE is subjected to a fourth gel column separation and a fourth high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound with a structure shown in formula IV; the mobile phase used in the fourth gel column separation is a dichloromethane-methanol system, the volume ratio of dichloromethane and methanol in the mobile phase used in the third gel column separation is 1:1, the mobile phase used in the fourth high performance liquid chromatography separation is an acetonitrile-water system, the volume ratio of acetonitrile and water in the mobile phase used in the fourth high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0.

[0046] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0047] The present invention extracts fennel with an organic solvent, and the obtained extract is concentrated to obtain an extract. In the present invention, the organic solvent is preferably a low-carbon alcohol solvent, and methanol is particularly preferred. The fennel is preferably a whole fennel plant; the water content of the whole fennel plant is preferably ≤5%. In the present invention, the whole fennel plant is preferably crushed before use to obtain a whole fennel plant powder, and the particle size of the whole fennel plant powder is preferably ≤100mm, more preferably 10-100mm. The extraction is preferably multiple extractions. The number of extractions is preferably 3-4 times. The extraction is preferably carried out under hot reflux conditions, and the hot reflux temperature is preferably 45-60°C, more preferably 50-60°C. The time for a single extraction is preferably 7-8h, more preferably 7-7.5h. The solid-liquid ratio of the whole fennel plant to the organic solvent during a single extraction is preferably 1kg:6-7L, more preferably 1kg:6.5-7L. After the leaching, the present invention further comprises concentrating the leaching solution obtained by the leaching to recover the extractant (organic solvent) to obtain an extract. The present invention has no special limitation on the concentration, and the concentration method well known to those skilled in the art can be used until no solvent flows out, such as vacuum distillation. In the present invention, the recovered extractant can be reused.

[0048] After obtaining the extract, the present invention dissolves the extract in water, adjusts the pH to acidic, and obtains an acidic solution; the acidic solution is first extracted with petroleum ether to obtain a petroleum ether phase and a first aqueous phase; the first aqueous phase is second extracted with ethyl acetate to obtain an ethyl acetate phase and a second aqueous phase; the second aqueous phase is adjusted to alkaline pH to obtain an alkaline solution, and the alkaline solution is third extracted with dichloromethane to obtain a dichloromethane phase; the dichloromethane is concentrated to obtain the total alkaloids of anise alkaloids. In the present invention, the temperature of the water used for the dissolution is preferably 35-40°C. The present invention does not specifically limit the amount of water used to dissolve the extract, as long as the extract can be fully dissolved. The pH value of the acidic solution is preferably 2-3, more preferably 2.5-3. The present invention does not specifically limit the acid used to adjust the pH value to acidic, and an acid well known to those skilled in the art can be used, such as hydrochloric acid; the mass concentration of the acid is preferably 3-5%, more preferably 4-5%.

[0049] In the present invention, the pH value of the alkaline solution is preferably 9 to 10, more preferably 9.5 to 10. The present invention has no particular limitation on the base used for adjusting the pH value to alkaline, and a base well known to those skilled in the art can be used, such as NaOH aqueous solution or ammonia water; the mass concentration of the base is preferably 9 to 10%, more preferably 9.5 to 10%.

[0050] In the present invention, the volume ratio of the organic phase to the aqueous phase of the first extraction, the second extraction, and the third extraction is preferably 1:1.5-2, and specifically preferably 1:1.5 or 1:2. The number of extractions of the first extraction, the second extraction, and the third extraction is preferably 3-4 times, and more preferably 3 times or 4 times. The present invention preferably also includes concentrating the organic phase obtained by the first extraction, the second extraction, and the third extraction to recover the extractant, and the third extraction organic phase is concentrated to obtain the total alkaloids of anise alkaloids. The present invention has no special limitation on the concentration, and the concentration method familiar to those skilled in the art can be used to concentrate until no solvent flows out, such as vacuum distillation. In the present invention, the recovered extractant can be reused. The present invention preferably achieves the purpose of removing chlorophyll through the first extraction and the second extraction.

[0051] After obtaining the total alkaloids of anise alkaloids, the present invention mixes the total alkaloids of anise alkaloids with silica gel, and the obtained mixture is subjected to the first silica gel column chromatography separation, the first silica gel column chromatography separation includes a first gradient elution and a second gradient elution, the first gradient elution is carried out according to the volume ratio from large to small, using a petroleum ether-acetone system with a volume ratio of petroleum ether and acetone of 60:1 to 1:1 for gradient elution, and the second gradient elution is carried out according to the volume ratio from large to small, using a dichloromethane-methanol system with a volume ratio of dichloromethane and methanol of 10:1 to 0:1 for gradient elution; the same fractions are combined to obtain five fractions, which are named Fr.Ⅰ, Fr.Ⅱ, Fr.Ⅲ, Fr.Ⅳ, and Fr.Ⅴ respectively. In the present invention, the mesh number of the mixed silica gel (mixing silica gel) is preferably 60 to 80 meshes. The first silica gel column chromatography separation preferably includes sequentially performing the first gradient elution and the second gradient elution. During the first gradient elution, the volume ratio of petroleum ether to acetone is preferably 60:1, 30:1, 15:1, 5:1, and 1:1. During the second gradient elution, the volume ratio of dichloromethane to methanol is preferably 10:1, 5:1, 3:1, 1:1, and 0:1.

[0052] After obtaining Fr.Ⅳ, the present invention performs a second silica gel column chromatography separation on Fr.Ⅳ. The second silica gel column chromatography separation is performed according to the volume ratio from large to small, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1 for gradient elution, and the same fractions are combined to obtain five sub-fractions, which are named Fr.ⅣA, Fr.ⅣB, Fr.ⅣC, Fr.ⅣD, and Fr.ⅣE. In the present invention, the elution mode of the second silica gel column chromatography separation is gradient elution, and the volume ratio of dichloromethane to methanol is preferably 40:1, 20:1, 15:1, 8:1, 5:1, 1:1, and 0:1, respectively.

[0053] After obtaining Fr.IVB, the present invention performs a first gel column separation and a first high performance liquid chromatography separation on Fr.IVB to obtain an isoquinoline alkaloid compound of the structure shown in Formula I; the mobile phase used in the first gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane and methanol in the mobile phase used in the first gel column separation is 1:1, and the mobile phase used in the first high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile and water in the mobile phase used in the first high performance liquid chromatography separation is 10:90 to 100:0, and water is not 0. In the present invention, the gel used in the first gel column separation is Sephadex LH-20. The chromatographic column used in the first high performance liquid chromatography separation is Waters X-bridge, with a specification of 5μm, 10mm×150mm. The volume ratio of acetonitrile and water in the mobile phase used in the first high performance liquid chromatography separation is preferably 30:70 to 60:40, and more preferably 45:55. The flow rate of the first HPLC separation is preferably 3-4.5 mL / min, preferably 3.5 mL / min; the column temperature is preferably 25-30°C, more preferably 30°C.

[0054] After obtaining Fr.IVD, the present invention performs a second gel column separation and a second high performance liquid chromatography separation on Fr.IVD to obtain an isoquinoline alkaloid compound of the structure shown in formula II; the mobile phase used in the second gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane and methanol in the mobile phase used in the second gel column separation is 1:1, and the mobile phase used in the second high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile and water in the mobile phase used in the second high performance liquid chromatography separation is 10:90 to 100:0, and water is not 0. In the present invention, the gel used in the second gel column separation is Sephadex LH-20. The chromatographic column used in the second high performance liquid chromatography separation is Waters X-bridge, with a specification of 5μm, 10mm×150mm. The volume ratio of acetonitrile and water in the mobile phase used in the second high performance liquid chromatography separation is preferably 25:75 to 60:40, and more preferably 40:60. The flow rate of the second HPLC separation is preferably 3-4.5 mL / min, more preferably 3.5 mL / min; the column temperature is preferably 25-30°C, more preferably 30°C.

[0055] After obtaining Fr.V, the present invention performs a third silica gel column chromatography separation on Fr.V. The third silica gel column chromatography separation is performed according to the volume ratio from large to small, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1 for gradient elution, and the same fractions are combined to obtain five sub-fractions, which are named Fr.VA, Fr.VB, Fr.VC, Fr.VD, and Fr.VE. In the present invention, the elution mode of the third silica gel column chromatography separation is gradient elution, and the volume ratios of dichloromethane and methanol used in the third silica gel column chromatography separation are preferably 40:1, 20:1, 12:1, 5:1, 1:1, and 0:1, respectively.

[0056] After obtaining Fr. VC, the present invention performs a third gel column separation and a third high performance liquid chromatography separation on Fr. VC to obtain an isoquinoline alkaloid compound of the structure shown in formula III; the mobile phase used in the third gel column separation is methanol, and the mobile phase used in the third high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile and water in the mobile phase used in the third high performance liquid chromatography separation is 10:90 to 100:0, and water is not 0. In the present invention, the gel used in the third gel column separation is Sephadex LH-20. The chromatographic column used in the third high performance liquid chromatography separation is Waters X-bridge, with a specification of 5μm, 10mm×150mm. The volume ratio of acetonitrile and water in the mobile phase used in the third high performance liquid chromatography separation is preferably 20:80 to 60:40, and more preferably 30:70. The flow rate of the third HPLC separation is preferably 3-4.5 mL / min, more preferably 3.5 mL / min; the column temperature is preferably 25-30°C, more preferably 30°C.

[0057] After obtaining Fr.IVE, the present invention performs a fourth gel column separation and a fourth high performance liquid chromatography separation on Fr.IVE to obtain an isoquinoline alkaloid compound of the structure shown in formula IV; the mobile phase used in the fourth gel column separation is a dichloromethane-methanol system, the volume ratio of dichloromethane and methanol in the mobile phase used in the third gel column separation is 1:1, and the mobile phase used in the fourth high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile and water in the mobile phase used in the fourth high performance liquid chromatography separation is 10:90 to 100:0, and water is not 0. In the present invention, the gel used in the fourth gel column separation is Sephadex LH-20. The chromatographic column used in the fourth high performance liquid chromatography separation is Waters X-bridge, with a specification of 5μm, 10mm×150mm. The volume ratio of acetonitrile and water in the mobile phase used in the fourth high performance liquid chromatography separation is preferably 20:80 to 60:40, and more preferably 25:75. The flow rate of the fourth HPLC separation is preferably 3-4.5 mL / min, more preferably 3 mL / min or 3.5 mL / min; the column temperature is preferably 25-30°C, more preferably 30°C.

[0058] The invention provides an isoquinoline alkaloid pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises the isoquinoline alkaloid compound described in the above technical scheme or a pharmaceutically acceptable salt, hydrate or solvent compound of the isoquinoline alkaloid compound described in the above technical scheme or an isoquinoline alkaloid compound obtained by the preparation method described in the above technical scheme or the isoquinoline alkaloid compound described in the above technical scheme.

[0059] In the present invention, the mass percentage of the active component in the isoquinoline alkaloid pharmaceutical composition is preferably 0.1 to 99%, more preferably 0.5 to 90%, and further preferably 1 to 50%.

[0060] In the present invention, the pharmaceutically acceptable excipient preferably includes a carrier and / or an excipient. The present invention has no particular limitation on the carrier and / or excipient, and any carrier and / or excipient well known to those skilled in the art may be used, such as one or more of a solid diluent, a semisolid diluent, a liquid diluent, a filler or a pharmaceutical product adjuvant.

[0061] In the present invention, the preparation types of the isoquinoline alkaloid pharmaceutical composition preferably include liquid preparations, solid preparations, sprays or mists; the liquid preparations preferably include injections, suspensions, emulsions, solutions or syrups; the solid preparations preferably include tablets, capsules, granules or granules. In the present invention, there is no particular limitation on the preparation method of the isoquinoline alkaloid pharmaceutical composition, and a preparation method well known in the art can be used.

[0062] In the present invention, the administration method of the isoquinoline alkaloid pharmaceutical composition is preferably injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection or subcutaneous injection.

[0063] In the present invention, the isoquinoline alkaloid pharmaceutical composition is preferably used in the form of a dosage per unit body weight; the dosage per unit body weight is preferably 0.5 to 20 mg / kg, more preferably 1 to 15 mg / kg, and further preferably 5 to 10 mg / kg.

[0064] The present invention provides the use of the isoquinoline alkaloid compound described in the above technical scheme, or the pharmaceutically acceptable salt, hydrate or solvate of the isoquinoline alkaloid compound described in the above technical scheme, or the isoquinoline alkaloid compound obtained by the preparation method described in the above technical scheme, or the isoquinoline alkaloid compound described in the above technical scheme, or the isoquinoline alkaloid pharmaceutical composition described in the above technical scheme in the preparation of drugs for treating cardiovascular diseases or the preparation of PCSK9 protein inhibitors.

[0065] In the present invention, the cardiovascular disease is a cardiovascular disease caused by an increase in low-density lipoprotein caused by PCSK9, and more preferably includes a cardiovascular disease caused by an increase in the level of PCSK9 and a decrease in the level of low-density lipoprotein receptors. The isoquinoline alkaloid compound provided by the present invention has a good affinity with PCSK9, inhibits the binding of PCSK9 to low-density lipoprotein receptors, and has potential application value in treating cardiovascular diseases by reducing low-density lipoprotein.

[0066] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1

[0068] 60kg of crushed (particle size ≤ 100mm) whole herb of Hypecoum erectum L. (water content ≤ 5%) was soaked and extracted in methanol reagent, refluxed, each extraction time was 8h, the number of extractions was 4, and the extracts were combined and concentrated to obtain an extract. After the extract was fully dissolved in warm water (35-40°C), the pH was adjusted to 2-3 with a 3wt% HCl aqueous solution, and then extracted with petroleum ether 3 times and ethyl acetate 3 times to achieve the purpose of removing chlorophyll; the pH of the aqueous phase was adjusted to 9-10 with a 10wt% NaOH aqueous solution, and extracted with dichloromethane for 3 times, and the dichloromethane phase was combined and concentrated to obtain 1kg of total alkali. The volume ratio of the extract to warm water was 1:2; the volume ratio of the organic phase to the aqueous phase during extraction was 1:1.5.

[0069] The total alkali was mixed with silica gel and separated by silica gel column chromatography with gradient elution. The volume ratios of petroleum ether / acetone were 60:1, 30:1, 15:1, 5:1, and 1:1, and the volume ratios of dichloromethane / methanol were 10:1, 5:1, 3:1, 1:1, and 0:1, to obtain 5 components, which were designated as Fr.Ⅰ, Fr.Ⅱ, Fr.Ⅲ, Fr.Ⅳ, and Fr.Ⅴ. The silica gel for mixing the sample was 60-80 mesh.

[0070] The Fr.Ⅳ segment was separated by silica gel column and gradient elution. The volume ratio of dichloromethane to methanol was 40:1, 20:1, 15:1, 8:1, 5:1, 1:1, and 0:1, respectively, to obtain five subcomponents, which were named Fr.ⅣA, Fr.ⅣB, Fr.ⅣC, Fr.ⅣD, and Fr.ⅣE, respectively. The Fr.ⅣB segment (5.0 g) was separated by Sephadex LH-20 dichloromethane-methanol 1:1 (v / v) gel column. High performance liquid chromatography was used for separation (chromatographic column was Waters X-bridge (5μm, 10mm×150mm), acetonitrile-water 45:55 (v / v), flow rate 3.5mL / min, column temperature 30°C, to obtain compound Ⅰ (6.2 mg, t R =32.6 min, yield 0.124%, purity 98%, white crystals).

[0071] Fr. IV D fragment (3.2 g) was separated by Sephadex LH-20 dichloromethane-methanol 1:1 (v / v) gel column, and then separated by high performance liquid chromatography (chromatographic column: Waters X-bridge (5 μm, 10 mm×150 mm), in the condition of acetonitrile-water 40:60 (v / v), flow rate 3.5 mL / min, column temperature 30°C to obtain compound II (17.0 mg, t R =17.5min, yield 0.531%, purity 98%, white crystals).

[0072] The present invention performs a third silica gel column separation on the FrⅤ segment, and the volume ratio of dichloromethane to methanol is 40:1, 20:1, 12:1, 5:1, 1:1, and 0:1, respectively, to obtain five subcomponents, which are sequentially recorded as Fr.VA, Fr.VB, Fr.VC, Fr.VD, and Fr.VE; the Fr.VC segment (6.0 g) is separated by a Sephadex LH-20 methanol gel column, and then separated by high performance liquid chromatography (the chromatographic column is Waters X-bridge (5 μm, 10 mm×150 mm), under the conditions of acetonitrile-water 30:67 (v / v), flow rate 3.5 mL / min, and column temperature 30°C, to obtain compound III (8.5 mg, t R = 23.0 min, yield 0.141%, purity 98%, yellow oil).

[0073] Fr. ⅣE segment (4.0 g) was separated by Sephadex LH-20 dichloromethane-methanol 1:1 (v / v) gel column, and then separated by high performance liquid chromatography (chromatographic column: Waters X-bridge (5 μm, 10 mm×150 mm), in the condition of acetonitrile-water 25:75 (v / v), flow rate 3 mL / min, column temperature 30°C to obtain compound Ⅳ (5.1 mg, t R =42.0 min, yield 0.127%, purity 98%, yellow oil).

[0074] Compound Ⅰ: white crystals; [α] 22.5D-10 (c 0.15, MeOH); UV (MeOH) λmax (logε) 210.0 (5.38), 241.5 (5.12), 312.0 (4.78), 357.5 (4.75) nm; IR (KBr) V max 3432,2912,1630,1558,1504,1485,1440cm -1 ; HRESIMS m / z 382.1283[M+H] + (calcd for C 21 H 19 NO6+H,382.1285).

[0075] Compound II: white needle-shaped crystals; [α] 22.8D-6 (c 0.14, MeOH); UV (MeOH) λmax (logε) 208.5 (5.23), 240.0 (4.95), 314.0 (4.69), 355.0 (4.59) nm; IR (KBr) V max 3432,2937,1626,1606,1564,1519,1461cm -1 ; HRESIMS m / z 398.1594[M+H] + (calcd for C 22 H 23 NO6+H,398.1598).

[0076] Compound III: yellow oil; [α] 22.6D-20 (c 0.07, MeOH); UV (MeOH) λmax (logε) 204.5 (5.06), 242.5 (4.69), 304.5 (4.43), 372.0 (4.30) nm; IR (KBr) V max 3432,2932,1709,1604,1564,1519,1644cm -1; HRESIMS m / z 396.1443[M+H] + (calcd for C 22 H 21 NO6+H,396.1442).

[0077] Compound IV: yellow oil; [α]22.4D-14 (c 0.08, MeOH); UV (MeOH) λ max (logε)206.0(5.49),245.5(5.12),300.5(4.84),373.0(4.66)nm; IR(KBr)ν max 3434,2924,1726,1630,1607,1503,1465cm -1 ; HRESIMS m / z 380.1134[M+H] + (calcd for C 21 H 17 NO6+H,380.1129).

[0078] Compound 1 HNMR, 13 C NMR data are shown in Tables 1 and 2.

[0079] Table 1 NMR data of compounds Ⅰ and Ⅱ

[0080]

[0081]

[0082] a 400MHz,CDCl3; b 125MHz,CDCl3; c 400MHz,CD3OD; d 125MHz,CD3OD

[0083] Table 2 NMR data of compounds III and IV

[0084]

[0085]

[0086] a 400MHz,CDCl3; b 125MHz,CDCl3.

[0087] Example 2

[0088] The isoquinoline alkaloids prepared in Example 1 and water for injection are uniformly mixed, and fine filtration and filling sterilization are performed in sequence to obtain an isoquinoline alkaloid injection, wherein the concentration of the isoquinoline alkaloids is 2-5%, specifically 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0089] Example 3

[0090] The isoquinoline alkaloid prepared in Example 1 is dissolved in sterile water for injection, stirred until the isoquinoline alkaloid is dissolved, filtered through a sterile suction filtration funnel and sterile fine filtration, and then packaged into ampoules, and then freeze-dried and aseptically sealed to obtain an isoquinoline alkaloid pharmaceutical composition powder injection, wherein the solid-liquid ratio of the isoquinoline alkaloid to the sterile water for injection is 1g:3-5mL, specifically 1g:3mL, 1g:3.5mL, 1g:4mL, 1g:4.5 or 1g:5mL.

[0091] Example 4

[0092] The isoquinoline alkaloids prepared in Example 1 were uniformly mixed with an excipient (starch) at a mass ratio of 9:1, and subjected to low-temperature freeze-drying and sterilization to obtain an isoquinoline alkaloid pharmaceutical composition powder.

[0093] Example 5

[0094] The isoquinoline alkaloids prepared in Example 1 and the excipient (starch) are uniformly mixed in a mass ratio of 1:5-10 (specifically 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10), granulated and tableted to obtain isoquinoline alkaloid pharmaceutical composition tablets.

[0095] Example 6

[0096] The isoquinoline alkaloids prepared in Example 1 were used in a conventional oral liquid preparation method to prepare an isoquinoline alkaloid pharmaceutical composition oral liquid.

[0097] Example 7

[0098] The isoquinoline alkaloids prepared in Example 1 and the excipients were uniformly mixed in a mass ratio of 5:1, and conventional preparation methods of capsules, granules and granules were respectively used to prepare isoquinoline alkaloid pharmaceutical composition capsules, isoquinoline alkaloid pharmaceutical composition granules and isoquinoline alkaloid pharmaceutical composition granules.

[0099] Example 8

[0100] The isoquinoline alkaloids prepared in Example 1 and the excipients were uniformly mixed in a mass ratio of 3:1, and conventional preparation methods of capsules, granules and granules were used to prepare isoquinoline alkaloid pharmaceutical composition capsules, isoquinoline alkaloid pharmaceutical composition granules and phthalide isoquinoline alkaloid pharmaceutical composition granules.

[0101] Test Example 1

[0102] Evaluation of biological activity of alkaloids

[0103] (1) Experimental instruments

[0104] Instrument model: Biacore T200;

[0105] Reagents: S series CM5 sensor chip; amino coupling kit; 10 mM sodium acetate solution (pH 4.0, pH 4.5, pH 5.0, pH 5.5); PBS-P buffer (20 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl and 0.05% (v / v) surfactant P20) supplemented with 5% DMSO (pH 7.4).

[0106] (2) Experimental methods

[0107] ① Protein preparation: Proprotein Convertase Subtilisin / Kexin Type 9 (Cat. No.: SRP6285, Lot: 7M06L72650), protein concentration: 40 μg / mL.

[0108] ② Ligand pre-enrichment: Ligand pre-enrichment was performed by manual injection to test the pre-enrichment of PCSK9 at a concentration of 40 μg / mL in 10 mM sodium acetate solution at pH 4.5 and pH 5.0 (pH 4.5 was selected for coupling).

[0109] ③Amino coupling:

[0110] The proteins were fixed to the Fc-4 channel of the CM5 chip by amino coupling, and the Fc-1 channel was activated and blocked as the reference channel of the Fc-4 channel.

[0111] ④Affinity analysis:

[0112] Analysis of the interaction between compounds I, II, III, and IV and protein PCSK9: The four compounds were prepared in a concentration gradient using running buffer, with a series of concentrations ranging from 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μM; flowed through Fc1-Fc4, and a solvent calibration curve (Solvent correction) was made at the same time to measure the interaction between the four compounds and PCSK9 fixed on the chip surface. Operation parameter settings: Flow path 4-1 (contact time 80 s, Flow rate 30 μl / min, Dissociation time 80 s), 25 ° C.

[0113] (3) Experimental results

[0114] Compounds Ⅰ, Ⅱ, Ⅲ, Ⅳ, positive control and protein PCSK9 interaction Figure 1 As shown, compounds Ⅰ and Ⅱ showed weak interactions, and Ⅲ and Ⅳ showed medium-strength interactions. The results are shown in Table 3:

[0115] Table 3 Results of interaction between compounds and PCSK9

[0116] Compound <![CDATA[K D Value(M)]]> Ⅰ 3.06E-04 Ⅱ 2.48E-04 Ⅲ 9.51E-05 Ⅳ 5.99E-05 PF-06446846 4.14E-05

[0117] In order to further study the interaction position of these alkaloids with PCSK9 protein, the molecular docking software Maestro11.9 was used to dock the small molecules with the crystal structure of PCSK9 protein (PDB ID: 6U3X), and the docking results were processed using Pymol. The docking results showed that the four small molecule compounds all had interaction forces with protein PCSK9. Figure 2 shown.

[0118] From the above examples, it can be seen that the isoquinoline alkaloid compounds provided by the present invention have good affinity with PCSK9, inhibit the binding of PCSK9 to low-density lipoprotein receptors, and have potential application value in treating cardiovascular diseases by reducing low-density lipoprotein. At the same time, the preparation method of the isoquinoline alkaloid compounds provided by the present invention has cheap raw materials and low preparation cost, which is convenient for large-scale promotion and use after drug development.

[0119] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An isoquinoline alkaloid compound, characterized in that: It has the structure shown in Formula I, Formula II, Formula III or Formula IV:

2. The method for preparing the isoquinoline alkaloid compound according to claim 1, characterized in that: The following steps are involved: Extract the fennel with methanol, and concentrate the obtained extract to obtain an extract; Dissolving the extract in water and adjusting the pH to acidic to obtain an acidic solution; The acidic solution is subjected to a first extraction with petroleum ether to obtain a petroleum ether phase and a first aqueous phase; The first aqueous phase is subjected to a second extraction with ethyl acetate to obtain an ethyl acetate phase and a second aqueous phase; the pH of the second aqueous phase is adjusted to alkaline to obtain an alkaline solution, and the alkaline solution is subjected to a third extraction with dichloromethane to obtain a dichloromethane phase; the dichloromethane is concentrated to obtain the total alkaloids of anise alkaloids; The total alkaloids of anise alkaloids and silica gel are mixed, and the obtained mixture is subjected to a first silica gel column chromatography separation, wherein the first silica gel column chromatography separation includes a first gradient elution and a second gradient elution, wherein the first gradient elution is carried out in a descending volume ratio, using a petroleum ether-acetone system with a volume ratio of petroleum ether to acetone of 60:1 to 1:1, and the second gradient elution is carried out in a descending volume ratio, using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 10:1 to 0:1; the same fractions are combined to obtain five fractions, which are named Fr.Ⅰ, Fr.Ⅱ, Fr.Ⅲ, Fr.Ⅳ, and Fr.Ⅴ respectively; Fr.Ⅳ was subjected to a second silica gel column chromatography separation, wherein the second silica gel column chromatography separation was performed in a gradient elution using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1, and the same fractions were combined to obtain five sub-fractions, which were named Fr.ⅣA, Fr.ⅣB, Fr.ⅣC, Fr.ⅣD, and Fr.ⅣE respectively; Fr.IVB is subjected to a first gel column separation and a first high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in Formula I; the mobile phase used in the first gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the first gel column separation is 1:1; the mobile phase used in the first high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the first high performance liquid chromatography separation is 10:90-100:0, and the water content is not 0; Fr.IVD is subjected to a second gel column separation and a second high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula II; the mobile phase used in the second gel column separation is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the mobile phase used in the second gel column separation is 1:1; the mobile phase used in the second high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the second high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0; Fr.V is subjected to a third silica gel column chromatography separation, wherein the third silica gel column chromatography separation is performed in a gradient elution using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 40:1 to 0:1 according to a large to small volume ratio, and the same fractions are combined to obtain five sub-fractions, which are named Fr.VA, Fr.VB, Fr.VC, Fr.VD, and Fr.VE respectively; Fr. VC is subjected to a third gel column separation and a third high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound of the structure shown in formula III; the mobile phase used in the third gel column separation is methanol, and the mobile phase used in the third high performance liquid chromatography separation is an acetonitrile-water system, and the volume ratio of acetonitrile to water in the mobile phase used in the third high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0; Fr.IVE is subjected to a fourth gel column separation and a fourth high performance liquid chromatography separation to obtain an isoquinoline alkaloid compound with a structure shown in formula IV; the mobile phase used in the fourth gel column separation is a dichloromethane-methanol system, the volume ratio of dichloromethane and methanol in the mobile phase used in the third gel column separation is 1:1, the mobile phase used in the fourth high performance liquid chromatography separation is an acetonitrile-water system, the volume ratio of acetonitrile and water in the mobile phase used in the fourth high performance liquid chromatography separation is 10:90 to 100:0, and the water content is not 0.

3. The preparation method according to claim 2, characterized in that: The horned fennel is the whole horned fennel plant; the water content of the whole horned fennel plant is ≤5%.

4. The preparation method according to claim 2 or 3, characterized in that: The extraction is multiple extractions, the extraction is carried out under hot reflux conditions, the hot reflux temperature is 45-60° C., the single extraction time is 7-8 hours, and the solid-liquid ratio of the whole fennel herb to the organic solvent is 1kg:6-7L during the single extraction.

5. The preparation method according to claim 2, characterized in that: The pH value of the acidic solution is 2-3; the pH value of the alkaline solution is 9-10.

6. The preparation method according to claim 2, characterized in that: The number of extractions of the first extraction, the second extraction and the third extraction is independently 3 to 4 times; the volume ratio of the organic phase to the aqueous phase when the first extraction, the second extraction and the third extraction are performed once is independently 1:1.5 to 2.

7. The preparation method according to claim 2, characterized in that: The volume ratio of acetonitrile to water in the mobile phase used in the first high performance liquid chromatography separation is 30:70 to 60:40; The volume ratio of acetonitrile to water in the mobile phase used in the second high performance liquid chromatography separation is 25:75 to 60:40; The volume ratio of acetonitrile to water in the mobile phase used in the third high performance liquid chromatography separation is 20:80 to 60:40; The volume ratio of acetonitrile to water in the mobile phase used in the fourth high performance liquid chromatography separation is 20:80 to 60:

40.

8. An isoquinoline alkaloid pharmaceutical composition, characterized in that: The invention comprises an active component and a pharmaceutically acceptable excipient; the active component comprises the isoquinoline alkaloid compound according to claim 1 or a pharmaceutically acceptable salt of the isoquinoline alkaloid compound according to claim 1 or an isoquinoline alkaloid compound obtained by the preparation method according to any one of claims 2 to 7.

9. Use of the isoquinoline alkaloid compound according to claim 1, or a pharmaceutically acceptable salt of the isoquinoline alkaloid compound according to claim 1, or an isoquinoline alkaloid compound obtained by the preparation method according to any one of claims 2 to 7, or an isoquinoline alkaloid pharmaceutical composition according to claim 8 in the preparation of drugs for treating cardiovascular diseases or the preparation of PCSK9 protein inhibitors.

10. The use according to claim 9, characterized in that: The cardiovascular disease is a cardiovascular disease caused by increased low-density lipoprotein caused by PCSK9.

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