Sponge-sourced pyridine inner salt and preparation method thereof

Abstract

The invention discloses a sponge-sourced pyridine inner salt. The chemical structure of the sponge-sourced pyridine inner salt is shown as a formula I: # imgabs0 #, and the sponge-sourced pyridine inner salt is sourced from Hippospongia lachne in South China Sea. The sponge-sourced pyridine inner salt disclosed by the invention is simple in preparation method, and medicine source materials are widely distributed in South China Sea and are easy to collect. The invention provides a new compound for marine drug research, and provides a scientific basis for development and utilization of marine medicinal resources in China.

Description

Technical Field

[0001] The invention belongs to the field of marine biology and medical technology, and in particular relates to a sponge-derived pyridine inner salt and a preparation method thereof. Background Art

[0002] Sponges are the most primitive multicellular animals. They are in the extreme environment of high pressure, high salt and hypoxia in the ocean. In order to adapt to this special environment, they are prone to produce secondary metabolites with rare structures and diverse activities. Therefore, they have always been an important direction for the study of marine natural products. The drug source material Hippospongia lachne belongs to the class Demospongiae, the order Dictyoceratida, and the family Hippospongia. It is produced in the South China Sea. Terpenes and their derivatives, long-chain aliphatics, polyketides, steroid compounds and other structural types of compounds have been isolated from this genus of sponges, but there has been no report on the isolation of pyridine salts from this genus of sponges. Summary of the invention

[0003] The purpose of the present invention is to provide a sponge-derived pyridine inner salt.

[0004] Another object of the present invention is to provide a method for preparing the sponge-derived pyridine inner salt.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect of the present invention, a sponge-derived pyridine inner salt is provided, and its chemical structure is shown in Formula I:

[0007]

[0008] The molecular formula of the compound of formula I is C 25 H 39 NO4.

[0009] The sponge-derived pyridinium salt is derived from the hairy horse sponge Hippospongia lachne in the South China Sea.

[0010] Another object of the present invention is to provide a method for preparing the sponge-derived pyridine inner salt, comprising the following steps:

[0011] The first step is to prepare the total extract

[0012] Cut the frozen (-20°C) hairy horse sponge Hippospongia lachne into small pieces (about 2cm*2cm), extract with 95% ethanol for at least three times, and obtain a total extract; disperse the total extract in water, extract with ethyl acetate for at least three times, and concentrate under reduced pressure to obtain a fat-soluble extract; suspend the fat-soluble extract in 90% methanol-water solution, defat with petroleum ether, dilute the 90% methanol-water solution with water to 60% methanol-water solution, extract with dichloromethane, and concentrate under reduced pressure to obtain a dichloromethane total extract;

[0013] The second step is separation and purification

[0014] 1) The total dichloromethane extract was separated by vacuum silica gel column chromatography (VLC), and gradient elution was performed using dichloromethane-methanol solution as the eluent. Similar fractions were combined according to the color development of thin layer chromatography (TLC) to obtain 7 components Fr.AG;

[0015] 2) The component Fr.F was separated by ODS medium pressure column chromatography, eluted with a methanol-water gradient, and similar fractions were combined according to TLC color development to obtain 7 components Fr.FA-FG;

[0016] 3) The component Fr.FC was separated by normal phase medium pressure silica gel column chromatography, eluted isocratically with dichloromethane-methanol solution, and similar fractions were combined according to TLC color development to obtain 7 components Fr.FCA-FCG;

[0017] 4) Fr.FCE is separated by reverse phase high performance liquid phase to obtain the pyridine inner salt derived from the sponge.

[0018] In the second step of separation and purification, in 1), the conditions for gradient elution using dichloromethane-methanol solution as the eluent are: elution with dichloromethane-methanol solutions with volume ratios of 100:1, 70:1, 50:1, 10:1, 5:1, 1:1, and 0:1, in sequence.

[0019] In the second separation and purification step, in step 2), the volume ratio of the methanol-water solution gradient elution is 10%→100%, and the time is 200 min.

[0020] In the second step of separation and purification, in step 3), the volume ratio of the isocratic elution of dichloromethane-methanol solution is 15:1.

[0021] In the second separation and purification step, in 4), the conditions for reversed-phase high performance liquid separation are: the eluent is acetonitrile-water solution with a volume ratio of 45:55, the chromatographic column is YMC-Pack Pro C18 RS (10×250mm, 5μm), the flow rate is 2.0mL / min, the detection wavelength is 276nm, and the retention time is 30.0min.

[0022] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0023] The pyridinium salt provided by the invention is separated from the hairy horse sponge Hippospongia lachne in the South China Sea. The preparation method of the pyridinium salt provided by the invention is simple, and the drug source material is widely distributed in the South my country Sea and is easy to collect, thus providing new compounds for marine drug research and providing a scientific basis for the development and utilization of my country's marine medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the DP4+ probability analysis of the pyridine inner salt of the present invention.

[0025] Figure 2 The diagram is a schematic diagram of the ECD spectrum of the complex of the pyridine inner salt and molybdenum tetraacetate recorded in DMSO. DETAILED DESCRIPTION

[0026] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0027] The present invention is a pyridine inner salt separated from the hairy horse sponge Hippospongia lachne collected from the South my country Sea. The present invention can also use other sponges to prepare the compound of the present invention or use an artificial synthesis method to prepare the compound of the present invention.

[0028] Example 1

[0029] Preparation of compounds of the present invention

[0030] The first step is to prepare the total extract

[0031] The hairy horse sponge Hippospongia lachne (wet weight 10kg) frozen in a -20℃ refrigerator was thawed and cut into small pieces (about 2cm*2cm), and cold-extracted with 95% ethanol (20L*3 times) to obtain a total extract (280g). The total extract was dispersed in water (1L), extracted 3 times with ethyl acetate (1L), and concentrated under reduced pressure to obtain a fat-soluble extract (141g). The fat-soluble extract was suspended in a 90% methanol-water solution (800mL), degreased with petroleum ether (800mL*3 times), and 400mL of water was added to the 90% methanol-water solution to dilute it to 60% methanol-water solution, and then extracted with dichloromethane (1.2L*3 times) and concentrated under reduced pressure to obtain a dichloromethane total extract (55g);

[0032] The second step is separation and purification

[0033] 1) The total dichloromethane extract was separated by vacuum silica gel column chromatography (VLC), and gradient elution was performed using dichloromethane-methanol solution (100:1, 70:1, 50:1, 10:1, 5:1, 1:1, 0:1) as the eluent, and similar fractions were combined according to the color development of thin layer chromatography (TLC) to obtain 7 components Fr.AG;

[0034] 2) The component Fr.F was separated by ODS medium pressure column chromatography, and gradient eluted with methanol-water solution (10%→100%, 200 min). Similar fractions were combined according to TLC color development to obtain 7 components Fr.FA-FG;

[0035] 3) The component Fr.FC was separated by normal phase medium pressure silica gel column chromatography, eluted isocratically with dichloromethane-methanol solution (15:1), and similar fractions were combined according to TLC color development to obtain 7 components Fr.FCA-FCG;

[0036] 4) Fr.FCE was separated by reverse phase high performance liquid phase separation, the eluent was acetonitrile-water solution (45:55), the chromatographic column was YMC-Pack Pro C18 RS (10×250 mm, 5 μm), the flow rate was 2.0 mL / min, the detection wavelength was 276 nm, the retention time was 30 min, and the compound of formula I of the present invention was obtained.

[0037] Step 3: Structural Identification

[0038] The molecular formula of the compound represented by formula I of the present invention is C 25 H 39 NO4, 1D NMR, 2D NMR, HRESIMS, DP4+ probability analysis, Snatzke method, determined that the compound C of formula I of the present invention 25 H 39 The chemical structure of NO4 is shown in Formula I:

[0039]

[0040] The compound C represented by formula I of the present invention 25 H 39 The physicochemical properties and NMR data of NO4 are as follows: white powder; +4.2(c 0.10,MeOH); UV(MeOH)λmax(logε)200(2.96),276(2.25)nm; IR(micro)ν max 3377,2924,2853,1710,1595,1530,1377,1079cm -1;ICD(DMSO)λmax(Δε)302(-0.13)nm; HRESIMS m / z 418.2957[M+H] + (calcd for C 25 H 40 NO4,418.2957). The nuclear magnetic resonance spectrum data are shown in Table 1:

[0041] Table 1 Compound C of Formula I of the present invention 25 H 39 NMR spectrum data of NO4 (CD3OD, 400MHz)

[0042] No. <![CDATA[δ H ,(JinHz)]]> <![CDATA[δ C ,type]]> <![CDATA[ 1 H- 1 HCOSY]]> HMBC(H→C) NOESY 2 157.6,C 3 7.89,d(8.4) 128.6,C 4 2,5,7 4 8.33,d(8.4) 147.4,CH 3 2,6 5 138.3,C 6 8.72,s 147.7,CH 2,5,24 7 3.13,m <![CDATA[33.2,CH2]]> 8a 2,9 8a 1.84,m <![CDATA[31.3,CH2]]> 7 8b 1.53,m 9 1.84,m <![CDATA[26.0,CH2]]> 10 3.39,d(9.2) 78.0,CH 9 11 75.3,C 12 1.54,m <![CDATA[39.6,CH2]]> 13b 13a 2.20,m <![CDATA[22.8,CH2]]> 13b 2.13,m 12,14 22 14 5.15,t(6.8) 126.0,CH 13b 22 16b 15 135.9,C 16a 2.10,m <![CDATA[40.9,CH2]]> 14 16b 2.00,m 17 14,15,17,22 14 17 2.10,m <![CDATA[27.8,CH2]]> 16b,18 16,19 21 18 5.10,t(6.8) 125.4,CH 17 16,20,21 20 19 132.1,C 20 1.67,s <![CDATA[25.9,CH3]]> 18,19,21 18 21 1.60,s <![CDATA[17.7,CH3]]> 18,19,20 17 22 1.63,s <![CDATA[16.1,CH3]]> 14,15,16 13b 23 1.13,s <![CDATA[21.8,CH3]]> 10,11,12 24 4.30,s <![CDATA[45.8,CH3]]> 2,6 25 3.65,s <![CDATA[41.8,CH2]]> 4,5,6,26 26 176.2,C

[0043] The relative configuration and absolute configuration of the vicinal diol fragment in the structure were determined by DP4+ probability analysis and Snatzke method, respectively. Figure 1 As shown, Figure 1 ) shows that the relative configurations of the vicinal diol fragments are 10R*, 11R* (Isomer 2) and 10S*, 11R* (Isomer 1), with probabilities of 100% and 0%, respectively, so the relative configurations of the vicinal diol fragments are determined to be 10R*, 11R*. The absolute configurations of the vicinal diol fragments determined by the Snatzke method are shown in FIG. Figure 2 As shown, Figure 2 The diagram is a schematic diagram of the ECD spectrum of the complex of the pyridine inner salt and molybdenum tetraacetate recorded in DMSO. The negative Cotton effect at 310 nm in the diagram indicates that the OCCO bond is a negative helix, thereby determining that the absolute configuration of the vicinal diol fragment is 10R, 11R.

[0044] The invention provides a new pyridine inner salt compound and a preparation method thereof for marine drug research, and provides a scientific basis for the development and utilization of my country's marine medicinal resources.

[0045] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A pyridine inner salt derived from a sponge, characterized in that: Its chemical structure is shown in Formula I:

2. The sponge-derived pyridine inner salt according to claim 1, characterized in that: The sponge-derived pyridinium salt is derived from the hairy horse sponge Hippospongia lachne in the South China Sea.

3. A method for preparing a sponge-derived pyridine inner salt according to claim 1 or 2, characterized in that: The following steps are involved: The first step is to prepare the total extract Cut the frozen hairy horse sponge Hippospongia lachne into small pieces (about 2cm*2cm), extract with 95% ethanol at least three times, and obtain a total extract; disperse the total extract in water, extract with ethyl acetate at least three times, and concentrate under reduced pressure to obtain a fat-soluble extract; suspend the fat-soluble extract in 90% methanol-water solution, defat with petroleum ether, dilute the 90% methanol-water solution with water to 60% methanol-water solution, extract with dichloromethane, and concentrate under reduced pressure to obtain a dichloromethane total extract; The second step is separation and purification 1) The total dichloromethane extract was separated by vacuum silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol solution as the eluent. Similar fractions were combined according to the color development of thin layer chromatography to obtain 7 components Fr.AG; 2) The component Fr.F was separated by ODS medium pressure column chromatography, eluted with a methanol-water gradient, and similar fractions were combined according to TLC color development to obtain 7 components Fr.FA-FG; 3) The component Fr.FC was separated by normal phase medium pressure silica gel column chromatography, eluted isocratically with dichloromethane-methanol solution, and similar fractions were combined according to TLC color development to obtain 7 components Fr.FCA-FCG; 4) Fr.FCE is separated by reverse phase high performance liquid phase to obtain the pyridine inner salt derived from the sponge.

4. The method for preparing the sponge-derived pyridine inner salt according to claim 3, characterized in that: In the second step of separation and purification, in 1), the conditions for gradient elution using dichloromethane-methanol solution as the eluent are: elution with dichloromethane-methanol solutions with volume ratios of 100:1, 70:1, 50:1, 10:1, 5:1, 1:1, and 0:1, in sequence.

5. The method for preparing the sponge-derived pyridine inner salt according to claim 3, characterized in that: In the second separation and purification step, in step 2), the volume ratio of the methanol-water solution gradient elution is 10%→100%, and the time is 200 min.

6. The method for preparing the sponge-derived pyridine inner salt according to claim 3, characterized in that: In the second step of separation and purification, in step 3), the volume ratio of the isocratic elution of dichloromethane-methanol solution is 15:

1.

7. The method for preparing the sponge-derived pyridine inner salt according to claim 3, characterized in that: In the second separation and purification step, in 4), the conditions for reversed-phase high performance liquid separation are: the eluent is acetonitrile-water solution with a volume ratio of 45:55, the chromatographic column is YMC-Pack Pro C18 RS, the flow rate is 2.0 mL / min, the detection wavelength is 276 nm, and the retention time is 30 min.

Images