CN111777512A

CN111777512A - Five diterpenoid compounds derived from euphorbia tirucalli as well as preparation method and application thereof

Info

Publication number: CN111777512A
Application number: CN201910269212.2A
Authority: CN
Inventors: 许婧; 郭远强; 梁月; 石照宇; 安莉君
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2020-10-16

Abstract

The invention relates to structures of five novel diterpenoid compounds (1-5), a preparation method and application thereof in anti-inflammatory drugs, wherein the novel compounds 1-5 have the following structures. The five novel compounds have NO inhibitory activity and can be used for development and application of anti-inflammatory drugs.

Description

Five diterpenoid compounds derived from euphorbia tirucalli as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a novel diterpenoid compound in euphorbia tirucalli as well as a preparation method and application thereof.

Background

NO (Nitric oxide) is an important messenger molecule, plays a key role in various physiological and biochemical processes, and has a magical physiological regulation function. In many tissues, although the amount of actual release is currently difficult to detect, it is well established that different concentrations of NO are released and that the change in concentration is closely related to the physiological function of the body. Many diseases, including inflammation, neurodegenerative diseases, cancer, etc., may be caused by abnormal release or regulation of NO.

In response to inflammation, inflammatory mediators or inflammatories induce or increase local NO synthesis and release, which in turn induces pro-inflammatory cytokine production, such as IL-1, TNF-αAnd the like. Excess NO promotes vasodilation, enhances vascular permeability and leakage, and produces cytotoxicity. Excessive NO cytotoxicity is non-specific, not only against microorganisms, but also causes damage to tissue and cells surrounding the cells. In view of the close relationship between inflammation and NO, inhibition of NO production has been one of the targets of anti-inflammatory drugs.

In the central nervous system, NO is a signal for neuroinflammatory responses. The inflammatory response is accompanied by activation of microglia and release of large amounts of NO, resulting in neurotoxicity, leading to neuronal degeneration and death, which in turn leads to neurodegenerative diseases. Therefore, the composition can inhibit the generation of NO in the central nervous system, treat neuroinflammation and has potential treatment effect on neurodegenerative diseases related to neuroinflammation, such as Alzheimer's disease, Parkinson's disease and the like.

Plants provide us with a natural product of diverse structure and biological activity. In order to find new NO inhibitors and further develop anti-inflammatory and neurodegenerative disease-treating drugs, we established a BV-2 cell screening model. The cells produce NO under stimulation by Lipopolysaccharide (LPS); the test drugs (compounds or plant extracts) are added while being stimulated by LPS, and the NO activity inhibition of the drugs is evaluated, so that the drugs for resisting inflammation and treating neurodegenerative diseases are discovered.

Disclosure of Invention

The invention aims to provide 5 novel diterpenoid compounds in euphorbia tirucalli and a preparation method and application thereof.

The novel compounds 1-5 provided by the invention belong to diterpenes, and the structures of the novel compounds are shown in figure 1.

The present invention also provides a process for the preparation of said novel compounds 1-5, which comprises the steps of:

(1) herba Euphorbiae Humifusae (herba Euphorbiae Humifusae)Euphorbia antiquorumL) extracting the stems with a solvent, and recovering the extract to obtain a crude extract;

(2) dissolving the crude extract obtained in the step (1) in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent to obtain an extract;

(3) separating the extract obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether/acetone or petroleum ether/ethyl acetate;

(4) separating the fraction obtained in the step (3) by MPLC (medium pressure liquid chromatography, ODS is chromatographic packing), and performing gradient elution by using methanol/water or acetonitrile/water mixed solvent as a mobile phase;

(5) and (3) carrying out HPLC-RI (high performance liquid-differential detection) chromatographic separation on the fractions obtained in the step (4), and eluting by taking methanol/water as a mobile phase or acetonitrile/water as a mobile phase to obtain the compounds 1-5.

The invention provides a preparation method of novel compounds 1-5, wherein the euphorbia is euphorbia (Euphorbiaceae) (namely euphorbia antiqua)Euphorbia antiquorumL.) extract of the stem.

The invention provides a preparation method of a new compound 1-5, the extraction method in the step (1) is heating reflux extraction or ultrasonic extraction, the used solvent is at least one of dichloromethane, chloroform, ethyl acetate, methanol and ethanol, and the medicinal materials: the weight-volume ratio of the solvent is 1: 5-1: 15.

The preparation method of the new compound 1-5 and the extraction method in the step (2) provided by the invention adopt any one of petroleum ether, dichloromethane, chloroform and ethyl acetate as an organic solvent, and the volume ratio of an aqueous solution to the organic solvent is 1: 1-1: 2.

According to the preparation method of the new compound 1-5, in the step (3), an elution solvent is a petroleum ether/acetone or petroleum ether/ethyl acetate mixed solvent, and the ratio of the petroleum ether/acetone to the petroleum ether/ethyl acetate mixed solvent is 100: 2-100: 30.

In the preparation method of the novel compound 1-5 provided by the invention, in the step (4), the ratio of the methanol/water mixed solvent is 6: 4-9: 1, preferably 7: 3-8: 2, or the ratio of the acetonitrile/water mixed solvent is 6: 4-8: 1, preferably 7: 3-8: 2.

According to the preparation method of the novel compound 1-5, the volume ratio of the mobile phase methanol and water mixed solvent or the acetonitrile and water mixed solvent in the step (5) is 6: 4-9: 1, preferably 7: 3-9: 1.

The five novel terpenoids provided by the invention have NO inhibitory activity. Can be used for preparing antiinflammatory drugs.

Drawings

FIG. 1 structural formulas of compounds 1-5 of the present invention;

FIG. 2 Process for preparation of Compound 1 of the present invention¹H NMR spectrum;

FIG. 3 preparation of Compound 1 of the present invention¹³C NMR spectrum;

FIG. 4 DEPT135 spectrum of inventive compound 1;

FIG. 5 HMQC spectra of Compound 1 of the invention;

FIG. 6 HMBC spectra of Compound 1 of the present invention;

FIG. 7 preparation of Compound 1 of the present invention¹H-¹H COSY spectrum;

FIG. 8 preparation of Compound 2 of the present invention¹H NMR spectrum;

FIG. 9 preparation of Compound 2 of the present invention¹³C NMR spectrum;

FIG. 10 HMQC spectra of Compound 2 of the invention;

FIG. 11 HMBC spectra of Compound 2 of the present invention;

FIG. 12 preparation of Compound 3 of the present invention¹H NMR spectrum;

FIG. 13 preparation of Compound 3 of the present invention¹³C NMR spectrum;

FIG. 14 HMQC spectra of Compound 3 of the invention;

FIG. 15 HMBC spectra of compound 3 of the present invention;

FIG. 16 preparation of Compound 4 of the present invention¹H NMR spectrum;

FIG. 17 preparation of Compound 4 of the present invention¹³C NMR spectrum;

FIG. 18 HMQC spectra of Compound 4 of the invention;

FIG. 19 HMBC spectra of Compound 4 of the present invention;

FIG. 20 preparation of Compound 5 of the present invention¹H NMR spectrum;

FIG. 21 preparation of Compound 5 of the present invention¹³C NMR spectrum;

FIG. 22 HMQC spectra of Compound 5 of the invention;

FIG. 23 HMBC spectra of compound 5 of the present invention;

FIG. 24 HMBC and compounds 1-5 of the present invention¹H-¹H COSY correlation signal diagram.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.

Example 1

(1) Extracting herba Euphorbiae Lathyridis stem 11.5 kg with methanol for 3 times (dosage 3 × 60L), and recovering extractive solution under reduced pressure to obtain crude extract;

(2) adding water into the methanol extract obtained in the step (1) to prepare suspension, and extracting with ethyl acetate and petroleum ether respectively to obtain ethyl acetate extract and petroleum ether extract;

(3) separating the petroleum ether extract in the step (2) by silica gel column chromatography, and sequentially eluting with petroleum ether, namely acetone 100:0, 100:2,100:4, 100:6, 100:8, 100:11, 100:16, 100:23 and 100: 30;

(3) separating the 100: 2-100: 23 parts of the petroleum ether and acetone obtained in the step (2) by using medium-pressure liquid chromatography (MPLC), and performing gradient elution by using methanol/water 6: 4-9: 1 as a mobile phase;

(4) separating methanol/water (6: 4) and (8: 2) fractions obtained in the step (3) by HPLC-RI, and eluting with methanol/water 60:40 to 90:10 as a mobile phase to obtain the new compounds 1 (yield 0.002%), 2 (yield 0.001%), 3 (yield 0.001%), 4 (yield 0.005%) and 5 (yield 0.001%).

The structures of the compounds 1 to 5 were identified based on their physicochemical properties and spectral data (the spectra of the compounds 1 to 5 are shown in FIGS. 2 to 23).

The structural identification data for compound 1 is as follows:

colorless oil [ alpha ]α]

+10.9 (c0.3, CH₂Cl₂); ECD (CH₃CN) 190 (Δ2.06), 217 (Δ-1.40), 307 (Δ1.47) nm; IR (KBr)ν _max2935, 2873, 1722, 1707, 1456, 1388,1263, 1177, 1096, 1031, 932, 732 cm^–1; ESIMSm/z425 [M + Na]⁺; HRESIMSm/z425.2302 [M + Na]⁺(calcd for C₂₄H₃₄NaO₅, 425.2304);¹³C NMR (100 MHz, CDCl₃) And¹H NMR (400 MHz, CDCl₃) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 23. The absolute configuration of the compound is determined by using a TDDFT (time density functional theory) method to calculate ECD (electronic circular dichroism), comparing an ECD spectrogram measured by an experiment with an ECD spectrogram of an enantiomer obtained by calculation, and determining that the absolute configuration of the compound is 4R, 5S, 8S, 9S, 10S, 12S, 13R。

The structural identification data for compound 2 is as follows:

colorless oil [ alpha ]α]

+13.3 (c0.3, CH₂Cl₂); ECD (CH₃CN) 190 (Δ1.92), 217 (Δ-1.94), 250 (Δ0.49), 252 (Δ0.50), 273 (Δ0.23) nm; IR (KBr)ν _max2930,2866, 1705, 1650, 1454, 1386, 1262, 1137, 1071, 976, 892, 732 cm^–1; ESIMSm/z437 [M + Na]⁺; HRESIMSm/z437.2301 [M + Na]⁺(calcd for C₂₅H₃₄NaO₅, 437.2304);¹³C NMR (100 MHz, CDCl₃) And¹H NMR (400 MHz, CDCl₃) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 23. The absolute configuration of the compound is determined by ECD, the compoundHas an absolute configuration of 4R, 5S, 8S,9S, 10S, 12S, 13R。

The structural identification data for compound 3 is as follows:

colorless oil [ alpha ]α]

-13.3 (c0.3, CH₂Cl₂); ECD (CH₃CN) 207 (Δ-0.80), 235 (Δ0.35), 281 (Δ-0.01), 320 (Δ0.12) nm; IR (KBr)ν _max2869, 2855, 1734,1704, 1451, 1376, 1182, 1079, 962, 733 cm^–1; ESIMSm/z415 [M + Na]⁺; HRESIMSm/z415.2458 [M + Na]⁺(calcd for C₂₃H₃₆NaO₅, 415.2460);¹³C NMR (100 MHz,CDCl₃) And¹H NMR (400 MHz, CDCl₃) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 23. The absolute configuration of the compound is determined by ECD and is 4R, 5S, 8S, 9R, 10S, 12S,16S。

The structural identification data for compound 4 is as follows:

colorless oil [ alpha ]α]

-4.8 (c0.3, CH₂Cl₂); ECD (CH₃CN) 204 (Δ-0.47), 236 (Δ0.15), 258 (Δ-0.12), 299 (Δ-0.22) nm; IR (KBr)ν _max2929, 2871, 1732,1705, 1450, 1376, 1254, 1166, 1041, 910, 733 cm^–1; ESIMSm/z429 [M + Na]⁺;HRESIMSm/z429.2616 [M + Na]⁺(calcd for C₂₄H₃₈NaO₅, 429.2617).;¹H NMR (400MHz, CDCl₃) and¹³C NMR (100 MHz, CDCl₃) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 23. The absolute configuration of the compound is determined by ECD and is 4R, 5S, 8S, 9R, 10S,12S, 16S。

The structural identification data for compound 5 is as follows:

white amorphous powderα]

+15.0 (c0.2, CH₂Cl₂); ECD (CH₃CN) 218 (Δ-0.67),307 (Δ0.76) nm; IR (film)ν _max2869, 2855, 1721, 1707, 1456, 1387, 1230,977, 893, 733 cm^–1; ESIMSm/z397 [M + Na]⁺; HRESIMSm/z397.1989 [M + Na]⁺(calcd for C₂₂H₃₀NaO₅, 397.1991);¹³C NMR (100 MHz, CDCl₃) And¹H NMR (400 MHz,CDCl₃) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 23. The absolute configuration of the compound is determined by ECD and is 4R, 5S, 8S, 9R, 10S, 13R, 16S。

Of compounds 1 to 5 of Table 1¹³C NMR data

TABLE 2 preparation of compounds 1 to 5¹H NMR data

Example 2

(1) Extracting herba Euphorbiae Lathyridis semen 8.0 kg with ethanol for 3 times (dosage 3 × 48L), and recovering extractive solution under reduced pressure to obtain crude extract;

(2) adding water into the ethanol extract obtained in the step (1) to prepare suspension, and extracting with ethyl acetate to obtain an ethyl acetate extract;

(3) separating by silica gel column chromatography, eluting by petroleum ether, namely acetone 100:4, 100:6, 100:8, 100:11, 100:16, 100:23 and 100:30 in sequence;

(3) separating the petroleum ether and ethyl acetate flow portions obtained in the step (2) by medium-pressure liquid chromatography (MPLC) in a ratio of 100: 2-100: 23, and performing gradient elution by using methanol/water as a mobile phase in a ratio of 7: 3-9: 1;

(4) separating methanol/water (8: 2) obtained in the step (3) by HPLC-RI, and eluting with methanol/water 70: 30-90: 10 as a mobile phase to obtain new compounds 1 (yield 0.002%), 2 (yield 0.001%), 3 (yield 0.002%), 4 (yield 0.005%) and 5 (yield 0.001%).

The structural identification of compounds 1-5 is shown in example 1.

Example 3

(1) Extracting herba Euphorbiae Lathyridis semen 8.0 kg with acetone for 3 times (dosage 3 × 48L), and recovering extractive solution under reduced pressure to obtain crude extract;

(2) adding water into the acetone extract obtained in the step (1) to prepare a suspension, and extracting with dichloromethane to obtain a dichloromethane extract;

(4) separating the 100: 2-100: 23 parts of the petroleum ether and acetone obtained in the step (3) by medium-pressure liquid chromatography (MPLC), and performing gradient elution by using methanol/water 7: 3-9: 1 as a mobile phase;

(5) separating methanol/water (8: 2) fractions obtained in the step (4) by HPLC-RI, and eluting with acetonitrile/water 70:30 to 90:10 as a mobile phase to obtain the new compounds 1 (yield 0.002%), 2 (yield 0.001%), 3 (yield 0.003%), 4 (yield 0.004%) and 5 (yield 0.001%).

The structural identification of compounds 1-5 is shown in example 1.

Example 4

Test of NO inhibitory Activity of novel Compounds 1-5 in Euphorbia antiquorum.

(1) Principle of experiment

The compound with NO inhibitory activity is closely related to inflammation, neurodegenerative diseases and the like, and is a potential drug for treating the neurodegenerative diseases such as inflammation, Alzheimer's disease, Parkinson's disease and the like. The experiment establishes a BV-2 cell model, and the cell generates NO under the stimulation of LPS; the compounds 1-5 were evaluated for NO inhibitory activity by addition of the test compound simultaneously with LPS stimulation, thereby finding potential drugs for anti-inflammatory and neurodegenerative diseases.

(2) Experimental methods

Firstly, culturing mouse microglia BV-2

A cell culture solution containing 10% fetal calf serum and 1% double antibody (penicillin: streptomycin =1: 1) is prepared on the basis of a DMEM high-sugar medium at 37 ℃ and 5% CO₂Culturing in an incubator, changing the culture solution once in 2-3 days until the cells are basically paved at the bottom of a culture bottle, and carrying out passage or experimental treatment.

Process for preparation of compounds

The test compound was dissolved in DMSO to prepare a stock solution at a concentration of 30 mM and stored at-20 ℃. It was diluted with DMEM medium at the time of use to 10 mM, 5 mM, 3 mM, 1mM, 0.1mM, 0.01mM in this order.

③ cytotoxicity of the test Compound

Adjusting the cell density of the cells in the logarithmic growth phase to 1 × 10⁵And (4) inoculating the cells/mL of the cells into a 96-well plate, placing the cells in a 5% incubator at 37 ℃, adding the test compound with different concentrations after culturing for 24 hours, observing the survival condition of the cells after 20 hours, and quantitatively detecting the toxicity of the compound on the cells by using an MTT method to determine the test concentration of the compound.

NO inhibitory Activity of Compound

The BV-2 cells in logarithmic growth phase were seeded in 96-well plates (5 × 10) at a controlled cell density⁴Cells/hole), culturing for 24 hours, adding compounds to be treated with different concentrations after the cells are completely attached to the wall, pretreating for 30 min, and adding LPS until the final concentration is 0.2μg/mL, after further culturing for 24 hours, 50 were takenμAdding 50% of the supernatant of the L cell cultureμL Griess reagent (solution A: solution B =1:1, solution A contains 1% sulfanilamide, 5% phosphoric acid, and solution B is 0.1%αNaphthyl ethylenediamine dihydrochloride, the solutions A and B which need to be stored in the dark), measuring the absorbance value at 550 nm according to the Griess method,and calculating the NO inhibition rate of each compound according to the absorbance value and the standard curve.

Fifthly, statistical method

All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, evaluation of global differences, mean between groups by One-Way ANOVA analysis for homogeneity of variance, and comparison between groups by Dunnett' test analysis. The homogeneity test of the multi-sample variance adopts Leven test whenp>0.05, the variance is uniform, and Dunnett's double-side T test is adopted to detect the difference of the mean values among the groups; when in usep<0.05, variance was irregular, and the mean differences between groups were examined using Dunnett T3.

⑥ IC₅₀Is calculated by

Calculating the IC of the compound for inhibiting NO by using nonlinear regression fitting on parameters such as each dosage and inhibition rate₅₀The value is obtained.

(3) The experimental results are as follows: IC of compound for inhibiting NO₅₀The values are shown in Table 3.

TABLE 3 IC50 values for NO inhibition by novel compounds 1-5

Sample name	IC₅₀ (μM)	Sample name	IC₅₀ (μM)
				Compound 1	>100	Compound 4	42.4
Compound 2	44.3	Compound 5	42.9
				Compound 3	76.6	SMT^a	3.1

^a2-Methyl-2-thiopseudouerea, Sulfate (SMT) was a positive control drug.

The results show that the novel compounds 2 to 5 prepared in examples 1 to 3 have NO inhibitory activity.

Claims

1. Five new diterpenoid compounds 1-5 in the field of euphorbia helioscopia are characterized in that: has the following structure.

2. A process for the preparation of a compound according to claim 1, characterized in that: the method comprises the following steps:

(1) herba Euphorbiae HumifusaeEuphorbia antiquorumL, extracting with a solvent, and recovering an extracting solution to obtain a crude extract;

(2) dissolving the crude extract obtained in the step (1) in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent under reduced pressure to obtain an extract;

(4) subjecting the fraction obtained in the step (3) to medium-pressure liquid chromatography, separating by using ODS as a chromatographic filler, and performing gradient elution by using methanol/water or acetonitrile/water mixed solvent as a mobile phase;

(5) and (4) separating the fractions obtained in the step (4) by high performance liquid chromatography-differential detection chromatography, and eluting by taking methanol/water as a mobile phase or acetonitrile/water as a mobile phase in a gradient manner to obtain the compounds 1-5.

3. A process for the preparation of a compound according to claim 2, characterized in that: said herba Euphorbiae Fischerianae is Euphorbia superba of EuphorbiaceaeEuphorbia antiquorumExtract of stem.

4. A process for the preparation of a compound according to claim 2, characterized in that: the extraction method in the step (1) is heating reflux extraction or ultrasonic extraction for 1-3 times, the used solvent is at least one of petroleum ether, cyclohexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol and ethanol, and the medicinal materials are as follows: the weight-volume ratio of the solvent is 1: 5-1: 15.

5. A process for the preparation of a compound according to claim 2, characterized in that: according to the extraction method in the step (2), the volume ratio of the aqueous solution to the organic solvent is 1: 1-1: 2, and the used extraction solvent is one of petroleum ether, dichloromethane, chloroform and ethyl acetate.

6. A process for the preparation of a compound according to claim 2, characterized in that: the ratio of the petroleum ether/acetone or petroleum ether/ethyl acetate mixed solvent used as the elution solvent in the step (3) is 100: 2-100: 30.

7. A process for the preparation of a compound according to claim 2, characterized in that: in the step (4), the ratio of the methanol/water or acetonitrile/water mixed solvent is 6: 4-9: 1.

8. A process for the preparation of a compound according to claim 2, characterized in that: the mobile phase methanol/water or acetonitrile/water mixed solvent in the step (5) is a mobile phase, and the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 1; the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 2; the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 3; the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 4; the ratio of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 5.

9. A pharmaceutical formulation comprising a compound or pharmaceutically acceptable salt as claimed in claim 1 and pharmaceutically acceptable adjuvants, diluents and carriers.

10. The use of the novel compounds of claim 1 for the preparation of a medicament for the prevention and treatment of inflammatory and neurodegenerative diseases.