CN110713473B

CN110713473B - Carborundum-reducing neolignan compound and medical application thereof

Info

Publication number: CN110713473B
Application number: CN201910686335.6A
Authority: CN
Inventors: 宋少江; 黄肖霄; 吕天铭; 郭睿
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2021-03-23
Anticipated expiration: 2039-07-29
Also published as: CN110713473A

Abstract

The invention belongs to the technical field of medicines, relates to a carbon-reducing neolignanoid compound and medical application thereof, and particularly relates to the carbon-reducing neolignanoid compound extracted from plant hawthorn fruits and application of the compound in preparation of anti-Parkinson Disease (PD) medicines. The invention provides four carbon-reducing neolignan compounds separated from Crataegus pinnatifida (Crataegus pinnatifida) of Crataegus of rosaceae, which have the following structures: for the four novel carbon-reduced neolignanoid compound pairs MPP⁺The neuroprotective effect of induced human SH-SY5Y nerve cell injury is investigated, and the in vitro cell test result shows that the compound 1a/1b-2a/2b is applied to MPP⁺The induced oxidative damage of human SH-SY5Y cells has obvious protective effect.

Description

Carborundum-reducing neolignan compound and medical application thereof

The technical field is as follows:

the invention belongs to the technical field of medicines, relates to a carbon-reducing neolignanoid compound and medical application thereof, and particularly relates to the carbon-reducing neolignanoid compound extracted from plant hawthorn fruits and application of the compound in preparation of anti-Parkinson Disease (PD) medicines.

Background art:

hawthorn (Crataegus pinnatifida): is a plant of Rosaceae (Rosaceae), Maloideae, and Crataegus. The fruit is slightly sour and astringent, the stone is hard, the pulp is slightly thin, and the fruit enters spleen, stomach and liver channels, and has the effects of promoting digestion, invigorating stomach, promoting qi circulation and removing blood stasis. Can be used for treating gastric distention, meat stagnation, arrhythmia, and congestive heart failure.

Parkinson's Disease (PD): one common degenerative disease of the nervous system, the most prominent pathological change of which is degenerative death of dopaminergic neurons in the midbrain substantia nigra, thus causing a marked reduction in striatal dopaminergic neuron content. PD is common in middle-aged and elderly people, and epidemiological investigation shows that PD currently affects more than 1000 million people all over the world, and the number of people suffering from diseases in China reaches more than 170. The exact etiology of this pathological change is still unclear, genetic factors, environmental factors, aging, oxidative stress, etc. may all be involved in the degenerative death process of dopaminergic neurons, but there is enough evidence that increased apoptosis due to oxidative stress is closely related to dopaminergic neuronal degenerative changes.

Research on PD has been receiving more attention for decades, but neither the pathogenesis nor the search for new therapeutic approaches leave the experimental model for PD. Establishing a stable and reliable dopaminergic neuron cell model is particularly necessary for studying the pathogenesis and treatment of PD. 1-methyl-4-phenylpyridine ion (MPP)⁺) Is an active metabolite of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP), can generate selective destruction effect on mesencephalic substantia nigra dopaminergic neuron, and thereby exerts toxic effect of inducing PD. MPP⁺Not only can induce whole animal model, but also can induce in vitro culture cell model, so that MPP⁺Cell models are gradually becoming a more ideal PD experimental model and are widely applied all over the world.

The invention content is as follows:

the invention provides four carbon-reducing neolignan compounds separated from Crataegus pinnatifida (Crataegus pinnatifida) of Crataegus of rosaceae, which have the following structures:

the preparation technical scheme of the invention comprises the following steps: (1) reflux-extracting dried fructus crataegi with ethanol, mixing extractive solutions, and concentrating to obtain extract;

(2) extracting the extract with ethyl acetate, subjecting the obtained components to silica gel column chromatography, performing gradient elution with dichloromethane/chloroform-methanol system 100:1-5:1, and collecting 4 fractions A, B, C, D;

(3) subjecting fraction A to HP-20 macroporous resin, eluting with 50% ethanol and 90% ethanol to obtain two components A₁、A₂。

(4) Two components A₁、A₂Gradient elution was performed using ODS column chromatography with methanol-water system 20:80-90:10 and combined by thin layer chromatography and automated analytical HPLC analysis to give further 5 fractions 1-5.

(5) The fraction 3 obtained is subjected to silica gel column chromatography and gradient elution with a dichloromethane-methanol system 100:1-3:1 to obtain 5 fractions 3.1-3.5.

(6) Fraction 3.3 was separated by preparative HPLC using a methanol-water system 45:55 to give 3.3.1-3.3.6.

(7) Elution of 3.3.5 by semi-preparative HPLC using acetonitrile-water system 30:70 gave compound (+ -) -1, and separation of 3.5.3 gave compound (+ -) -2 using the same procedure. And (3) carrying out chiral resolution on the (+/-) -1 compound by using a Daicel Chiralpak IG chiral chromatographic column and an n-hexane-isopropanol system 1:1 to obtain the compounds 1a and 1 b. And (3) carrying out chiral resolution on the compound (+/-) -2 by using a Daicel Chiralpak IG chiral chromatographic column to obtain compounds 2a and 2 b.

The preparation method comprises the step of extracting for 3-5 times in a refluxing manner for 2-3 hours each time.

The preparation method uses hawthorn as Crataegus pinnatifida (Crataegus pinnatifida) of Crataegus of Rosaceae.

The compound obtained is identified by the system structure as follows:

the plane structures of the

compounds

1 and 2 are identified by ultraviolet spectrum, high-resolution mass spectrum and one-dimensional and two-dimensional NMR technology. And determining the absolute configuration of the split optically pure compound 1a/1b-2a/2b by utilizing actually measured ECD, calculated ECD comparison and calculated nuclear magnetic technology.

Compound 1 is a pale yellow oily compound, and HRESIMS shows that the peak of the excimer ion is M/z 457.1472[ M + Na ]]⁺(calculated as C)₂₂H₂₆NaO₉457.1469), in combination¹H,¹³C-NMR data confirm that the molecular formula is C₂₂H₂₆O₉The unsaturation was calculated to be 10.¹H NMR(400MHz,CDCl₃) The spectra show 2 sets of aromatic proton signals [ delta 6.31(1H, d, J ═ 2.0Hz, H-2), 6.78(1H, d, J ═ 8.2Hz, H-5),6.48(1H, dd, J ═ 8.2,2.0Hz, H-6)]And [ delta.6.44 (2H, br s, H-2 '/H-6')]Suggested are a1, 3, 4-trisubstituted aromatic ring and a1, 3,4, 5-tetrasubstituted symmetric aromatic ring system. In addition, the¹The H NMR spectrum showed 4 aliphatic proton signals [ δ 3.41(1H, d, J ═ 5.7Hz, H-7),4.02(1H, d, J ═ 8.4Hz, H-7 '), 2.69(1H, ddd, J ═ 8.4,5.7,2.6Hz, H-8 '), 5.52(1H, J ═ 2.6Hz, H-9 ')]And 5 methoxy proton signals [ delta 3.26(3H, s),3.55(3H, s),3.74(3H, s),3.81(6H, s)]。¹³The C NMR spectrum and the HSQC spectrum indicate that the 22-carbon signal of the compound 1 corresponds to 5 methoxy groups, 4 methine groups, 1 ester carbonyl carbon and 2 benzene ring fragments respectively. The direct hydrocarbon correlation assignment of the compound is carried out by using HSQC spectrum, and the correlation of H-7, H-8 'and H-9' with C-8 in HMBC shows the existence of gamma-butyrolactone fragments. Delta.2.69 (1H, ddd, J. 8.4,5.7,2.6Hz, H-8 ') correlates with C-1/C-7/C-1 '/C-8 ' to show gamma-butyrolactone [ C₇-C₈-O-C_9′-C_8′]The fragment is linked to C-7 'via C-8' and to the phenyl ring via C-1. Thus, it is presumed that Compound 1 has C₆C₃-C₂C₆The skeleton of the compound is a carbon-reduced neolignan compound. CH (CH)₃O-3/C-3,CH₃O-3′/C-3′, CH₃O-5′/C-5′,CH₃O-7 '/C-7' and CH₃HMBC correlation of O-9 '/C-9' shows that these methoxy groups are linked to C-3, C-3 ', C-5', C-7 ', C-9', respectively, thereby defining the planar structure of Compound 1.

The relative configuration of compound 1 was determined by NOESY spectroscopy and nuclear magnetic calculations. H-8' and H-2/6 and CH were observed in the NOESY spectra₃The NOE of O-9 'was correlated, indicating that H-7 was oriented in trans with H-8', and H-8 'was oriented in trans with H-9'. In addition, the relative configuration of C-7 ' was confirmed by nuclear magnetic calculations and theoretical analysis of 2 possible isomers (7S, 7 ' S,8 ' S,9 ' R) -1 and (7S, 7 ' R,8 ' S,9 ' R) -1 using DP4+ probability analysis¹³The reliability of the results of the C NMR chemical shift calculations was evaluated. By using GIAO at B3LYP/6-311+ G (d, p) levelsGaussian 09 software, using continuous polarization model in methanol to make theory¹³C NMR chemical shift calculation, and comparing the obtained result with the actual measurement¹³C NMR comparison and analysis of the results using DP4+ probability analysis showed a reliability of approximately 100% for the relative configurations 7S,7 ' R,8 ' S,9 ' R. The relative configuration of compound 1 was thus determined to be 7S,7 ' R,8 ' S,9 ' R. Considering the potential chirality of the norneolignan compounds existing in nature, the compound 1 is subjected to chiral resolution to obtain an optically pure compound. Subsequently, compound 1 was resolved into a pair of enantiomers 1a and 1b (ratio about 1:1) using a Daicel Chiralpak IG chiral column. Their ECD spectra exhibited a mirror image Cotton effect and opposite rotation (1a:

1b:

). The absolute configuration of the compound is determined by comparing the calculated ECD spectrum with the measured ECD spectrum. The absolute configuration of the compounds 1a and 1b is determined to be 7S,7 'R, 8' S,9 'R and 7R, 7' S,8 'R, 9' S by comparison.

Compound 2, light yellow oily compound HRESIMS gives the peak M/z 487.1572[ M + Na ] of quasi-molecular ion]⁺(calculated as C)₂₃H₂₈NaO₁₀487.1575), in combination¹H,¹³C-NMR data confirm that the molecular formula is C₂₃H₂₈O₁₀The unsaturation degree was 10.¹H(400MHz,CDCl₃) And¹³C NMR(100MHz,CDCl₃) The spectrum shows 6 methoxy groups, 4 methine groups, 1 estercarbonyl carbon and 12 aromatic carbons (8 of which are quaternary carbons). Analysis of NMR spectrum data shows that compound 2 has the same parent nucleus of the norneolignan compound as compound 1, except that compound 2 additionally has a methoxy group at the C-5 position of compound 1. The planar structure of compound 2 was determined.

The relative configuration of compound 2 was determined in the same manner as compound 1. NOESY spectra observed for H-8' and H-2/6 and CH₃O-9′Indicates that H-7 is in trans orientation with H-8 ', and H-8 ' is in trans orientation with H-9 '. The relative configuration of C-7 'of compound 2 was confirmed using the same nuclear magnetic calculation method as compound 1, and thus the relative configuration of compound 2 was determined to be 7S, 7' R,8 'S, 9' R. The specific optical rotation of compound 2 was close to zero and there was no significant Cotton effect on the ECD, presumably a racemic mixture. Chiral resolution of compound 2 was performed using a Daicel Chiralpak IG chiral column, resulting in a pair of enantiomers (2a:

2b:

). Their absolute configuration was determined by comparing experimental ECD spectra with calculated ECD spectra. 2a and 2b, the Cotton effect peak in the experimental ECD spectrum can be better matched with the Cotton effect peak in the calculated ECD spectrum with preset configurations of 7S,7 'R, 8' S,9 'R and 7R, 7' S,8 'R, 9' S respectively. Thus, the absolute configurations of compounds 2a and 2b can be determined to be 7S,7 'R, 8' S,9 'R and 7R, 7' S,8 'R, 9' S, respectively. The nuclear magnetic data of 1a/1b-2a/2b are shown in the following table:

TABLE 11 a/1b-2a/2b in CDCl₃In¹H (400MHz) and¹³c (100MHz) NMR data

For the four novel carbon-reduced neolignanoid compound pairs MPP⁺The neuroprotective effect of induced human SH-SY5Y nerve cell injury is investigated, and the in vitro cell test result shows that the compound 1a/1b-2a/2b is applied to MPP⁺The induced oxidative damage of human SH-SY5Y cells has obvious protective effect. Therefore, the novel carbon-reduced neolignan compound has a novel medical application of an anti-PD effect.

Description of the drawings:

figure 1 UV spectrum of compound 1;

FIG. 2 HRESIMS spectra of Compound 1;

FIG. 3 HMBC spectra (600MHz, CDCl) of Compound 1₃)；

FIG. 4 HSQC spectra (600MHz, CDCl) of Compound 1₃)；

FIG. 5 NOESY spectrum (600MHz, CDCl) of Compound 1₃)；

FIG. 6 chiral resolution chromatogram of Compound 1;

FIG. 7 UV spectrum of Compound 2;

FIG. 8 HRESIMS spectrum of Compound 2;

FIG. 9 HMBC spectra (600MHz, CDCl) of Compound 2₃)；

FIG. 10 HSQC spectra (600MHz, CDCl) of Compound 2₃)；

FIG. 11 NOESY spectrum (600MHz, CDCl) of Compound 2₃)；

Figure 12 chiral resolution chromatogram of compound 2;

FIG. 13 is a comparison of measured ECD and calculated ECD for compounds 1a/1b-2a/2 b;

FIG. 14 HMBC correlation plot for compounds 1-2;

FIG. 15 NOESY correlation plots for compounds 1-2;

fig. 16(7S, 7 'S, 8' S,9 'R) -1 and (7S, 7' R,8 'S, 9' R) -1, (7S, 7 'S, 8' S,9 'R) -2 and (7S, 7' R,8 'S, 9' R) -2 calculated carbon spectra and DP4+ confidence test.

The specific implementation mode is as follows:

the examples set out below are intended to assist the person skilled in the art in a better understanding of the invention, but do not limit it in any way.

Example 1: preparation of Compounds 1a/1b-2a/2 b.

50kg of dried hawthorn (C.pinnatifida) fruits are extracted by reflux with 70% ethanol for 3 times, 2 hours each time. 3800g of crude ethanol extract is obtained, the extract is extracted by ethyl acetate and n-butanol, components (600g) obtained by ethyl acetate extraction are subjected to silica gel column chromatography, gradient elution is carried out by a dichloromethane-methanol system at a ratio of 100:1-5:1, and 4 fractions A, B, C, D are collected in total. Subjecting fraction A to HP-20 chromatographic column, eluting with 50% ethanol-water and 90% ethanol-water to obtain two components A₁(40g)、A₂(80g) In that respect Two components are utilizedODS column chromatography was gradient eluted with an alcohol-water system 20:80-90:10, and the pooled bottles were further analyzed by thin layer chromatography and automatic analytical HPLC to give 5 fractions 1-5. The resulting fraction 3(10g) was subjected to silica gel column chromatography with a gradient elution using a dichloromethane-methanol system 100:1-3:1 to give 5 fractions 3.1-3.5. Fraction 3.3 was separated by preparative HPLC using a methanol-water system 45:55 to give 3.3.1-3.3.6. Elution of 3.3.5 by semi-preparative HPLC using acetonitrile-water system 30:70 gave compound (+ -) -1(9.4mg, t)_R43.4min), 3.5.3 was isolated using the same procedure to give compound (+ -) -2(7.9mg, t)_R47.4 min). And the compound (+/-) -1 is subjected to chiral resolution (n-hexane/isopropanol, 1:1, flow rate of 0.5ml/min) by using Daicel Chiralpak IG chiral chromatographic column to obtain a compound 1a (4mg, t)_R17.6min) and 1b (4.3mg, t)_R20.1 min). Chiral resolution (n-hexane/isopropanol, 1:1, flow rate 0.5ml/min) is carried out on the compound (+/-) -2 by a Daicel Chiralpak IG chiral chromatographic column to obtain a compound 2a (3.9mg, t)_R39.5min) and 2b (3.6mg, t)_R 47.8min)。

Example 2: compounds 1a/1b-2a/2b for in vitro treatment of MPP⁺And (3) the research of the protective effect of the induced human SH-SY5Y nerve cell damage.

Investigating compound pair MPP by MTT experiment⁺Protection against induced SH-SY5Y cell damage. The cells were placed in a 96-well plate, left to stand for 12h in 100. mu.L of culture medium, SH-SY5Y nerve cells were pretreated for 1h with different concentrations of compound 1a/1b-2a/2b (12.5,25, 50. mu.M), and MPP⁺Cells were treated (1mM) for 36 h. The culture broth was then replaced with phosphate buffer solution containing 0.5mg/mL MTT and left at 37 ℃ for 4 h. The supernatant was removed and DMSO (150 mL/well) was added with MPP⁺(1mM) cells treated alone were used as a control, and different concentrations of treated cells were detected using an ultraviolet spectrophotometer at 490nm (Thermo Scientific Multiskan MK3, Shanghai, China). The degree of survival of the cells was expressed as percent survival and analyzed using GraphPad Prism 6 software. The results show that the compounds 1a/1b-2a/2b show obvious protective effect under different concentrations, particularly have the strongest effect under the concentration of 50 mu M, and are compared with 50 of the positive drug5 +/-3.31%, the cell survival rate under the 1a/1b-2a/2b treatment reaches 87.5 +/-3.05%, 84.2 +/-2.06%, 86.3 +/-2.61% and 83.8 +/-1.33%, respectively.

TABLE 2 percentage of cell survival after dosing

Percent cell survival in model group compared to control group^###P<0.001; percent cell survival in experimental versus model groups<0.01,***P<0.01。

Claims

1. A compound or salt thereof represented by the following structure:

。

2. a process for producing the compound or the salt thereof according to claim 1,

(1) reflux-extracting dried fructus crataegi with ethanol, mixing extractive solutions, and concentrating to obtain extract;

(2) extracting the extract with ethyl acetate, subjecting the obtained components to silica gel column chromatography, performing gradient elution with dichloromethane/chloroform-methanol, and collecting 4 fractions A, B, C, D;

(3) eluting fraction A with HP-20 macroporous resin, 50% ethanol and 90% ethanol to obtain two components A1 and A2;

(4) the two components A1 and A2 are subjected to gradient elution by using ODS column chromatography in a methanol-water 20:80-90:10 system, and are further combined by thin layer chromatography and automatic analytical HPLC analysis to obtain 5 components 1-5;

(5) gradient elution is carried out on the obtained component 3 by a silica gel column chromatography by a dichloromethane/trichloromethane-methanol system to obtain 5 components 3.1-3.5;

(6) separating the fraction 3.3 by preparative HPLC using a methanol-water system 45:55 to give 3.3.1-3.3.6;

(7) eluting 3.3.5 with acetonitrile-water system 30:70 by semi-preparative HPLC to obtain compound (+/-) -1, and separating 3.5.3 by the same method to obtain compound (+/-) -2; chiral resolution is carried out on the compound (+/-) -1 by using a chiral chromatographic column to obtain compounds 1a and 1 b; chiral separation is carried out on the (+/-) -2 compound by using a chiral chromatographic column to obtain the compounds 2a and 2 b.

3. The method for producing a compound or a salt thereof according to claim 2, wherein the extraction in the step (1) is reflux extraction, and the number of times of extraction is 3 to 5, each for 2 to 3 hours.

4. The process for producing a compound or a salt thereof according to claim 2, wherein the gradient of dichloromethane/chloroform-methanol in the step (2) is 100:1 to 5: 1.

5. The method of claim 2, wherein the gradient of dichloromethane/chloroform-methanol in step (5) is from 100:1 to 3: 1.

6. The process for producing a compound or a salt thereof according to claim 2, wherein the chiral resolution conditions in the step (7) are n-hexane/isopropanol 1: 1.

7. A pharmaceutical composition comprising a compound of claim 1 or a salt thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of the compound or the salt thereof according to claim 1 for the preparation of an anti-parkinson's disease drug.

9. The use of the pharmaceutical composition of claim 7 for the preparation of an anti-parkinson's disease medicament.