CN111018876B

CN111018876B - Application of phthalide dimer in preparation of vasodilation drugs

Info

Publication number: CN111018876B
Application number: CN201911422088.5A
Authority: CN
Inventors: 谭玉柱; 彭成; 周厚成; 胡昌江; 唐飞; 王利霞
Original assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd; Chengdu University of Traditional Chinese Medicine
Current assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd; Chengdu University of Traditional Chinese Medicine
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-08-04
Anticipated expiration: 2039-12-31
Also published as: CN111018876A

Abstract

The invention aims to provide an application of a phthalide dimer compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof in preparing vasodilation drugs. The phthalide dimer compound is formed by cycloaddition of two phthalide monomers through [ 2+2 ], and the formed phthalide dimer comprises 1 or 2 spiro atoms, wherein the phthalide monomers are selected from the following structures. The compound has excellent vasodilation activity, is superior to a monophthalide compound, plays a role in vasodilation by antagonizing the Cav1.2 receptor, provides a new choice for clinically screening and/or preparing vasodilation medicaments, provides a new molecular template for designing novel calcium antagonist medicaments, and has excellent clinical application prospect.

Description

Application of phthalide dimer in preparation of vasodilation drugs

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a phthalide dimer in preparation of a vasodilator medicine.

Background

Cardiovascular disease (Cardiovascular Disease) is the disease with the highest global morbidity and mortality at present, seriously affects the quality of life of patients and creates a huge economic and social burden. The disease is a heart and vascular disease, which is generally referred to as ischemic or hemorrhagic disease of heart and whole body tissue caused by hyperlipidemia, atherosclerosis, hypertension and the like, and comprises various diseases such as coronary heart disease, myocardial infarction, cerebral vasospasm and the like.

With the progressive younger age of the cardiovascular disease onset population, morbidity and mortality are also increasing, and control of the disease state is becoming increasingly important. At present, most of medicines show obvious toxic and side effects when treating diseases, often damage liver and kidney, and easily cause complications such as heart, brain, kidney and the like when serious, and have obvious toxic and side effects. For example, prolonged administration of dihydropyridines may cause arrhythmia or myocardial infarction, and muscle poisoning may be caused by administration of other drugs. Therefore, there is an urgent need to find an innovative medicament with good curative effect and little toxic and side effects. Therefore, searching for monomer components with better curative effects from natural plants such as traditional Chinese medicines becomes an important source of the innovative medicines, and many traditional Chinese medicine extracts and the monomer components thereof have been screened to have antihypertensive and vasodilatory effects at present, wherein part of monomer compounds have been studied as potential therapeutic agents for treating hypertension and related complications thereof.

Calcium channel blockers are one of the most effective drugs currently used to treat primary hypertension. Long-term drugs, such as amlodipine, can continuously and gently lower blood pressure levels. Cav1.2 is the primary gating site for calcium ions to enter vascular cells. Calcium ions enter the cell through the cav1.2 channel, acting as a second messenger, regulating various physiological functions of the cell, such as contraction and gene expression. In resistance-type blood vessels, the cav1.2 channel plays an important role in blood pressure regulation. The Cav1.2 channel is widely expressed in blood vessels and hearts, and plays roles in regulating and controlling blood pressure, heart rate, contraction and the like. L-type calcium channel blockers such as dihydropyridines have been shown to be effective drugs for treating hypertension.

Ligusticum wallichii is a dried rhizome of Ligusticum wallichii (Ligusticum chuanxiong Hort.) belonging to Umbelliferae, and is a well-known Chuan-producing area medicinal material. Has the effects of activating blood circulation to dissipate blood stasis, promoting qi circulation to resolve depression, dispelling wind and relieving pain, is clinically used for treating headache, liver depression and qi stagnation syndrome, various blood stasis syndromes and the like, and is recorded in Bencao Meng ban: ascending head and eyes, descending blood sea, activating liver meridian and qi in blood. Modern researches have shown that Ligusticum wallichii has remarkable pharmacological activity on respiratory system, cardiovascular system, urinary system and the like. The main components of the rhizoma ligustici wallichii comprise phthalide, alkaloid, organic phenolic acid, polysaccharide and the like. The phthalide compound is used as the main active ingredient of the ligusticum wallichii, has obvious regulation effect on cardiovascular and cerebrovascular systems, nervous systems and the like, and is studied and presumed to be the main material foundation of the ligusticum wallichii for treating blood stasis or cardiovascular and cerebrovascular diseases. Moreover, researches show that the phthalide medicine can penetrate through the blood brain barrier, which is probably the drug effect substance foundation of the ligusticum chuanxiong, which is especially good for treating headache. However, vasodilation is a main treatment mode for cardiovascular diseases such as coronary heart disease, angina pectoris and myocardial infarction at present, and researches show that ligustilide, senkyunolide A and other monophthalide have vasodilation activity, but the related activity of the phthalide dimer is not reported yet, and needs to be further researched.

The traditional medicinal parts of the ligusticum wallichii are rhizome, and overground parts (stems and leaves) accounting for 75 percent of the fresh weight of the whole plant are discarded, so that not only is the resource waste and the environmental pollution caused, but also the sustainable utilization of the traditional Chinese medicine resource is not facilitated. However, the stem and leaf of Ligusticum wallichii is used in ancient times, the stem and leaf of Ligusticum wallichii is called Mi rui, shen nong Ben Cao Jing (Shen nong's herbal medicine meridian) records "main cough and reverse, calm the convulsion, expel evil and dislike, remove the parasitic and ghost to fully recover, remove three worms and activate the spirit after long-term administration. Based on the long history of folk eating of the stem leaves of the ligusticum wallichii and the activity characteristics of the phthalide components, the invention starts from the vasodilatation effect of the phthalide components of the stem leaves of the ligusticum wallichii, so as to screen the phthalide active components with novel structure and obvious activity, lay a foundation for the comprehensive development and utilization of non-medicinal parts of the ligusticum wallichii, and realize the waste of traditional Chinese medicine resources.

Disclosure of Invention

The invention aims to provide an application of a phthalide dimer compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof in preparing vasodilation drugs.

The invention provides a use of a phthalide dimer compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, in the preparation of a vasodilator drug, the phthalide dimer compound comprising 1 or 2 spiro atoms, the phthalide dimer compound being formed by cycloaddition of two phthalide monomers by [ 2+2 ], wherein the two phthalide monomers are each independently selected from the following structures:

further, the phthalide dimer compound comprises a structure shown in the following formulas I to III:

further, the phthalide dimer compound comprises a structure shown in the following formulas IV and V:

the invention provides a method for preparing the phthalide dimer compound, which comprises the following steps:

step 1: pulverizing stem and leaf of rhizoma Ligustici Chuanxiong, extracting with 95% ethanol, concentrating the ethanol extractive solution under reduced pressure to obtain extract;

step 2: dispersing the extract in the step 1 with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate and n-butanol parts;

step 3: separating the petroleum ether part in the step 2 by an MCI chromatographic column, eluting with methanol and water=90:10, concentrating the eluent, passing through a 200-300 mesh silica gel column, gradient eluting with petroleum ether and ethyl acetate=99:1, 98:2,95:5,90:10,80:20,60:40,50:50 and 1:99, detecting and combining the same spot-like components by TLC, and concentrating to obtain components Fr.1-8;

step 4: separating the component Fr3 containing the compound of the formula I obtained in the step 3 by a silica gel column, performing gradient elution by using normal hexane to acetone=10:0, 8:2, 6:4, 4:6, 2:8 and 0:10, detecting and combining similar components by using TLC, and concentrating to obtain a component Fr.3.1-3.6;

step 5: separating and purifying the component Fr.3.5 containing the compound of the formula I in the step 4 by semi-preparative HPLC, and concentrating to obtain the compound of the formula I;

step 6: passing the component Fr5 containing the compound of formula II in step 3 through a gel chromatographic column as eluent: CHCl (CHCl) ₃ Meoh=2:3, separation, TLC detection, combining similar fractions to give three fractions fr.5.1-fr.5.3;

step 7: subjecting the component Fr5.2 containing the compound of formula II in step 6 to semi-preparative HPLC to obtain the compound of formula II;

step 8: passing component Fr5.3 containing the compound of formula III in step 6 through C ₁₈ Medium pressure preparative chromatography eluting with MeOH H ₂ O=70:30-100:0, gradient elution, TLC detection, combining similar components to obtain fr.5.3.1-fr.5.3.4;

step 9: the fraction fr.5.3.4 containing the compound of formula III in step 8 is purified and separated by semi-preparative HPLC to give the compound of formula III.

Further, in step 5, the mobile phase is: CH (CH) ₃ CN:H ₂ O=70:30, flow rate of 3.0mL/min; detection wavelengths are 208 and 254nm; the retention times of the compounds of formula I are: 27.6min;

in step 7, the mobile phase is: CH (CH) ₃ CN:H ₂ O=70:30, flow rate of 3.0mL/min; detection wavelengths are 208 and 254nm; chemical conversion of IIThe retention time of the compound was: 32.4min;

in step 9, the mobile phase is: CH (CH) ₃ CN:H ₂ O=70:30, flow rate of 3.0mL/min; detection wavelengths are 208 and 254nm; the retention time of the compound of formula III is: 33.8min.

The invention also provides a method for preparing the phthalide dimer compound, and the compound of the formula II is separated by chiral HPLC to obtain the compound of the formula IV and the compound of the formula V.

Further, the chiral column is: CHIRALPAK IC,4.6 μm, 250X 4.6mm; the mobile phase is: n-hexane: ethanol=90:10, flow rate: 0.8mL/min; the retention time of the compound of formula IV is: 21.93min, retention time of the compound of formula V: 24.34min.

Further, the vasodilator in the application of the invention is a calcium ion channel Cav1.2 inhibitor.

Further, the vasodilator in the application of the invention is a medicine for treating cardiovascular diseases.

Further, the cardiovascular disease is hyperlipidemia, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction and hypertension.

The compounds of the formulas I-V have the function of dilating blood vessels, and have better activity than the contrast monophthalide compound. Meanwhile, the chiral resolution is carried out on the compound of the formula II, and the obtained compounds of the formula IV and the formula V have remarkable effect of inhibiting Cav1.2, wherein the activity of the compound of the formula IV for inhibiting Cav1.2 is superior to that of the compound of the formula V. The compound can be used for preparing a Cav1.2 inhibitor, can also be used for preparing a vasodilation medicament, and can be further used for preparing a medicament for treating cardiovascular diseases, and the compound plays a role in vasodilation by antagonizing the action of a Cav1.2 receptor, so that a new choice is provided for clinically screening and/or preparing the vasodilation medicament, and a new molecular template is provided for designing a novel calcium antagonist medicament, thereby having wide clinical application prospect.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

The term "spiro atom" as used herein refers to two rings sharing only one atom, and the shared atom is a spiro atom.

The "[ 2+2 ] cycloaddition" refers to a compound with four-membered stable ring formed by combining two molecules of olefin, small molecules are not eliminated, delta bonds are only generated, and the delta bonds are not broken. Specific examples in the present invention are phthalide dimers in which the double bonds in two phthalide monomers are joined by a four-membered ring formed by [ 2+2 ] cycloaddition.

The compound of the formula I is represented by a code CX31 in a study, the code CX51 in a compound of the formula II in a study, the code CX59 in a compound of the formula III in a study, the code CX51a in a compound of the formula IV in a study, and the code CX51b in a compound of the formula V in a study.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 chiral resolution chromatograms of compounds of formula IV and formula V;

FIG. 2 is an IR diagram of a compound of formula I;

FIG. 3 is a hydrogen spectrum of a compound of formula I;

FIG. 4 is a carbon spectrum of a compound of formula I;

FIG. 5 DEPT spectra of compounds of formula I;

FIG. 6 HSQC spectra of compounds of formula I;

FIG. 7 is a HMBC spectrum of a compound of formula I;

FIG. 8 Compounds of formula I ¹ H- ¹ H COSY profile;

FIG. 9 NOSEY spectra of compounds of formula I;

FIG. 10 is a high resolution mass spectrum of a compound of formula II;

FIG. 11 is an IR diagram of a compound of formula II;

FIG. 12 is a hydrogen spectrum of a compound of formula II;

FIG. 13 carbon spectrum of compound of formula II;

FIG. 14 DEPT spectra of compounds of formula II;

FIG. 15 HSQC spectra of compounds of formula II;

FIG. 16 is a HMBC profile of a compound of formula II;

FIG. 17 compounds of formula II ¹ H- ¹ H COSY profile;

FIG. 18 NOSEY spectrum of a compound of formula II;

FIG. 19 is a two-dimensional correlation diagram of compounds of formula II;

FIG. 20 is a high resolution mass spectrum of a compound of formula III;

FIG. 21 is an IR diagram of a compound of formula III;

FIG. 22 is a hydrogen spectrum of a compound of formula III;

FIG. 23 carbon spectrum of a compound of formula III;

FIG. 24 DEPT spectra of compounds of formula III;

FIG. 25 HSQC spectra of compounds of formula III;

FIG. 26 is a HMBC profile of a compound of formula III;

FIG. 27 compounds of formula III ¹ H- ¹ H COSY profile;

FIG. 28 NOSEY spectra of compounds of formula III;

FIG. 29 is a two-dimensional correlation diagram of compounds of formula III;

FIG. 30 ECD diagrams of compounds of formula IV and formula V;

FIG. 31 changes in the diastolic rate and vascular tone of KCl pre-contracted vascular rings with Compounds 3-Butylidenephthalide, CX31 (Compound of formula I), CX51 (Compound of formula II) and CX59 (Compound of formula III), P compared to the model group<0.05,**P<0.01,***P<0.001(n＝6)；

FIG. 32 Compound of formula IV (Compound CX51 a) versus Cav1.2 Current inhibition effect curve (IC ₅₀ )；

FIG. 33 is a graph of the current-inhibiting effect of a compound of formula IV (compound CX51 a) on different cells;

FIG. 34 Current-inhibiting effect of Compound of formula V (Compound CX51 b) on different cells;

FIG. 35 Current-inhibiting effect of the compound of formula III (compound CX 59) on different cells.

Detailed Description

EXAMPLE 1 preparation of Compounds of formulas I-V

1. Experimental instrument and reagent

1 laboratory apparatus

Avance 400MHz superconducting nmr (Bruker, germany); avance 600MHz superconducting nmr (Bruker, germany); a Synapt G2 HDMS mass spectrometer (Waters company); perkin-Elmer 341plus polarimeter (Perkin Elmer Co., U.S.A.); LC50 medium-high pressure liquid phase (sieboldii beijing technologies limited); chromatographic column (COSMOS, choiester,10id×250 nm); electronic balance (model FA2004B, shanghai plain scientific instruments limited); an ex vivo tissue organ constant temperature perfusion system (PL 3508B6/C-V Panlab 8Chamber Organ Bath System); ultra-pure water of the UpUPT series (Chengdu Upelectronic products Co., ltd.); adjustable pipettor: (Thermo Fisher Scinentific).

2 experiment reagent

Sephadex LH-20 chromatographic packing (Pharmacia, switzerland); MCI resin (mitsubishi chemical company of japan); silica gel GF254 thin layer plate (Qingdao ocean chemical plant); 200-300 mesh column chromatography silica gel (Qingdao ocean chemical plant); chromatographic pure methanol (TEDIA company, usa); deuterated reagent (CIL Co., USA). Other reagents were analytically pure (colone chemicals limited, adult city); golopami (Me) (Methoxyverapamil, me, jiangsu Rui year front drug Co., ltd., approval paper number: H32020683); SD rats, SPF grade, both male and female, weight 200-240 g, supplied by Sichuan Chengdu Shuo Biotech Co., ltd (SCXK 2015-030). The raising temperature is 20-25 ℃, and the lighting and ventilation environment are naturally regulated.

Stem and leaf of Ligusticum wallichii (Ligusticum chuanxiong Hort.) was collected from Dongpo area of Mitsui, sichuan, 4 months in 2018, and the specimen was found in the university of Chengdu traditional Chinese medicine, traditional Chinese medicine chemical 1001 laboratory.

2. Experimental details

1 preparation of Compounds

1.1 extraction and isolation of Compounds (preparation of Compounds of formulae I to III)

1) Pulverizing natural and shade-dried stem and leaf of Ligusticum chuanxiong (20 kg)) into coarse powder, percolating with 95% ethanol, and concentrating under reduced pressure to obtain extract.

2) Sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate and n-butanol.

3) Petroleum ether fraction (0.5 kg) was eluted with methanol/water=90:10 by MCI chromatography, and the decolorized, concentrated solution was eluted with a gradient of silica gel column (200-300 mesh, PE-EtOAc (99:1, 98:2,95:5,90:10,80:20,60:40,50:50,1:99, v/v) to give fractions Fr.1-8, respectively.

4) Wherein, component fr.3 (40 g) was subjected to gradient elution by silica gel column separation (n-hexane: acetone=10:0, 8:2, 6:4, 4:6, 2:8, 0:10 gradient elution) to obtain 6 parts (fr.3.1-fr.3.6).

5) Fr.3.5 fractions were subjected to semi-preparative HPLC (CH ₃ CN-H ₂ O,70:30, v/v,3.0mL/min; detection wavelength: 208,254 nm) to give a compound of formula I, CX31 (t) _R 27.6min,8.4mg)。

6) Component Fr.5 (60 g) was purified by gel chromatography (CHCl) ₃ MeOH, 2:3, v/v) gives three components Fr.5.1-Fr.5.3.

7) Fr.5.2 by semi-preparative HPLC (MeOH-H ₂ O,70:30, v/v,3.0mL/min; detection wavelength: 208 and 254 nm) to give a compound of formula II, CX51 (t) _R 32.4min,8.6mg)。

8) Fr.5.3 fraction was taken through C ₁₈ Medium pressure preparative chromatography (MeOH: H) ₂ O,70:30-100:0, v/v) gradient elution to give fr.5.3.1-fr.5.3.4.

9) Fr.5.3.4 fractions were purified by semi-preparative HPLC (CH ₃ CN-H ₂ O,70:30, v/v,3.0mL/min; detection wavelength: 208 and 254 nm) to give a compound of formula III, CX59 (t) _R 33.8min，8.6mg)。

1.2 chiral resolution of Compounds of formula II CX51 (preparation of Compounds of formulae IV and V)

Resolution of compound CX51 of formula II by chiral HPLC gives compound CX51a of formula IV and compound CX51b of formula V.

The chiral column is: CHIRALPAK IC (Lot. No. IC00CE-XC 014), 4.6 μm, 250X 4.6mm. The mobile phase is: n-hexane-ethanol (90:10) at a flow rate of 0.8mL/min. CX51a retention time is: 21.93min, CX51b retention time of 24.34min. (the chromatogram is shown in FIG. 1)

2 structural identification

2.1 Compounds of formula I CX31

Basic physical and chemical properties: colorless crystals, molecular formula: c (C) ₂₄ H ₂₆ O ₄ Molecular weight: 378.1988, is easily dissolved in organic solvents such as methanol, dichloromethane, ethyl acetate and the like.

Structural data and identification:

optical rotation data: [ alpha ]] ²⁵ _D ＝-2.5°(c 0.05,MeOH)。

Ultraviolet spectral data: UV (MeOH) λmax (log epsilon) =230 (0.22) nm

Infrared spectral data: IR:2961,2923,2853,2813,1751,1645,1576,1463,1411,1260,1061cm ^-1 (the map is shown in FIG. 2).

The nuclear magnetic data and attribution are shown in Table 1 (the patterns are shown in figures 3-9).

TABLE 1 Nuclear magnetic data and assignment of Compounds CX31 of formula I

The above spectral data are consistent with reported compound Diangeliphthalide A of the literature (Zou J, chen G D, zhao H, et al, triangeliphtalides A-D: bioactive phthalide trimers with new skeletons from Angelica sinensis and their production mechanism [ J ]. Chemical Communications,2019,55.).

2.2 Compounds of formula II CX51

Basic physical and chemical properties: yellow oil, molecular formula: c (C) ₂₄ H ₂₈ O ₄ Molecular weight: 380.20, is easily dissolved in organic solvents such as methanol, dichloromethane, ethyl acetate and the like.

Structural data and identification:

[α] ²⁵ _D ＝-3.38°(c 0.04,MeOH)。

HRESIMS：m/z 381.2060[M+H] ⁺ prompt molecular formula C ₂₄ H ₂₈ O ₄ (calcd for C ₂₄ H ₂₉ O ₄ 381.2060) (see figure 10 for a map).

UV(MeOH)λmax(logε)＝286(0.51)nm。

Infrared spectral data: IR:2981,2903,1748, 1720, 1402,1250,1067,894cm ^-1 (see FIG. 11 for a map).

The nuclear magnetic data and attribution are shown in table 2 (maps are shown in fig. 12-18), and the two-dimensional correlation diagram is shown in fig. 19. The planar structure is similar to that of the compound tokiaerialide reported in the literature (Uto, T.; tung, N.H.; taniyama, R.; miyanowaki, T.; morinaga, O.; shoyama, Y.; anti-inflammatory Activity of Constituents Isolated from Aerial Part of Angelica acutiloba Kitagawa. Photon Res 2015,29 (12), 1956-1963.)

TABLE 2 Nuclear magnetic data and assignment of Compounds CX51 of formula II

2.3 Compounds of formula III CX59

Basic physical and chemical properties: yellow oil, molecular formula: c (C) ₂₄ H ₂₈ O ₄ Molecular weight: 380.19, is easily dissolved in organic solvents such as methanol, dichloromethane, ethyl acetate and the like.

Structural data and identification:

[α] ²⁵ _D ＝-5.38°(c 0.04,MeOH)。

UV(MeOH)λmax(logε)＝286(0.50)nm。

HRESIMS m/z 381.2057[M+H] ⁺ prompt molecular formula C ₂₄ H ₂₈ O ₄ (calcd for C ₂₄ H ₂₉ O ₄ 381.2060) (see figure 20 for a map).

Infrared spectral data: IR:2981,2903,1748, 1720, 1402,1250,1067,894cm ^-1 (see FIG. 21 for a map).

The nuclear magnetic data and attribution are shown in Table 3 (maps are shown in FIGS. 22-28), and the two-dimensional correlation map is shown in FIG. 29. The planar structure is similar to that of the reported compound neodiligustilide. ( Chen, q.c.; lee, j.; jin, w.; you, u.; kim, h.; lee, i.s.; zhang, x.; song, k.; seong, y; bae, k., cytotoxic constituents from angelicaesinensis radix. Arch Pharm Res 2007,30 (5), 565-9. )

TABLE 3 Nuclear magnetic data and assignment of Compounds CX-59 of formula III

2.4 Compounds CX51a and CX51b of formula IV

2.4.1 Compounds of formula IV

[α] ²⁵ _D ＝-23.4°(c 0.04,MeOH),

ECD(c 0.01,MeOH)＝Δε ₂₁₄ +26.37,Δε ₂₄₇ –5.77。

2.4.2 Compounds of formula V

[α] ²⁵ _D ＝+68.1°(c 0.04,MeOH),

ECD(c 0.01,MeOH)＝Δε ₂₁₄ –15.37,Δε ₂₄₉ +3.72 (map see FIG. 31).

From the above data, and FIG. 30, it can be determined that compound CX51a of formula IV is configured as (3R, 8S,3'aR,7' aR) -51a, whereas compound 51b of formula V is configured as: (3S, 8R,3'aS,7' aS) as follows.

/>

Test example 1 vasodilation experiment

1 method

The rat cervical dislocation is sacrificed, the thoracic aorta is taken out after the thoracic cavity is opened, and the thoracic aorta is cut into a vascular ring with the length of about 4mm and placed in O ₂ Saturated 4 ℃ K-H solution [ (g/L), naCl 6.92,KCl 0.35,CaCl ₂ 0.28,KH ₂ PO ₄ 0.16,MgSO ₄ 0.14,NaHCO ₃ 2.00,Glucose 1.82,EDTA 0.009]Constant temperature of 37 ℃. Connecting an in-vitro tissue perfusion model and a Power Lab data analysis system, regulating the resting tension to be 1g, and continuously introducing 94.6% O ₂ +5.4％CO ₂ The K-H solution is replaced every 15min, the process is repeated for two times, after balancing for 60min, KCl (60 mmol/L) is used for stimulating vascular ring, the K-H solution is used for flushing for three times when the tension reaches the plateau value, each time is separated for 5min, after the state is restored to the basic state, the process is stabilized for 30min, and the process is repeated for 2 times. After the 3 rd time of pre-shrinking of the vascular ring to reach a plateau value and stabilizing, adding 2.4-12 mu mol/L of samples in a bath in a cumulative way, adding 2 mu L of total volume, and adding an equal volume of DMSO into a blank group. The vasodilation effect profile of the test compounds (3-Butylidenephthalide, CX, CX51, CX 59) was recorded with golopamide (Me) as a positive drug control and repeated 6 times per group.

2 statistical method

The effect of each concentration of the compound on the magnitude of vasoconstriction was calculated with the maximum systolic tension due to KCl (60 mmol/L) being 100%. The experimental data adopts the change of blood vessel tension value to reflect the action of the medicine, and the unit is g. SPSS 21.0 statistical software is adopted for analysis and treatment, and P is less than 0.05, so that the method has statistical significance.

Calculation formula of vasodilation rate:

results of vasodilatory Activity

The blank group had no significant effect on potassium chloride (KCl) -induced rat thoracic aortic annulus contractions (P)>0.05 Each of the administration groups was able to lower the tension value of the vascular ring. Each concentration of golopamide (Me) can significantly relax the vascular ring (P)<0.001 The highest concentration diastolic rate was 120%. The test compounds all have the effect of dilating the vascular ring and exhibit concentration dependence. The highest concentration (12. Mu. Mol/L) of the reference Z-3-butenyl phthalide (abbreviated Bu) has a diastole of 40%, wherein the highest concentration (12. Mu. Mol/L) of CX31 has a diastole of 65.1%, and IC ₅₀ =8.81 μmol/L; CX51 maximum concentration (12. Mu. Mol/L) diastole 96.1%, IC ₅₀ =5.87 μmol/L; CX59 maximum concentration (12. Mu. Mol/L) diastole 93.4%, IC ₅₀ =6.19 μmol/. See fig. 31.

From the above results, the phthalide dimer compounds of the present invention have vasodilating effect in all of the formulae I to V (formulae IV and V are chiral resolution of formula II). And the activity of the phthalide dimer compound is better than that of the monophthalide (Z-3-butenyl phthalide).

Test example 2 electrophysiological experiments

1 instrument and reagents

All instruments and reagents for this experiment are commercially available.

2 cell culture

In this experiment, a CHO cell line stably expressing cav1.2 channels was used, and the cell line was constructed by the laboratory of biotechnology, inc.

(1) The CHO cell line stably expressing cav1.2 channels was cultured in DMEM medium containing 10% fetal bovine serum and 0.8mg/mL G418 at 37 ℃ and carbon dioxide concentration of 5%.

(2)Cell passage: the old medium was removed and washed once with PBS, then 1mL of TrypLE was added ^TM Express solution, incubated at 37℃for 0.5min (CHO cells 1.5 min). When the cells were detached from the bottom of the dish, 5mL of complete medium, pre-warmed at 37℃was added. The cell suspension was gently swirled with a pipette to separate the aggregated cells. The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000rpm for 5min to collect the cells. Expanding or maintaining culture, inoculating cells into 6cm cell culture dishes, wherein the cell culture dishes are inoculated with 2.5×10 cells ⁵ cells (final volume: 5 mL).

(3) To maintain the cell's electrophysiological activity, the cell density must not exceed 80%.

(4) Patch clamp detection, prior to experiment, cells were assayed with TrypLE ^TM Express separation, 3 x 10 ³ Cells were spread on cover slips and cultured in 24 well plates (final volume: 500. Mu.L), and after 24 hours, experimental detection was performed.

3 Whole cell patch clamp recordings

Extracellular fluid: 140mM TEA-Cl,2mM MgCl ₂ ·6H ₂ O，10mM CaCl ₂ ·2H ₂ O,10mm hepes,5mm md-glucose, TEA-OH adjusts ph=7.4.

Intracellular fluid: 120mMCsCl,1mM MgCl ₂ ·6H ₂ O，10mM HEPES，10mM EGTA，0.3mM Na ₂ GTP,4mM Mg-ATP, csOH adjusts ph=7.2.

The voltage stimulation protocol for patch clamp recording whole cell cav1.2 calcium current is as follows: after the whole cell seal is formed, the cell membrane voltage is clamped at-60 mV. The clamping voltage is divided from-60 mV to +10mV for 0.3s, data are repeatedly collected every 20s, and the effect of the drug on the current peak of Cav1.2 is observed. Experimental data was collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

Capillary glass tubes (BF 150-86-10,Sutter Instruments) were drawn into recording electrodes using a microelectrode drawing instrument (P97, sutter Instruments). Manipulation of the microelectrode manipulator (MP 285, sutter Instuments/card-C-S, MCI instruments) under a microscope (IX 71, olympus/AE31EF-INV, motic (China)) the recording electrode was contacted with the cell, and negative pressure aspiration was applied to form a GΩ seal. After forming G omega sealing, carrying out rapid capacitance compensation, then continuing to give negative pressure, sucking and breaking cell membranes, and forming a whole cell recording mode. Then, compensation of the slow capacitor is performed, the film capacitor and the series resistance are recorded, and no leakage compensation is given.

When the administration was started after the current recorded by the whole cells was stabilized, the next concentration was detected after each drug concentration was applied to 5min (or the current was stabilized), and the test compounds (CX 59, CX51a, CX51 b) were detected at a plurality of concentrations. The coverslip with cells laid thereon was placed in a recording cell in an inverted microscope, and the test compound and the external liquid containing no compound were sequentially flowed through the recording cell from low concentration to high concentration by gravity perfusion to act on the cells, and liquid exchange was performed in the recording using a vacuum pump. The current detected by each cell in the compound-free external fluid served as its own control. Multiple cells were independently and repeatedly tested. All electrophysiological experiments were performed at room temperature.

4 data quality criteria

The following criteria are used to determine whether the data is acceptable:

(1) Electrode resistance <5MΩ

(2) Sealing resistance >1G omega

(3) The start of the access resistance is <15MΩ

(4) The end of the access resistance is less than 15MΩ

(5) There is no significant spontaneous decay in current

5 data analysis

The current after each drug concentration was first normalized to the current for the blankThe inhibition rate or increase ratio corresponding to each drug concentration is then calculated. Average and standard errors were calculated for each concentration and the semi-inhibitory concentration for each compound was calculated using the following equation: />

Non-linear fitting of dose-dependent effects was performed with the above equation, where C represents drug concentration, IC ₅₀ H represents the Hill coefficient for the half-inhibitory concentration. Curve fitting and IC ₅₀ Is done using IGOR software.

6 electrophysiological experimental results

The present study uses whole cell patch clamp to record ion channel currents of CHO cell lines stably expressing cav1.2 and to analyze the effect of the compounds of the invention on cav1.2 channels. The experimental results show that the compound (CX 51 a) in the formula IV and the compound (CX 51 b) in the formula V have remarkable Cav1.2 inhibition, wherein the CX51a has remarkable inhibition, and the IC50 is 7.5 mu M+/-1.6 mu M; other compounds do not act significantly. The results are shown in tables 4 and 5, and FIGS. 32 to 35.

TABLE 4 inhibition ratio of samples (CX 59, CX51a, CX51 b) to Cav1.2 current

TABLE 5 inhibition ratio of test sample (CX 51 a) to Cav1.2 current and IC ₅₀ Results

In conclusion, the compounds of the present invention, formulas I-V, all have vasodilatory effects and are more active than the control monophthalide compounds. Meanwhile, the chiral resolution is carried out on the compound of the formula II, and the obtained compounds of the formula IV and the formula V have remarkable effect of inhibiting Cav1.2, wherein the activity of the compound of the formula IV for inhibiting Cav1.2 is superior to that of the compound of the formula V. The compound can be used for preparing a Cav1.2 inhibitor, can also be used for preparing a vasodilation medicament, and further can be used for preparing a medicament for treating cardiovascular diseases, and has wide clinical application prospect.

Meanwhile, the compound provides a direction for searching lead compounds for treating cardiovascular diseases. Through development and utilization research on overground biological resources of ligusticum wallichii, the chemical component library of the stem and leaf of ligusticum wallichii is further enriched, a foundation is laid for screening vascular expanding seedling compounds, the 'integrated dual-purpose' of traditional Chinese medicine resources is realized, and the sustainable development of the traditional Chinese medicine resources is facilitated.

Claims

1. Use of a phthalide dimer compound or a salt thereof in the preparation of a vasodilator drug, wherein the phthalide dimer compound has a structure shown in the following formulas I-III:

2. use according to claim 1, characterized in that: the phthalide dimer compound has a structure shown in a formula IV and a formula V:

3. a process for preparing the phthalide dimer compound of claim 1, characterized in that:

step 1: pulverizing stem and leaf of Ligusticum chuanxiong, extracting with 95% v/v ethanol, concentrating the ethanol extractive solution under reduced pressure to obtain extract;

step 3: separating the petroleum ether part in the step 2 by an MCI chromatographic column, eluting by adopting a methanol-water volume ratio of 90:10, concentrating an eluent, passing through a 200-300 mesh silica gel column, carrying out gradient elution by adopting petroleum ether-ethyl acetate volume ratios of 99:1,98:2,95:5,90:10,80:20,60:40,50:50 and 1:99, detecting and combining the same spot-like components by utilizing TLC, and concentrating to obtain components Fr.1-8;

step 4: separating the component Fr3 containing the compound of the formula I obtained in the step 3 by a silica gel column, performing gradient elution by using n-hexane to acetone in a volume ratio of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10, detecting and combining similar components by using TLC, and concentrating to obtain a component Fr.3.1-3.6;

step 6: passing the component Fr5 containing the compound of formula II in step 3 through a gel chromatographic column as eluent: CHCl (CHCl) ₃ MeOH volume ratio 2:3, separation, TLC detection, combining similar components to obtain three components Fr.5.1-Fr.5.3;

step 8: passing component Fr5.3 containing the compound of formula III in step 6 through C ₁₈ Medium pressure preparative chromatography eluting with MeOH H ₂ The O volume ratio is 70:30-100:0, gradient elution and TLC detection are carried out, and similar components are combined to obtain Fr.5.3.1-Fr.5.3.4;

step 9: purifying and separating the component Fr.5.3.4 containing the compound of the formula III in the step 8 by semi-preparative HPLC to obtain the compound of the formula III;

in step 5, the mobile phase is: CH (CH) ₃ CN:H ₂ The O volume ratio is 70:30, and the flow rate is 3.0mL/min; detection wavelengths are 208 and 254nm; the retention times of the compounds of formula I are: 27.6min;

in step 7, the mobile phase is: CH (CH) ₃ CN:H ₂ The O volume ratio is 70:30, and the flow rate is 3.0mL/min; detection wavelengths are 208 and 254nm; the retention time of the compound of formula II is: 32.4min;

in step 9, the mobile phase is: CH (CH) ₃ CN:H ₂ The O volume ratio is 70:30, and the flow rate is 3.0mL/min; detection wavelengths are 208 and 254nm; the retention time of the compound of formula III is: 33.8min.

4. A process for preparing the phthalide dimer compound of claim 2, characterized in that: resolving the compound of formula II according to claim 1 by chiral HPLC to give a compound of formula IV and a compound of formula V;

the chiral column is: CHIRALPAK IC,4.6 μm, 250X 4.6mm; the mobile phase is: n-hexane: ethanol=90:10, flow rate: 0.8mL/min; the retention time of the compound of formula IV is: 21.93min, retention time of the compound of formula V: 24.34min.

5. Use according to claim 1 or 2, characterized in that: the vasodilator is a calcium ion channel Cav1.2 inhibitor.

6. Use according to claim 1 or 2, characterized in that: the vasodilator is a medicine for treating cardiovascular diseases.

7. Use according to claim 6, characterized in that: the cardiovascular diseases are hyperlipidemia, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction and hypertension.