CN109970738B

CN109970738B - Caragana N-isoflavone compound and preparation method and application thereof

Info

Publication number: CN109970738B
Application number: CN201910147340.XA
Authority: CN
Inventors: 尹小英; 冯煌迪; 陈玲艳
Original assignee: Shanghai University of Engineering Science
Current assignee: Shanghai University of Engineering Science
Priority date: 2019-02-27
Filing date: 2019-02-27
Publication date: 2021-07-09
Anticipated expiration: 2039-02-27
Also published as: CN109970738A

Abstract

The invention relates to a cytisine N-isoflavonoid compound and a preparation method and application thereof, wherein the compound has the structural formula as follows:

wherein R is¹One selected from hydroxyl, alkoxy, amido or halogen radical, R²One selected from hydrogen, alkyl or aryl, R³One or more of hydroxyl, alkoxy, amino or halogen group. Compared with the prior art, the isoflavone compound is spliced to the N of the 12 th site of the cytisine by using a combinatorial synthesis technology as a structural unit, so that a series of cytisine-isoflavone derivatives are synthesized, and a new active pharmacological action is exerted.

Description

Caragana N-isoflavone compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of drug synthesis, and particularly relates to a cytisine N-isoflavone compound and a preparation method and application thereof.

Background

Cytisine (cytisine) is first separated from cytisis Laburnum Med seeds in 1865 by Husemann and Marme, and is mostly distributed in plants of Jinhua, Jueming, Genistian and Sophora. The alkaloid has pharmacological effects of enhancing myocardial contraction force, exciting respiration, enhancing motor ability, improving cognitive function, resisting depression, relieving pain, resisting tumor, etc., especially has strong anticancer activity. The cytisine belongs to quinolizine alkaloid, and the structural modification of the cytisine focuses on the derivatization of 12-bit N, and mainly comprises alkylation, acylation, benzylation, benzoylation, glycosylation and the like.

Isoflavone compounds (isoflavanoid) are compounds of which the basic parent nucleus is 3-phenylchromone, are widely present in leguminous plants, pasture and the like, and are secondary metabolites of plants. In recent decades, with the development of modern separation technology and structure research methods, people separate various isoflavone compounds from plants, animals or marine organisms, and many researches find that the compounds have various biological activities and good pharmacological effects, such as anti-tumor, anti-hyperglycemia, antifungal, antiviral, anti-inflammatory, antioxidant, cardiovascular diseases and the like, and are valuable medicaments.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a cytisine N-isoflavone compound and a preparation method and application thereof.

The purpose of the invention can be realized by the following technical scheme: a laburnine N-isoflavonoid compound has the following structural formula:

wherein R is¹One selected from hydroxyl, alkoxy, amido or halogen radical, R²One selected from hydrogen, alkyl or aryl, R³One or more of hydroxyl, alkoxy, amino or halogen group.

Molecular simulation is carried out by using the DISCOVERY study 4.5 software, and from the results, the compound molecules are basically positioned in the active cavity center of HER2 receptor protein and are surrounded by a plurality of amino residues such as ASP26, PRO360, ALA233, HIS229 and LEU275, and strong hydrophobic effect is formed. These residues form the active pocket of the HER2 protein around the substrate molecule. Wherein substrate molecules 5-OH and 7-OH form double hydrogen bonds with ASP26 and ALA233 amino acid residues, which are main pharmacophores, so that 5-OR2 is a functional group capable of forming hydrogen bonds with ASP26 amino acid residues.

The compound also comprises a salt corresponding to the structural formula, wherein the salt is prepared by reacting a substance corresponding to the structural formula with an inorganic acid or an organic acid, the inorganic acid comprises one of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid or phosphoric acid, and the organic acid is one of citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzoic acid, 2-acetoxybenzoic acid or isethionic acid.

A preparation method of the cytisine N-isoflavonoid compound comprises the following steps:

(1) in that

Adding N, N-dimethyl acetal, and reacting to obtain

Then react under the action of elemental iodine to generate

(2) Prepared by the step (1)

With commercially available

Mixing and reacting to obtain

(3) Prepared in the step (2)

Reacting with cytisine and formalin under the catalysis of DMAP to obtain the product

Preferably, in the step (1),

the ratio of the molar mass of N, N-dimethyl acetal to the volume of N, N-dimethyl acetal is 1mmol: (5-7) mL, the reaction temperature is 70-80 ℃, and the reaction time is 4-8 h;

the molar ratio of the iodine to the elemental iodine is 1: (1.1-2.0), the reaction temperature is 10-35 ℃, and the reaction time is 12-48 h.

Preferably, in the step (2),

in a molar ratio of 1: (1.0-2.0), the reaction temperature is 10-35 ℃, and the reaction time is 4-8 h.

The reaction of (1) takes alkali and palladium acetate as catalysts, and the alkali is sodium carbonate or potassium carbonate.

Preferably, in step (3),

the molar ratio of the obtained product to the cytisine and formalin is 1: (0.3-0.6): (0.4-0.8), the reaction temperature is 60-80 ℃, and the reaction time is 4-9 h.

An application of the cytisine N-isoflavonoid compound in preparing the medicine for treating tumor, such as breast cancer.

Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:

(1) the preparation method is efficient, easy to operate and high in product purity;

(2) can effectively inhibit the migration of cancer cells and has good cancer treatment effect.

Drawings

FIG. 1 is a hydrogen spectrum of the compound obtained in example 1;

FIG. 2 is a carbon spectrum of the compound obtained in example 1;

FIG. 3 is a mass spectrum of the compound prepared in example 1;

FIG. 4 is a graph showing the results of testing the MDA-MB-231 breast cancer cell migration inhibition ability of the compounds prepared in examples 1-3 at different concentrations;

FIG. 5 is a graph showing the results of tests on the inhibition of the migration ability of 4T1 breast cancer cells by the compounds prepared in examples 1-3 at different concentrations;

FIG. 6 is a graph showing the effect of the compound prepared in example 1 on HER2 receptor protein.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

Synthesis of compound CNF1, structural formula:

the preparation steps are as follows:

(1) synthesizing 7-methoxy methyl ether-5-hydroxy-3-iodochromone; (2) synthesizing genistein; (3) the genistein and the genistein are subjected to Mannich reaction to synthesize a target substance.

The specific process comprises the following steps:

(1) 2,4, 6-trihydroxyacetophenone is taken as a raw material, and 2.1 times of equivalent of K is added₂CO₃The hydroxyl groups at the 4 and 6 positions are selectively dissociated, then 2.12 times of equivalent of chloromethyl methyl ether is added to obtain 2-hydroxyl-4, 6-dimethyl methyl ether acetophenone, then N, N-dimethyl acetal is added to synthesize the aminoketene at the temperature of 74 ℃ with the yield of 88 percent, and finally, the synthesis of 5, 7-dimethyl methyl ether-3-iodine chromone is completed with the simple cyclization reaction in one step, namely, with the yield of 75 percent under the action of simple substance iodine. Dissolving 3-iodine chromone 4 in dichloro, placing on a stirrer, stirring at medium speed for 4 hours, and removing 5-methyl ether to obtain 3-iodine chromone 5; the structural formula varies as follows:

(2) the isoflavone 7 is synthesized by the compounds 5 and 6 under the Suzuki coupling reaction, methyl ether at the 7 position is removed under 3MHCl, and then methoxy is removed under boron tribromide to obtain genistein 9, wherein the structural formula changes as follows:

(3) the cytisine-N-methylene- (5,7, 4' -trihydroxy) isoflavone CNF1 is synthesized by cytisine, genistein and formalin under the catalysis of DMAP, and the structural formula is changed as follows:

to a solution of compound 9(0.5mmol) in isopropanol (5ml) were added cytisine (0.625mmol) and formalin (0.25ml, 37%), DMAP (1.2mg), and placed in an oil bath under nitrogen for reaction 3 h-overhead. The reaction was monitored by TCL for completion and purified by flash column chromatography to give compound CNF 1.

The prepared CNF1 was subjected to nuclear magnetic and mass spectrometry, and the obtained hydrogen spectrum, carbon spectrum, and mass spectrum were shown in fig. 1, 2, and 3, respectively.

¹H NMR(400MHZ，DMSO)cytisine protons：7.26(1H,dd,J＝6.8；8.8,H-4)， 6.15(1H,dd,J＝1.6；9.2,H-3)，6.01(1H,dd,J＝1.2；7.2,H-5)，3.65,3.80(2H,2d,J＝14.8, CH₂-10)，2.84-3.04(3H,m,H-11,13,7)，2.38-2.50(3H,m,H-11,13,9)，1.70-1.83(2H,m, CH₂-8)。Isoflavone protons：8.18(1H,s,H-2)，7.37(2H,d,J＝8.8,H-2',6')，6.81(2H, d,J＝8.8,H-3’,5’)，6.14(1H,s,H-6)，3.68(2H,s,CH₂-8)。¹³C NMR(100MHZ，DMSO) 180.8,164.1,162.6,161.2,157.8,155.8,154.1,151.8,139.1,130.6,122.4,121.6,116, 115.5,104.5,104.3,100.8,98.9,59.9,59.3,55.3,50.5,49.8,34.8,27.7,25.2。 HRMS(ESI):m/z＝471.16(calcd.471.16for C₂₇H₂₃N₂O₆[M-H]^-)。M.P.＝ 237-239℃。

From this we can analyse: from the above hydrogen spectrum, carbon spectrum and mass spectrum, it can be confirmed that the structure is laburnine-N-methylene- (5,7, 4' -trihydroxy) isoflavone compound, i.e. compound CNF 1.

The prepared CNF1 is subjected to molecular simulation by using the DISCOVERY study 4.5 software, and the result is shown in figure 6, and as can be seen from the figure, the CNF1 molecule is basically positioned in the active cavity center of HER2 receptor protein and is surrounded by multiple amino residues such as ASP26, PRO360, ALA233, HIS229 and LEU275, and a strong hydrophobic effect is formed. These residues form the active pocket of the HER2 protein around the substrate molecule. Wherein substrate molecules 5-OH and 7-OH form double hydrogen bonds with ASP26 and ALA233 amino acid residues, which are main pharmacophores, so that 5-OR2 is a functional group capable of forming hydrogen bonds with ASP26 amino acid residues.

Example 2

Synthesis of compound CNF2, structural formula:

wherein R is¹＝OH,R²＝H，R³＝R⁴＝OCH₃. The preparation method comprises the following specific steps:

(1) the isoflavone intermediate is prepared by the following synthetic route:

carrying out Suzuki coupling reaction on the compound 19 and 3, 4-dimethoxy phenylboronic acid to obtain an isoflavone compound 21, and specifically comprising the following steps:

polyethylene glycol 10000(21g), Pd (OAc)₂(0.025g, 0.11mmol) was added to a solution of sodium carbonate (0.548g, 5.17mmol) in methanol (25ml) and stirred under nitrogen in an oil bath at 50 ℃ until the system became black, immediately after which a mixture of compound 19(0.820g,2.17mmol) and 3, 4-dimethoxyphenylboronic acid (1.395g, 5.41mmol) was addedAdding into the system. After 3h of reaction, TCL showed the reaction was complete. Adding water to separate PEG10000 into solid, filtering, washing the solid with ethyl acetate, combining ethyl acetate washing liquid and water phase, extracting with ethyl acetate, drying organic layer with anhydrous sodium sulfate, removing solvent under reduced pressure, and performing flash column chromatography (PE: EA ═ 8:1) to obtain white solid compound 21 (93%)

Compound 21(0.2g, 0.43mmol) was added to the methanol solution, 3M HCl was added and the reaction was heated to reflux for 2h, and TCL monitored for completion. Water was added, and the mixture was extracted with dichloro, washed with water, concentrated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give a residue, flash column chromatography (PE: EA 4:1) was performed to give isoflavone compound 21(0.11mg, 91%)

(2) The cytisine, isoflavone 21 and formalin are catalyzed by DMAP to synthesize the cytisine-N-methylene-isoflavone CNF 2.

To a solution of compound 21(0.5mmol) in isopropanol (5ml) were added cytisine (0.625mmol) and formalin (0.25ml, 37%), DMAP (1.2mg), and placed in an oil bath under nitrogen for reaction 3 h-overhead. The reaction was monitored by TCL for completion and purified by flash column chromatography to give compound CNF 2.

Example 3

Synthesis of compound CNF3, structural formula:

wherein R is¹＝OH,R²＝H，R³＝H，R⁴＝OCH₃The preparation method comprises the following specific steps:

(1) the isoflavone intermediate is prepared by the following synthetic route:

the specific steps are basically the same as the second embodiment, the key intermediate is replaced by the p-methoxyphenylboronic acid, the synthesis conditions are slightly adjusted, as shown in the technical scheme,

(2) the cytisine, isoflavone 20 and formalin are catalyzed by DMAP to synthesize the cytisine-N-methylene-isoflavone CNF 3.

The specific steps are the same as the second embodiment, the isoflavone is replaced by the compound 20, and the rest is unchanged.

And (3) detecting the pharmacological activity:

the experimental results of the anti-tumor metastasis activities of the three compounds CNF1-CNF3 are as follows:

research on influence of three samples on migration capacity by using two tumor cells and cell scratch experimental models

Respectively uniformly seeding MDA-MB-231 cells and 4T1 cells of breast cancer in logarithmic growth state in a Costar6 well plate, wherein the final volume of each well is 50% of the total volume, and the cell density is controlled to be about 1.5 multiplied by 10⁵One per ml. Each well was provided with 3 replicates and the experiment was repeated 5 times. After the cells were about to be confluent, the supernatant was discarded, a line (wound area) simulating the wound was drawn longitudinally at the center of the bottom of each well with a sterile inoculating loop, and the width of the wound area was measured under a microscope. Washing with new culture medium for 3 times, washing off floating cells, and stirring into serum-free culture medium without drug. Samples were dissolved in serum-free medium and solutions of three compound samples were added to the experimental group to final concentrations of 10. mu.M, 30. mu.M and 90. mu.M, respectively. The wound area of each group was photographed under a microscope and the width of the wound area was measured under a microscope at the time when the line was drawn to disappear after the sample addition and the negative control group, respectively, and the migration distance and the migration ratio (tumor cell migration distance/initial width of the wound area) were calculated. All three compounds can inhibit MDA-MB-231 cell migration, tumor cell migration distance in wound area is shortened, and migration rate of each administration group is obviously reduced compared with blank group<0.05,**P<0.01) and the migration rate decreased more with increasing compound dose, indicating that the inhibition is dose-dependent (as shown in figure 4). The migration inhibitory effect of the three compound samples on breast cancer 4T1 cells is shown in fig. 5, with a significant decrease in migration rate. As can be seen from FIG. 4 and FIG. 5, the anti-tumor cell migration effect of the 3 compounds is close to that of the positive drug, and the 3 compounds have better in vitro anti-breast cancer tumor cell metastasis activity.

Claims

1. A cytisine N-isoflavonoid compound or a pharmaceutically acceptable salt thereof is characterized in that the structural formula of the compound is as follows:

2. the cytisine N-isoflavonoid compound or pharmaceutically acceptable salt thereof according to claim 1, wherein said pharmaceutically acceptable salt is prepared by reacting a substance corresponding to the above formula with an inorganic acid or an organic acid, wherein said inorganic acid comprises one of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid or phosphoric acid, and said organic acid is selected from citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzoic acid, 2-acetoxybenzoic acid or isethionic acid.

3. A process for the preparation of the cytisine N-isoflavonoids of claim 1 wherein the compound is

The preparation process comprises the following steps:

4. the use of the cytisine N-isoflavonoid compound or the pharmaceutically acceptable salt thereof as claimed in claim 1 or2 for the preparation of a medicament for the treatment of tumors.