CN115160170B - Caffeic acid micromolecule active probe and preparation method and application thereof - Google Patents

Caffeic acid micromolecule active probe and preparation method and application thereof Download PDF

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CN115160170B
CN115160170B CN202210898631.4A CN202210898631A CN115160170B CN 115160170 B CN115160170 B CN 115160170B CN 202210898631 A CN202210898631 A CN 202210898631A CN 115160170 B CN115160170 B CN 115160170B
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张迎
唐欢
王继刚
邱梁佳
郭秋岩
朱银华
刘丹丹
王晨
夏斐
高鹏
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Abstract

The embodiment of the invention discloses a preparation method of a caffeic acid micromolecule active probe, which has a structure shown in the following formula, wherein n is an integer of 0-6, and the preparation method comprises the following steps: adding caffeic acid and alkynylamine into a solvent, and reacting in the presence of a catalyst to obtain a mixed product; and extracting and column-chromatography are carried out on the reacted solution, and the caffeic acid micromolecular active probe is obtained through separation. The caffeic acid micromolecule active probe is used as a biological orthogonal marking probe and is used for researchingPharmacological mechanisms in aspects of anti-inflammatory, antiviral, antibacterial, antitumor, antioxidant, neuroprotection and the like of caffeic acid are explored, and the protein action target point of caffeic acid is explored, so that the action mechanism of caffeic acid is deeply known. It has certain anti-inflammatory and/or anti-tumor effects, and can be directly used for preparing anti-inflammatory and/or anti-tumor drugs. The method for preparing the caffeic acid micromolecular active probe is simple to operate, the probe yield is high, the purity is more than or equal to 98%, and the pharmacopoeia standard is achieved.

Description

Caffeic acid micromolecule active probe and preparation method and application thereof
Technical Field
The invention relates to a caffeic acid micromolecular active probe, and belongs to the technical field of pharmaceutical chemistry.
Background
Caffeic acid, also called 3, 4-dihydroxycinnamic acid, is a natural phenolic compound with a hydroxy-benzoic acid structure, and widely exists in foods and traditional Chinese medicinal materials such as honeysuckle, valerian, thyme and the like. In recent years, in order to reduce the irritation of caffeic acid to gastrointestinal tract and improve bioavailability, various novel high-efficiency low-toxicity medicines such as acetyl caffeic acid, caffeic acid phenethyl ester, rosmarinic acid and salvianolic acid A are developed. Recent pharmacological studies show that caffeic acid and its derivatives have various biological activities such as anti-inflammatory, antiviral, antibacterial, antitumor, antioxidant and neuroprotection. Although caffeic acid is used as a safe and effective natural compound at present, caffeic acid and derivatives thereof play an important role in various aspects such as anti-tumor, antioxidation, anti-inflammatory, immunoregulation and the like, the specific protein target and action mechanism in the body are not clear.
Therefore, the development of a molecular probe with similar properties to caffeic acid has important significance, can be marked and captured, and has fundamental effect on researching the action mechanism of caffeic acid.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a caffeic acid micromolecule active probe prepared by a one-step synthesis method, the preparation method is simple, the multi-step synthesis method in the prior art is broken through, the basically equivalent or even higher synthesis yield is achieved, the prepared caffeic acid micromolecule active probe can be used as a biological orthogonal mark probe, is used for determining target proteins and exploring pharmacological mechanisms of caffeic acid and derivatives thereof, has anti-inflammatory, anti-tumor and other activities, and can be used for preparing corresponding medicaments.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
the technical purpose of the first aspect of the invention is to provide a preparation method of a caffeic acid small molecule active probe, which comprises the following steps:
adding caffeic acid and alkynylamine into a solvent, and reacting in the presence of a catalyst to obtain the caffeic acid micromolecule active probe; the alkynylamine is a compound having a structure represented by formula ii:
wherein n is an integer of 0 to 6;
the caffeic acid small molecule active probe has a compound with a structure shown in a formula I:
wherein n is an integer of 0 to 6.
Further, preferably, n is 1,2,3 or 4.
Further, in the above reaction, the molar ratio of caffeic acid to alkynylamine is 1: a further preferred molar ratio is from 0.8 to 5, from 1:1 to 3.
Further, the solvent is selected from acetonitrile, N-butanol, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (CH) 2 Cl 2 ) At least one of chlorobenzene and dimethylsulfoxide.
Further, the catalyst is selected from cesium fluoride, sodium fluoride, potassium fluoride, silver hexafluoroantimonate, (C) 4 H 9 ) 4 N(PF 6 ) At least one of potassium hexafluorophosphate and sodium hexafluorophosphate, more preferably (C) 4 H 9 ) 4 N(PF 6 )。
Further, the molar ratio of the catalyst to caffeic acid is 0.3-3: 1.
Further, the reaction temperature is 70 to 160 ℃, preferably 110 to 130 ℃.
Further, the reaction time is 8-72h.
Further, the preparation method further comprises the step of separating and purifying the caffeic acid micromolecular active probe from the solution after the reaction, and specifically comprises the following steps: and sequentially adding water and an organic extractant into the reacted solution, merging organic phases after multiple extractions, concentrating, and performing column chromatography to obtain white crystals, thus obtaining the caffeic acid micromolecule active probe.
Further, the organic extractant is an organic solvent such as ethyl acetate, petroleum ether or diethyl ether.
Further, the developing agent used in the column chromatography is at least one selected from petroleum ether, ethyl acetate, methylene chloride and methanol. Preferably, the developing agent is selected from the group consisting of a combination of two or three solvents: petroleum ether and ethyl acetate, methylene chloride and methanol, petroleum ether, ethyl acetate and methanol.
The technical purpose of the second aspect of the invention is to provide the caffeic acid micromolecular active probe prepared by the preparation method.
The technical purpose of the third aspect of the invention is to provide the application of the caffeic acid micromolecule active probe as a caffeic acid labeling probe.
The small molecule active probe of caffeic acid is used as a labeled probe of caffeic acid and is used for researching the pharmacological action mechanism of caffeic acid. In particular, the pharmacological mechanisms of caffeic acid in the aspects of anti-inflammation, antivirus, bacteriostasis, anti-tumor, antioxidation, neuroprotection and the like are studied. Wherein the anti-inflammatory is anti-sepsis and/or enteritis.
The technical purpose of the fourth aspect of the invention is to provide the application of the caffeic acid small molecule active probe in preparing anti-inflammatory and/or anti-tumor drugs.
The implementation of the embodiment of the invention has the following beneficial effects:
(1) The invention adopts a one-step method to successfully prepare the caffeic acid micromolecular active probe, and has high synthesis yield. Because caffeic acid contains hydroxyl structure, in the synthetic reaction, generally adopt the way of first acetylating and then deacetylating to protect, and specifically to the reaction process of the invention, the prior art is conventionally thought to be through four steps of acetylating, carboxyl group activating, aminating and deacetylating, each step of reaction needs to be carried out the separation and purification of the product, the synthetic step is very complicated, and the cost is high; the invention breaks the routine, and the synthetic system can be created by selecting a proper catalyst to obtain the caffeic acid micromolecule active probe by adopting one-step reaction, so that the yield reaches or even exceeds the level of a four-step method, and the synthetic steps are greatly simplified.
(2) The caffeic acid micromolecular active probe prepared by the method has high yield and purity of more than or equal to 98 percent, and meets the pharmacopoeia standard.
(3) The caffeic acid micromolecule active probe can be used as a biological orthogonal labeling probe, especially used for labeling caffeic acid, is used for researching pharmacological mechanisms in the aspects of caffeic acid anti-inflammatory, antiviral, antibacterial, antitumor, antioxidant, neuroprotection and the like, explores a protein action target point of caffeic acid, is beneficial to understanding the action mechanism of caffeic acid in depth, and is beneficial to designing safer and more efficient anti-inflammatory and antitumor caffeic acid derivative drugs.
(4) The caffeic acid micromolecular active probe has a certain anti-inflammatory and/or anti-tumor effect and the like, and can be directly used for preparing anti-inflammatory and/or anti-tumor medicaments and the like.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
FIG. 1 is a nuclear magnetic resonance diagram of a small molecule active probe of caffeic acid prepared in example 1;
FIG. 2 is a graph showing the effect of caffeic acid and small molecule caffeic acid activity probes on TNF- α expression in LPS-stimulated lung macrophages (RAWs) in example 7, wherein CA is caffeic acid and CA-P is a small molecule caffeic acid activity probe;
FIG. 3 is a graph showing the comparison of NO-scavenging efficiency of caffeic acid and small molecule active probes of example 8.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1) 0.2mmol of caffeic acid and 0.24mmol of 1-amino-2-propyne are dissolved in 2mM LDMF, 0.1mmol of potassium hexafluorophosphate is added, and the mixture is reacted at 120 ℃ for 12 hours to obtain a product mixture.
2) To the mixture obtained in 1), a small amount of water was added, extraction was performed three times with ethyl acetate, and the organic layers were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=10:1) to give white crystals with a yield of 75%.
The reaction formula is as follows:
1H NMR(400MHz,MeOD)δ7.32(d,J＝15.7Hz,1H),6.91(d,J＝2.0Hz,1H),6.83–6.79(m,1H),6.67(d,J＝8.2Hz,1H),6.25(d,J＝15.7Hz,1H),3.96(d,J＝2.5Hz,2H),2.50(t,J＝2.6Hz,1H)。
the nuclear magnetic diagram is shown in figure 1.
Example 2
1) 0.2mmol of caffeic acid and 0.3mmol of 1-amino-3-butyne are dissolved in DMF, 0.2mmol of sodium hexafluorophosphate is added and reacted at 120℃for 24 hours to obtain a mixture.
2) To the mixture in 1) was added a small amount of water, extracted three times with 8mL of ethyl acetate, and the organic layers were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=8:1) to give white crystals with a yield of 66%.
Example 3
1) 0.3mmol of caffeic acid and 0.4mmol of 1-amino-4-pentyne were dissolved in DMF, and 0.1mmol of silver hexafluoroantimonate was added thereto to react at 90℃for 24 hours to obtain a mixture.
2) To the mixture in 1) was added a small amount of water, extracted three times with 8mL of ethyl acetate, and the organic layers were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=6:1) to give white crystals with a yield of 58%.
Example 4
1) 0.1mmol of caffeic acid and 0.3mmol of 1-amino-3-butyne were dissolved in DMF, and 0.3mmol of cesium fluoride was added to react at 80℃for 48 hours to obtain a mixture.
2) To the mixture of 1) was added a small amount of water, extracted three times with 4mL of ethyl acetate, and the organic phases were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=15:1) to give white crystals with a yield of 48%.
Example 5
1) 0.15mmol of caffeic acid and 0.3mmol of 1-amino-2-propyne are dissolved in 4mLCH 2 Cl 2 To the mixture, 0.15mmol of sodium hexafluorophosphate was added and the mixture was reacted at 100℃for 12 hours to obtain a mixed solution.
2) To the mixture of 1) was added a small amount of water, extracted three times with 10mL of ethyl acetate, and the organic phases were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=10:1) to give white crystals with a yield of 68%.
Example 6
1) Dissolving 0.3mmol of caffeic acid and 0.6mmol of 1-amino-5-hexyne in CH 2 Cl 2 To which 0.2mmol (C 4 H 9 ) 4 N(PF 6 ) Reacting at 120 ℃ for 48 hours to obtain a mixed solution.
2) To the mixture in 1) was added a small amount of water, extracted three times with 12mL of ethyl acetate, and the organic phases were combined.
3) The organic phase was concentrated, column chromatographed (dichloromethane: methanol=6:1) to give white crystals with a yield of 70%.
Comparative example 1
For such reactions, the prior art generally considers that hydroxyl is protected by acetylation and then deacetylated, and specifically, the product of the invention is obtained through four steps of reactions:
1) Acetylation: caffeic acid (0.5 g) was acetylated overnight at room temperature by acetic anhydride (1 mL) in anhydrous pyridine (2 mL) to yield di-O-Ac-caffeic acid (579 mg, 90%).
2) Carboxylic acid group activation: di-O-Ac-caffeic acid was converted to di-O-Ac-caffeic acid N-hydroxysuccinimide ester by reaction with N, N' -disuccinimidyl carbonate (1.28 g) in N, N-dimethylformamide (2 mL).
3) Amination: after removal of the solvent by rotary evaporation under reduced pressure, the residue containing N-hydroxysuccinimide ester of di-O-Ac-caffeic acid was treated with propargylamine (320. Mu.L, 275 mg) at room temperature for 8 hours.
4) Deacetylation: the reaction mixture was treated with 3 equivalents of NaOMe/MeOH for 2 hours. After completion of the reaction according to TLC detection, the reaction mixture was purified by gel chromatography on a silica column to give PACA, pale yellow powder, 325mg,60%.
The caffeic acid probes used in examples 7-9 below were the products prepared in example 1.
Example 7
The effect of caffeic acid and caffeic acid probes on LPS-stimulated TNF- α expression in RAW cells was compared in this example:
1) Cell digestion, counting, 2X 10 per well in 6-well plate 5 Individual cells were plated and placed at 37 ℃.5% CO 2 Culturing in an incubator for 24 hours.
2) The medium was changed, washed with PBS, 1mL of complete medium containing LPS (500 ng/mL) was added, and another well was set as a control well, and only complete medium was added. Placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours.
3) The medium was changed, washed with PBS, and 1mL of complete medium containing different concentrations of caffeic acid and caffeic acid probes (0, 50, 100. Mu.M) was added to the LPS-stimulated group, and another well was set as the model group, with only complete medium added. Placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours.
4) The supernatant was discarded, washed three times with PBS, cells were collected with a cell scraper, centrifuged at 1000g for 3min, and the supernatant was discarded. mu.L RIPA/0.1% cocktail lysate was added, incubated on ice for 30min, centrifuged at 20000g for 15min, and the supernatant was BCA quantified and the concentration of each group normalized to 20. Mu.g.
5) Adding 5 μl of 5 Xloading buffer, boiling at 95deg.C for 5min, and loading on polyacrylamide gel electrophoresis. The initial voltage of electrophoresis is 80V, after the dye enters the separation gel, the voltage is increased to 120V, the voltage is stabilized, the sample loading buffer solution is used for electrophoresis out of the bottom of the gel for about 80 min.
6) Immersing a polyvinylidene fluoride (PVDF) film in methanol for about 10s for activation, immersing a sponge, filter paper and PVDF film in a transfer buffer solution in advance for 5min, removing bubbles, and sequentially placing the materials in the order of negative electrode-sponge-filter paper-gel-PVDF film-filter paper-sponge-positive electrode; clamping the layers by using a plastic bracket, placing the layers into a transfer inner tank and an outer tank, injecting ice-precooled transfer buffer solution, placing one side of a PVDF film close to a positive electrode and one side of a polyacrylamide gel close to a negative electrode, and carrying out ice bath film transfer at 120V for 80 min.
7) After the transfer is completed, taking out the PVDF membrane, placing the PVDF membrane on filter paper, sealing the PVDF membrane by using sealing liquid (5% skimmed milk), and oscillating for 2 hours at room temperature; TNF- α antibodies were diluted with Tris buffer followed by overnight incubation at 4 ℃.
8) Diluting the secondary antibody according to the proportion of 1:5000 by using Tris buffer solution, incubating for 1.5h at room temperature, and washing the membrane for 3 times by using the Tris buffer solution for 10min each time; according to the dark preparation of developer (ESL), the film is placed on a black plate, a proper amount of developer is added dropwise, and the automatic exposure time is directly used for shooting.
The results are shown in FIG. 2, which can be seen: the caffeic acid probe can inhibit the expression of inflammatory factor TNF-alpha, and the effect is not significantly different from caffeic acid. It can be concluded that this biotin labeling method has no significant effect on the function of caffeic acid in anti-inflammatory factor production.
Example 8
The efficiency of caffeic acid and caffeic acid probes to scavenge NO was compared in this example:
1) Cell digestion, counting, 2X 10 per well in 6-well plate 5 Individual cells were plated. Placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours.
2) The medium was changed, washed with PBS, and 1mL of complete medium containing 500ng/mL LPS was added. Placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours.
3) The medium was changed, washed with PBS, and 1mL of complete medium containing different concentrations of caffeic acid and caffeic acid probes (0, 1, 10, 50, and 100. Mu.M) was added to the LPS-stimulated group, and another well was set as the model group, and only complete medium was added. Placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 24 hours.
4) The supernatant was taken and the NO standard curve was prepared using the same medium without LPS, with concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 0 μm in this order.
5) After 50 mu L of standard solution and 50 mu L of sample are added respectively, 50 mu L of solution I and 50 mu L of solution II are added sequentially according to a detection kit, three compound holes are arranged, absorbance is measured at 540nm on an enzyme-labeled instrument, and NO clearance is calculated.
The results are shown in FIG. 3. The effect of the caffeic acid probe on NO clearance rate is equivalent to that of caffeic acid, and the NO clearance rate of the caffeic acid probe is higher at low concentration, so that the caffeic acid probe can be further used as an anti-inflammatory drug.
Example 9
In this example, the experiment of caffeic acid and caffeic acid probe-tagged proteins was compared:
1) RAW cells were cultured at 1X 10 6 After plating 6 well plates and incubation for 24 hours, lipopolysaccharide was added at a final concentration of 1. Mu.g/mL for 24 hours. Cells were collected after 4h of reaction with caffeic acid at final concentrations of 0, 1, 5, 10, 50, 100. Mu.M, respectively. Then, caffeic acid probe was added to the mixture to a final concentration of 50. Mu.M, and no probe was added to the blank.
2) Cell lysis: before harvesting the cells, unreacted drug and probe were washed off with 1 XPBS, lysed directly with lysis solution (0.1% triton/PBS) and scraped off with a spatula. Each sample tube was sonicated under ice bath conditions at 12,000rpm at 4℃and centrifuged for 10min, and the supernatant was taken in a fresh EP tube. The BCA method measures protein concentration and adjusts the protein concentration to a system with equal volume and other total protein concentration according to the protein concentration of a sample.
3) click reaction. Preparation of click reagents (50. Mu.M TAMRA-azide,1mM TCEP/NaVc, 100. Mu.M TBTA/THPTA,1mM CuSO) 4 ) And adding the mixed click reaction reagent into each sample according to the reaction system, and rotating and shading the reaction at room temperature for 2 hours. After the Click reaction was completed, 1mL of glacial acetone was added to each sample tube to precipitate the protein, and the supernatant acetone was discarded. To each sample tube was added 30. Mu.L of loading buffer and ice bath sonication was used to solubilize the precipitated proteins. After the ultrasonic treatment, the sample tube is heated at 95 ℃ for 10min, and is vibrated every 5 min.
4) 15. Mu.L of SDS-PAGE gel was run for each sample (one charge and one control for control at loading). After running, the gel was removed and observed under fluorescence (Protein sample, multi-green channel). The gel was stained with coomassie blue for 10min, then washed with deionized water overnight, and the washed gel was photographed.
The experimental results show that: by co-incubating the probe with RAW cells cultured in vitro, it can bind to protein, fluorescence intensity increases with increasing dose, and some clear protein bands can be seen by SDS-PAGE. When the caffeic acid is pre-incubated with RAW cells, the fluorescence intensity of the probe is obviously reduced, which indicates that the caffeic acid competes with target protein bound by the probe in situ, and the specificity of the binding of the probe and the protein is reflected. Therefore, the protein screened by the probe can be determined to be highly reliable caffeic acid target protein, and a theoretical basis is provided for further research on the mechanism of caffeic acid.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims (6)

1. The preparation method of the caffeic acid micromolecular active probe comprises the following steps:
adding caffeic acid and alkynylamine into a solvent, and reacting in the presence of a catalyst to obtain the caffeic acid micromolecule active probe; the alkynylamine is a compound having a structure represented by formula ii:wherein n is 1;
the caffeic acid small molecule active probe is a compound with a structure shown in a formula I:
wherein n is 1;
the catalyst is at least one selected from silver hexafluoroantimonate, potassium hexafluorophosphate and sodium hexafluorophosphate;
the solvent is at least one selected from N, N-dimethylformamide and dichloromethane.
2. The method according to claim 1, wherein the molar ratio of caffeic acid to alkynylamine is 1:0.8-5.
3. The method according to claim 1, wherein the catalyst has a molar ratio to caffeic acid of 0.3 to 3: 1.
4. The preparation method according to claim 1, wherein the reaction temperature is 70-160 ℃ and the reaction time is 8-72h.
5. The method according to claim 1, further comprising the step of separating and purifying the caffeic acid small molecule active probes from the post-reaction solution, in particular: and sequentially adding water and an organic extractant into the reacted solution, merging organic phases after multiple extractions, concentrating, and performing column chromatography to obtain white crystals.
6. The use of a small molecule caffeic acid probe prepared by the method for preparing a small molecule caffeic acid active probe according to claim 1 as a labeled caffeic acid probe.
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