CN115160170A

CN115160170A - Caffeic acid small molecule active probe and preparation method and application thereof

Info

Publication number: CN115160170A
Application number: CN202210898631.4A
Authority: CN
Inventors: 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
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-10-11
Anticipated expiration: 2042-07-28
Also published as: CN115160170B

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 caffeic acid micromolecule active probe is prepared by a one-step method: adding caffeic acid and alkynylamine into a solvent, and reacting in the presence of a catalyst to obtain a mixed product; and then extracting and carrying out column chromatography on the reacted solution, and separating to obtain the caffeic acid micromolecule active probe. The caffeic acid small molecule active probe of the invention is used as a bioorthogonal label probeThe injection is used for researching pharmacological mechanisms of caffeic acid in the aspects of anti-inflammation, antivirus, bacteriostasis, anti-tumor, antioxidation, neuroprotection and the like, exploring the protein action target of caffeic acid and helping to deeply understand the action mechanism of caffeic acid. The compound has certain anti-inflammatory and/or anti-tumor effects and can be directly used for preparing anti-inflammatory and/or anti-tumor medicines. The method for preparing the caffeic acid micromolecule active probe has simple operation, high probe yield, purity of more than or equal to 98 percent and reaches the pharmacopoeia standard.

Description

Caffeic acid small molecule active probe and preparation method and application thereof

Technical Field

The invention relates to a caffeic acid micromolecule active probe, belonging to the technical field of pharmaceutical chemistry.

Background

Caffeic acid, also known as 3,4-dihydroxycinnamic acid, is a natural phenolic compound with a structure of hydroxyphenylenic acid, and widely exists in food and Chinese medicinal materials such as honeysuckle, valerian, thyme and the like. In recent years, in order to reduce the stimulation of caffeic acid to gastrointestinal tract and improve bioavailability, a plurality of novel high-efficiency low-toxicity medicaments are developed, such as acetyl caffeic acid, caffeic acid phenethyl ester, rosmarinic acid and salvianolic acid A. Recent pharmacological research shows that caffeic acid and derivatives thereof have multiple biological activities of resisting inflammation, resisting virus, inhibiting bacteria, resisting tumor, resisting oxidation, protecting nerves and the like. Although caffeic acid is taken as a safe and effective natural compound at present, caffeic acid and derivatives thereof play important roles in various aspects such as tumor resistance, oxidation resistance, inflammation resistance, immunoregulation and the like, specific protein targets and action mechanisms in vivo are not clear.

Therefore, the development of a molecular probe with similar properties to caffeic acid is of great significance, can be labeled and captured, and has a fundamental role in researching the action mechanism of caffeic acid.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides the caffeic acid micromolecule active probe prepared by a one-step synthesis method, the preparation method is simple, the multistep 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 bio-orthogonal marker probe, is used for determining target protein and researching pharmacological mechanisms of caffeic acid and derivatives thereof, and has activities of resisting inflammation, resisting tumors and the like, and can be used for preparing corresponding medicines.

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 the structure shown in formula II:

wherein n is an integer of 0 to 6;

the caffeic acid small molecule active probe is 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 1:0.8 to 5, and a further preferred molar ratio is 1:1-3.

Further, the solvent is selected from acetonitrile, N-butanol, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and 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 and (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 the caffeic acid is 0.3-3:1 is added.

Further, the reaction temperature is 70-160 ℃, preferably 110-130 ℃.

Further, the reaction time is 8-72h.

Further, the preparation method also comprises the step of separating and purifying the caffeic acid small molecule active probe from the reacted solution, and specifically comprises the following steps: and sequentially adding water and an organic extracting agent into the reacted solution, extracting for multiple times, combining organic phases, concentrating, and performing column chromatography to obtain white crystals, wherein the white crystals are 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 solvent used in the column chromatography is at least one selected from the group consisting of petroleum ether, ethyl acetate, dichloromethane, and methanol. Preferably, the developing solvent is selected from the following combinations of two or three solvents: petroleum ether and ethyl acetate, dichloromethane and methanol, petroleum ether, ethyl acetate and methanol.

The technical purpose of the second aspect of the invention is to provide a caffeic acid small molecule 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 small molecule active probe as a caffeic acid labeled probe.

The caffeic acid small molecule active probe is used as a labeling probe of caffeic acid and is used for researching the pharmacological action mechanism of the caffeic acid. In particular to research on pharmacological mechanisms of caffeic acid in the aspects of anti-inflammation, antivirus, bacteriostasis, anti-tumor, antioxidation, neuroprotection and the like. Wherein the anti-inflammatory agent 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 embodiment of the invention has the following beneficial effects:

(1) The invention successfully prepares the caffeic acid micromolecule active probe by adopting a one-step method, and has high synthesis yield. Because caffeic acid contains a hydroxyl structure, the synthesis reaction is generally protected by adopting a mode of acetylation followed by deacetylation, and particularly, in the reaction process of the invention, the conventional thought in the prior art is to carry out four steps of acetylation, carboxyl group activation, amination and deacetylation, the product needs to be separated and purified after each step of reaction, the synthesis steps are extremely complicated, and the cost is high; the invention breaks the routine, and by selecting a proper catalyst, the created synthesis system can obtain the caffeic acid micromolecule active probe by adopting one-step reaction, the yield reaches or even exceeds the level of a four-step method, and the synthesis steps are greatly simplified.

(2) The caffeic acid micromolecule active probe prepared by the method has high yield, the purity is more than or equal to 98 percent, and the standard of pharmacopoeia is reached.

(3) The caffeic acid micromolecule active probe can be used as a bio-orthogonal labeling probe, is particularly used for labeling caffeic acid, is used for researching pharmacological mechanisms of the caffeic acid in the aspects of anti-inflammation, anti-virus, bacteriostasis, anti-tumor, antioxidation, neuroprotection and the like, probes protein action targets of the caffeic acid, is favorable for deeply knowing the action mechanism of the caffeic acid, and is favorable for designing safer and more efficient caffeic acid derivative medicines with anti-inflammation and anti-tumor effects.

(4) The caffeic acid small molecule active probe has certain anti-inflammatory and/or anti-tumor effects and the like, and can be directly used for preparing anti-inflammatory and/or anti-tumor drugs and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Wherein:

FIG. 1 is a nuclear magnetic diagram of a caffeic acid small molecule active probe prepared in example 1;

FIG. 2 is a graph comparing 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, where CA is caffeic acid and CA-P is a small molecule caffeic acid activity probe;

FIG. 3 is a graph of the efficiency of NO scavenging for caffeic acid and small molecule active probes of caffeic acid in example 8.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1) 0.2mmol of caffeic acid and 0.24mmol of 1-amino-2-propyne are dissolved in 2ml of DMF, 0.1mmol of potassium hexafluorophosphate is added, and the mixture is reacted for 12 hours at 120 ℃ to obtain a product mixed solution.

2) Adding a small amount of water to the mixture obtained in 1), extracting three times with ethyl acetate, and combining the organic layers.

3) The organic phase is concentrated and subjected to column chromatography (dichloromethane: methanol = 10) gave 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 FIG. 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 the mixture reacts for 24 hours at the temperature of 120 ℃ to obtain a mixed solution.

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 is concentrated and subjected to column chromatography (dichloromethane: methanol = 8:1) gave white crystals with a yield of 66%.

Example 3

1) 0.3mmol caffeic acid and 0.4mmol 1-amino-4-pentyne are dissolved in DMF, 0.1mmol silver hexafluoroantimonate is added, and the mixture is reacted for 24 hours at 90 ℃ to obtain a mixed solution.

3) The organic phase is concentrated and subjected to column chromatography (dichloromethane: methanol = 6:1) gave white crystals in 58% yield.

Example 4

1) 0.1mmol of caffeic acid and 0.3mmol of 1-amino-3-butyne are dissolved in DMF, 0.3mmol of cesium fluoride is added, and the mixture reacts for 48 hours at 80 ℃ to obtain a mixed solution.

2) To the mixture in 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 is concentrated and subjected to column chromatography (dichloromethane: methanol = 15) gave white crystals with a yield of 48%.

Example 5

1) 0.15mmol of caffeic acid and 0.3mmol of 1-amino-2-propyne in 4mLCH ₂ Cl ₂ In (1),0.15mmol of sodium hexafluorophosphate is added to react for 12h at 100 ℃ to obtain a mixed solution.

2) To the mixture in 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 is concentrated and subjected to column chromatography (dichloromethane: methanol = 10) gave white crystals with a yield of 68%.

Example 6

1) 0.3mmol caffeic acid and 0.6mmol 1-amino-5-hexyne in CH ₂ Cl ₂ In (1), 0.2mmol (C) of ₄ H ₉ ) ₄ N(PF ₆ ) And reacting for 48 hours at 120 ℃ 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 is concentrated and subjected to column chromatography (dichloromethane: methanol = 6:1) gave white crystals with a yield of 70%.

Comparative example 1

For such reactions, the prior art generally considers the protection of hydroxyl groups by acetylation and the subsequent deacetylation, and specifically obtains the product of the invention by 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,. About.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 the N-hydroxysuccinimide ester of di-O-Ac-caffeic acid was treated with propargylamine (320. Mu.L, 275 mg) for 8 hours at room temperature.

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 as a 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 TNF- α expression in LPS stimulated RAW cells was compared in this example:

1) The cells were digested and counted in 6 well plates at 2X 10 per well ⁵ The individual cells were plated and placed at 37 ℃.5% of CO ₂ The culture was carried out in an incubator for 24 hours.

2) Medium was changed, washed with PBS, and 1mL of complete medium containing LPS (500 ng/mL) was added, and one well was set as a control well and only complete medium was added. Standing at 37 deg.C, 5% CO ₂ The culture was carried out 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 each LPS-stimulated group, and only complete medium was added to another well as the model group. Standing at 37 deg.C, 5% CO ₂ The culture was carried out 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. Add 100. Mu.L RIPA/0.1% cocktail lysate, incubate 30min on ice, centrifuge 15min at 20000g, take supernatant, BCA quantification, normalize each group concentration to 20. Mu.g.

5) 5 μ L of 5 Xloading buffer was added, boiled at 95 ℃ for 5min, and loaded 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 for about 80min, and the sample-carrying buffer solution is electrophoresed out of the bottom of the gel.

6) Soaking a polyvinylidene fluoride (PVDF) membrane in methanol for about 10s for activation, then soaking a sponge, a filter paper and the PVDF membrane in a transfer buffer solution for 5min in advance, removing bubbles, and sequentially placing the materials according to the sequence of a negative electrode, sponge, filter paper, gel, the PVDF membrane, the filter paper, the sponge and a positive electrode; clamping the layers by using a plastic bracket, placing the layers into an inner transfer groove and an outer transfer groove, injecting a transfer buffer solution precooled by ice, enabling one side of the PVDF film to be close to a positive electrode and one side of the polyacrylamide gel to be close to a negative electrode, and carrying out ice-bath film transfer at 120V for 80min.

7) After the transfer is finished, taking out the PVDF membrane, putting the PVDF membrane on filter paper, sealing the PVDF membrane by sealing liquid (5% skim milk), and oscillating for 2 hours at room temperature; according to the dilution of TNF-. Alpha.antibody with Tris buffer followed by overnight incubation at 4 ℃.

8) According to the following steps, secondary antibodies are diluted by using Tris buffer solution according to the proportion of 1; and preparing a developing solution (ESL) according to light shielding, placing the film on a black plate, dropwise adding a proper amount of the developing solution, and directly shooting by using the automatic exposure time.

The results are shown in FIG. 2, where it can be seen that: the caffeic acid probe can inhibit the expression of inflammatory factor TNF-alpha, and the effect of the caffeic acid probe is not obviously different from that of caffeic acid. It can be concluded that this biotin labeling had no significant effect on the function of caffeic acid in anti-inflammatory factor production.

Example 8

The efficiency of scavenging NO by caffeic acid and caffeic acid probes was compared in this example:

1) The cells were digested and counted in 6 well plates at 2X 10/well ⁵ Individual cells were plated. Standing at 37 deg.C, 5% CO ₂ The culture was carried out in an incubator for 24 hours.

2) Medium was changed, washed with PBS, and 1mL of complete medium prepared to contain 500ng/mL LPS was added. Standing at 37 deg.C, 5% CO ₂ The culture was carried out in an incubator for 24 hours.

3) The medium was changed, washed with PBS, and LPS-stimulated groups were supplemented with 1mL of complete medium containing different concentrations of caffeic acid and caffeic acid probes (0, 1, 10, 50, and 100. Mu.M), respectively, and a separate well was set as a model group, to which only complete medium was added. Standing at 37 deg.C, 5% CO ₂ The culture was carried out in an incubator for 24 hours.

4) Supernatants were taken and NO standard curves were prepared using the same medium without LPS, with concentrations set to 100, 50, 25, 12.5, 6.25, 3.125 and 0 μ M in that order.

5) Respectively adding 50 mu L of standard solution and 50 mu L of sample, sequentially adding 50 mu L of solution I and solution II according to a detection kit, setting three multiple holes, measuring absorbance at 540nm on an enzyme-labeling instrument, and calculating NO clearance.

The results are shown in FIG. 3. It can be seen that the effect of caffeic acid probe on NO clearance is equivalent to that of caffeic acid, and the NO clearance of caffeic acid probe is higher at low concentration, further indicating that caffeic acid probe can be used as anti-inflammatory drug.

Example 9

Experiments comparing caffeic acid and caffeic acid probe-labeled proteins in this example:

1) RAW cells were 1X 10 ⁶ After incubation in 6-well plates for 24 hours, lipopolysaccharide was added to a final concentration of 1. Mu.g/mL for 24 hours. Cells were collected after 4h reaction by adding caffeic acid to final concentrations of 0, 1, 5, 10, 50, 100. Mu.M, respectively. Then, caffeic acid probes were added to the final concentration of 50. Mu.M, and no probes were added to the blank.

2) Cell lysis: before harvesting cells, unreacted drug and probe were washed with 1 × PBS, lysed by adding lysis buffer directly (0.1% triton/PBS), and scraped off with a spatula. Each sample tube was sonicated under ice bath conditions, centrifuged at 12,000rpm,4 ℃ for 10min, and the supernatant was taken in a fresh EP tube. And (3) measuring the protein concentration by using the BCA method, and adjusting the protein concentration to a system with the same volume and the same total protein concentration according to the protein concentration of the sample.

3) click reaction. Click reaction reagent (50. Mu.M TAMRA-azide,1mM TCEP/NaVc, 100. Mu.M TBTA/THPTA,1mM CuSO) was prepared ₄ ) And adding the mixed click reaction reagent into each sample according to the reaction system, and rotating and keeping out of the light at room temperature for 2 hours. After the Click reaction was completed, 1mL of glacial acetone was added to each sample tube to precipitate proteins, and the supernatant acetone was discarded. To each sample tube, 30. Mu.L of loading buffer was added and the precipitated protein was solubilized by sonication in ice bath. After sonication the sample tube was heated at 95 ℃ for 10min and shaken every 5 min.

4) For each sample, 15. Mu.L of each sample was run on SDS-PAGE gel (one dose for loading and one control for control). After running the gel, the gel was removed and observed under fluorescence (Protein sample, multi-green channel). The gels were stained with Coomassie Brilliant blue for 10min, then washed overnight with deionized water and the washed gels photographed.

The experimental results show that: using this probe and in vitro cultured RAW cells incubated with it, it was able to bind to proteins, the fluorescence intensity increased with increasing dose, and some clear protein bands were visible by SDS-PAGE. When caffeic acid is incubated with RAW cells in advance, the fluorescence intensity of the probe is obviously reduced, which shows that caffeic acid competes for the target protein combined by the probe in situ, and the specificity of combining the probe and the protein is embodied. Therefore, the protein screened by the probe can be determined to be a highly credible caffeic acid target protein, and a theoretical basis is provided for further research on the mechanism of caffeic acid.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Claims

1. A preparation method of a caffeic acid small molecule active probe comprises the following steps:

wherein n is an integer of 0 to 6;

wherein n is an integer of 0 to 6.

2. The process according to claim 1, wherein the molar ratio between caffeic acid and alkynylamine is 1:0.8-5.

3. The method according to claim 1, wherein the solvent is at least one selected from the group consisting of N-butanol, acetonitrile, N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chlorobenzene, and dimethyl sulfoxide.

4. The process according to claim 1, wherein 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.

5. The method according to claim 4, wherein the molar ratio of the catalyst to caffeic acid is from 0.3 to 3:1 is added.

6. The method according to claim 1, wherein the reaction temperature is 70-160 ℃ and the reaction time is 8-72 hours.

7. The method for preparing according to claim 1, further comprising a step of separating and purifying caffeic acid small molecule active probes from the post-reaction solution, in particular: and sequentially adding water and an organic extracting agent into the reacted solution, extracting for multiple times, combining organic phases, concentrating, and performing column chromatography to obtain white crystals, wherein the white crystals are the caffeic acid micromolecule active probe.

8. The caffeic acid small molecule active probe prepared by the preparation method of any one of claims 1-7.

9. Use of the caffeic acid small molecule active probe of claim 8 as a caffeic acid labeled probe.

10. The use of the caffeic acid small molecule active probe of claim 8 in the preparation of anti-inflammatory and/or anti-tumor drugs.