CN115368265A

CN115368265A - Sofalcone small molecule active probe and preparation method and application thereof

Info

Publication number: CN115368265A
Application number: CN202211304094.2A
Authority: CN
Inventors: 唐欢; 张迎; 王继刚; 宋阳; 褚铮; 周利润; 朱银华; 马昂; 王晨; 刘丹丹; 张书杰
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2022-11-22
Anticipated expiration: 2042-10-24
Also published as: CN115368265B

Abstract

The embodiment of the invention discloses a sofalcone small molecule active probe which has a structure shown in the following formula, wherein n is an integer of 0-6. The sofalcone small-molecule active probe is obtained by adding sofalcone and alkynylamine into a solvent and reacting in the presence of a condensing agent and an alkaline reagent. The sofalcone small molecule active probe can be used as a living creatureThe orthogonal labeling probe is particularly used for labeling sofalcone, is used for researching pharmacological mechanisms of sofalcone in the aspects of anti-inflammation, bacteriostasis and the like, probes the protein action target of the sofalcone, is favorable for deeply knowing the action mechanism of the sofalcone and is favorable for designing safer and more efficient anti-inflammatory and antibacterial sofalcone derivative drugs. It also has certain antiinflammatory and antibacterial effects, and can be directly used for preparing antiinflammatory and antibacterial medicines.

Description

Sofalcone small molecule active probe and preparation method and application thereof

Technical Field

The invention relates to a sofalcone small molecule active probe, belonging to the technical field of medicinal chemistry.

Background

Sofalcone (sofalcone), also known as sufaracin, is a compound isolated from the traditional botanical drug subprostrate sophora and is chemically (E) -2- [5- (3-methyl-2-butenyloxy) -2- [3- [4- (3-methyl-2-butenyloxy) ] phenyl ] acryloyl ] acetic acid (I).

Sofalcone is the only prostaglandin catabolic enzyme inhibitor drug approved to be on the market in China for treating gastritis and gastric ulcer at present, and has pharmacological effects of increasing prostaglandin E2 content, promoting gastric mucosa repair, resisting ulcer and the like. The content of prostaglandin E2 in stomach tissues is increased by inhibiting 15-hydroxy prostaglandin dehydrogenase, and the prostaglandin E2 plays an important role in protecting gastric mucosa; has better treatment effect on gastric ulcer, duodenal ulcer and chronic gastritis; can inhibit the activity of the pyrori strain, leads to pyrori deformation, inhibits the adhesion of H.pyrori to gastric mucosa, inhibits lipolysis, and has been clinically proven to have long-term use safety.

Although sofalcone as a safe and effective medicine plays an important role in many aspects at present, the specific protein target and action mechanism of sofalcone in vivo are not clear.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a sofalcone small molecule active probe which can be used as a bioorthogonal labeling probe, is used for determining target protein and researching the pharmacological mechanism of sofalcone and derivatives thereof, has activities such as anti-inflammation 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 sofalcone small molecule active probe, which is a compound with a structure shown in a formula I:

Ⅰ

wherein n is an integer of 0 to 6.

More preferably, n is 1,2,3 or 4.

The technical purpose of the second aspect of the invention is to provide a preparation method of a sofalcone small molecule active probe, which comprises the following steps:

adding sofalcone and alkynylamine into a solvent, and reacting in the presence of a condensing agent and an alkaline reagent to obtain the sofalcone small-molecule active probe; the alkynylamine is a compound having a structure represented by formula II:

Ⅱ

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 sofalcone to alkynylamine is 1:0.8 to 5, and a further preferred molar ratio is 1:1-3.

Further, the reaction time is 12-72h, and the reaction temperature is 0-60 ℃, preferably 20-50 ℃.

Further, the solvent is selected from water, acetonitrile, methanol, ethanol, ethyl acetate, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and dichloromethane (CH) ₂ Cl ₂ ) At least one of chloroform and dimethylsulfoxide.

Further, the condensing agent is selected from at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium Hexafluorophosphate (HCTU), and 4-Dimethylaminopyridine (DMAP). More preferably, the condensing agent is a combination of two solvents: EDCI and HOBT.

Further, the molar ratio of the condensing agent to the sofalcone is 0.5-10:1, preferably 1.5 to 8:1.

Further, the basic agent is at least one selected from the group consisting of N, N-Diisopropylethylamine (DIPEA), N-diethylethylamine (TEA), potassium tert-butoxide, sodium hydride, potassium hydride, pyridine, diazabicyclo (DBU), sodium ethoxide, sodium methoxide, sodium bicarbonate, and sodium carbonate.

Further, the molar ratio of the alkaline reagent to the sofalcone is 0.5-6:1 is added.

Further, the preparation method also comprises the step of separating and purifying the sofalcone small molecule 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, extracting for multiple times, combining organic phases, concentrating, and carrying out column chromatography to obtain white crystals, wherein the white crystals are the sofalcone 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 third aspect of the invention is to provide the application of the small molecule active sofalcone probe as a sofalcone labeled probe.

The small molecule active sofalcone probe is used as a labeled probe of sofalcone and is used for researching the pharmacological action mechanism of the sofalcone. In particular to research of pharmacological mechanisms of sofalcone in the aspects of anti-inflammation, antibiosis and the like.

The technical purpose of the fourth aspect of the invention is to provide an application of the sofalcone small molecule active probe in preparing anti-inflammatory and/or antibacterial drugs. Wherein, the application in preparing the medicine for treating gastritis or gastric ulcer and the application in preparing the medicine for resisting staphylococcus aureus are provided.

The embodiment of the invention has the following beneficial effects:

(1) The small molecule active sofalcone probe can be used as a bioorthogonal labeling probe, particularly used for labeling sofalcone, researching pharmacological mechanisms of sofalcone in the aspects of anti-inflammation, bacteriostasis and the like, researching protein action targets of the sofalcone, being beneficial to deeply knowing the action mechanism of the sofalcone and being beneficial to designing safer and more efficient anti-inflammatory and antibacterial sofalcone derivative drugs.

(2) The sofalcone small molecule active probe has certain anti-inflammatory and antibacterial effects and can be directly used for preparing anti-inflammatory and antibacterial medicines.

(3) The sofalcone micromolecule active probe prepared by the method has high purity and reaches the pharmacopoeia standard.

(4) The invention adopts a one-step reaction method to prepare the sofalcone small-molecule active probe, and the synthetic method has simple operation and mild synthetic conditions.

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 small molecule active sofalcone probe prepared in example 1;

FIG. 2 is a graph showing the in vitro antibacterial efficiency of the small molecule active sofalcone probe of example 7;

FIG. 3 shows the results of in situ competition between the active small molecule sofalcone probe and sofalcone in live Staphylococcus aureus of example 9.

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 sofalcone and 0.24 mmol of 1-amino-2-propyne in 2 mLCH ₂ Cl ₂ Adding condensing agent 0.6mmol EDCI and 0.6mmol HOBT, and alkaline reagent 0.6mmol DIPEA, reacting at 30 deg.C for 12h to obtain product mixture.

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) to obtain white crystals as the sofalcone small molecule active probe, with a yield of 76%.

1H NMR (500 MHz, CDCl ₃ ) δ 8.14 (s, 1H), 7.59 (dd, J = 16.9, 12.2 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.11 (d, J = 15.7 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 6.54 (dd, J = 8.6, 1.9 Hz, 1H), 6.40 (d, J = 1.9 Hz, 1H), 5.41 (dd, J = 3.2, 1.4 Hz, 2H), 4.49 (t, J = 7.6 Hz, 4H), 3.94 (dd, J = 5.4, 2.4 Hz, 2H), 2.08 (t, J = 2.4 Hz, 1H), 1.74 (d, J = 4.8 Hz, 6H), 1.69 (d, J = 6.4 Hz, 6H).

The nuclear magnetic diagram is shown in FIG. 1.

Example 2

1) Dissolving 0.2mmol of sofalcone and 0.3mmol of 1-amino-3-butyne in DMF, adding 0.3mmol of HATU and 0.3mmol of DIPEA, and reacting at 40 ℃ for 24h 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 70%.

Example 3

1) 0.3mmol of sofalcone and 0.4mmol of 1-amino-4-pentyne are dissolved in DMF, 0.9mmol of HATU and 0.9mmol of DIPEA are added, and the mixture is reacted for 36h at 50 ℃ to obtain a mixed solution.

3) The organic phase is concentrated and subjected to column chromatography (dichloromethane: methanol = 6:1) gave white crystals in 72% yield.

Example 4

1) 0.1mmol sofalcone and 0.3mmol 1-amino-3-butyne were dissolved in DMF, 0.3mmol HATU and 0.3mmol DIPEA were added, and 48h was reacted at 20 ℃ 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 60%.

Example 5

1) Dissolving 0.15mmol sofalcone and 0.3mmol 1-amino-2-propyne in 4mLCH ₂ Cl ₂ 0.6mmol EDCI and 0.6mmol HOBT, 0.45mmol DIPEA as alkaline reagent were added and reacted at 25 ℃ for 72 hours to obtain a mixture.

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 65%.

Example 6

1) 0.3mmol of sofalcone and 0.6mmol of 1-amino-5-hexyne in CH ₂ Cl ₂ 0.9mmol EDCI and 0.9mmol HOBT, and 0.9mmol DIPEA as alkaline reagent were added and reacted at 50 deg.C for 48h to obtain a mixture.

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%.

The sofalcone probes used in examples 7-9 below were the products prepared in example 1.

Example 7

In this example, the in vitro antibacterial Minimum Inhibitory Concentration (MIC) of the sofalcone small molecule active probe was determined, and the used strain was Staphylococcus aureus.

Adding 50 μ L of the bacterial liquid into 700 μ L of LB liquid medium, and culturing in 200 rpm/min 37 deg.C full-temperature oscillator until OD600 reaches 0.8. Adding 100 μ L of liquid culture LB medium into No. 1-12 holes of B-G row, diluting with Kong Jiaru 97.44.44 μ LLB medium and 2.56 μ L of stock solution (the concentration of the drug in the holes is 128 μ G/mL) sequentially in No. 2-11 holes of B-G row, wherein the drug concentration in each hole is 128 μ G/mL, 64 μ G/mL, 32 μ G/mL, 16 μ G/mL, 8 μ G/mL, 4 μ G/mL, 2 μ G/mL, 1 μ G/mL, 05. Mu.g/mL, 0.25. Mu.g/mL. Then, 1. Mu.L of the bacterial suspension was added to the wells 1 to 11 on the rows B to G. And setting the No. 1 hole of the B-G row as a negative control, adding only bacteria liquid and not adding medicines, and setting the No. 12 hole of the B-G row as a blank control, and not adding the bacteria liquid and the medicines. And finally placing the mixture in a constant-temperature incubator at 37 ℃ to incubate 16 h, and measuring OD600 value by naked eyes and a multifunctional microplate reader to obtain a result. The bacteria inhibition rates of strains corresponding to different drug concentrations are shown in figure 2; the lowest antibacterial compound concentration in the holes without significant difference with the OD value of the negative control group is the Minimum Inhibitory Concentration (MIC) ₅₀ And MIC ₉₀ The results are shown in Table 1.

TABLE 1

Example 8

In the embodiment, the strength and specificity of the sofalcone small molecule active probe combined with the staphylococcus aureus protein are explored:

1) Shaking the bacteria overnight until the cells enter a stationary period of 1h, collecting the bacteria, centrifuging at 8000 rpm at low temperature (4 ℃) for 10 min, then washing with PBS once, removing the PBS, adding PBS to resuspend the bacteria, taking 100 mu L, adding into a new EP tube, and marking as 0 mu M, 1 mu M, 5 mu M, 10 mu M, 25 mu M and 50 mu M respectively.

2) Preparing a sofalcone probe solution: the sofalcone probe was weighed and added with DMSO to prepare a 100mM stock solution for use. Then, the sofalcone probe mother liquor is diluted into a solution with the concentration gradient of 5mM, 2.5 mM, 1mM, 0.5 mM, 0.1mM and 0mM by using DMSO for standby.

3) 1 mu L of the sofalcone probe solution with the concentration gradient is respectively added into an EP tube, the concentration of the drug probe in the system is respectively 50 mu M, 25 mu M, 10 mu M, 5 mu M, 1 mu M and 0 mu M, the mixture is vortexed and uniformly mixed, then the mixture is instantaneously centrifuged for a plurality of seconds, and the incubation is carried out for 12 hours at the room temperature of 500 rpm/min.

4) 12h, centrifuging at 8000 rpm at low temperature (4 ℃) for 5min, washing with PBS for three times, removing PBS, adding 200 mu L of bacterial lysate into each tube of EP tube, shaking on a vortex oscillator for 30sec to fully suspend the thalli, placing the tubes on a shaking table at room temperature, shaking gently for 2h, or placing the tubes into a refrigerator or liquid nitrogen for repeated freeze thawing, centrifuging at 12000 rpm at low temperature (4 ℃) for 5min, and taking the supernatant for subsequent experiments.

5) Preparing a Click working solution: the bacterial protein solution was quantified by BCA method and diluted to 1.6mg/mL using bacterial lysate. Per 100 uL reaction add 6. Mu.L of 1 XTTA (1.7 mM), 2. Mu.L of 50 mM CuSO ₄ After 0.6. Mu.L of 10 mM rhodomine-N3 and 2. Mu.L of LTCEP (concentration 14.4mg/mL,50 mM ready for use in the present preparation), 12. Mu.L of the mixture was pipetted and rapidly added to each sample, and after vortexing and centrifugation, 4h was reacted at 29 ℃;

6) Acetone precipitation: after the Click reaction is finished, 1mL glacial acetone is added into each sample tube to precipitate protein (the acetone is put to-20 ℃ in advance), and the mixture is kept stand at-20 ℃ for 20-30 min or stays at-20 ℃ overnight. Centrifuging the ice-cooled sample tube at 12000 rpm (or highest rotation speed) at 4 deg.C for 10 min, and removing supernatant acetone;

7) Ultrasonic redissolution and glue running: adding 30 mu L of 1X loading buffer into each sample tube, and carrying out water bath ultrasound; after the ultrasonic treatment, the sample tube is placed at 95 ℃ to be boiled for 5min, then is taken out to be slightly shaken, and is boiled for 5min again, and the total time is 10 min. 20 μ L of each sample was run on SDS-PAGE gels and subsequently fluorescence imaged. The gels were stained with Coomassie Brilliant blue and then rinsed, and the washed gels were examined for total protein.

The results show that the synthesized sofalcone probe can bind to the labeled bacterial protein, and the effect is dose-dependent. Further indicates that the sofalcone probe can be used as an anti-inflammatory drug.

Example 9

In this example, the experiment of in-situ competition between small molecule active probes of sofalcone and sofalcone in live staphylococcus aureus is shown as follows:

1) Shake bacteria overnight until cells enter stationary phase 1h, collect bacteria, centrifuge at 8000 rpm for 10 min at low temperature (4 ℃), wash with PBS once, remove PBS, add PBS to resuspend bacteria, take 1mL to add to new EP tube, label blank, probe set, 1.

2) Preparing a sofalcone raw pesticide: the technical product of sofalcone is weighed and added with DMSO to prepare a mother solution with the concentration of 100mM for later use. 1, adding 2.5 mu L of a technical sofalcone solution into 10 groups of EP tubes, wherein the concentration of the medicine in the system is 250 mu M respectively, performing vortex mixing, then performing instant centrifugation for several seconds, and incubating for 4 hours at room temperature and 500 rpm/min.

3) Preparation of sofalcone probe solution: the sofalcone probe was weighed and added with DMSO to prepare a 100mM stock solution for use. Then DMSO is used to dilute the sofalcone mother liquor into a solution with the concentration of 25 mM for standby.

4) Adding 1 mu L of the sofalcone probe solution with the concentration gradient into a probe set and a 1; after 8h, centrifuging at 8000 rpm at low temperature (4 ℃) for 5min, washing with PBS for three times, removing PBS, adding 200 mu L of bacterial lysate into each tube of EP tube, oscillating on a vortex oscillator for 30sec to fully suspend the thalli, placing on a shaking table at room temperature, gently oscillating for 1h, ultrasonically cracking, centrifuging at 12000 rpm at low temperature (4 ℃) for 5min, and taking the supernatant for subsequent experiments.

5) Protein quantification: the bacterial protein solution was quantified by BCA method and diluted to 2mg/ML using bacterial lysate. Per 100 uL reaction add 6. Mu.L of 1 XTTA (1.7 mM), 2. Mu.L of 50 mM CuSO ₄ After 0.6. Mu.L of 20 mM rhodomine-N3 and 2. Mu.L of TCEP (concentration of 14.4mg/ml,50 mM ready-to-use ready-made) are mixed, 13. Mu.L of the mixture is absorbed and rapidly added into each sample respectively, and the mixture is mixed, vortexed and centrifuged to react for 4 hours at 29 ℃;

7) Ultrasonic redissolution: adding 50 mu L of 1X loading buffer into each sample tube, and carrying out water bath ultrasound;

8) Glue running: after the ultrasonic treatment, the sample tube is placed at 95 ℃ to be boiled for 5min, then is taken out to be slightly shaken, and is boiled for 5min again, and the total time is 10 min. A15. Mu.L aliquot of each sample was run on SDS-PAGE gels and subsequently fluorescence imaged. After photographing under fluorescence, the gel was stained with Coomassie brilliant blue, and then rinsed, and the total amount of protein was observed by photographing the washed gel.

The results are shown in FIG. 3. Fig. 3 shows that the small molecule active sofalcone probe prepared in example 1 can specifically bind to a protein bound by sofalcone, and it is proved that the probe has almost no difference compared with the original drug, and can substitute the original drug to perform binding research on related protein targets, thereby providing a theoretical basis for further research on the mechanism of sofalcone.

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 sofalcone small molecule active probe is characterized in that the sofalcone small molecule active probe is a compound with a structure shown in a formula I:

Ⅰ

wherein n is an integer of 0 to 6.

2. The sofalcone small molecule active probe according to claim 1, wherein n in formula I is 1,2,3 or 4.

3. A preparation method of a sofalcone small molecule active probe comprises the following steps:

adding sofalcone and alkynylamine into a solvent, and reacting in the presence of a condensing agent and an alkaline reagent to obtain the sofalcone small-molecule active probe; the alkynylamine is a compound having the structure shown in formula II:

Ⅱ

wherein n is an integer of 0 to 6.

4. The process according to claim 3, wherein the molar ratio of sofalcone to alkynylamine is 1:0.8-5.

5. The method according to claim 3, wherein the solvent is at least one selected from the group consisting of water, acetonitrile, methanol, ethanol, ethyl acetate, N-dimethylformamide, dimethyl sulfoxide, methylene chloride, chloroform and dimethyl sulfoxide.

6. The method according to claim 3, wherein the condensing agent is at least one selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, O-benzotriazol-tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, and 4-dimethylaminopyridine.

7. The method according to claim 3, wherein the basic reagent is at least one selected from the group consisting of N, N-diisopropylethylamine, N-diethylethylamine, potassium tert-butoxide, sodium hydride, potassium hydride, pyridine, diazabicyclo, sodium ethoxide, sodium methoxide, sodium bicarbonate and sodium carbonate.

8. The method according to claim 3, further comprising a step of extracting and purifying the sofalcone small molecule active probe from the solution after the reaction.

9. The use of the sofalcone small molecule active probe of claim 1 as a sofalcone labeled probe.

10. The sofalcone small molecule active probe of claim 1 is applied to preparing antibacterial and anti-inflammatory drugs, wherein the anti-inflammatory drugs are drugs for treating gastritis or gastric ulcer, and the antibacterial drugs are anti-staphylococcus aureus drugs.