CN114456085B - Preparation method and application of 2-azido-3-hydroxypropionic acid - Google Patents

Preparation method and application of 2-azido-3-hydroxypropionic acid Download PDF

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CN114456085B
CN114456085B CN202210130042.1A CN202210130042A CN114456085B CN 114456085 B CN114456085 B CN 114456085B CN 202210130042 A CN202210130042 A CN 202210130042A CN 114456085 B CN114456085 B CN 114456085B
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hydroxypropionic acid
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CN114456085A (en
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易强英
张宇佳
吴尧
康珂
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Sichuan University
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    • C—CHEMISTRY; METALLURGY
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    • C07C247/00—Compounds containing azido groups
    • C07C247/02—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C247/12—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
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Abstract

The invention discloses a preparation method and application of 2-azido-3-hydroxypropionic acid, wherein the preparation method comprises the following steps: (1) Under the ice bath condition, blending an aqueous solution of sodium azide with tert-butyl methyl ether, then adding 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate, stirring for reaction at room temperature, then standing at room temperature, and separating and collecting an organic phase to obtain a product; (2) And (2) mixing the product obtained in the step (1) with N, N-dimethylformamide, L-serine and a potassium bicarbonate aqueous solution, stirring at room temperature for reaction, adding ethyl acetate into the reaction product, adjusting the pH value of an aqueous phase to 1-2, separating liquid and an organic phase, washing and drying to obtain the compound. 2-azido-3-hydroxypropionic acid has toxic effect on cancer cells, and is metabolized to cell membranes in the form of azido phosphatidylserine after being taken in by the cancer cells, so that the permeability of the membranes is changed, and the cells are killed.

Description

Preparation method and application of 2-azido-3-hydroxypropionic acid
Technical Field
The invention relates to the technical field of medical chemistry, in particular to a preparation method and application of 2-azido-3-hydroxypropionic acid.
Background
The azido functional group has extremely important and wide application in organic synthesis, but the compounds have very high energy and have great potential safety hazards in the processes of synthesis, storage and transportation, so that the synthesis and the use are difficult. Researchers have used the diazo transfer mechanism to synthesize the corresponding azide, but the known overlap transfer reagents have the following disadvantages: the synthesis is not easy; substances with explosion risks can be generated in the reaction process; heavy metals are required for diazo transfer reactions as catalysts, etc. However, there are very few reports on the preparation and use of 2-azido-3-hydroxypropionic acid, which is an azide compound, and it is necessary to study the preparation and use of 2-azido-3-hydroxypropionic acid.
Disclosure of Invention
In order to solve the above-mentioned disadvantages of the prior art, the present invention aims to provide a preparation method of 2-azido-3-hydroxypropionic acid and an application thereof, wherein the 2-azido-3-hydroxypropionic acid has a toxic effect on cancer cells, and is metabolized to cell membranes in the form of azido phosphatidylserine after being taken in by the cancer cells, so that the membrane permeability changes, which results in cell death.
The technical scheme for solving the technical problems is as follows: the preparation method of the 2-azido-3-hydroxypropionic acid comprises the following steps:
(1) Under the ice bath condition, mixing an aqueous solution of sodium azide with an organic solvent, adding 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate, stirring and reacting at room temperature for 8-15min, standing at room temperature for 10-30min, separating, and collecting an organic phase, namely a product; wherein the molar ratio of sodium azide to 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate is 1;
(2) And (2) mixing the product obtained in the step (1) with N, N-dimethylformamide, L-serine and an alkali solution, stirring and reacting at room temperature for 20-40min, adding ethyl acetate into the reaction product, adjusting the pH value of a water phase to 1-2, separating liquid and separating an organic phase, washing and drying to obtain the 2-azido-3-hydroxypropionic acid.
On the basis of the technical scheme, the invention can be further improved as follows:
further, the concentration of the aqueous sodium azide solution in the step (1) is 0.3 to 0.6M, and preferably the concentration of the aqueous sodium azide solution is 0.5M.
Further, the organic solvent is tert-butyl methyl ether, ethyl acetate or chloroform.
Further, in the step (1), the volume ratio of the sodium azide aqueous solution to the tert-butyl methyl ether is 1 to 3, preferably 1.
Further, adding 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate into the step (1), stirring at room temperature for 10min, reacting at a stirring speed of 600r/min, and standing at room temperature for 10min.
Further, still include: and (3) standing the organic phase for at least 12h, removing the aqueous phase, and then carrying out the reaction in the step (2).
Further, the molar ratio of the product obtained in the step (1), L-serine and alkali is 1-2.
Further, the alkali solution in the step (2) is a potassium bicarbonate aqueous solution or a sodium hydroxide solution (pH value is 8.4-8.6), preferably a potassium bicarbonate aqueous solution, and the concentration of the potassium bicarbonate aqueous solution is 2-5M.
Further, in the step (2), the reaction is carried out for 30min under stirring at room temperature, and the stirring speed is 600r/min.
Further, the washing process in the step (2) is as follows: the mixture was washed with a saturated aqueous sodium chloride solution, dilute hydrochloric acid, and a saturated aqueous sodium chloride solution in this order for 3 times.
Application of 2-azido-3-hydroxypropionic acid in preparation of antitumor drugs
The invention has the following beneficial effects:
the intermediate product FSO can be generated by sodium azide and 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate 2 N 3 (diazo transfer agent), in particular when a biphasic system of water and immiscible organic solvent is used, the sodium azide in the aqueous phase reacts rapidly with the 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-triflate to form FSO in the organic phase 2 N 3 . Subsequent intermediate FSO 2 N 3 Under alkaline conditions, 2-amino on L-serine in the system can be converted into an azide group to form the final product 2-azido-3-hydroxypropionic acid. The method is completed in an alkaline environment, so that explosive azide acid can be effectively prevented from being generated in the reaction process; in addition, the method can directly prepare the 2-azido-3-hydroxypropionic acid by utilizing the L-serine in one step without protecting other non-reactive groups (1-carboxyl and 3-hydroxyl) of the L-serine by functional groups, thereby saving the cost.
The 2-azido-3-hydroxypropionic acid prepared by the invention has toxic effect on cancer cells, and the cancer cells are metabolized to cell membranes in the form of azido phosphatidylserine after being taken in, so that the change of membrane permeability is caused, and the cell death is caused. The 2-azido-3-hydroxypropionic acid prepared by the invention can be used as micromolecule antitumor drugs, antitumor auxiliary drugs and the like.
Drawings
FIG. 1 is a nuclear magnetic spectrum of 2-azido-3-hydroxypropionic acid.
FIG. 2 shows the survival of each cell line after incubation for 12 hours with different concentrations of drug.
FIG. 3 shows the survival rate of each cell line after 24 hours incubation with different concentrations of drug.
FIG. 4 shows the labeling of azide groups on the cell membrane surface of MCF-7 after incubation for 24 hours at 50ug/mL drug concentration under an inverted fluorescent microscope.
FIG. 5 shows the rupture of cancer cell membrane under confocal microscope (633 nm laser).
Detailed Description
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1:
2-azido-3-hydroxypropionic acid, the structural formula is as follows:
Figure BDA0003502088050000031
the preparation method of the 2-azido-3-hydroxypropionic acid comprises the following steps:
(1) Aqueous sodium azide solution (0.5mL, 40mL, containing 1.3g,20mmol of NaN) 3 ) Blend with tert-butyl methyl ether (MTBE, 40 mL) in a plastic bottle under ice bath conditions. 1- (Fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-triflate (Compound 1,7.9g, 24mmol) was dissolved in acetonitrile (4 mL) and added quickly to a plastic bottle. The ice bath was removed and the mixture was stirred at 600r/min for 10 minutes at room temperature. After the reaction, the plastic bottle was allowed to stand at room temperature for 10 minutes, followed by liquid separation to collect the organic phase. And continuously standing the organic phase for 12 hours, and absorbing and discarding the separated water phase to obtain an organic phase I.
(2) The organic phase was placed in a flask and then added sequentiallyN, N-dimethylformamide (DMF, 40 mL), L-serine (2.1 g), aqueous potassium bicarbonate (3M, 27mL), and stirred at room temperature for 0.5 h. After the reaction was complete, ethyl acetate (400 mL) was added to the mixture and the pH of the aqueous phase was adjusted to 1-2 with dilute hydrochloric acid (1M). The organic phase was separated by liquid separation, washed 3 times with a saturated aqueous sodium chloride solution (containing 0.1M hydrogen chloride, 0.1M lithium chloride, 400 mL), diluted hydrochloric acid (1M, 400mL) and a saturated aqueous sodium chloride solution (containing 0.1M hydrogen chloride, 400 mL) in this order, and the organic phase was collected by liquid separation. The organic phase was then dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to give the product (2-azido-3-hydroxypropionic acid) as a yellow oil in 70% yield, which is shown in FIG. 1 for the nuclear magnetic spectrum, with specific nuclear magnetic data: 1 H NMR(400MHz,CDCl 3 ):δ＝1.26(s,CHCH 2 OH)，3.95(s,CHCH 2 OH)，4.04–4..51(m,-COOH and-OH)。
(1)
Figure BDA0003502088050000041
(2)
Figure BDA0003502088050000042
experimental example 1:
cell lines in logarithmic growth phase were removed separately: human breast cancer cells (MCF-7), mouse skin melanoma cells (B16-F10) and mouse fibroblasts (L929) were plated in 96 well plates at approximately 2X 10 cells per well 4 And (4) cells. After 12 hours of incubation in the incubator, complete medium containing 2-azido-3-hydroxypropionic acid was added to the culture wells at a gradient concentration set to: 0. 20, 40, 60, 80, 100. Mu.g/mL. After incubation for 12 hours and 24 hours, cell viability was measured using a cell counting kit (CCK-8), and the results are shown in FIGS. 2 and 3.
Wherein, FIG. 2 shows the survival rate of each cell line when the drug with different concentrations is incubated for 12 hours, and FIG. 3 shows the survival rate of each cell line when the drug with different concentrations is incubated for 24 hours.
As can be seen from fig. 1 and 2, 2-azido-3-hydroxypropionic acid has an inhibitory effect on cancer cells, has a potential anticancer effect, and can be used as a small-molecule antitumor drug, an antitumor adjuvant drug, and the like.
Experimental example 2:
after incubating complete medium (500. Mu.M) containing 2-azido-3-hydroxypropionic acid with human breast cancer cells (MCF-7) for 24h, 2-azido-3-hydroxypropionic acid on the cell membrane was labeled with DBCO-Cy5 dye (10 ug/mL,37 ℃,30 min). The results were observed by using an inverted fluorescence microscope and a confocal microscope, and are shown in FIGS. 4 and 5.
As can be seen from FIG. 4, the azide group on the cell membrane surface of MCF-7 was labeled with DBCO-Cy5 fluorescent dye (10 ug/mL,37 ℃ C., 30 min) in advance. Under an inverted fluorescence microscope, the surface of the cell membrane has obvious fluorescence signals (the left image of a figure 4 is a bright field image, and the right image is a fluorescence field image), and the fact that the azide groups are metabolized to the cell membrane after the cancer cells take in the 2-azido-3-hydroxypropionic acid is proved.
As can be seen from FIG. 5, the presence of membrane rupture in cells was observed under confocal microscope (633 nm laser), specifically: after uptake by cancer cells, the azide phosphatidylserine is metabolized to the cell membrane, causing a change in membrane permeability, resulting in cell death.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (9)

  1. The application of 2-azido-3-hydroxypropionic acid in preparing antitumor drugs, wherein the 2-azido-3-hydroxypropionic acid is prepared by the following method:
    (1) Under the ice bath condition, mixing an aqueous solution of sodium azide with an organic solvent, adding 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate, stirring and reacting at room temperature for 8-15min, standing at room temperature for 10-30min, separating, and collecting an organic phase, namely a product; wherein the molar ratio of sodium azide to 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate is 1;
    (2) And (2) mixing the product obtained in the step (1) with N, N-dimethylformamide, L-serine and an alkali solution, stirring and reacting for 20-40min at room temperature, adding ethyl acetate into the reaction product, adjusting the pH value of a water phase to 1-2, separating liquid and an organic phase, washing and drying to obtain the 2-azido-3-hydroxypropionic acid.
  2. 2. The use according to claim 1, wherein the concentration of the aqueous sodium azide solution in step (1) is 0.3 to 0.6M.
  3. 3. The use according to claim 1, wherein the organic solvent in step (1) is t-butyl methyl ether, ethyl acetate or chloroform.
  4. 4. The use according to claim 1, wherein the 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazole-3-trifluoromethanesulfonate is added in step (1), and then the mixture is stirred at room temperature for 10min, the stirring speed is 600r/min, and then the mixture is allowed to stand at room temperature for 10min.
  5. 5. The method for producing 2-azido-3-hydroxypropionic acid according to claim 1, further comprising: and (3) standing the organic phase for at least 12h, removing the aqueous phase, and then carrying out the reaction in the step (2).
  6. 6. The use according to claim 1, wherein the molar ratio of the product obtained in step (1), L-serine and base is 1.
  7. 7. The use according to claim 1, wherein in step (2), the reaction is stirred at room temperature for 30min, and the stirring speed is 600r/min.
  8. 8. The use according to claim 1, wherein the alkaline solution in step (2) is an aqueous solution of potassium bicarbonate or sodium hydroxide, and the concentration is 2-5M.
  9. 9. The use according to claim 1, wherein the washing process in step (2) is: the reaction mixture was washed with a saturated aqueous sodium chloride solution, dilute hydrochloric acid, and a saturated aqueous sodium chloride solution in this order for 3 times.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012035436A1 (en) * 2010-09-15 2012-03-22 Tokyo University Of Pharmacy And Life Sciences Plinabulin prodrug analogs and therapeutic uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012035436A1 (en) * 2010-09-15 2012-03-22 Tokyo University Of Pharmacy And Life Sciences Plinabulin prodrug analogs and therapeutic uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Design of Glycosyltransferase Inhibitors: Serine Analogues as Pyrophosphate Surrogates?;Shuai Wang 等;《ChemPlusChem》;20150714;第80卷(第10期);第1525页图解1和Supporting第S3-S4页 *
Modular click chemistry libraries for functional screens using a diazotizing reagent;Genyi Meng 等;《Nature》;20191003;第574卷(第7776期);第86页图1和Supporting1的第S8页第1段过程3-1，第87页图2和第S13页过程4-1以及第S38页过程6-1，第S39-S51页的Plate1-14系列化合物 *
Water-Soluble Prodrug of Antimicrotubule Agent Plinabulin: Effective Strategy with Click Chemistry;Fumika Yakushiji 等;《Chemistry-A European Journal》;20110929;第17卷(第45期);Supporting第3页第2段 *

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