CN115974689A - Tritium-labeled pleuromutilin and synthetic method thereof - Google Patents

Abstract

The invention provides a synthesis method of tritium-labeled pleuromutilin, which comprises the steps that an olefinic bond at the tail end of pleuromutilin forms an aldehyde group under the action of ozone and dimethyl sulfide, the aldehyde group reacts with (1-diazo-2-oxo-propanol) -dimethyl phosphonate to form an acetylene bond, and the acetylene bond and tritium gas undergo selective addition reaction under the action of an inert catalyst to obtain the tritium-labeled pleuromutilin; the invention fills the vacancy of the domestic pleurin drug isotope labeling route, and provides a solution for the metabolic research of the pleurin drugs in animal bodies.

Description

A kind of tritium-labeled pleuromutilin and its synthesis method

technical field

The invention relates to the technical field of radioactive labeling of pleuromutilin, in particular to a tritium-labeled pleuromutilin and a synthesis method thereof.

Background technique

Radioactive tracer technology is the main means of absorption, distribution, transformation and excretion (ADME) of research drugs recommended by the US Food and Drug Administration (FDA) and the International Veterinary Drug Registration Harmonization (VICH). In the non-clinical research stage of antibiotics, metabolism and excretion studies are an important part of determining the maximum residue limit of drugs in target animals, and determine the residual target tissues and residual markers.

Radioactive tracer technology has the advantages of high sensitivity, less interference, and easy operation. When studying the metabolism of veterinary drugs in animals, it can locate and quantitatively analyze labeled drugs and their metabolites, and obtain the distribution and elimination rules of each metabolite in different tissues. , and then obtain the residual target tissue and residual markers of the drug in the animal body. ³ H (tritium) and ¹⁴ C are the most commonly used radioactive tracer nuclides, which will not affect the biological activity of the drug after labeling, and have the advantages of long half-life, low radiation energy, high safety, and high detection sensitivity. ³ H labeling has the characteristics of easy tritium source, convenient synthesis and low price, so it is widely used in biotechnology, pharmaceutical development and agriculture and other fields.

Pleuromutilin drugs started in the 1990s and have good antibacterial activity against Gram-positive bacteria and mycoplasma. Pleuromutilins are widely used. As veterinary medicines, tiamulin and warnemulin are used to prevent and treat diseases such as swine dysentery and mycoplasma pneumonia caused by pathogenic microorganisms, but the metabolism research in animals is not detailed. In recent years, major breakthroughs have been made in the progress of this type of drug in human medicine, among which retapamulin is mainly used to treat short-term impetigo caused by local skin infection outside the human body or wound infection caused by staphylococcus and streptococcus. Lefamorelin is a special drug for the treatment of community-acquired bacterial pneumonia, and its clinical efficacy is not inferior to that of moxifloxacin.

In addition, the metabolism study of drugs in animals is an important part of evaluating drug safety. When the exposure ratio level is higher than that of the parent drug, a nonclinical safety evaluation of the metabolite is required. In order to obtain the above data, it is internationally recommended to use radioactive tracer technology to study the metabolic process of drugs in experimental animals, but the isotope labeling route of pleuromutilin drugs has not been reported in detail, which limits the development of pleuromutilin drugs with application. Therefore, it is necessary to develop a method for isotope (tritium) labeling on the parent ring structure of pleuromutilin drugs, which provides a solution for the metabolism research of this type of drugs in animals.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art and provide a method for synthesizing tritiated-labeled pleuromutilin.

The present invention is achieved like this:

The invention provides a synthetic method of tritiated pleuromutilin, which comprises forming an aldehyde group under the action of ozone and dimethyl sulfide at the terminal olefinic bond of pleuromutilin, and the aldehyde group is substituted with (1-diazo-2-oxo -propanol)-phosphonic acid dimethyl ester reacts to form an alkyne bond, and the alkyne bond undergoes selective addition reaction with tritium gas under the action of an inert catalyst to obtain tritium-labeled pleuromutilin.

Further, the synthetic route is as follows:

Further, the specific steps are as follows:

The first step, pleuromutilin 1 is dissolved in an organic solvent. In a low temperature environment, ozone is added to form an epoxide. After removing excess ozone with nitrogen, dimethyl sulfide is added, and then the epoxide is hydrolyzed to form an aldehyde. base compound, intermediate 2 is obtained after extraction and drying;

In the second step, intermediate 2 is under alkaline conditions, and the aldehyde group reacts with (1-diazo-2-oxo-propanol)-phosphonic acid dimethyl ester for carburization, and is extracted, concentrated and recrystallized to obtain Intermediate 3 of the terminal alkyne bond;

In the third step, the alkyne bond of the intermediate 3 undergoes a selective addition reaction with tritium gas under the action of an inert catalyst, and the target product 4 is obtained by lyophilization after liquid phase preparation.

Further, the organic solvent is dichloromethane, methanol or ethanol.

Further, in the first step, the low temperature environment is -60°C to -70°C.

Further, in the second step, the reagent used in the alkaline environment is C (potassium carbonate).

Further, in the third step, the inert catalyst is 5% Pd/CaCO ₃ .

The present invention provides tritium-labeled pleuromutilin synthesized according to the above method.

The present invention also provides the application of the tritiated-labeled pleuromutilin drugs prepared from the tritiated-labeled pleuromutilins in animal metabolism research.

The present invention has the following beneficial effects:

1. The present invention fills the vacancy of the isotope labeling route of pleuromutilin drugs in China, and provides a solution for the metabolism research of such drugs in animals;

2. The structure of the markers prepared by the present invention is stable, which improves the accuracy and scientificity of this type of drug metabolism research.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the proton nuclear magnetic resonance NMR spectrogram of the compound 1 provided in the embodiment of the present invention;

Fig. 2 is the proton nuclear magnetic resonance NMR spectrogram of intermediate 3 provided in the embodiment of the present invention;

Figure 3 is a mass spectrum information diagram of Intermediate 3 provided in the Examples of the present invention;

Figure 4 is a chemical purity diagram of tritiated pleuromutilin provided in the examples of the present invention;

Fig. 5 is a graph showing the radiochemical purity of tritiated pleuromutilin provided in the examples of the present invention;

Figure 6(A) shows the stability of the chemical purity of tritiated pleuromutilin provided in the examples of the present invention;

Fig. 6(B) shows the radiochemical purity stability of tritiated pleuromutilin provided in the examples of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

The specific steps of the synthesis of tritium-labeled pleuromutilin in the present invention are as follows:

first step:

Take 10 g of compound 1 pleuromutilin (light yellow, 26.42 mmoL), dissolve it in 200 mL of dichloromethane, and cool to -70 °C. At the same temperature, add ozone until the solution turns from light yellow to blue (30 minutes), and control the temperature at -60°C to -70°C. After the solution turned blue, the mixture was then added with ozone for 2-5 minutes. Then, the blue solution was re-dosed with _N2 until it turned back to light yellow (10 min), then _N2 was added for 2-5 min. Then, Me2S (8.21 g, 132.10 mmol, 9.70 mL) was added dropwise to the light yellow solution. After the addition, the reaction was heated to 20°C-25°C and stirred for 2-2.5h. The starting material was consumed as monitored by TLC, and the reaction solution was diluted with dichloromethane (250 mL), extracted with water (100 mL), and brine (150 mL). The aqueous phase was extracted with dichloromethane (150mL), the organic phase was collected, dried with anhydrous sodium sulfate for 4-6 hours, concentrated and evaporated to dryness to obtain the product intermediate 2 (11.0g, 17.35mmoL, 65.66% yield, 60% purity) directly into the next step reaction without further purification;

Step two:

Take 10.0g (15.77mmoL) of the light yellow solid product of the first step and 3.92g (20.50mmoL) (1-diazo-2-oxo-propanol)-phosphonic acid dimethyl ester, dissolve in 132mL methanol, dissolve Then add 4.36g (31.54mmoL) potassium carbonate to the above solution. The above mixed solution was stirred at 20°C-25°C for 14-16h. TLC (PE:EA=1:1, KMnO4, dinitrophenylhydrazine) showed that the starting material was consumed. The reaction solution was washed with ethyl acetate (30 mL), filtered, and the filtrate was collected and concentrated to give a yellow gum. The above substances were dissolved in ethyl acetate (500 mL), extracted with water (150 mL), the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, and dried over anhydrous sodium sulfate. Concentrate to obtain a light yellow gum, mix the solid with a mixed solution of n-hexane/acetone = 30:1 (50 mL), collect the filter residue and wash it with hexane (10 mL), freeze-dry the filter residue to obtain 3.80 g of white powder of Intermediate 3 , the above solid was added to ethyl acetate for recrystallization, filtered after cooling, and the filter residue was collected and dried to obtain 2.2 g of white solid (5.54 mmol, yield 35.13%, purity 96%);

third step:

Take 2g (5mmoL) of the white solid in the second step, dissolve it in 40mL of ethanol, add 100mg of an inert catalyst (5%Pd/CaCO ₃ ), pass tritium gas at 25°C and stir for 1.5 hours. After the disappearance of the raw material point was monitored by TLC, the reaction solution was concentrated and decomposed with 50 mL of dichloromethane, and 20 mL of water was added for extraction, and the organic phase was collected and concentrated to obtain a colloidal liquid. Add 10mL of ethyl acetate to it, heat to dissolve, then transfer to -20°C and stir for 20h, crystals can be observed to precipitate, filter and dry to obtain the final product 1.34g white powder-like solid, the obtained tritium-labeled Pleurotus truncated The pleuromutilin was prepared using a liquid phase preparative chromatography system (SepaBean machine T) to improve the chemical purity of tritiated pleuromutilin. See Table 1 for the liquid phase conditions. The collected fractions were monitored for TIC purity, the collected fractions with higher purity were mixed and the organic phase was evaporated to dryness, an equal volume of dichloromethane was added for multiple extractions, the organic phases were collected, and tritium-labeled cut-off was obtained after freeze-drying. Pleuromutilin (3.51moL, yield 70.2%, chemical purity 98.2%, radiochemical purity ≥ 97%, specific activity 28.07Ci/g).

Table 1

Example 2

The specific steps of the synthesis of tritium-labeled pleuromutilin in the present invention are as follows:

first step:

Take 10 g of compound 1 pleuromutilin (light yellow, 26.42 mmoL), dissolve it in 200 mL of ethyl acetate, and cool to -50 °C. At the same temperature, add ozone until the solution turns from light yellow to blue, and control the temperature at -50°C to -600°C. When the solution turned blue, _N2 was added again until it turned back to pale yellow. Then, Me2S (8.21 g, 132.10 mmol, 9.70 mL) was added dropwise to the light yellow solution. After the addition, the reaction was heated to 20°C-25°C and stirred for 5-8h. The starting material was consumed as monitored by TLC, and the reaction solution was diluted with dichloromethane (250 mL), extracted with water (100 mL), and brine (150 mL). The aqueous phase was extracted with ethyl acetate (150 mL), the organic phase was collected, dried with anhydrous sodium sulfate for 4-6 hours, concentrated and evaporated to dryness to obtain the product intermediate 2 as a pale yellow solid, which was directly put into the next reaction without further purification;

Step two:

Take 10.0g (15.77mmoL) of the light yellow solid product of the first step and 3.92g (20.50mmoL) (1-diazo-2-oxo-propanol)-phosphonic acid dimethyl ester, dissolve in 130mL ethanol, dissolve Then add 3.3g (31.5mmoL) sodium carbonate to the above solution. The above mixed solution was stirred at 20°C-25°C for 6-8h. TLC (PE:EA=1:1, KMnO4, dinitrophenylhydrazine) showed that the starting material was consumed. The reaction solution was washed with dichloromethane (30 mL), filtered, and the filtrate was collected and concentrated to give a yellow gum. The above substances were dissolved in dichloromethane (500 mL), extracted with water (150 mL), the aqueous phase was extracted with dichloromethane (100 mL), the organic phases were combined, and dried over anhydrous sodium sulfate. Concentrate to obtain a pale yellow gum, mix the solid with a mixed solution of n-hexane/acetone=30:1 (50 mL), collect the filter residue and wash it with hexane (10 mL), freeze-dry the filter residue to obtain intermediate 3, and the above solid It was added to isopropyl acetate for recrystallization, filtered after cooling, and the filter residue was collected and dried to obtain a white solid.

third step:

Take 2g (5mmoL) of the white powder-like product in the second step, dissolve it in 40mL of ethanol, add 150mg of an inert catalyst (5% Pd/CaCO ₃ ), and pass tritium gas at 35°C and stir for 2.0 hours. TLC monitors that the raw material point disappears and then filters, and the filtrate is concentrated and dissolved with 50 mL of tert-butyl methyl ether. Add 20 mL of water for extraction. The organic phase was collected and concentrated to obtain a colloidal liquid. Add 10mL of ethyl acetate to it, heat to dissolve it, then transfer to -20°C and stir for 10h, crystals can be observed to precipitate, filter and dry to obtain the final product as a white powder-like solid, and use the obtained tritiated pleuromutilin Liquid phase preparative chromatography system (SepaBean machine T) was used for preparation to improve the chemical purity of tritiated pleuromutilin. See Table 1 for liquid phase conditions. The collected fractions were monitored for TIC purity, the collected fractions with higher purity were mixed and the organic phase was evaporated to dryness, an equal volume of dichloromethane was added for multiple extractions, the organic phases were collected, and tritium-labeled cut-off was obtained after freeze-drying. pleuromutilin. (3.51 mol, yield 70.2%, purity 98.2%).

Test case

Compound 1 (pleuromutilin) and intermediate 3 were analyzed by NMR nuclear magnetic resonance, and intermediate 3 was analyzed by mass spectrometry. As can be seen from Figures 1 to 3, the end of compound 3 has an obvious alkyne bond structure. The olefinic bond was reduced to the acetylenic bond, and finally the selective addition reaction of tritium gas on the acetylenic bond was used to complete the labeling of pleuromutilin with tritium isotope.

The chemical purity, radiochemical purity, specific activity and stability of the tritiated pleuromutilin prepared in Example 1 were determined:

Analysis of the chemical purity of tritiated pleuromutilin:

The obtained tritium-labeled pleuromutilin was detected by high performance liquid chromatography, and a solvent blank was set at the same time, and the chemical purity of tritium-labeled pleuromutilin was calculated by the area normalization method. The results are shown in Figure 4, showing that its chemical purity is higher than 98 %. (Phase chromatography detection condition is: chromatographic column XDB-C18; Mobile phase: phosphate buffer: acetonitrile: methanol: pure water = 5:4:2:1; isocratic elution 20min; detection wavelength 210nm; flow velocity 1.0mL/ min).

Radiochemical Purity Analysis of Tritiated Pleuromutilin:

The total radioactivity value of the end of the chromatographic column collected within each minute was detected by LSC, and the results are shown in Figure 5. The radiochemical purity of tritiated warnemulin calculated by the area normalization method was higher than 97%.

Specific activity analysis of tritiated pleuromutilin:

The corresponding chemical content is 0.00801 μg when the radioactive value detected by LSC and liquid phase is 492314 dpm. The specific activity is the ratio of radioactive value to chemical mass in a certain volume, and the specific activity calculated in this study is 28.07Ci/g.

Stability analysis of tritiated pleuromutilin

The chemical and radiochemical purity results of tritiated pleuromutilin during the storage period of December are shown in Figure 6. The results showed that the chemical purity and radiochemical purity of tritiated pleuromutilin reached over 97% within 6 months, and maintained above 96% and 95% within 12 months. It shows that the synthesized tritiated pleuromutilin can maintain good stability at -20℃.

The tritium-labeled pleuromutilin synthesized by the present invention has high purity, good radioactive tracer performance, a radiochemical purity higher than 97%, and a specific activity of 28.07Ci/g, which fills the gap in the isotope labeling route of pleuromutilin drugs in China. It can play a very helpful role in the metabolism research of pleuromutilin drugs in animals, and the synthesized tritium-labeled pleuromutilin has a stable structure, which also improves the accuracy and scientificity of the drug metabolism research.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

1. A synthesis method of tritium-labeled pleuromutilin is characterized by comprising the steps of forming aldehyde group by an olefinic bond at the tail end of pleuromutilin under the action of ozone and dimethyl sulfide, reacting the aldehyde group with (1-diazo-2-oxo-propanol) -dimethyl phosphonate to form an acetylene bond, and carrying out selective addition reaction on the acetylene bond and tritium gas under the action of an inert catalyst to obtain the tritium-labeled pleuromutilin.

2. A method of synthesis of tritiated pleuromutilins according to claim 1, wherein: the synthetic route is as follows:

3. a method of synthesis of a tritiated pleuromutilin according to claim 1 or 2, characterized in that: the method comprises the following specific steps:

dissolving pleuromutilin 1 in an organic solvent, adding ozone to enable an olefinic bond to form an epoxy compound in a low-temperature environment, removing excessive ozone by using nitrogen, adding dimethyl sulfide, hydrolyzing the epoxy compound to generate an aldehyde compound, and extracting and drying to obtain an intermediate 2;

secondly, under the catalysis of alkali, the aldehyde group of the intermediate 2 and (1-diazo-2-oxo-propyl) -dimethyl phosphonate undergo a recarburization reaction, and an intermediate 3 with an acetylene bond at the end is obtained through extraction, concentration and recrystallization;

and thirdly, carrying out selective addition reaction on the acetylene bond of the intermediate 3 and tritium gas under the action of an inert catalyst, and freeze-drying after liquid phase preparation to obtain a target product 4.

4. A method of synthesizing a tritiated pleuromutilin according to claim 3, wherein: the organic solvent is dichloromethane, methanol or ethanol.

5. A method of synthesizing a tritiated pleuromutilin according to claim 3, wherein: in the first step, the low-temperature environment is-60 ℃ to-70 ℃.

6. A method of synthesizing a tritiated pleuromutilin according to claim 3, wherein: in the second step, the reagent used in the alkaline environment is C (potassium carbonate).

7. A method of synthesizing a tritiated pleuromutilin according to claim 3, wherein: in the third step, the inert catalyst is 5% by weight of Pd/CaCO ₃ 。

8. A tritium-labeled pleuromutilin, comprising: a method of synthesis of tritiated pleuromutilins according to any one of claims 1 to 7.

9. Use of a tritium-labeled pleuromutilin drug made from a tritium-labeled pleuromutilin according to claim 8 for in vivo animal metabolism studies.

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