Preparation method and application of deuterium-labeled sphingosine compound and deuterium-labeled ceramide compound

Abstract

The invention provides a preparation method and application of a deuterium-labeled sphingosine compound and a deuterium-labeled ceramide compound. Firstly, deuterium-labeled fatty aldehyde at the tail end of an alkyl chain is successfully synthesized; according to the method, deuterium-labeled sphingosine is synthesized through the steps of reaction with dimethyl methylphosphonate, reaction with fatty aldehyde, reduction reaction, deprotection group removal and the like, and then various deuterium-labeled ceramides are synthesized through condensation of different fatty acid active esters and the deuterium-labeled sphingosine. Each deuterium-labeled intermediate is used as a key node in the synthesis process, so that the gradual evolution of a molecular structure is embodied, the stability of isotope abundance is also ensured, and finally, the synthesis of various deuterium-labeled compounds with important biomedical significance is realized; a powerful tool and technical support are provided for studying the action mechanism and metabolic pathway of fatty aldehyde, sphingosine and ceramide in a living body and diagnosing and treating related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and in particular to a preparation method and application of a deuterium-labeled sphingosine compound and a deuterium-labeled ceramide compound. Background Art

[0002] In the research process of biomedicine and related fields, fatty aldehydes, sphingosine and ceramide have attracted much attention due to their key biological functions. As important biological signaling molecules, fatty aldehydes are deeply involved in energy metabolism and material synthesis, and occupy a key synthon position in the drug synthesis pathway. Long-chain fatty aldehydes are an important link in the synthesis of sphingosine and ceramide and sphingomyelin metabolism. As the cornerstone of the sphingolipid family, sphingosine itself has the function of cell information substances. They play a very important role in basic biological processes such as cell proliferation, differentiation, and receptor function. Ceramide (Cer) is the basic skeleton of various complex sphingolipids. Cer is composed of a sphingosine skeleton and is connected to fatty acid chains of different lengths (C14-C36) through amide bonds. Ceramide plays an important role in the sphingolipid metabolism pathway, affects cell signaling pathways, regulates cell and body metabolism, and is related to the occurrence and development of a variety of diseases. Ceramide or a combination of different types of ceramides may be used as a new biomarker for clinical auxiliary diagnosis, risk assessment and prognosis judgment of a variety of diseases.

[0003] In recent years, with the rapid development of mass spectrometry technology, the use of liquid chromatography-tandem mass spectrometry (LC-MS / MS) to conduct qualitative and quantitative analysis of the corresponding target substances has become the current mainstream means. Isotope dilution mass spectrometry and stable isotope tracer technology have become highly promising methods and technologies in the fields of clinical pharmacokinetic research, molecular biology, food safety, and environmental science. For example, the use of isotope-labeled ceramide can accurately and quickly quantify the level of ceramide in samples such as plasma and serum. The development and promotion of the above-mentioned mass spectrometry technology based on stable isotope-labeled compounds are inseparable from the isotope-labeled compounds of the target to be detected. How to efficiently and economically prepare the relevant labeled compounds has become a key factor in breaking the deadlock.

[0004] In the prior art, there are many methods for synthesizing naturally abundant sphingosine and ceramide reported in the literature, but there are few reports on deuterium-labeled synthesis. Regarding the preparation of deuterium-labeled ceramide compounds, the literature (Engberg, Oskar, Angewandte Chemie-International Edition, 2020, 17383-17387) reported a method for synthesizing C24 ceramide-D28 directly using very expensive deuterium-labeled tetracosanol as a raw material, and the synthesis economy of the target product was poor. In the literature (J. Label Compd Radiopharm 2016; 59: 531-542; European Journal of Organic Chemistry, 2023, 26; Bulletin de la Societe Chimique de France, 1993, 130, 575-583), deuterium sources such as deuterium gas or heavy water are used to directly exchange naturally abundant side chain fatty acids under the action of catalysts to obtain fully deuterium-labeled deuterated fatty acids, and finally construct deuterated ceramide. However, on the one hand, the synthetic operating conditions of this method are harsh or dangerous, and on the other hand, the introduction of deuterium atoms in the product is very irregular. Not only is its isotopic abundance only about 95% at most, but also the deuteration at the target position is incomplete, and the labeling site is non-selective. The above-mentioned document also reports a method for synthesizing a D3 compound, wherein the synthesis route of the method requires 9-10 chemical reactions, and the steps are cumbersome. Each step of the reaction requires strict control of the reaction conditions, product separation and purification operations, which is not only time-consuming and laborious, but also reduces the total yield due to the accumulated material loss and incomplete reaction. In addition, this method is limited to the construction of D3, and the position of the introduced deuterium is limited, which makes it difficult to meet the diverse deuterium labeling requirements. The preparation method of deuterium-labeled ceramide in the above-mentioned document has the disadvantages of high cost, poor economy, inflexible deuterium labeling position, poor selectivity, and unfriendly safety and operation, and is not suitable for large-scale preparation of deuterium-labeled ceramide internal standards.

[0005] Therefore, it is urgently necessary to develop a method for preparing deuterium-labeled sphingosine compounds and deuterium-labeled ceramide compounds. By carefully designing a multi-step organic synthesis route and taking common and inexpensive raw materials as starting materials, high-abundance and high-purity deuterium-labeled sphingosine and a variety of deuterium-labeled ceramide internal standards are successfully synthesized, which is conducive to in-depth research on the metabolic mechanism and physiological and pathological functions of sphingosine and ceramide in organisms, and accurate analysis of their role in key biological processes such as cell signaling and cell apoptosis; provide highly accurate detection tools for clinical diagnosis, and assist in accurate screening and disease monitoring at the early stage of the disease; effectively promote the drug research and development process, improve the reliability of drug efficacy evaluation and the efficiency of drug target research, and lay the foundation for the development of new therapeutic drugs; and in the field of biological sample analysis and detection, enhance the credibility and repeatability of quantitative detection, ensure the quality of detection data, and promote the high-quality development of biomedical research. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a preparation method and application of deuterium-labeled sphingosine compounds and deuterium-labeled ceramide compounds, verifies the feasibility of the synthesis route of deuterium-labeled sphingosine from deuterium-labeled fatty aldehydes at the end of the alkyl chain, and lays a foundation for the construction of various ceramide internal standards. At the same time, the deuterium-labeled sphingosine compounds synthesized above are condensed with different fatty acid active esters to synthesize various deuterium-labeled ceramides, and can provide key precursor raw materials for deuterium-labeled dihydrosphingosine, deuterium-labeled dihydroceramide, and deuterium-labeled sphingomyelin, thereby quickly constructing a ceramide internal standard library and a sphingomyelin internal standard library, which are used for accurate and rapid quantitative determination of ceramide levels in samples such as plasma and serum, and study the role of ceramide in the sphingolipid metabolic pathway, as well as its regulatory role in cell signaling pathways, cell and body metabolism, and provide a basis for clinical auxiliary diagnosis, risk assessment and prognosis judgment of various diseases.

[0007] In one aspect, the present invention provides an alkyl chain terminal deuterium-labeled fatty aldehyde compound, wherein the alkyl chain terminal deuterium-labeled fatty aldehyde compound has the following chemical structure:

[0008] ;

[0009] Wherein, R1 includes any one or more combinations of H or alkyl; n is an integer including 0, 1, 2, 3, ...

[0010] On the other hand, the present invention provides a method for preparing an alkyl chain terminal deuterium-labeled fatty aldehyde compound, wherein the alkyl chain terminal deuterium-labeled fatty aldehyde compound is prepared using an aliphatic methyl ketone compound as a raw material by a DH exchange method; the synthetic route is as follows:

[0011] .

[0012] In the present invention, the aliphatic methyl ketone compounds refer to a class of organic compounds containing a carbonyl (C=O) structure, whose molecular structure contains a methyl (CH3) group and a keto (C=O) group. The general molecular formula of this type of compound is HOCH 2- RC(O)-CH3, wherein R represents a fatty group or an alkyl group, which may be a straight chain or branched alkane group, usually containing 2 or more carbon atoms. Such compounds include 14-hydroxy-2-tetradecanone, 8-hydroxy-2-octanone, 10-hydroxy-2-decanone and the like.

[0013] Due to the growing demand for stable isotope labeled compounds in the fields of biomedical research, analytical testing, etc., especially in the study of fatty aldehydes, sphingosine and ceramide related metabolic pathways and disease mechanisms, there is an urgent need for efficient and economical preparation methods of labeled compounds. The present invention plans to design an innovative preparation technology for alkyl chain terminal deuterium labeled fatty aldehyde compounds. Through in-depth research on the chemical structure, biological metabolic pathways and internal standard design requirements of sphingosine and ceramide, an innovative synthesis route is proposed. The stable isotope atom deuterium (D) is selected as the labeling atom for introduction. At the same time, considering the abundance stability of the internal standard, the risk of isotope abundance dilution of the prepared intermediates and internal standards is avoided. Deuterium labeling is selected at its "inert" position, that is, the terminal carbon chain with inactive chemical properties, and 5-7 D atoms are designed to be introduced. In the early stage of the experiment, it was attempted to directly use expensive iodopropane-D7 as a raw material to synthesize alkyl chain terminal deuterium labeled fatty aldehydes, but it was found through experiments that this method has the problem of low product abundance, which is only about 90%, far lower than the requirements for product abundance in the field of standard research.

[0014] Therefore, in the selection of raw materials, the later experiments abandoned the expensive iodopropane-D7, which has the risk of carbon cation rearrangement during the synthesis process, and used common and cheap raw materials, such as aliphatic methyl ketone 14-hydroxy-2-tetradeconone, etc. Through key steps such as hydroxyl protection, DH exchange, reaction with p-toluenesulfonyl hydrazide, deprotection group, oxidation reaction, and a series of subsequent synthesis steps, the problems of high preparation cost of deuterium-labeled fatty aldehydes at the end of the alkyl chain, low product abundance, and difficulty in deriving and synthesizing deuterium-labeled sphingosine and ceramide series compounds were successfully solved. Through the precise control and synergistic effect of each step, the efficient synthesis from the starting material to the final target product is ensured, and the isotopic abundance of each intermediate and target product is above 98%. The process is controllable and will not cause serious dilution of isotopic abundance. At the same time, the synthesized compounds can not only be used to study the mechanism and metabolic pathway of fatty aldehydes, sphingosine and ceramide in the body, but also can be used as internal standards for quantitative detection, which is of great significance in clinical diagnosis, drug development and other fields.

[0015] Furthermore, in the process of synthesizing compound 4 from compound 3, compound 4 is prepared by a "one-pot method".

[0016] In some embodiments, in order to verify the necessity of the "one-pot method" of the present invention, a comparative verification test was conducted, and the results showed that only when the "one-pot method" was adopted, the obtained compound 4 could have the highest yield, deuteration rate and isotope abundance. This is because the "one-pot method" synthesis of the present invention has significant advantages, which does not require heating and energy consumption, simplifies the operation process, reduces the loss of raw materials and products during the separation process, reduces the experimental operation steps, and reduces the risk of introducing impurities, thereby ensuring that the purity and isotope abundance of the product are both the highest, and significantly improves the synthesis efficiency.

[0017] Furthermore, the preparation method comprises the following steps: step (1) hydroxyl protection, step (2) DH exchange, step (3) reaction with p-toluenesulfonyl hydrazide and reduction, step (4) deprotection group, and step (5) oxidation reaction; in step (3), compound 3 is reacted with p-toluenesulfonyl hydrazide under the action of a reducing agent to be converted into compound 4, and the reducing agent comprises any one or more combinations of sodium borodeuteride, sodium cyanoborodeuteride, and lithium aluminum hydride deuteride.

[0018] In some embodiments, in order to verify the necessity of using "sodium cyanoborodeuteride" as a reducing agent in the present invention, a comparative verification test was conducted, and the results showed that when "sodium cyanoborodeuteride" was used as a reducing agent, the yield and isotopic abundance of the prepared compound 4 could be optimized. This is because when "sodium cyanoborodeuteride" was used as a reducing agent, its reaction activity was more moderate than that of other reducing agents, which was beneficial to improving the selectivity of the reaction and reducing the occurrence of side reactions, thereby helping to significantly improve the purity and isotopic abundance of the product.

[0019] Furthermore, the molar ratio of the compound 3 to the reducing agent is 1:(1.2~3).

[0020] In some embodiments, when "sodium cyanoborodeuteride" is selected as the reducing agent, its molar ratio to compound 3 is 1:1.2~3, and compound 3 reacts smoothly with p-toluenesulfonylhydrazide and is converted into compound 4 with high selectivity, providing high-quality intermediates for subsequent steps and ensuring smooth progress of the synthetic route.

[0021] Furthermore, in the step (1), aliphatic methyl ketone compounds are used as raw materials and reacted with a protecting group reagent to obtain compound 2; in the step (2), compound 2 is subjected to DH exchange to obtain compound 3; in the step (4), compound 4 is deprotected to obtain compound 5; in the step (5), compound 5 is oxidized by an oxidant to generate compound 6, and compound 6 is a fatty aldehyde compound with deuterium labeled at the end of the alkyl chain;

[0022] ;

[0023] Wherein, R1 includes any one or more combinations of H or alkyl; R2 includes any one or more combinations of -Bn, -TBDS, -MOM; and n is an integer including 0, 1, 2, 3, ...

[0024] Furthermore, in step (1), the protecting group reagent includes any one or more combinations of BnBr, TBDSCl, and MOMCl.

[0025] Furthermore, the aliphatic methyl ketone compound and the protecting group reagent are reacted with a base to prepare compound 2, and the base includes an inorganic base or an organic base, including any one or more combinations of sodium bicarbonate, potassium carbonate, triethylamine, and N,N-diisopropylethylamine.

[0026] Furthermore, the molar ratio of the compound 1, the protecting group reagent and the base is 1:(1.2~2):(1.2~2).

[0027] In some embodiments, when MOMCl is selected as a protecting group reagent, the molar ratio of the compound 1, the protecting group reagent, and the base is 1: (1.2~2): (1.2~2), and the reaction is carried out in a dichloromethane solvent, which can effectively protect the hydroxyl group of the compound 1, avoid interference with subsequent reactions, and provide a suitable substrate for subsequent DH exchange reactions; when MOMCl is used in combination with N,N-diisopropylethylamine, at a suitable molar ratio, the reaction conditions are mild, the requirements for the reaction equipment are low, and on the basis of protecting the hydroxyl group, the active sites of the subsequent reactions can be made easier to control, thereby ensuring that the entire synthesis route proceeds in the expected direction and improving the synthesis efficiency and purity of the target product.

[0028] Furthermore, in the step (2), compound 2 is subjected to DH exchange under the action of a base to obtain compound 3; the base comprises any one or more combinations of potassium carbonate, potassium tert-butoxide, sodium methoxide, and sodium bicarbonate; and the deuterium source for DH exchange comprises any one or more combinations of heavy water, deuterated methanol, and deuterated ethanol.

[0029] Furthermore, the molar ratio of the compound 2 to the base is 1:(2-7).

[0030] In the second step reaction of the synthesis of the fatty aldehyde labeled with deuterium at the end of the alkyl chain of the present invention, the preparation of compound 3 mainly depends on the DH exchange reaction, and the base and the deuterium source play a key role in this process; the combination of different bases and deuterium sources will produce different yields and deuterium substitution rates due to their own properties and substrate properties. Therefore, in some embodiments, the combination of different bases and deuterium sources is compared and screened, and the experimental results show that the selection of bases and deuterium sources is greatly related to the yield and isotopic abundance. When sodium bicarbonate is selected as a base, sodium bicarbonate is weak in alkalinity, which is not enough to achieve the hydrogen dissociation of the carbonyl α position, thereby causing the deuterium substitution rate to be too low, so it is not suitable as a reagent for deuterium substitution reaction, and when the base selects potassium carbonate and the deuterium source selects heavy water, the yield of compound 2 converted to compound 3 is the highest, which is 66.7%, and the deuterium substitution rate is the highest, which is 98.8 atom% D, and the operability is better, which should be preferred.

[0031] Furthermore, in the step (4), compound 4 is subjected to removal of the protecting group under certain conditions to obtain compound 5; when R2 is -Bn, the "certain conditions" include the use of a catalyst, and the catalyst includes any one or more combinations of Pd / C and H2; when R2 is -TBDS or MOM, the "certain conditions" include the use of any one or more combinations of fluorine reagents or acid reagents.

[0032] Furthermore, the fluorine reagent or acid reagent includes any one or more combinations of ammonium fluoride, tetrabutylammonium fluoride, hydrochloric acid, sulfuric acid, and trifluoroacetic acid.

[0033] Furthermore, the mass ratio of the compound 4 to the catalyst is 1:(0.05~1).

[0034] Furthermore, in the oxidation reaction of step (5), the oxidant includes any one or more combinations of pyridinium chlorochromate, Dess-Martin periodinane, tetramethylpiperidinium oxide, dimethyl sulfoxide, and sulfur trioxide-pyridine complex.

[0035] In the fifth step reaction of the synthesis of the fatty aldehyde labeled with deuterium at the end of the alkyl chain of the present invention, in the process of converting compound 5 to compound 6, different oxidants are crucial to the yield of compound 6. Therefore, in some embodiments, different oxidants are compared and screened. The experimental results show that when different oxidants are selected, there are obvious differences in the yield of the final compound 6, and when the preferred oxidant is a Dess-Martin oxidant, the yield of the compound 6 obtained at this time reaches the highest. This is because different oxidants have different adaptability to the substrate, which in turn affects the reaction conversion rate.

[0036] Furthermore, the molar ratio of the compound 5 to the oxidant is 1:(1.2~3).

[0037] In another aspect, the present invention provides a use of a fatty aldehyde compound labeled with deuterium at the end of an alkyl chain for preparing a reagent for synthesizing sphingosine and ceramide internal standards, wherein the fatty aldehyde compound labeled with deuterium at the end of an alkyl chain is prepared by the preparation method described in any one of the above technical schemes.

[0038] In some embodiments, the alkyl chain terminal deuterium-labeled fatty aldehyde compound is used as a key precursor for synthesizing sphingosine and ceramide internal standard reagents, and is synergistically reacted with specific phosphorus-containing reagents, bases and solvents under strictly controlled reaction conditions, such as accurately setting the reaction temperature, time and reagent ratio, and efficiently converted into sphingosine and ceramide internal standards through multi-step organic reactions. The prepared internal standard, with its stable isotope labeling characteristics, can accurately correct the errors in sample processing and detection in mass spectrometry and other detection technologies, significantly improve the accuracy and reliability of the determination of sphingosine and ceramide content in biological samples, strongly support biomedical research and clinical diagnosis, and provide key technical support for in-depth exploration of its metabolic mechanism, disease association and drug development.

[0039] In another aspect, the present invention provides a sphingosine compound labeled with deuterium at the end of an alkyl chain, having the following chemical structure:

[0040] ;

[0041] Wherein, n is an integer including 0, 1, 2, 3, ...

[0042] In another aspect, the present invention provides a deuterium-labeled ceramide compound, which is prepared by using the alkyl chain terminal deuterium-labeled sphingosine compound described in the above technical solution, and has the following chemical structure:

[0043] ;

[0044] Wherein, R5 includes any one or more combinations of H or aliphatic alkanes (straight chain or branched chain); n is an integer including 0, 1, 2, 3, ...

[0045] In another aspect, the present invention provides a method for preparing a deuterium-labeled sphingosine compound, wherein the deuterium-labeled sphingosine compound is prepared by using an alkyl chain terminal deuterium-labeled fatty aldehyde compound, wherein the alkyl chain terminal deuterium-labeled fatty aldehyde compound is prepared using an aliphatic methyl ketone compound as a raw material and adopting DH exchange and other reaction steps; the synthetic route is as follows:

[0046] .

[0047] Furthermore, the deuterium-labeled sphingosine compound is obtained by a series of chemical reactions using an amino acid methyl ester compound with protected hydroxyl and amino groups and a deuterium-labeled fatty aldehyde compound at the end of an alkyl chain as key raw materials; the synthesis route is as follows:

[0048] ;

[0049] Wherein, R3 includes any one or more combinations of -TBDS, -MOM, -Trt; R4 includes any one or more combinations of -Boc, -Fmoc, -Cbz, -Alloc, -Pht; R5 includes a C1~C25 alkyl chain (straight chain or branched chain).

[0050] Furthermore, the preparation method comprises the following steps:

[0051] (1) Reaction with dimethyl methylphosphonate: Intermediate 7 is used as a raw material and reacted with dimethyl methylphosphonate to obtain compound 8;

[0052] (2) Reaction with fatty aldehydes: Compound 8 reacts with deuterium-labeled fatty aldehydes to generate compound 9;

[0053] (3) Reduction reaction: Compound 9 reacts with a reducing agent to generate compound 10;

[0054] (4) Removal of protecting groups: Compound 10 is subjected to removal of protecting groups to obtain deuterium-labeled sphingosine compounds;

[0055] Furthermore, in the step (1), compound 7 reacts with dimethyl methylphosphonate under certain conditions to obtain compound 8, and the "certain conditions" include using a base substance, and the base substance includes any one or more combinations of lithium bis(trimethylsilyl)amide, lithium diisopropylamide, and n-butyl lithium.

[0056] In some embodiments, the entire synthesis process starts with an amino acid methyl ester compound, and deuterium-labeled sphingosine is synthesized by reacting with dimethyl methylphosphonate, reacting with deuterium-labeled fatty aldehydes, reducing reactions, and removing protecting groups. Finally, a variety of deuterium-labeled ceramides are synthesized by condensing fatty acid active esters with deuterium-labeled sphingosine. Each step is closely linked, and the product of the previous step provides the necessary raw materials or structural basis for the next step, and together constructs a complete synthesis system. The key steps play an irreplaceable role in introducing deuterium labels, constructing molecular structures, and realizing functional group transformations. Each deuterium-labeled intermediate, as a key node in the synthesis process, not only reflects the gradual evolution of the molecular structure, but also ensures the stability of the isotope abundance, and finally realizes the synthesis of a variety of deuterium-labeled compounds with important biomedical significance, which provides powerful tools and technical support for studying the mechanism of action, metabolic pathways, and diagnosis and treatment of related diseases of fatty aldehydes, sphingosine, and ceramides in vivo. At the same time, the synthesis method of the present invention has significant advantages in raw material selection, synthesis route design and ease of operation, overcomes the problems of high cost and low abundance in the prior art, and has broad application prospects.

[0057] Furthermore, the molar ratio of dimethyl methylphosphonate to the base is 1:(1.2-1.5).

[0058] Furthermore, in the step (2), compound 8 reacts with aliphatic aldehyde under certain conditions to generate compound 9, and the "certain conditions" include using lithium chloride and triethylamine, and the molar ratio of lithium chloride to triethylamine is 1:(1.2~1.5).

[0059] In some embodiments, the reaction conditions of step (2) are compared and screened, and the experimental results show that different reaction conditions are closely related to the yield of compound 9. The results show that when the reaction conditions are a combination of an inorganic lithium salt reagent and a base, the final compound 9 has a better yield, and when a combination of lithium chloride and triethylamine is preferred, the yield of the compound 9 obtained at this time is significantly improved.

[0060] Furthermore, in step (3), the reducing agent includes any one or more of sodium borohydride, lithium tri-tert-butoxyaluminum hydride, and lithium aluminum hydride, and the molar ratio of compound 9 to the reducing agent is 1:(1.2-5).

[0061] In some embodiments, the reducing agent of step (3) was screened comparatively, and the experimental results showed that: in the synthesis process of compound 10, the selection of the reducing agent has a great correlation with the yield of compound 10. When different reducing agents are selected, the yield of the final compound 10 is significantly different, and when the preferred reducing agent is lithium tri-tert-butoxyaluminum hydride, the yield of the compound 10 obtained at this time reaches the highest.

[0062] Furthermore, in the step (4), compound 10 is subjected to certain conditions to remove the protecting group to obtain a deuterium-labeled sphingosine compound. When R3 is -TBDS and R4 is -Boc, "certain conditions" include acidic conditions.

[0063] In another aspect, the present invention provides a deuterium-labeled sphingosine compound prepared by the preparation method described in any one of the above technical schemes.

[0064] In another aspect, the present invention provides a method for preparing a deuterium-labeled ceramide compound, wherein a deuterium-labeled sphingosine compound is reacted with an active ester of a fatty acid to obtain a deuterium-labeled ceramide compound, wherein the deuterium-labeled sphingosine compound is prepared by the preparation method described in any one of the above technical schemes.

[0065] In some embodiments, the aforementioned synthesized D-sphingosine-D7 is used to condense with different fatty acid active esters to synthesize a variety of deuterium-labeled ceramides, and a ceramide internal standard library is constructed to accurately and quickly quantify the ceramide level in samples such as plasma and serum, study the role of ceramide in sphingolipid metabolic pathways, and its regulatory role in cell signaling pathways, cell and body metabolism, and provide a basis for clinical auxiliary diagnosis, risk assessment and prognosis judgment of various diseases. The preparation of a variety of ceramide internal standards including C16 ceramide-D7, C20 ceramide-D7, C22 ceramide-D7, C22:1 ceramide-D7, etc. is achieved, and a ceramide internal standard library is constructed to meet different research and detection needs.

[0066] Furthermore, in the preparation method, the fatty acid is first subjected to a condensation reaction with N-hydroxysuccinimide under certain conditions to obtain a corresponding active ester intermediate, and then the active ester intermediate is condensed again with a deuterium-labeled sphingosine compound under certain conditions to obtain a deuterium-labeled ceramide compound; the certain conditions for the reaction of the fatty acid with N-hydroxysuccinimide include the use of an alkaline substance and a condensing agent; the alkaline substance includes any one or more combinations of triethylamine and diisopropylethylamine.

[0067] Furthermore, in the fatty acid, the number of carbon atoms includes any one or more combinations of 2 to 26.

[0068] Furthermore, the fatty acid includes any one or more combinations of palmitic acid, eicosanoic acid, and docosanoic acid; the prepared deuterium-labeled ceramide compound includes any one or more combinations of C16 ceramide-D7, C20 ceramide-D7, C22 ceramide-D7, or C22:1 ceramide-D7.

[0069] Furthermore, when the fatty acid is palmitic acid, palmitic acid active ester is obtained, and the palmitic acid active ester is condensed with D-sphingosine-D7 to obtain C16 ceramide-D7.

[0070] Furthermore, when the fatty acid is eicosanoic acid, eicosanoic acid active ester is obtained, and the eicosanoic acid active ester is condensed with D-sphingosine-D7 to obtain C20 ceramide-D7.

[0071] Furthermore, when the fatty acid is behenic acid, behenic acid active ester is obtained, and the behenic acid active ester is condensed with D-sphingosine-D7 to obtain C22 ceramide-D7.

[0072] Further, the condensing agent includes any one or more combinations of dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / 1-hydroxybenzotriazole (EDCI / HOBT), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (HATU).

[0073] 1-Hydroxybenzotriazole HOBT is generally not used as a condensation agent, and is generally used as a catalyst in combination with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI.

[0074] In some embodiments, the combination of different bases and condensing agents has a great correlation with the yield of deuterium-labeled ceramide compounds. When the preferred base is triethylamine and the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the yield of the deuterium-labeled ceramide compounds obtained at this time is significantly improved.

[0075] Furthermore, the certain conditions for the reaction of the active ester intermediate with the deuterium-labeled sphingosine compound include using any one or more combinations of dichloromethane and tetrahydrofuran.

[0076] Furthermore, the molar ratio of the fatty acid, the condensing agent and the base is 1:1~1.5:1.2~1.5.

[0077] Beneficial effects achieved by the present invention:

[0078] 1. The present invention provides a preparation method and application of deuterium-labeled sphingosine compounds and deuterium-labeled ceramide compounds, innovatively designs a multi-step organic synthesis route, starts with common and inexpensive raw materials, cleverly uses a variety of common reagents to participate in the reaction, and accurately controls the reaction conditions of each step, successfully overcoming the previous difficulties of high preparation cost, cumbersome steps, and expensive raw materials. Successfully synthesized high-abundance, high-purity deuterium-labeled sphingosine (such as D-sphingosine-D7), realized the preparation of a variety of ceramide internal standards including C16 ceramide-D7, C20 ceramide-D7, C22 ceramide-D7, C22:1 ceramide-D7, etc., constructed a ceramide internal standard library, and met different research and detection needs.

[0079] 2. The preparation method and application of a deuterium-labeled sphingosine compound and a deuterium-labeled ceramide compound provided by the present invention verify the feasibility of the synthetic route of deuterium-labeled sphingosine from deuterium-labeled fatty aldehyde at the end of the alkyl chain, successfully synthesize deuterium-labeled sphingosine, provide necessary intermediates for the subsequent synthesis of ceramide, promote the synthesis process of the entire deuterium-labeled ceramide series compounds, and lay the foundation for the construction of various ceramide internal standards.

[0080] 3. The various deuterium-labeled compounds prepared by the present invention, as key nodes in the synthesis process, not only reflect the gradual evolution of the molecular structure, but also ensure the stability of the isotope abundance, and finally realize the synthesis of various deuterium-labeled compounds with important biomedical significance, and provide powerful tools and technical support for studying the mechanism of action, metabolic pathways and diagnosis and treatment of related diseases of fatty aldehydes, sphingosine and ceramide in vivo. At the same time, the synthesis method of the present invention has significant advantages in terms of raw material selection, synthetic route design and ease of operation, overcomes the problems of high cost and low abundance in the prior art, and has broad application prospects. At the same time, as an ideal tracing tool, it provides key support for in-depth exploration of the dynamic changes and transformation paths of the complex metabolic network of fatty aldehydes, sphingosine and ceramide in vivo. It can accurately analyze its mechanism of action in key biological processes such as cell signaling and apoptosis, dig key clues for the study of the mechanism of disease occurrence and development, and effectively promote the discovery of disease diagnostic markers and the determination of new therapeutic targets, accelerating the progress of medical research.

[0081] 4. The deuterium-labeled sphingosine compounds and deuterium-labeled ceramides synthesized in the present invention can be used as key precursor materials for deuterium-labeled dihydrosphingosine, deuterium-labeled dihydroceramide and deuterium-labeled sphingomyelin, thereby quickly constructing a ceramide internal standard library and a sphingomyelin internal standard library. When applied to quantitative detection technologies such as LC-MS / MS, they can effectively calibrate the errors caused by various factors in the detection process, greatly improve the accuracy, precision and reliability of the determination of sphingosine and ceramide contents in biological samples, provide a solid guarantee for the precision of clinical diagnosis, help improve the efficiency and quality of drug research and development, and at the same time strengthen the quality control system of biomedical research, lay a solid foundation for promoting the efficient transformation of scientific research results into clinical practice, and promote the vigorous development and technological innovation in the biomedical field in all aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 :Synthesis process of fatty aldehyde compounds labeled with deuterium at the end of alkyl chain of the present invention

[0083] Figure 2 :NMR spectrum of compound 2a

[0084] Figure 3 :NMR spectrum of compound 3a

[0085] Figure 4A :NMR spectrum of compound 4a (one-pot method)

[0086] Figure 4B :NMR spectrum of compound 4a (non-one-pot method)

[0087] Figure 5 :NMR spectrum of compound 6a

[0088] Figure 6 :The synthesis process of deuterium-labeled sphingosine and ceramide of the present invention

[0089] Figure 7 :NMR spectrum of compound D-sphingosine-D7

[0090] Figure 8 :NMR spectrum of compound C16 ceramide-D7

[0091] Fig. 9 :NMR spectrum of compound C20 ceramide-D7

[0092] Fig.10 :NMR spectrum of compound C22 ceramide-D7

[0093] Fig.11 :NMR spectrum of compound C22:1 ceramide-D7 DETAILED DESCRIPTION

[0094] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. "Including" and similar words used in this article mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0095] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0096] In the present invention, "yield" refers to the ratio of the amount of the target product actually obtained to the amount of the target product that can be obtained theoretically. The calculation method is: yield = (actual yield / theoretical yield) × 100%.

[0097] “Isotopic abundance” refers to the percentage of the number of atoms of a particular isotope in a mixture of isotopes of an element to the total number of atoms of that element, expressed as atom% A, where A is the code for the element symbol of the particular isotope.

[0098] In the deuterium-labeled compound of the present invention, the isotope abundance indicates the percentage of the number of deuterium atoms in the total number of hydrogen atoms in the target molecule, which can generally be measured by mass spectrometry. If the MS response of the product is weak, it can also be measured by NMR. For example, the isotope abundance of compound 3a was measured to be 98.8 atom% D, which means that the relative content of deuterium atoms at a specific position of the compound has reached a high level, indicating that the synthesis method of the present invention has high selectivity and efficiency when introducing deuterium atoms, can effectively control the degree of isotope labeling, and meet the requirements for high isotope abundance of the labeled compound, thereby ensuring its accuracy and reliability in subsequent applications such as as an internal standard.

[0099] Example 1: Optimal Synthesis Method of Alkyl Chain Terminal Deuterium-Labeled Fatty Aldehydes, Deuterium-Labeled Sphingosine and Ceramide of the Present Invention

[0100] Fatty aldehydes play an important role in the field of organisms, environment and medicine, and deuterium-labeled fatty aldehydes are important intermediates for the synthesis of sphingosine and ceramide, and their preparation is of key significance for the study of related biological processes and diseases. At the same time, the development of mass spectrometry technology based on stable isotope-labeled compounds depends on efficient and economical methods for preparing labeled compounds. Therefore, this embodiment aims to find a new method for preparing fatty aldehydes labeled with deuterium at the end of an alkyl chain to solve the problem of high preparation cost and unsuitability for economical and rapid preparation of internal standards for sphingosine and ceramide in the prior art.

[0101] During the development of the present invention, an initial attempt was made to directly use alkynes and expensive iodopropane-D7 as a synthesis method for starting materials. However, experiments have found that this method has the problem of low product abundance, with an abundance of only about 90 atom% D. Theoretical analysis and experimental verification have speculated that the main reasons for this phenomenon are the following two aspects: first, in a strong base reaction environment, the deuterium atom (D) at the α position of the alkynyl group will exchange with the hydrogen atom (H) in the reaction system environment; second, the iodopropane molecule is very likely to undergo carbon cation rearrangement during the reaction process. During this process, the deuterium atom (D) in the iodopropane-D7 molecule will exchange with the hydrogen atom (H) in the system environment, thereby reducing the product abundance. In the field of standard product research, there are strict requirements for product abundance. Generally, an abundance of 95% is regarded as the basic bottom line, and a more ideal abundance value should be above 98%. Therefore, the initial attempt at the synthesis method obviously cannot meet the expected standards.

[0102] Based on the above research background and problems, this example proposes an innovative synthesis method of alkyl chain terminal deuterium labeled fatty aldehydes based on comprehensive consideration of the chemical structure, biological metabolic pathways and internal standard design requirements of sphingosine and ceramide. The internal standard molecule design follows the quality by design (QBD) concept, fully considering the stability, reliability and economy of the target molecule. In the present invention, the design and synthesis of fatty aldehyde, sphingosine and ceramide labeled compounds complement each other. Through the analysis and research of the synthetic route of sphingosine, the synthesis of deuterium (D) labeled fatty aldehyde is also a key intermediate for the synthesis of deuterium labeled sphingosine. On the other hand, considering that ceramide (Cer) is usually further metabolized into sphingosine and fatty acids with different carbon chain lengths and double bond numbers in the body, the design and introduction of labeled atoms on the "sphingosine" carbon chain skeleton (i.e., fatty carbon chain) of ceramide will make the rapid construction of the ceramide internal standard library extremely convenient and economical. Considering that the raw materials for constructing carbon 13 labeling are extremely expensive and the synthesis process is difficult, the stable isotope atom deuterium (D) is selected as the labeling atom for introduction. In addition, in order to ensure that the molecular weight of the internal standard and the analyte (natural abundance Cer) has a certain difference so as to be more conducive to detection, the number of deuterium labels is designed to introduce 5-7 D atoms.

[0103] The specific synthesis method is as follows:

[0104] 1. Synthesis of fatty aldehydes labeled with deuterium at the end of the alkyl chain

[0105]

[0106] The specific synthesis process is as above Figure 1 As shown, R1 can be H or alkyl; R2 can be -Bn, -TBDS, -MOM, etc.; n can be 0, 1, 2, 3, ..., etc.

[0107] In the present invention, taking compound 2 as an example, intermediate 2 is compound 2, compound 2 is a class of compounds, and compound 2a is a specific compound, see Figure 1 In compound 2, R1 can be H or alkyl; R2 can be -Bn, -TBDS, -MOM, etc.; n can be 0, 1, 2, 3, etc. In compound 2a, R1=H, R2=Mom, etc.; n=10.

[0108] 1. The first step: protecting the exposed hydroxyl group of compound 1. In this step, compound 1 can be reacted with a common protecting group reagent (BnBr, TBDSCl, MOMCl, etc.) under the action of a base to obtain intermediate 2 (compound 2a). The base can be an inorganic base or an organic base, such as sodium bicarbonate, potassium carbonate, triethylamine, N,N-diisopropylethylamine, etc. The organic solvent used can be dichloromethane, tetrahydrofuran, etc. The molar ratio of the compound 1, the protecting group reagent, and the base is 1: (1.2~2): (1.2~2).

[0109] The synthesis of compound 2a is as follows:

[0110] At room temperature, 10.0 g of 14-hydroxy-2-tetradecanone (aliphatic methyl ketone compound, Chengdu Kamel Pharmaceutical Technology Co., Ltd.), 100 mL of DCM, 7.4 g of DIEA, and 4.3 g of MomCl were added to a 250 mL single-mouth bottle. After the addition, the reaction was carried out at 25 °C for 16 hours. After the reaction was complete, the reaction solution was poured into water, extracted with dichloromethane, and concentrated under reduced pressure to obtain a crude product, which was separated and purified by Flash column chromatography to obtain intermediate 2a, 9.0 g, with a yield of 75.6%. 1 H NMR (400 MHz, Chloroform-d) δ 4.61 (s, 2H), 3.51 (t, J = 6.7 Hz, 2H), 3.35 (s, 3H), 2.41 (t, J = 7.5Hz, 2H), 2.13 (s, 3H), 1.67 - 1.49 (m, 4H), 1.39 - 1.18 (m, 19H).

[0111] The NMR spectrum of compound 2a is as follows: Figure 2 shown.

[0112] 2. Step 2: Intermediate 2 (Compound 2a) is subjected to DH exchange under the action of a base to obtain intermediate 3 (Compound 3a), wherein the base can be potassium carbonate, potassium tert-butoxide, sodium methoxide, sodium bicarbonate, etc., and the deuterium source can be heavy water, deuterated methanol, deuterated ethanol, etc. The molar ratio of the intermediate 2 to the base is 1:2-7.

[0113] The synthesis of compound 3a is as follows:

[0114] At room temperature, 5.0 g of compound 2a was dissolved in 100 mL of deuterated water and 100 mL of 1,4-dioxane, and 15.6 g of potassium carbonate was added. The reaction was allowed to proceed for 16 hours. After the reaction was completed, ethyl acetate was added for separation and extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. Flash column chromatography was used for separation and purification to obtain 4.6 g of intermediate 3a with a yield of 90.3% and an isotopic abundance of 98.8 atom% D. 1 H NMR (400 MHz, Chloroform-d) δ 4.62 (s, 2H), 3.51 (t, J = 6.6 Hz, 2H), 3.36 (s, 3H), 1.65 - 1.48 (m, 4H), 1.45 - 1.16 (m,16H).

[0115] The NMR spectrum of compound 3a is as follows: Figure 3 shown.

[0116] 3. Step 3: Intermediate 3 (compound 3a) is converted into intermediate 4 (compound 4a) under specific conditions with p-toluenesulfonyl hydrazide. The specific conditions here can be sodium borohydride, sodium borodeuteride, sodium cyanoborohydride, sodium cyanoborodeuteride, lithium aluminum hydride, lithium deuterated aluminum hydride, etc. The molar ratio of the intermediate 3 to the reducing agent is 1:1.2~3.

[0117] The synthesis of compound 4a is as follows:

[0118] 5.0 g of p-toluenesulfonyl hydrazide and 7.5 g of compound 3a were dissolved in 45 mL of CD3OD, and 3.6 g of sodium cyanoborodeuteride was added to react for 16 hours. After the reaction, dichloromethane and water were added for separation and extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was separated and purified by Flash column chromatography to obtain 5.8 g of intermediate 4a with a yield of 80.6% and an isotopic abundance of 98.6 atom% D. 1H NMR (400 MHz, Chloroform-d) δ 4.62 (s, 2H), 3.51 (t, J = 6.7 Hz, 2H), 3.36 (s, 3H), 1.64 - 1.52 (m, 2H), 1.40 - 1.18 (m, 18H).

[0119] The NMR spectrum of compound 4a is as follows: Figure 4A shown.

[0120] 4. Step 4: The intermediate 4 (compound 4a) is deprotected under certain conditions. If R2 is -Bn, the "certain conditions" here can be "common hydrogenation debenzylation methods such as Pd / C / H2". The mass ratio of the intermediate 4 to the catalyst is 1:0.05~0.1. If R2 is -TBDS or MOM, the "certain conditions" here include the use of fluorine reagents or acid reagents, such as ammonium fluoride, tetrabutylammonium fluoride, hydrochloric acid, sulfuric acid, trifluoroacetic acid, etc.

[0121] The synthesis of compound 5a is as follows:

[0122] 5.2 g of compound 4a was dissolved in 50 mL of methanol, and 5.2 mL of concentrated hydrochloric acid was added. After the addition, the mixture was reacted at 65°C for 16 hours. After the reaction, the mixture was diluted with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by flash column chromatography to obtain 4.0 g of a colorless oil with a yield of 92.3% and an isotopic abundance of 98.2 atom%D. 1 H NMR (400 MHz, Chloroform-d) δ 3.64 (t, J = 6.7 Hz, 2H), 1.62 - 1.51 (m,2H), 1.43 - 1.17 (m, 18H).

[0123] 5. Step 5: conventional oxidation reaction, intermediate 5 (compound 5a) is oxidized by an oxidant to generate intermediate 6 (compound 6a), the oxidant can be pyridinium chlorochromate, Dess-Martin oxidant, tetramethylpiperidinium oxide, dimethyl sulfoxide, sulfur trioxide-pyridine complex, etc. The molar ratio of intermediate 5 to oxidant is 1:1.2-3.

[0124] The synthesis of compound 6a (deuterium-labeled fatty aldehyde at the end of the alkyl chain) is as follows:

[0125] 3.0 g of compound 5a was dissolved in 50 mL of dichloromethane, and 8.5 g of DMP was added. The mixture was reacted at 25°C for 3 hours. After the reaction, the reaction solution was poured into an aqueous sodium sulfite solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. Flash column chromatography was used for separation and purification to obtain 2.0 g of a colorless oil with a yield of 67.3% and an isotopic abundance of 98.8 atom% D. 1 H NMR (400 MHz, Chloroform-d) δ 9.76 (d, J = 2.0 Hz,1H), 2.41 (td, J = 7.4, 1.9 Hz, 2H), 1.61 (q, J = 7.1 Hz, 2H), 1.39 - 1.16(m, 18H).

[0126] The NMR spectrum of compound 6a is as follows: Figure 5 shown.

[0127] 2. Synthesis of Deuterium-labeled Sphingosine and Ceramide

[0128]

[0129] The specific synthesis process is as above Figure 6 As shown, R3 includes any one or more combinations of -TBDS, -MOM, -Trt; R4 includes any one or more combinations of -Boc, -Fmoc, -Cbz, -Alloc, -Pht; R5 includes a C1-C25 alkyl chain (straight or branched); n is an integer including 0, 1, 2, 3, ...

[0130] 1. The first step: Compound 7 reacts with dimethyl methylphosphonate under certain conditions to prepare intermediate 8 (Compound 8a). The "certain conditions" here include the use of a base substance, which can be lithium bistrimethylsilylamide, lithium diisopropylamide, n-butyl lithium, etc. The molar ratio of dimethyl methylphosphonate to the base is 1:1.2-1.5.

[0131] Synthesis of compound 8a:

[0132] 74.0 g of dimethyl methylphosphonate was dissolved in 600 mL of THF, cooled to -65°C, and 120 mL of n-butyl lithium was added dropwise. After the addition, the system was reacted at this temperature for 0.5 hours, and 60.0 g of compound 7a was added, and the reaction was continued at this temperature for 3 hours. TLC monitored the reaction to be complete, and the reaction solution was poured into an ice-cold saturated aqueous solution of ammonium chloride, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain a crude product, which was separated and purified by flash column chromatography to obtain 43.0 g of a colorless oil with a yield of 56.1%. 1HNMR (400 MHz, Chloroform-d) δ 5.52 (d, J = 7.4 Hz, 1H), 4.45 (dt, J = 7.8,4.1 Hz, 1H), 4.07 (dd, J = 10.7, 4.0 Hz, 1H), 3.93 – 3.74 (m, 7H), 3.41 (dd,J = 22.0, 14.6 Hz, 1H), 3.13 (dd,J = 22.1, 14.6 Hz, 1H), 1.45 (s, 9H), 0.86(s, 9H), 0.04 (d, J = 1.0 Hz, 6H).

[0133] 2. Step 2: Intermediate 8 (Compound 8a) reacts with aliphatic aldehyde under specific conditions to generate intermediate 9 (Compound 9a). The specific conditions here are lithium chloride and triethylamine conditions. The molar ratio of lithium chloride to triethylamine is 1:1.2-1.5.

[0134] Synthesis of compound 9a:

[0135] 3.7 g of compound 8a, 1.0 g of compound 6a, 1.3 g of triethylamine, 0.6 g of lithium chloride and 50 mL of tetrahydrofuran were mixed in a single-mouth bottle and reacted at 20°C for 16 hours. After the reaction, the reaction solution was concentrated and purified by flash column chromatography to obtain 1.8 g of colorless oil with a yield of 81.8% and an isotopic abundance of 98.8 atom% D. 1H NMR (400MHz, Chloroform-d) δ 6.96 (dt, J = 15.7, 6.9 Hz, 1H), 6.27 (d, J = 15.7 Hz, 1H), 5.54 (d, J = 7.7 Hz, 1H), 4.60 – 4.53 (m, 1H), 3.97 (dd, J = 10.3, 3.5Hz, 1H), 3.83 (dd, J = 10.2, 4.7 Hz, 1H), 2.27 – 2.14 (m, 2H), 1.42 (s, 11H), 1.25 (s, 16H), 0.84 (s, 9H), 0.10 – -0.06 (m, 6H).

[0136] 3. The third step: conventional reduction reaction, the reducing agent can be sodium borohydride, lithium tri-tert-butoxyaluminum hydride, etc. The molar ratio of the intermediate 9 to the reducing agent is 1:1.2-5.

[0137] Synthesis of compound 10a:

[0138] In a three-necked flask, 1.8 g of compound 9a was dissolved in 20 mL of ethanol, cooled to -65°C, and 1.8 g of lithium tri-tert-butoxyaluminum hydride was added. The mixture was reacted at this temperature for 1 hour. After the reaction was completed as monitored by TLC, the reaction solution was poured into 0.1 N dilute hydrochloric acid, extracted with ethyl acetate, and concentrated under reduced pressure to obtain a crude product. Flash column chromatography was used for separation and purification to obtain 1.4 g of a colorless oil with a yield of 77.8% and an isotopic abundance of 98.9 atom% D. 1 H NMR (400 MHz, Chloroform-d) δ5.74 (ddd, J = 13.6, 7.6, 1.4 Hz, 1H), 5.56 – 5.45 (m, 1H), 5.24 (d, J = 8.4Hz, 1H), 4.19 (t, J = 5.1 Hz, 1H), 3.94 (dd, J = 10.3, 3.0 Hz, 1H), 3.75 (dd,J = 10.4, 3.3 Hz, 1H), 3.57 (s, 1H), 2.05 (d, J = 6.4 Hz, 2H), 1.48 – 1.15(m, 27H), 0.90 (s, 9H), 0.07 (d, J = 1.3 Hz, 6H).

[0139] 4. Step 4: The intermediate 10 is deprotected under certain conditions. If R3 is -TBDS and R4 is -Boc, the "certain conditions" here can be common methods such as acidic conditions.

[0140] Synthesis of compound D-sphingosine-D7:

[0141] 1.0 g of compound 10a was dissolved in 20 mL of dichloromethane, and 9 mL of trifluoroacetic acid was added. After the reaction was complete, the reaction solution was poured into a sodium hydroxide solution, the aqueous phase was extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. Flash column chromatography was used to separate and purify the crude product to obtain 0.3 g of a colorless oil with a yield of 50.8% and an isotopic abundance of 99.0 atom% D. 1 H NMR (400 MHz, Methanol-d4) δ 5.74 (dt, J = 14.2, 6.7Hz, 1H), 5.49 (ddt, J = 15.3, 7.4, 1.4 Hz, 1H), 3.97 (t, J = 6.8 Hz, 1H), 3.67 (dd, J = 10.9, 4.5 Hz, 1H), 3.49 (dd, J = 10.9, 6.9 Hz, 1H), 2.84 – 2.71(m, 1H), 2.08 (q, J = 7.1 Hz, 2H), 1.53 – 1.13 (m, 18H).

[0142] The NMR spectrum of compound D-sphingosine-D7 is as follows: Figure 7 shown.

[0143] 5. Step 5: The fatty acid (C2~C26) is subjected to a condensation reaction with N-hydroxysuccinimide under certain conditions to obtain the corresponding active ester intermediate. The certain conditions here include the use of alkaline substances and condensation agents; the alkaline substances include bases such as triethylamine and diisopropylethylamine, and the condensation agents include dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and other condensation agents. The molar ratio of the fatty acid, condensation agent and base is: 1:1~1.5:1.2~1.5. The above-obtained active ester is reacted with D-sphingosine-D7 under certain conditions to obtain different ceramide-D7, and the molar ratio of D-sphingosine-D7 to the active ester is: 1:1~1.5. In the certain conditions here, the organic solvent used can be dichloromethane, tetrahydrofuran, N,N-dimethylformamide and other solvents, and the reaction temperature is 20~50°C.

[0144] Synthesis of Compound C16 Ceramide-D7:

[0145] 0.1 g of sphingosine-D7 and 0.3 g of the active ester corresponding to palmitic acid were dissolved in 3 mL of dichloromethane and reacted at 20°C for 16 hours. After the reaction, the reaction mixture was concentrated to obtain a crude product, which was separated and purified by Flash column chromatography to obtain 0.12 g of a white solid with a yield of 67.4% and an isotopic abundance of 99.0 atom% D. 1 H NMR (400 MHz, Methanol-d4) δ 5.75 – 5.64 (m, 1H), 5.51 – 5.41 (m, 1H), 4.04 (t, J = 7.5 Hz, 1H), 3.89 – 3.81 (m, 1H), 3.69 (d, J = 5.0 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 2.04(q, J = 7.1 Hz, 2H), 1.29 (s, 44H), 0.96 – 0.85 (m, 3H).

[0146] The NMR spectrum of compound C16 ceramide-D7 is as follows Figure 8 shown.

[0147] Synthesis of Compound C20 Ceramide-D7:

[0148] 0.1 g of sphingosine-D7 and 0.48 g of the active ester corresponding to the compound eicosanoic acid were dissolved in 3 mL of dichloromethane and reacted at 20°C for 16 hours. After the reaction, the reaction mixture was concentrated to obtain a crude product, which was separated and purified by Flash column chromatography to obtain 0.14 g of a white solid with a yield of 71.4% and an isotopic abundance of 99.0 atom% D. 1 H NMR (400 MHz, Methanol-d4) δ 5.70 (dt, J = 15.5, 6.7 Hz, 1H), 5.47 (ddt, J = 15.4, 7.5, 1.5Hz, 1H), 4.04 (t, J = 7.5 Hz, 1H), 3.90 – 3.81 (m, 1H), 3.69 (d, J = 5.0 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 2.04 (q, J = 7.8, 7.3 Hz, 2H), 1.29 (s, 52H), 0.97 – 0.84 (m, 3H).

[0149] The NMR spectrum of compound C20 ceramide-D7 is as follows Fig. 9 shown.

[0150] Synthesis of Compound C22 Ceramide-D7:

[0151] 0.15 g of sphingosine-D7 and 0.32 g of the active ester corresponding to the compound docosanoic acid were dissolved in 15 mL of dichloromethane and reacted at 20°C for 16 hours. After the reaction, the reaction mixture was concentrated to obtain a crude product, which was separated and purified by Flash column chromatography to obtain 0.24 g of a white solid with a yield of 77.9% and an isotopic abundance of 99.0 atom% D. 1 H NMR (400MHz, Methanol-d4) δ 5.71 (dt, J = 14.1, 6.6 Hz, 1H), 5.48 (ddt, J = 15.2,7.4, 1.4 Hz, 1H), 4.57 (s, 1H), 4.06 (t, J = 7.5 Hz, 1H), 3.87 (dt, J = 7.6,5.0 Hz, 1H), 3.71 (d, J = 5.0 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 2.06 (q, J =6.9 Hz, 2H), 1.31 (s, 56H), 0.99 – 0.86 (m, 3H).

[0152] The NMR spectrum of compound C22 ceramide-D7 is as follows Fig.10 shown.

[0153] Synthesis of Compound C22:1 Ceramide-D7:

[0154] 0.12 g of sphingosine-D7 and 0.26 g of the active ester corresponding to the compound docosanoic acid were dissolved in 15 mL of dichloromethane and reacted at 20°C for 16 hours. After the reaction, the reaction mixture was concentrated to obtain a crude product, which was separated and purified by Flash column chromatography to obtain 0.19 g of a white solid with a yield of 79.2% and an isotopic abundance of 99.0 atom% D. 1H NMR (400MHz, Methanol-d4) δ 5.75 – 5.64 (m, 1H), 5.46 (ddt, J = 15.4, 7.5, 1.4 Hz, 1H), 5.34 (td, J = 4.6, 2.3 Hz, 2H), 4.04 (t, J = 7.5 Hz, 1H), 3.85 (dt, J =7.6, 5.0 Hz, 1H), 3.69 (d, J = 5.0 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 2.13 –1.95 (m, 6H), 1.66 – 1.53 (m, 2H), 1.51 – 1.14 (m, 46H), 0.97 – 0.84 (m, 3H).

[0155] The NMR spectrum of compound C22:1 ceramide-D7 is as follows Fig.11 shown.

[0156] In this embodiment, through the two-step key reaction of DH exchange and deuterium labeling reduction, the hydrogen atoms on the saturated carbon chain are directional deuterium replacement, and an efficient preparation method of deuterium-labeled fatty aldehydes at the end of an alkyl chain is successfully established, as well as a complete process for further synthesizing deuterium-labeled sphingosine and various ceramides based on this, the method is simple to operate and the synthesis cycle is short. Each deuterium-labeled intermediate involved is a new compound. At the same time, the synthesis of deuterium-labeled compounds and their derivatives such as deuterium-labeled alkyl alcohols and deuterium-labeled carboxylic acids with different carbon chain lengths or containing branched structures is realized, and the synthesis scheme is brand new. And the raw materials and reagents used in the scheme are common commercial products and are easy to obtain. The best scheme provided in this embodiment ensures that the isotopic abundance of each intermediate and target product synthesized is more than 98%, the process is controllable, and no serious dilution of isotopic abundance will be caused. The deuterium-labeled fatty aldehydes at the end of the alkyl chain synthesized at the same time can not only be used as key synthons of sphingosine and ceramide internal standards, but can also be used in the synthesis of other popular compounds, such as the synthesis of peptide drugs. At the same time, the idea and method of introducing deuterium labeled atoms on the terminal carbon chain of the present invention can also be used as a reference for the design of internal standards for other compounds with saturated fatty carbon chains.

[0157] Example 2: Necessity of using "aliphatic methyl ketone compounds" as raw materials in the present invention

[0158] In the preparation method of the present invention, different raw materials are used to synthesize the alkyl chain terminal deuterium-labeled fatty aldehyde, deuterium-labeled sphingosine and ceramide, which has a great influence on the yield, deuteration rate and overall quality of the product. In order to verify the necessity of using "aliphatic methyl ketone compounds" as raw materials in the present invention, a comparative verification was carried out in this example, and the specific experimental process is as follows:

[0159] 14-Hydroxy-2-tetradeconone (the best aliphatic methyl ketone compound of the present invention) and tetracosanoic acid (Shanghai Sai Ke Rui Biotechnology Co., Ltd., commercially available fatty acids disclosed in the literature (J. Label Compd Radiopharm 2016; 59: 531-542; European Journal of Organic Chemistry, 2023, 26; Bulletin de la Societe Chimique de France, 1993, 130, 575-583)) were used as raw materials, and compound 6a was prepared according to the best preparation method in Example 1 of the present invention, and the abundance of the target labeled compounds obtained under different raw materials was calculated respectively. The results are shown in Table 1.

[0160] Table 1. Abundance of deuterium-labeled fatty aldehydes at the end of alkyl chains prepared from different raw materials

[0161]

[0162] As can be seen from Table 1, the scheme of preparing compound 6a using 14-hydroxy-2-tetradeconone (aliphatic methyl ketone compound) as a raw material through multi-step chemical reactions such as DH exchange has a higher isotopic abundance.

[0163] At the same time, when commercially available tetradecanoic acid is used as a raw material, the DH exchange full labeling (non-selective) method makes the final product abundance only about 94%. This method lacks the ability to accurately locate the target site, resulting in strong randomness of hydrogen-deuterium exchange, and it is difficult to achieve high selectivity and high abundance of specific deuterium-labeled product synthesis. At the same time, in the prior art, the number of deuterium labels is generally preferred to be more than 3 and less than 10, because a large increase in the number of deuterium will change the electron cloud distribution, spatial conformation and polarity of the molecule. Physical and chemical properties, such as affecting the retention time in chromatographic analysis, ionization efficiency and fragmentation pattern in mass spectrometry detection, etc., thereby affecting the accuracy of detection. Therefore, in this embodiment, aliphatic methyl ketone compounds (14-hydroxy-2-tetradeconone) are preferred as raw materials.

[0164] At the same time, it has been explained in the background technology of the present invention that directly using deuterium-labeled fatty acids as raw materials is usually costly. Therefore, in this embodiment, cost-effectiveness is comprehensively considered, and deuterium-labeled fatty acids as raw materials are not elaborated in detail, aiming to explore more cost-effective and technically advantageous raw material alternatives to improve the overall process feasibility and economic value.

[0165] Example 3: Comparative verification of the necessity of the "one-pot method" used in the present invention

[0166] In the best preparation method of the present invention, the "one-pot method" is used for the experiment. Specifically, during the reaction process, there is no need to first condense the methyl ketone raw material with hydrazine to generate a hydrazone intermediate, and then add a reducing agent for reduction, and there is no need to separate and treat the hydrazone intermediate generated in the reaction system before the next reduction reaction. The method of the present invention is to add the methyl ketone raw material, hydrazine, and the reducing agent in a reaction container at one time, and then place it at room temperature for reaction. In order to verify the necessity of the "one-pot method" for the experiment of the present invention, the following comparative verification test is carried out:

[0167] 1. Method 1: The process of synthesizing compound 4a from compound 3a by a "one-pot method" is as follows: 5.0 g of p-toluenesulfonyl hydrazide and 7.5 g of compound 3a are dissolved in 45 mL of CD3OD, and 3.6 g of sodium cyanoborodeuteride is added, and the reaction is carried out for 16 hours. After the reaction is completed, dichloromethane and water are added for liquid separation and extraction, and the organic phase is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which is separated and purified by Flash column chromatography to obtain 5.8 g of intermediate 4a, with a yield of 80.6% and an isotopic abundance of 98.6atom% D. 1 H NMR (400 MHz, Chloroform-d) δ 4.62 (s, 2H), 3.51 (t, J = 6.7 Hz,2H), 3.36 (s, 3H), 1.64 - 1.52 (m, 2H), 1.40 - 1.18 (m, 18H).

[0168] The NMR spectrum of compound 4a is as follows: Figure 4A shown.

[0169] 2. Method 2: Instead of using the "one-pot method", the methyl ketone raw material is first condensed with p-toluenesulfonyl hydrazide to generate a hydrazone intermediate, which is then separated and then reduced with a reducing agent (sodium cyanoborodeuteride) to obtain compound 4a. The remaining steps are the same as those in method 1 of this embodiment. The specific NMR spectrum of compound 4a is as follows: Figure 4B shown.

[0170] The yield and isotope abundance of compound 4a prepared by different methods were calculated respectively. The results are shown in Table 2.

[0171] Table 2. Yield and isotopic abundance of compound 4a prepared by different methods

[0172]

[0173] As can be seen from Table 2, only when the "one-pot method" is adopted can the obtained compound 4a have the highest yield and isotopic abundance. This is because the "one-pot method" synthesis of the present invention has significant advantages, which simplifies the operation process, reduces the loss of raw materials and products in the separation process, reduces the experimental operation steps, and reduces the risk of introducing impurities, thereby ensuring that the purity and isotopic abundance of the product are both the highest, significantly improving the synthesis efficiency, and on the other hand, there is no need to heat and consume energy and increase related equipment and facilities.

[0174] Therefore, it can be seen that the present invention preferably adopts a "one-pot method" to prepare the alkyl chain terminal deuterium-labeled fatty aldehyde.

[0175] Example 4: Comparative verification of the necessity of using "sodium cyanoborodeuteride" as a reducing agent in the present invention

[0176] In the third step of the present invention, compound 3a is converted into compound 4a under specific conditions, and the specific conditions preferably use "sodium cyanoborodeuteride" as a reducing agent. In order to verify the advantage of using "sodium cyanoborodeuteride" as a reducing agent in the present invention, the following comparative verification test is performed. The reaction is carried out according to the best method of Example 1, except that different reducing agents are optimized and screened, and the yield and isotopic abundance of compound 4a are measured. The selected reducing agents and the corresponding results are shown in Table 3 (at the same time, since the DH exchange reaction mechanism adopted in the present invention has specific requirements on the deuterated characteristics of the reducing agent, in this embodiment, only reducing agents with deuterated structures and that can effectively promote the target conversion and ensure the isotopic abundance and yield of the product in the reaction system are screened. Conventional non-deuterated reducing agents such as sodium borohydride, sodium cyanoborohydride, and lithium aluminum hydride are not included in the scope of this screening).

[0177] Table 3. Yield and isotopic abundance of compound 4a prepared by different methods

[0178]

[0179] As can be seen from Table 3, when sodium cyanoborodeuteride, sodium borodeuteride and lithium aluminum hydride deuteride are used as reducing agents, the final obtained compound 4a has good yield and isotopic abundance. This is because when "sodium cyanoborodeuteride" is used as a reducing agent, its reaction activity is more moderate than other reducing agents, which is conducive to improving the selectivity of the reaction and reducing the occurrence of side reactions, thereby helping to significantly improve the yield and isotopic abundance of the product.

[0180] Therefore, the present invention preferably uses "sodium cyanoborodeuteride" as the reducing agent.

[0181] Example 5: DH exchange study under alkali

[0182] In the second step of the present invention, the preparation of compound 3a mainly depends on the DH exchange reaction, and the base and the deuterium source play a key role in this process; different combinations of bases and deuterium sources will produce different yields and deuteration rates due to their own properties and the properties of the substrate.

[0183] In this example, the DH exchange reaction of compound 2a to compound 3a was studied. The reaction was carried out according to the best method of Example 1, except that the base and deuterium source were changed, and the yield and deuteration rate of the reaction were measured. The selected base and deuterium source are shown in Table 4. Only 7 combinations in the table can make the DH exchange reaction proceed. The combinations of other bases and deuterium sources have been tested to have extremely low deuteration rates and low yields. The yields and deuteration rates of the 7 combinations are shown in Table 4.

[0184] Table 4. Selection and yield of base and deuterium source, and isotope abundance results

[0185]

[0186] According to the experimental results, the selection of base and deuterium source has a great correlation with the yield and isotopic abundance. When sodium bicarbonate is selected as the base, the alkalinity of sodium bicarbonate is weak, which is not enough to achieve the dissociation of hydrogen at the α position of the carbon group, resulting in too low deuteration rate. Therefore, it is not suitable as a reagent for deuteration reaction. When potassium carbonate is selected as the base and heavy water is selected as the deuterium source, the yield of compound 2 to compound 3 is the highest, which is 66.7%, and the deuteration rate is the highest, which is 98.8 atom%. D should be preferred.

[0187] Therefore, in the second step of reacting compound 2a to synthesize compound 3a, it is preferred that the base is potassium carbonate and the deuterium source is heavy water.

[0188] Example 6: Study on the screening of reagents in different steps of the present invention

[0189] In the synthesis process of the present invention, the combination of different reagents in each step will have a great influence on the synthesis of the corresponding compound. Therefore, in this embodiment, the combination of reagents in different steps is optimized and screened, as follows:

[0190] 1. Synthesis of fatty aldehydes labeled with deuterium at the end of the alkyl chain

[0191] 1. Step 5: In the fifth step, the reaction of converting compound 5a to compound 6a was studied. The reaction was carried out according to the best method of Example 1, except that different oxidants were optimized and screened, and the yield and isotopic abundance of compound 6a (deuterium-labeled fatty aldehyde at the end of the alkyl chain) were determined. The selected oxidants and the corresponding results are shown in Table 5.

[0192] Table 5. Effects of different oxidants on the synthesis of compound 6a

[0193]

[0194] According to the experimental results, in the synthesis process of compound 6a, the selection of oxidant has a great correlation with the yield of compound 6a. The results show that when different oxidants are selected, there are obvious differences in the yield of the final compound 6a. When the preferred oxidant is Dess-Martin oxidant, the yield and isotopic abundance of the compound 6a obtained at this time are both the highest. This is because different oxidants have different adaptability to the substrate, which in turn affects the reaction conversion rate.

[0195] Therefore, in the fifth step of the reaction to synthesize compound 6a, the preferred oxidant is a Dess-Martin periodinane, and the compound 6a obtained at this time has the highest yield and isotopic abundance.

[0196] 2. Synthesis of Deuterium-labeled Sphingosine and Ceramide

[0197] 1. Step 2: In the second step, the process of compound 8a reacting with labeled fatty aldehyde to generate compound 9a was studied. The reaction was carried out according to the best method of Example 1, except that different reaction conditions were used and the yield of the reaction was measured. The different reaction conditions selected and the corresponding results are shown in Table 6.

[0198] Table 6. Effect of different reaction conditions on the yield of compound 9a

[0199]

[0200] According to the test results, different reaction conditions are closely related to the yield of compound 9a. The results show that when the reaction conditions are a combination of inorganic lithium salt reagents and bases, the final compound 9a has a good yield, and when the combination of lithium chloride and triethylamine is preferred, the yield of compound 9a is optimal. This may be because lithium chloride is an inorganic lithium salt reagent and triethylamine is an organic base reagent. When the two work synergistically, the reaction efficiency and selectivity can be significantly improved. Lithium chloride can accurately activate the reaction sites of compound 8a through ion complexation, effectively reducing the activation energy of the target reaction; triethylamine, as an organic base, can accurately capture the protons generated in the reaction, maintain the stability of the alkaline environment of the reaction system, and ensure that the reaction continues to proceed in the forward direction. And limiting the appropriate molar ratio of the two (1:1.2-1.5) is conducive to strictly controlling the reaction rate and degree, avoiding side reactions caused by excessive base or incomplete reactions caused by insufficient base, thereby greatly improving the product yield and isotopic abundance.

[0201] Therefore, in the process of synthesizing compound 9a, the preferred reaction conditions are a combination of an inorganic lithium salt reagent and an organic base reagent, and when a combination of lithium chloride and triethylamine is preferred, the yield of compound 9a obtained at this time is optimal.

[0202] 2. Step 3: In the third step, the reaction of converting compound 9a to compound 10a was studied. The reaction was carried out according to the best method of Example 1, except that different reducing agents were optimized and screened, and the yield and isotopic abundance of compound 10a were measured. The selected reducing agents and the corresponding results are shown in Table 7.

[0203] Table 7. Effects of different reducing agents on the synthesized compound 10a

[0204]

[0205] According to the test results, in the synthesis process of compound 10a, the selection of reducing agent has a great correlation with the yield of compound 10a. The results show that when different reducing agents are selected, the yield of the final compound 10a is significantly different, and when the preferred reducing agent is lithium tri-tert-butoxyaluminum hydride, the yield of compound 10a obtained at this time reaches the highest. This may be because lithium tri-tert-butoxyaluminum hydride has a higher steric effect and has better stereo-induction selectivity in the reduction process, thereby efficiently converting the target configuration product.

[0206] Therefore, in the third step of the reaction to synthesize compound 10a, the preferred reducing agent is lithium tri-tert-butoxyaluminum hydride, and the compound 10a obtained in this case has the highest yield.

[0207] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A sphingosine compound labeled with deuterium at the end of an alkyl chain, characterized in that: It has the following chemical structure: ； Wherein, n is an integer including 0, 1, 2, 3, ...

2. A deuterium-labeled ceramide compound, characterized in that: The deuterium-labeled ceramide compound is prepared by using the alkyl chain terminal deuterium-labeled sphingosine compound as claimed in claim 1, and has the following chemical structure: ； Wherein, R5 includes any one or more combinations of H or aliphatic alkanes (straight chain or branched chain); n is an integer including 0, 1, 2, 3, ...

3. A method for preparing a deuterium-labeled sphingosine compound, characterized in that: The deuterium-labeled sphingosine compound is prepared by using an alkyl chain terminal deuterium-labeled fatty aldehyde compound, and the alkyl chain terminal deuterium-labeled fatty aldehyde compound is prepared using an aliphatic methyl ketone compound as a raw material through a DH exchange step; the synthetic route is as follows: 。 4. The preparation method according to claim 3, characterized in that: The deuterium-labeled sphingosine compound is obtained by reaction using an amino acid methyl ester compound in which both the hydroxyl group and the amino group are protected and a fatty aldehyde compound with deuterium labeled at the end of the alkyl chain as key raw materials; the synthesis route is as follows: ； Wherein, R3 includes any one or more combinations of -TBDS, -MOM, -Trt; R4 includes any one or more combinations of -Boc, -Fmoc, -Cbz, -Alloc, -Pht; R5 includes a C1~C25 alkyl chain.

5. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: (1) Reaction with dimethyl methylphosphonate: Compound 7 is used as a raw material and reacted with dimethyl methylphosphonate to obtain compound 8a; (2) Reaction with deuterium-labeled fatty aldehyde: Compound 8 reacts with fatty aldehyde to generate compound 9; (3) Reduction reaction: Compound 9 reacts with a reducing agent to generate compound 10; (4) Removal of protecting groups: Compound 10 is subjected to removal of protecting groups to obtain deuterium-labeled sphingosine compounds.

6. The preparation method according to claim 5, characterized in that: In the step (1), compound 7 reacts with dimethyl methylphosphonate under certain conditions to obtain compound 8, and the "certain conditions" include using a base substance, and the base substance includes any one or more combinations of lithium bistrimethylsilylamide, lithium diisopropylamide, and n-butyl lithium.

7. The preparation method according to claim 5, characterized in that: In the step (2), compound 8 reacts with a fatty aldehyde under certain conditions to generate compound 9, and the "certain conditions" include using lithium chloride and triethylamine.

8. The preparation method according to claim 5, characterized in that: In the step (3), the reducing agent includes any one or more combinations of sodium borohydride, lithium tri-tert-butoxyaluminum hydride, and lithium aluminum hydride.

9. The preparation method according to claim 5, characterized in that: In the step (4), compound 10 is subjected to certain conditions to remove the protecting group to obtain a deuterium-labeled sphingosine compound. When R3 is -TBDS and R4 is -Boc, "certain conditions" include acidic conditions.

10. A method for preparing a deuterium-labeled ceramide compound, characterized in that: A deuterium-labeled sphingosine compound is reacted with an active ester of fatty acid to obtain a deuterium-labeled ceramide compound, wherein the deuterium-labeled sphingosine compound is prepared by the preparation method according to any one of claims 3 to 9.

Images