An alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound, its preparation method and application

Abstract

The present invention provides an alkyl-chain-terminal deuterium-labeled aliphatic aldehyde compound, a preparation method thereof and an application thereof. In view of the demand for labeled compounds in the research of aliphatic aldehydes, sphingosine and ceramides, the existing preparation technologies for alkyl-chain-terminal deuterium-labeled aliphatic aldehydes have defects such as high cost, complex steps and expensive raw materials. The present invention uses aliphatic methyl ketone compounds as raw materials, and through multiple steps of reactions such as hydroxyl protection, D-H exchange, reaction with p-toluenesulfonyl hydrazine, deprotection group removal, oxidation reaction, etc., and optimizes the conditions of each step, such as the ratio of specific protecting group reagents to bases, precise reaction material ratios, appropriate reaction temperatures, etc., and successfully solves the preparation problem. High-abundance and high-quality target compounds are synthesized, which can be used to study metabolic pathways and disease mechanisms in organisms, serve as internal standards to improve the accuracy of quantitative detection, provide strong support for related research and the synthesis of various popular compounds, and promote the development of the field.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and particularly relates to an alkyl chain end deuterium-labeled aliphatic aldehyde compound, a preparation method thereof and an application thereof. Background Art

[0002] Aliphatic aldehydes are aldehyde compounds in which the aldehyde group is connected to an aliphatic hydrocarbon group (or a hydrogen atom). They can serve as biological signal molecules that regulate apoptosis, inflammatory responses, etc. They play a variety of extremely important physiological functions in living organisms, participating in energy metabolism and substance synthesis; in the environment, aliphatic aldehydes generated as industrial and transportation emissions, pesticides, etc. are also important environmental pollutants; at the same time, they play an important role as important synthons or building blocks in the synthesis of synthetic drugs in the pharmaceutical field. For example, they can be further converted into fatty acids and participate in the synthesis of drugs or lipid substances containing amide bonds, such as ceramide (Cer) composed of a sphingosine backbone and fatty acid chains of different lengths (C14-C36) connected by amide bonds. At the same time, long-chain aliphatic aldehydes are also important intermediates in the synthesis of sphingosine and ceramide, and are also one of the important metabolic degradation products of sphingomyelin in the body. In recent years, with the rapid development of mass spectrometry technology, qualitative and quantitative analysis of corresponding target substances by liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the current mainstream method. Isotope dilution mass spectrometry and stable isotope tracer technology have become very promising methods and technologies in the fields of clinical pharmacokinetics research, molecular biology, food safety, environmental science, etc. For example, isotope-labeled aliphatic aldehydes can be used for quantitative analysis of metabolites to help study metabolic pathways and metabolic changes in living organisms. The development and popularization of the above-mentioned mass spectrometry technology based on stable isotope-labeled compounds are inseparable from the isotope-labeled compounds of the substances to be detected. How to efficiently and economically prepare relevant labeled compounds has become the key factor to break the deadlock.

[0003] In the prior art, fatty alcohols, fatty aldehydes, and fatty acids can be chemically converted through certain technical routes. There are many reported synthetic methods for natural abundance fatty aldehydes or fatty acids in the literature, but there are few reported synthetic literatures on deuterium-labeled compounds. Regarding the preparation of fatty alcohols, fatty aldehydes, or fatty acids with deuterium-labeled at the end of the alkyl chain, a method for preparing 1-heptadecanol-D7 with deuterium-labeled at the end of the alkyl chain was reported in the literature (SN Crane, Journal of Labelled Compounds and Radiopharmaceuticals, 2006, 1273-1285). Using deuterium-labeled butanol as the raw material, the 1-heptadecanol-D7 compound was synthesized through eight-step chemical reactions, and further oxidized to obtain the corresponding aldehyde-labeled compound. However, the deuterium-labeled raw material used in this method is expensive and the synthesis steps are long. 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 1-tetracosanol as the raw material, and the synthesis economy of the target product is poor.

[0004] 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 were used to directly perform D-H exchange on natural abundance fatty acids under the action of a catalyst to obtain fully deuterium-labeled deuterated fatty acids. On the one hand, the synthesis operation 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 the isotope abundance only about 95% at most, but also the deuteration at the target position is incomplete, there is no selectivity for the labeled sites, and the target alkyl chain end deuterium-labeled aliphatic compounds cannot be obtained, which cannot meet the application requirements. A method for synthesizing D3 compounds was also reported in the above literature. The synthetic route of this method requires 9-10 steps of chemical reactions, and the steps are cumbersome. Each step of the reaction requires strict control of the reaction conditions and operations for product separation and purification. It is not only time-consuming and laborious, but also reduces the total yield due to cumulative material losses and incomplete reactions. Moreover, this method is limited to constructing D3, and the position of introducing deuterium is limited, making it difficult to meet diverse deuterium-labeling requirements. The preparation methods for deuterium-labeled fatty alcohols, fatty aldehydes, and fatty acids in the above literature have disadvantages such as high cost, poor economy, poor flexibility and selectivity of deuterium-labeled positions, and unfriendly safety operability, and are not suitable for the large-scale preparation of alkyl chain end deuterium-labeled fatty aldehydes and related derivatives with different structures or carbon chain lengths.

[0005] Therefore, it is urgent to develop a synthesis and preparation technology for deuterium-labeled fatty aldehydes at the end of an alkyl chain. The present invention designs a suitable organic synthesis route, follows the basic chemical reaction principle, uses commonly used deuterium sources and deuterium labeling reagents, uses aliphatic methyl ketone compounds as starting materials, and introduces deuterium-labeled atoms at specific sites to synthesize a deuterium-labeled fatty aldehyde compound at the end of an alkyl chain. The compound can not only realize the chemical transformation of related fatty alcohols and fatty acids, but also can be used as a key synthon to realize the synthesis of various drugs and clinical detection markers or metabolites, such as sphingosine and ceramide, for studying related metabolic pathways and disease mechanisms in organisms, and as an internal standard for quantitative detection, etc., providing strong support for related research and applications. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a deuterium-labeled fatty aldehyde compound at the end of an alkyl chain and a preparation method and application thereof. Through a carefully designed organic synthesis route, aliphatic methyl ketone compounds are used as starting materials, and deuterium-labeled fatty aldehydes at the end of an alkyl chain are synthesized through multi-step reactions such as hydroxyl protection, DH exchange, reduction, oxidation, and further deuterium-labeled sphingosine and ceramide are synthesized. High-abundance, high-quality target compounds are successfully synthesized, which can be used as important intermediates for studying metabolic pathways and disease mechanisms in organisms, as internal standards for quantitative detection, and can also provide references for the synthesis of other popular compounds and the design of internal standards, playing an important role in many fields.

[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] Among them, R 1 It 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 compound refers to a class of organic compounds containing a carbonyl (C=O) structure, and its molecular structure contains a methyl (CH 3group and a keto (C=O) group, and the general molecular formula of such compounds is HOCH 2- R-C(O)-CH 3 , where R represents an aliphatic group or an alkyl group, which can be a straight-chain or branched-chain 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 other compounds.

[0013] Due to the increasing demand for stable isotope-labeled compounds in the fields of biomedical research, analytical detection, etc., especially when studying the metabolic pathways and disease mechanisms related to fatty aldehydes, sphingosine and ceramides, there is an urgent need for an efficient and economical method for preparing labeled compounds. The present invention plans to design an innovative preparation technology for deuterium-labeled fatty aldehyde compounds at the end of the alkyl chain. By deeply studying the chemical structures, biological metabolic pathways and internal standard design requirements of sphingosine and ceramides, an innovative synthetic route is proposed. The stable isotope atom deuterium (D) is selected as the labeling atom to be introduced. At the same time, considering the abundance stability of the internal standard, the risk of isotope abundance dilution of the prepared intermediates and internal standard is avoided. Deuterium labeling is carried out at its "inert" site, that is, the chemically inert terminal carbon chain, and 5-7 D atoms are designed to be introduced. In the initial stage of the experiment, expensive 1-iodopropane-D7 was directly used as the raw material to synthesize deuterium-labeled fatty aldehyde at the end of the alkyl chain. However, it was found through experiments that this method had the problem of low product abundance, and the abundance was only about 90%, far lower than the requirements for product abundance in the field of standard products.

[0014] Therefore, in the later experiments, in terms of raw material selection, expensive 1-iodopropane-D7 with the risk of carbocation rearrangement during the synthesis process was abandoned, and common and inexpensive raw materials such as aliphatic methyl ketone 14-hydroxy-2-tetradecanone were used. Through key steps such as hydroxyl protection, D-H exchange, reaction with p-toluenesulfonylhydrazine, deprotection group, oxidation reaction, and a series of subsequent synthesis steps, the problems of high preparation cost, low product abundance of deuterium-labeled fatty aldehyde at the end of the alkyl chain, and difficulty in derivatively synthesizing deuterium-labeled sphingosine and ceramide series compounds were successfully solved. Through the precise control and synergy of each step, the efficient synthesis from the starting material to the final target product was ensured, and the isotope abundances of each intermediate and target product were above 98%. The process was controllable and would not cause serious dilution of the isotope abundance. At the same time, the synthesized compounds can not only be used to study the action mechanisms and metabolic pathways of fatty aldehydes, sphingosine and ceramides in vivo, but also be used as internal standards for quantitative detection, which is of great significance in the fields of clinical diagnosis, drug research and development, etc.

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

[0016] In some embodiments, in order to verify the necessity of using the "one-pot method" in the present invention, a comparative verification test was carried out. The results showed that only when the "one-pot method" was used, could the obtained compound 4 have the highest yield, deuteration rate and isotope abundance. This is because the "one-pot method" of the present invention has significant advantages. It does not require heating and consuming energy, 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, thus ensuring that the purity and isotope abundance of the product reach the highest, and significantly improving the synthesis efficiency.

[0017] Further, the preparation method includes the following steps: step (1) hydroxyl protection, step (2) D-H exchange, step (3) reaction with p-toluenesulfonylhydrazide and reduction, step (4) deprotection group, step (5) oxidation reaction; in step (3), compound 3 reacts with p-toluenesulfonylhydrazide under the action of a reducing agent to be converted into compound 4, and the reducing agent includes any one or a combination of sodium borodeuteride, sodium cyanoborohydride, and lithium aluminum hydride-d.

[0018] In some embodiments, in order to verify the necessity of using "sodium cyanoborohydride" as a reducing agent in the present invention, a comparative verification test was carried out. The results showed that when "sodium cyanoborohydride" was used as a reducing agent, the yield and isotope abundance of the obtained compound 4 could reach the best. This is because when "sodium cyanoborohydride" is used as a reducing agent, compared with other reducing agents, its reaction activity is more moderate, which is beneficial to improving the selectivity of the reaction and reducing the occurrence of side reactions, thus contributing to significantly improving the purity and isotope abundance of the product.

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

[0020] In some embodiments, when "sodium cyanoborohydride" is selected as the reducing agent, its molar ratio to compound 3 is 1:1.2 - 3, and the reaction of compound 3 with p-toluenesulfonylhydrazide proceeds smoothly, and is highly selectively converted into compound 4, providing a high-quality intermediate for the subsequent steps and ensuring the smooth progress of the synthesis route.

[0021] Further, in step (1), an aliphatic methyl ketone compound is used as a raw material and reacts with a protecting group reagent to obtain compound 2; in step (2), compound 2 undergoes D-H exchange to obtain compound 3; in step (4), compound 4 is deprotected to obtain compound 5; in step (5), compound 5 is oxidized by an oxidizing agent to generate compound 6, and compound 6 is an aliphatic aldehyde compound with a deuterium label at the end of the alkyl chain;

[0022] ; wherein, R 1Comprising any one or more combinations of H or alkyl; R 2 Comprising any one or more combinations of -Bn, -TBDS, -MOM; n is an integer including 0, 1, 2, 3, ….

[0023] Furthermore, in the step (1), the protecting group reagent comprises any one or more combinations of BnBr, TBDSCl, MOMCl.

[0024] Furthermore, the aliphatic methyl ketone compound and the protecting group reagent are used to prepare compound 2 under the action of a base, and the base comprises an inorganic base or an organic base, including any one or more combinations of sodium bicarbonate, potassium carbonate, triethylamine, N,N-diisopropylethylamine.

[0025] Furthermore, the molar ratio of the compound 1, the protecting group reagent, and the base is 1: (1.2~2): (1.2~2).

[0026] In some embodiments, when MOMCl is selected as the 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 dichloromethane solvent, which can effectively protect the hydroxyl group of the compound 1, avoid interference in subsequent reactions, and provide a suitable substrate for subsequent D-H exchange and other reactions; when using a combination of MOMCl and N,N-diisopropylethylamine, under a suitable molar ratio, the reaction conditions are mild, the requirements for reaction equipment are low, and on the basis of protecting the hydroxyl group, the subsequent reaction active sites are easier to control, ensuring that the entire synthesis route proceeds in the expected direction and improving the synthesis efficiency and purity of the target product.

[0027] Furthermore, in the step (2), compound 2 undergoes D-H 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, sodium bicarbonate; the deuterium source for D-H exchange comprises any one or more combinations of heavy water, deuterated methanol, deuterated ethanol.

[0028] Furthermore, the molar ratio of the compound 2 and the base is 1: (2~7).

[0029] In the second step of the synthesis of the deuterium-labeled fatty aldehyde at the end of the alkyl chain of the present invention, the preparation of compound 3 mainly depends on the D-H exchange reaction, and the base and 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. Therefore, in some embodiments, different combinations of bases and deuterium sources are compared and screened. The experimental results show that there is a large correlation between the selection of bases and deuterium sources and the yield and isotope abundance. When sodium bicarbonate is selected as the base, since sodium bicarbonate has a weak alkalinity and is not sufficient to dissociate the hydrogen at the α-position of the carbonyl group, the deuteration rate is too low. Therefore, it is not suitable as a reagent for the deuteration reaction. When potassium carbonate is selected as the base and heavy water is selected as the deuterium source, the conversion rate of compound 2 to compound 3 is the highest, which is 66.7%, the deuteration rate is the highest, which is 98.8 atom% D, and the operability is better, so it should be preferred.

[0030] Further, in the step (4), compound 4 is deprotected under certain conditions to obtain compound 5; when R 2 is -Bn, the "certain conditions" include using a catalyst, and the catalyst includes any one or a combination of Pd / C, H 2 ; when R 2 is -TBDS or MOM, the "certain conditions" include using any one or a combination of fluorine reagents or acid reagents.

[0031] Further, the fluorine reagent or acid reagent includes any one or a combination of ammonium fluoride, tetrabutylammonium fluoride, hydrochloric acid, sulfuric acid, trifluoroacetic acid.

[0032] Further, the mass ratio of compound 4 to the catalyst is 1:(0.05 - 1).

[0033] Further, in the oxidation reaction of the step (5), the oxidant includes any one or a combination of pyridinium chlorochromate, Dess-Martin oxidant, tetramethylpiperidine oxide, dimethyl sulfoxide, sulfur trioxide-pyridine complex.

[0034] In the fifth step of the synthesis of the deuterium-labeled fatty aldehyde 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 for 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 finally prepared compound 6, and when the preferred oxidant is Dess-Martin oxidant, the yield of the prepared compound 6 reaches the highest at this time. This is because different oxidants have different adaptabilities to the substrate, which in turn affects the reaction conversion rate.

[0035] Further, the molar ratio of compound 5 to the oxidant is 1:(1.2 - 3).

[0036] On the other hand, the present invention provides the use of an alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound as a reagent for preparing synthetic sphingosine and ceramide internal standards, and the alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound is prepared by the preparation method described in any one of the above technical solutions.

[0037] In some embodiments, the alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound is used as a key precursor for the reagent of synthetic sphingosine and ceramide internal standards. Under strictly controlled reaction conditions, such as precisely setting the reaction temperature, time, and reagent ratio, it acts synergistically with specific phosphorus-containing reagents, bases, and solvents, and is efficiently converted into sphingosine and ceramide internal standards through multiple-step organic reactions. The prepared internal standards, by virtue of their stable isotope labeling characteristics, can accurately correct the errors in sample processing and detection in detection techniques such as mass spectrometry analysis, significantly improving the accuracy and reliability of the determination of sphingosine and ceramide contents in biological samples, strongly supporting biomedical research and clinical diagnosis work, and providing key technical support for in-depth exploration of their metabolic mechanisms, disease associations, and drug development.

[0038] On the other hand, the present invention provides an alkyl chain-terminated deuterium-labeled sphingosine compound, having the following chemical structure:

[0039] ;

[0040] wherein n is an integer including 0, 1, 2, 3,....

[0041] On the other hand, the present invention provides a deuterium-labeled ceramide compound, which is prepared from the alkyl chain-terminated deuterium-labeled sphingosine compound described in the above technical solution and has the following chemical structure:

[0042] ;

[0043] wherein R5 includes any one or a combination of more than one of H or aliphatic alkanes (linear or branched); n is an integer including 0, 1, 2, 3,....

[0044] On the other hand, the present invention provides a preparation method of a deuterium-labeled sphingosine compound, wherein the deuterium-labeled sphingosine compound is prepared from an alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound, and the alkyl chain-terminated deuterium-labeled aliphatic aldehyde compound is prepared from an aliphatic methyl ketone compound through reaction steps such as D-H exchange; the synthesis route is as follows:

[0045] ;

[0046] Furthermore, the deuterium-labeled sphingosine compound is obtained from key raw materials including an amino acid methyl ester compound with both hydroxyl and amino groups protected and a deuterium-labeled fatty aldehyde compound at the end of the alkyl chain through a series of chemical reactions. The synthetic route is as follows:

[0047] ;

[0048] Among them, R3 includes any one or a combination of more than one of -TBDS, -MOM, -Trt; R4 includes any one or a combination of more than one of -Boc, -Fmoc, -Cbz, -Alloc, -Pht; R5 includes an alkyl chain with 1 to 25 carbon atoms (linear or branched).

[0049] Furthermore, the preparation method includes the following steps:

[0050] (1) Reaction with dimethyl methylphosphonate: Using intermediate 7 as the raw material, reacting with dimethyl methylphosphonate to obtain compound 8;

[0051] (2) Reaction with fatty aldehyde: Compound 8 reacts with the deuterium-labeled fatty aldehyde to generate compound 9;

[0052] (3) Reduction reaction: Compound 9 reacts with a reducing agent to generate compound 10;

[0053] (4) Deprotection: Compound 10 is deprotected to obtain the deuterium-labeled sphingosine compound;

[0054] Furthermore, in the step (1), compound 7 reacts with dimethyl methylphosphonate under certain conditions to obtain compound 8. The "certain conditions" include using an alkali substance, and the alkali substances include any one or a combination of more than one of lithium bis(trimethylsilyl)amide, lithium diisopropylamide, and n-butyllithium.

[0055] In some embodiments, the entire synthesis process starts from an amino acid methyl ester compound and synthesizes deuterium-labeled sphingosine through steps such as reaction with dimethyl methylphosphonate, reaction with deuterium-labeled fatty aldehyde, reduction reaction, and deprotection of groups. Finally, various 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, jointly constructing a complete synthesis system. The key steps play an irreplaceable role in introducing deuterium labeling, constructing molecular structures, and realizing functional group transformation. As key nodes in the synthesis process, each deuterium-labeled intermediate not only reflects the gradual evolution of the molecular structure but also ensures the stability of the isotope abundance, ultimately achieving the synthesis of various deuterium-labeled compounds with important biomedical significance, providing powerful tools and technical support for studying the action mechanisms, metabolic pathways of fatty aldehydes, sphingosine, and ceramides in vivo, as well as the diagnosis and treatment of related diseases. At the same time, the synthesis method of the present invention has significant advantages in terms of raw material selection, synthesis route design, and operational simplicity, overcomes the problems of high cost and low abundance existing in the prior art, and has broad application prospects.

[0056] Further, the molar ratio of the dimethyl methylphosphonate to the base is 1:(1.2 - 1.5).

[0057] Further, in step (2), compound 8 reacts with a fatty aldehyde to form compound 9 under certain conditions. The "certain conditions" include using lithium chloride and triethylamine, and the molar ratio of the lithium chloride to the triethylamine is 1:(1.2 - 1.5).

[0058] In some embodiments, the reaction conditions of step (2) are compared and screened. The experimental results show that there is a great correlation between different reaction conditions and the yield of compound 9. The results show that when the combination of an inorganic lithium salt reagent and a base is selected as the reaction condition, the finally prepared compound 9 has a better yield, and when the combination of lithium chloride and triethylamine is preferably selected, the yield of the prepared compound 9 is significantly increased.

[0059] Further, in step (3), the reducing agent includes any one or a combination 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).

[0060] In some embodiments, the reducing agents in step (3) are compared and screened. The experimental results show that in the synthesis process of compound 10, the selection of the reducing agent is greatly correlated with the yield of compound 10. When different reducing agents are selected, there are obvious differences in the yields of the finally prepared compound 10, and when the reducing agent is preferably lithium tri-tert-butoxyaluminum hydride, the yield of the prepared compound 10 reaches the highest.

[0061] Further, in the step (4), compound 10 is deprotected under certain conditions to obtain a deuterium-labeled sphingosine compound. When R 3 is -TBDS and R 4 is -Boc, the "certain conditions" include using acidic conditions.

[0062] On the other hand, the present invention provides a deuterium-labeled sphingosine compound, which is prepared by using the preparation method described in any one of the above technical solutions.

[0063] On the other hand, the present invention provides a preparation method of a deuterium-labeled ceramide compound. A deuterium-labeled ceramide compound is obtained by reacting a deuterium-labeled sphingosine compound with a fatty acid active ester, and the deuterium-labeled sphingosine compound is prepared by using the preparation method described in any one of the above technical solutions.

[0064] In some embodiments, the synthesized D-sphingosine-D7 is condensed with different fatty acid active esters to synthesize a variety of deuterium-labeled ceramides, constructing a ceramide internal standard library for accurately and rapidly quantifying the ceramide levels in samples such as plasma and serum, studying the role of ceramide in the sphingolipid metabolic pathway, as well as its regulatory role in cell signaling pathways, cell and body metabolism, and providing a basis for the 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 realized, and a ceramide internal standard library is constructed to meet different research and detection requirements.

[0065] Further, in the preparation method, first, a fatty acid is condensed with N-hydroxysuccinimide under certain conditions to obtain a corresponding active ester intermediate, and then the active ester intermediate is condensed 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 using an alkali substance and a condensing agent; the alkali substance includes any one or a combination of triethylamine and diisopropylethylamine.

[0066] Further, in the fatty acid, the number of carbon atoms includes any one or a combination of 2 to 26.

[0067] Further, the fatty acid includes any one or a combination of palmitic acid, eicosanoic acid, and docosanoic acid; the prepared deuterium-labeled ceramide compounds include any one or a combination of C16 ceramide-D7, C20 ceramide-D7, C22 ceramide-D7, or C22:1 ceramide-D7.

[0068] Further, when the fatty acid is palmitic acid, a palmitic acid active ester is prepared, and the palmitic acid active ester is condensed with D-sphingosine-D7 to prepare C16 ceramide-D7.

[0069] Further, when the fatty acid is arachidic acid, an arachidic acid active ester is prepared, and the arachidic acid active ester is condensed with D-sphingosine-D7 to prepare C20 ceramide-D7.

[0070] Further, when the fatty acid is behenic acid, a behenic acid active ester is prepared, and the behenic acid active ester is condensed with D-sphingosine-D7 to prepare C22 ceramide-D7.

[0071] Further, the condensing agent includes any one or a combination of more of dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / 1-hydroxybenzotriazole (EDCI / HOBT), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU).

[0072] 1-Hydroxybenzotriazole HOBT is generally not used as a condensing agent and is generally used in combination with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI as a catalyst.

[0073] In some embodiments, the combination of different bases and condensing agents is significantly related to 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 prepared at this time is significantly increased.

[0074] Further, certain conditions for the reaction of the active ester intermediate with deuterium-labeled sphingosine compounds include using any one or a combination of more of dichloromethane and tetrahydrofuran.

[0075] Further, the molar ratio of the fatty acid, condensing agent, and base is: 1: 1~1.5: 1.2~1.5.

[0076] The beneficial effects obtained by the present invention:

[0077] 1. The present invention provides an alkyl-chain terminal deuterium-labeled fatty aldehyde compound, its preparation method and application, which have the following advantages: (1) In the preparation process of deuterium-labeled intermediates, the present invention does not use expensive deuterium-labeled raw materials such as isotope-labeled iodopropane, iodobutane, butanol, etc., nor does it use relatively dangerous or unsafe reaction reagents such as deuterium gas for deuterium labeling of substrate molecules. Instead, it uses relatively inexpensive basic deuterium-labeled raw materials such as heavy water and sodium borohydride-d for labeling target molecules, which has obvious safety and economy; (2) The present invention selects to design and introduce labeled atoms on the common unit of ceramide series compounds, that is, the carbon chain skeleton of "sphingosine", to prepare this key synthon of alkyl-chain terminal deuterium-labeled fatty aldehyde, which is extremely convenient and economical for the rapid construction of a ceramide internal standard library. And it selects to perform deuterium labeling at its "inert" site, that is, the chemically inactive terminal carbon chain, to avoid the risk of isotope abundance dilution of the prepared intermediates and internal standards; (3) The design and synthesis scheme of the present invention has no literature reports. The designed synthetic route is easy to operate and can introduce deuterium atoms directionally at specific positions, taking into account the stability, reliability and economy of the target molecule; (4) The present invention has realized the preparation of dozens of ceramide internal standards including sphingosine-D7, C16 ceramide-D7 (C16 Ceramide-d7), C20 ceramide-D7 (C20 Ceramide-d7), C22 ceramide-D7 (C22 Ceramide-d7), C22:1 ceramide-D7 (C22:1 Ceramide-d7), etc.

[0078] 2. The alkyl-chain terminal deuterium-labeled fatty aldehyde compounds and their derivatives prepared by the present invention, due to their stable isotope labeling characteristics, can be used as ideal tracer tools in the study of metabolic pathways related to fatty aldehydes, sphingosine and ceramides, helping to accurately analyze their dynamic changes and transformation paths in the complex metabolic network in vivo, providing key clues for in-depth exploration of the disease occurrence and development mechanism, and strongly promoting the discovery of disease diagnostic markers and new therapeutic targets.

[0079] 3. In the field of analytical detection, the compounds prepared by the present invention are used as internal standards in quantitative detection techniques such as LC-MS / MS, which can significantly improve the accuracy and precision of the detection method, effectively reduce the adverse effects of factors such as sample matrix interference and instrument signal fluctuation on the measurement results, ensure the reliability and repeatability of the determination data of the relevant compound content in biological samples, and build a solid technical support for clinical diagnosis, drug research and development and quality control of biomedical research.

[0080] 4. The optimal synthetic route provided by the present invention is synergistically efficient after optimization. The raw materials are common, easy to obtain, and the reaction conditions are mild and easy to control, significantly shortening the synthesis cycle, reducing production costs, and improving production efficiency. It is conducive to large-scale industrial production, laying a foundation for the stable supply of high-quality labeled compounds, fully meeting the growing scientific research and application needs, and effectively promoting the efficient transformation of scientific research results in related fields into practical applications. Description of the Drawings

[0081] Figure 1 : Synthesis process of the deuterium-labeled aliphatic aldehyde compounds with alkyl chain end of the present invention

[0082] Figure 2 : Nuclear magnetic resonance spectrum of compound 2a

[0083] Figure 3 : Nuclear magnetic resonance spectrum of compound 3a

[0084] Figure 4A : Nuclear magnetic resonance spectrum of compound 4a (one-pot method)

[0085] Figure 4B : Nuclear magnetic resonance spectrum of compound 4a (non-one-pot method)

[0086] Figure 5 : Nuclear magnetic resonance spectrum of compound 6a

[0087] Figure 6 : Synthesis process of the deuterium-labeled sphingosine and ceramide of the present invention

[0088] Figure 7 : Nuclear magnetic resonance spectrum of compound D-sphingosine-D7

[0089] Figure 8 : Nuclear magnetic resonance spectrum of compound C16 ceramide-D7

[0090] Figure 9 : Nuclear magnetic resonance spectrum of compound C20 ceramide-D7

[0091] Figure 10 : Nuclear magnetic resonance spectrum of compound C22 ceramide-D7

[0092] Figure 11 : Nuclear magnetic resonance spectrum of compound C22:1 ceramide-D7 Detailed Embodiments

[0093] To make the objectives, 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 accompanying drawings of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meaning as understood by those of ordinary skill in the field to which the present invention pertains. The words such as "including" used herein mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items.

[0094] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0095] 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 could theoretically be obtained. The calculation method is: yield = (actual output / theoretical output) × 100%.

[0096] "Isotope abundance" refers to the percentage of the number of atoms of a specific isotope in the isotope mixture of an element, expressed as atom% A, where A is the code of the element symbol of the specific isotope.

[0097] In the deuterium-labeled compounds of the present invention, the isotope abundance represents the percentage of the number of deuterium atoms in the total number of hydrogen atoms in the target molecule. Generally, it can be measured by mass spectrometry means. If the MS response of the product is weak, it can also be determined by NMR means. For example, the isotope abundance of compound 3a was measured to be 98.8 atom% D, which means that at a specific position of this compound, the relative content of deuterium atoms has reached a relatively high level, indicating that the synthesis method of the present invention has high selectivity and efficiency in introducing deuterium atoms, can effectively control the degree of isotope labeling, meet the requirements for high isotope abundance of labeled compounds, and thus ensure the accuracy and reliability in subsequent applications such as internal standards.

[0098] Example 1: The optimal synthesis methods of deuterium-labeled fatty aldehydes, deuterium-labeled sphingosine and ceramides at the end of the alkyl chain of the present invention

[0099] In the fields of organisms, environment, medicine, etc., fatty aldehydes play an important role. As an important intermediate for the synthesis of sphingosine and ceramide, the preparation of deuterium-labeled fatty aldehydes is crucial for studying 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 example aims to find a new method for preparing deuterium-labeled fatty aldehydes at the end of the alkyl chain to solve the problems of high preparation cost and inapplicability for the economical and rapid preparation of sphingosine and ceramide internal standards in the prior art.

[0100] During the research and development process of the present invention, an initial attempt was made to directly use alkynes and expensive iodopropane-D7 as starting materials for the synthesis method. However, through experiments, it was found that this method had the problem of low product abundance, with the abundance being only about 90 atom% D. Through theoretical analysis and experimental verification, it was speculated that the reasons for this phenomenon were mainly the following two aspects: First, in a strong base reaction environment, the deuterium atom (D) at the α-position of the alkynyl group would undergo an exchange reaction with the hydrogen atom (H) in the reaction system environment; Second, the iodopropane molecule was very likely to undergo a carbocation rearrangement during the reaction process. During this process, the deuterium atom (D) in the iodopropane-D7 molecule would exchange with the hydrogen atom (H) in the system environment, thereby resulting in a decrease in product abundance. In the field of standard product research, there are strict requirements for product abundance. Usually, 95% abundance is regarded as the basic bottom line, and a more ideal abundance value should be above 98%. Therefore, the initial attempted synthesis method clearly could not meet the expected standards.

[0101] Based on the above research background and problems faced, this example proposes an innovative synthesis method for deuterium-labeled fatty aldehydes at the end of the alkyl chain based on a comprehensive consideration of the chemical structures, biological metabolic pathways, and internal standard design requirements of sphingosine and ceramide. Among them, 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 synthesis 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 in vivo into sphingosine and fatty acids with different carbon chain lengths and double bond numbers, choosing to introduce labeled atoms on the "sphingosine" carbon chain skeleton of ceramide (i.e., the fatty carbon chain) will be extremely convenient and economical for the rapid construction of the ceramide internal standard library. Considering the extremely high cost of the raw materials for constructing carbon-13 labeling and the great difficulty of the synthesis process, the stable isotope atom deuterium (D) is selected as the labeled atom for introduction. In addition, to ensure that the molecular weight of the internal standard has a certain difference from that of the analyte (natural abundance Cer) to be more conducive to detection, 5 - 7 D atoms are designed and introduced for the deuterium labeling number.

[0102] The specific synthesis method is as follows:

[0103] I. Synthesis of aliphatic aldehyde with deuterium labeled at the end of the alkyl chain

[0104]

[0105] The specific synthesis process is as above Figure 1 as shown, where R 1 can be H or an alkyl group; R 2 can be -Bn, -TBDS, -MOM, etc.; n can be 0, 1, 2, 3, ….

[0106] 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. For details, see Figure 1 In compound 2, R 1 can be H or an alkyl group; R 2 can be -Bn, -TBDS, -MOM, etc.; n can be 0, 1, 2, 3, …. In compound 2a, R 1 =H, R 2 =Mom, etc.; n = 10.

[0107] 1. First step: Protect the exposed hydroxyl group of compound 1. This step of the reaction can be carried out by reacting compound 1 with common protecting group reagents (such as 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 compound 1, the protecting group reagent, and the base is 1:(1.2 - 2):(1.2 - 2).

[0108] The synthesis of compound 2a is specifically as follows:

[0109] At room temperature, add 10.0 g of 14 - hydroxy - 2 - tetradecanone (an aliphatic methyl ketone compound, Chengdu Camel Pharmaceutical Technology Co., Ltd.), 100 mL of DCM, 7.4 g of DIEA, and 4.3 g of MomCl into a 250 mL single - necked flask. After addition, react at 25 °C for 16 hours. After the reaction is complete, pour the reaction solution into water, extract with dichloromethane, concentrate under reduced pressure to obtain a crude product, and purify it by Flash column chromatography to obtain intermediate 2a, 9.0 g, with a yield of 75.6%. 11H 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).

[0110] The nuclear magnetic resonance spectrum of compound 2a is specifically as follows Figure 2 shown.

[0111] 2. Second step: Intermediate 2 (compound 2a) undergoes D-H exchange under the action of a base to obtain Intermediate 3 (compound 3a), where 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 Intermediate 2 to the base is: 1:2 - 7.

[0112] The synthesis of compound 3a is specifically as follows

[0113] At room temperature, 5.0 g of compound 2a was dissolved in 100 mL of heavy water and 100 mL of 1,4-dioxane, and 15.6 g of potassium carbonate was added. After the addition, the reaction was carried out for 16 hours. After the reaction was completed, ethyl acetate was added for liquid-liquid 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 4.6 g of Intermediate 3a, with a yield of 90.3% and an isotope abundance of 98.8 atom% D. 1 1H 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).

[0114] The nuclear magnetic resonance spectrum of compound 3a is specifically as follows Figure 3 shown.

[0115] 3. Third step: Intermediate 3 (compound 3a) is converted into Intermediate 4 (compound 4a) with p-toluenesulfonylhydrazide under specific conditions, and the specific conditions here can be sodium borohydride, sodium borodeuteride, sodium cyanoborohydride, sodium cyanoborodeuteride, lithium aluminum hydride, deuterated lithium aluminum hydride, etc. The molar ratio of Intermediate 3 to the reducing agent is: 1:1.2 - 3.

[0116] The synthesis of compound 4a is specifically as follows

[0117] Dissolve 5.0 g of p-toluenesulfonylhydrazide and 7.5 g of compound 3a in 45 mL of CD 3 OD, add 3.6 g of sodium cyanoborodeuteride, and react for 16 hours. After the reaction, add dichloromethane and water for liquid separation and extraction. The organic phase is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product is separated and purified by Flash column chromatography to obtain 5.8 g of intermediate 4a with a yield of 80.6% and an isotope abundance of 98.6 atom% 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).

[0118] The nuclear magnetic resonance spectrum of compound 4a is specifically as Figure 4A shown.

[0119] 4. Fourth step: Intermediate 4 (compound 4a) removes the protecting group under certain conditions. If R 2 is -Bn, the "certain conditions" here can be "Pd / C / H 2 and other common methods for hydrogenation debenzylation". The mass ratio of the intermediate 4 to the catalyst is: 1:0.05 - 0.1. If R 2 is -TBDS or MOM, the "certain conditions" here include using fluorine reagents or acid reagents, such as ammonium fluoride, tetrabutylammonium fluoride, hydrochloric acid, sulfuric acid, trifluoroacetic acid, etc.

[0120] The synthesis of compound 5a is as follows:

[0121] Dissolve 5.2 g of compound 4a in 50 mL of methanol, add 5.2 mL of concentrated hydrochloric acid. After adding, react at 65 °C for 16 hours. After the reaction, dilute with water, extract with dichloromethane, dry with anhydrous sodium sulfate and concentrate under reduced pressure to obtain a crude product. The crude product is separated and purified by Flash column chromatography to obtain 4.0 g of a colorless oil with a yield of 92.3% and an isotope 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).

[0122] 5. Step 5: Conventional oxidation reaction. Intermediate 5 (Compound 5a) is oxidized by an oxidant to form Intermediate 6 (Compound 6a). The oxidant can be pyridinium chlorochromate, Dess-Martin periodinane, 2,2,6,6-tetramethylpiperidine 1-oxyl, dimethyl sulfoxide, sulfur trioxide-pyridine complex, etc. The molar ratio of Intermediate 5 to the oxidant is 1:1.2 - 3.

[0123] The synthesis of Compound 6a (alkyl chain end deuterium-labeled fatty aldehyde) is as follows:

[0124] Dissolve 3.0 g of Compound 5a in 50 mL of dichloromethane, and add 8.5 g of DMP. React at 25 °C for 3 hours. After the reaction is completed, pour the reaction solution into an aqueous sodium sulfite solution, extract with dichloromethane, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure to obtain a crude product, and purify by Flash column chromatography to obtain 2.0 g of a colorless oil, with a yield of 67.3% and an isotope 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).

[0125] The nuclear magnetic resonance spectrum of Compound 6a is specifically as Figure 5 shown.

[0126] II. Synthesis of Deuterium-Labeled Sphingosine and Ceramide

[0127]

[0128] The specific synthesis process is as above Figure 6 shown, where R3 includes any one or a combination of more than one of -TBDS, -MOM, -Trt; R4 includes any one or a combination of more than one of -Boc, -Fmoc, -Cbz, -Alloc, -Pht; R5 includes an alkyl chain (linear or branched) of C1 - C25; n is an integer including 0, 1, 2, 3, ….

[0129] 1. Step 1: Compound 7 reacts with dimethyl methylphosphonate under certain conditions to prepare Intermediate 8 (Compound 8a). The "certain conditions" here include using basic substances, and the basic substances can be lithium bis(trimethylsilyl)amide, lithium diisopropylamide, n-butyllithium, etc. The molar ratio of dimethyl methylphosphonate to the base is 1:1.2 - 1.5.

[0130] Synthesis of Compound 8a:

[0131] 74.0 g of dimethyl methylphosphonate was dissolved in 600 mL of THF, and the temperature was lowered to -65 °C. 120 mL of n-butyllithium was added dropwise. After the addition was complete, the system was reacted at this temperature for 0.5 h, 60.0 g of compound 7a was added, and the reaction was continued at this temperature for 3 h. The reaction was monitored by TLC until completion. The reaction solution was poured into ice-cold saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by Flash column chromatography to obtain 43.0 g of a colorless oil, with a yield of 56.1%. 1H NMR (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).

[0132] 2. Second step: Intermediate 8 (Compound 8a) reacts with an aliphatic aldehyde under specific conditions to form 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.

[0133] Synthesis of Compound 9a:

[0134] 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-necked flask and reacted at 20 °C for 16 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was separated and purified by Flash column chromatography to obtain 1.8 g of a colorless oil, with a yield of 81.8% and an isotope abundance of 98.8 atom% D. 11H NMR (400 MHz, 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, 3.5 Hz, 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).

[0135] 3. 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.

[0136] Synthesis of compound 10a:

[0137] 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. After the addition, the reaction was carried out at this temperature for 1 hour. After monitoring the reaction to completion by TLC, the reaction solution was poured into 0.1 N dilute hydrochloric acid, extracted with ethyl acetate, concentrated under reduced pressure to obtain a crude product, and purified by Flash column chromatography to obtain 1.4 g of a colorless oil, with a yield of 77.8% and an isotope abundance of 98.9 atom% D. 1 1H 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 = 8.4 Hz, 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).

[0138] 4. Fourth step: The intermediate 10 is deprotected under certain conditions. If R 3 is -TBDS, R4 It is - Boc, and the "certain conditions" here can be common methods such as acidic conditions.

[0139] Synthesis of compound D - sphingosine - D7:

[0140] Dissolve 1.0 g of compound 10a in 20 mL of dichloromethane, and add 9 mL of trifluoroacetic acid. After the reaction is complete, pour the reaction solution into sodium hydroxide solution, extract the aqueous phase with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product. Flash column chromatography is used for separation and purification to obtain 0.3 g of colorless oil, with a yield of 50.8% and an isotope 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).

[0141] The nuclear magnetic resonance spectrum of compound D - sphingosine - D7 is specifically as Figure 7 shown.

[0142] 5. The fifth step: Condense fatty acids (C2 - C26) with N - hydroxysuccinimide under certain conditions to obtain the corresponding active ester intermediate. The certain conditions here include using basic substances and condensing agents; the basic substances include bases such as triethylamine and diisopropylethylamine, and the condensing agents include condensing agents such as dicyclohexylcarbodiimide, 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide, and 1 - hydroxybenzotriazole. The molar ratio of the fatty acid, condensing agent, and base is: 1:1 - 1.5:1.2 - 1.5. React the above - obtained active ester with D - sphingosine - D7 under certain conditions to obtain different ceramides - D7. The molar ratio of D - sphingosine - D7 to the active ester is: 1:1 - 1.5. In the certain conditions here, the organic solvents used can be solvents such as dichloromethane, tetrahydrofuran, and N,N - dimethylformamide, and the reaction temperature is 20 - 50 °C.

[0143] Synthesis of compound C16 ceramide - D7:

[0144] Dissolve 0.1 g of sphingosine-D7 and 0.3 g of the corresponding active ester of palmitic acid in 3 mL of the solvent dichloromethane, and react 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 isotope 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).

[0145] The NMR spectrum of compound C16 ceramide-D7 is specifically as Figure 8 shown.

[0146] Synthesis of compound C20 ceramide-D7:

[0147] Dissolve 0.1 g of sphingosine-D7 and 0.48 g of the corresponding active ester of eicosanoic acid in 3 mL of the solvent dichloromethane, and react 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 isotope 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, 1.5 Hz, 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).

[0148] The NMR spectrum of compound C20 ceramide-D7 is specifically as Figure 9 shown.

[0149] Synthesis of Compound C22 Ceramide-D7:

[0150] Dissolve 0.15 g of sphingosine-D7 and 0.32 g of the active ester corresponding to docosanoic acid in 15 mL of dichloromethane as the solvent, and react at 20 °C for 16 hours. After the reaction, the reaction mixture is concentrated to obtain a crude product, which is separated and purified by Flash column chromatography to obtain 0.24 g of a white solid, with a yield of 77.9% and an isotope 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).

[0151] The nuclear magnetic resonance spectrum of Compound C22 Ceramide-D7 is specifically as Figure 10 shown.

[0152] Synthesis of Compound C22:1 Ceramide-D7:

[0153] Dissolve 0.12 g of sphingosine-D7 and 0.26 g of the active ester corresponding to docosanoic acid in 15 mL of dichloromethane as the solvent, and react at 20 °C for 16 hours. After the reaction, the reaction mixture is concentrated to obtain a crude product, which is separated and purified by Flash column chromatography to obtain 0.19 g of a white solid, with a yield of 79.2% and an isotope abundance of 99.0 atom% D. 11H NMR (400 MHz, 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).

[0154] The nuclear magnetic resonance spectrum of compound C22:1 ceramide-D7 is specifically as follows Figure 11 shown

[0155] In this example, through two key reactions of D-H exchange and deuterium-labeled reduction, the hydrogen atoms on the saturated carbon chain were directionally deuterium-substituted, and an efficient preparation method for deuterium-labeled aliphatic aldehydes with a deuterium label at the end of the alkyl chain was 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 has a short synthesis cycle. Each deuterium-labeled intermediate involved is a new compound. At the same time, the synthesis of deuterium-labeled compounds and their derivatives with different carbon chain lengths or containing branched-chain structures, such as deuterium-labeled alkyl alcohols and deuterium-labeled carboxylic acids, was achieved, and the synthesis scheme is brand new. Moreover, the raw materials and reagents used in the scheme are all common commercially available products and are easy to obtain. The best scheme provided in this example ensures that the isotope abundances of each intermediate and target product synthesized are above 98%, the process is controllable, and there will be no serious dilution phenomenon of isotope abundance. The deuterium-labeled aliphatic aldehyde synthesized at the end of the alkyl chain can not only be used as a key synthon for the internal standards of sphingosine and ceramide, but also be used in the synthesis of other popular compounds, such as the synthesis of polypeptide drugs. At the same time, the idea and method of introducing deuterium-labeled atoms on the terminal carbon chain in the present invention can also be used as a reference for the design of internal standards for other compounds with saturated fatty carbon chains.

[0156] Example 2: The necessity of using "aliphatic methyl ketone compounds" as raw materials in the present invention

[0157] In the preparation method of the present invention, different raw materials are used to synthesize deuterium-labeled fatty aldehydes, deuterium-labeled sphingosine, and ceramides at the end of the alkyl chain, which have a great impact on the yield, deuteration rate, and overall quality of the products. To verify the necessity of using "aliphatic methyl ketone compounds" as raw materials in the present invention, in this example, a comparative verification was carried out, and the specific experimental process is as follows:

[0158] 14-Hydroxy-2-tetradecanone (the best aliphatic methyl ketone compound of the present invention) and tetracosanoic acid (a commercially available fatty acid 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) of Shanghai Sigma-Aldrich Biotechnology Co., Ltd.) were respectively used as raw materials, and compound 6a was prepared according to the best preparation method in Example 1 of the present invention, and the abundances of the target labeled compounds prepared under different raw materials were calculated respectively. The results are shown in Table 1.

[0159] Table 1. Abundances of deuterium-labeled fatty aldehydes at the end of the alkyl chain prepared under different raw materials

[0160]

[0161] As can be seen from Table 1, the scheme of using 14-hydroxy-2-tetradecanone (aliphatic methyl ketone compound) as a raw material to prepare compound 6a through multiple chemical reactions such as D-H exchange has a relatively high isotope abundance.

[0162] At the same time, when using commercially available tetracosanoic acid as a raw material, through the method of D-H exchange full labeling (non-selective), the abundance of the finally prepared product is only about 94%. This method lacks the ability to accurately locate the target site, resulting in strong randomness of hydrogen-deuterium exchange and difficulty in synthesizing specific deuterium-labeled products with high selectivity and high abundance. At the same time, in the prior art, the number of deuterium labels is generally preferably more than 3 and less than 10. Because a large increase in the number of deuteriums will change the physicochemical properties such as the electron cloud distribution, spatial conformation, and polarity of the molecule, such as affecting the retention time in chromatographic analysis, ionization efficiency and fragmentation pattern in mass spectrometry detection, etc., thus affecting the accuracy of detection use. Therefore, in this example, aliphatic methyl ketone compounds (14-hydroxy-2-tetradecanone) are preferably used as raw materials.

[0163] 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.

[0164] Example 3: Comparative verification of the necessity of the "one-pot method" used in the present invention

[0165] 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:

[0166] 1. Method 1: The process of synthesizing compound 4a from compound 3a using the 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 CD 3 OD, 3.6 g of sodium cyanoborodeuteride was added and the reaction was carried out for 16 hours. After the reaction was completed, dichloromethane and water were added for separation and extraction, and 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 98.6atom% 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).

[0167] The NMR spectrum of compound 4a is as follows: Figure 4A shown.

[0168] 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.

[0169] The yields and isotope abundances of compound 4a prepared by different methods were calculated separately, and the results are shown in Table 2.

[0170] Table 2. Yields and isotope abundances of compound 4a prepared by different methods

[0171]

[0172] As can be seen from Table 2, only when the "one-pot method" is adopted can the highest yield and isotope abundance of the prepared compound 4a be obtained. This is because the "one-pot method" of the present invention has significant advantages. It simplifies the operation process, reduces the losses of raw materials and products during the separation process, reduces the experimental operation steps, and reduces the risk of introducing impurities, thus ensuring that both the purity and isotope abundance of the product reach the highest, significantly improving the synthesis efficiency. On the other hand, it does not require heating, thus saving energy and avoiding the need for additional equipment.

[0173] Therefore, it can be known that the present invention preferably adopts the "one-pot method" to prepare deuterium-labeled aliphatic aldehydes at the end of the alkyl chain.

[0174] Example 4: Comparative verification on the necessity of using "sodium cyanoborodeuteride" as a reducing agent in the present invention

[0175] In the third step of the present invention, during the process of converting compound 3a into compound 4a under specific conditions, the specific conditions preferably use "sodium cyanoborodeuteride" as a reducing agent. To verify the advantages of using "sodium cyanoborodeuteride" as a reducing agent in the present invention, the following comparative verification tests were carried out. The reaction was carried out according to the best method of Example 1, with the difference being the optimization and screening of different reducing agents. The yields and isotope abundances of compound 4a were measured. The selected reducing agents and the corresponding results are shown in Table 3 (meanwhile, due to the D-H exchange reaction mechanism adopted in the present invention having specific requirements for the deuterated characteristics of the reducing agent, in this example, only reducing agents with deuterated structures that can effectively promote the target conversion, ensure the isotope abundance and yield of the product in this reaction system were screened. Conventional non-deuterated reducing agents such as sodium borohydride, sodium cyanoborohydride, and lithium aluminum hydride were not included in this screening range).

[0176] Table 3. Yields and isotope abundances of compound 4a prepared by different methods

[0177]

[0178] As can be seen from Table 3, when sodium cyanoborodeuteride, sodium borodeuteride, and lithium aluminum deuteride are used as reducing agents respectively, the finally prepared compound 4a has good yields and isotope abundances. This is because when using "sodium cyanoborodeuteride" as the reducing agent, compared with other reducing agents, its reaction activity is more moderate, which is conducive to improving the selectivity of the reaction, reducing the occurrence of side reactions, and thus helping to significantly improve the yield and isotope abundance of the product.

[0179] Therefore, the present invention preferably uses "sodium cyanoborodeuteride" as the reducing agent.

[0180] Example 5: Study on D-H exchange under the action of a base

[0181] In the second step reaction of the present invention, the preparation of compound 3a mainly depends on the D-H exchange reaction, and the base and 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 substrates.

[0182] In this example, the D-H exchange reaction for the conversion of compound 2a to compound 3a was studied. The reaction was carried out according to the best method of Example 1, with the difference being the change of the base and deuterium source. The yields and deuteration rates of the reaction were measured. The selected bases and deuterium sources are shown in Table 4. Only the 7 combinations in the table can enable the D-H exchange reaction to proceed. The combinations of other bases and deuterium sources have been proven to have extremely low deuteration rates and low yields through experiments. The results of the yields and deuteration rates of the 7 combinations are shown in Table 4.

[0183] Table 4. Selection of bases, deuterium sources and results of yields and isotope abundances

[0184]

[0185] According to the experimental results, there is a great correlation between the selection of bases, deuterium sources and yields and isotope abundances. When sodium bicarbonate is selected as the base, since sodium bicarbonate has a weak alkalinity and is not sufficient to dissociate the hydrogen at the α-position of the carbonyl group, the deuteration rate is too low, so it is not suitable as a reagent for the deuteration reaction. When potassium carbonate is selected as the base and heavy water is selected as the deuterium source, the highest yield for the conversion of compound 2 to compound 3 is 66.7%, and the highest deuteration rate is 98.8 atom% D, which should be preferred.

[0186] Therefore, in the process of synthesizing compound 3a from compound 2a in the second step reaction, potassium carbonate is preferably used as the base and heavy water is used as the deuterium source.

[0187] Example 6: Study on reagent screening in different steps of the present invention

[0188] In the synthesis process of the present invention, the combination of different reagents in each step will have a great impact on the synthesis of the corresponding compound. Therefore, in this example, the combination of reagents in different steps was optimized and screened as follows:

[0189] I. Synthesis of Fatty Aldehydes with Deuterium Label at the End of the Alkyl Chain

[0190] 1. Fifth Step: In the fifth-step reaction, the reaction of converting compound 5a to compound 6a was studied. The reaction was carried out according to the optimal method of Example 1, with the difference being the optimization and screening of different oxidants. The yields and isotope abundances of compound 6a (fatty aldehyde with deuterium label at the end of the alkyl chain) were determined. The selected oxidants and the corresponding results are shown in Table 5 below.

[0191] Table 5. Effects of Different Oxidants on the Synthesized Compound 6a

[0192]

[0193] According to the test results, in the synthesis process of compound 6a, the selection of the oxidant is highly correlated with the yield of compound 6a. The results show that when different oxidants are selected, there are significant differences in the yields of the finally prepared compound 6a. Moreover, when the preferred oxidant is Dess-Martin periodinane, both the yield and isotope abundance of the prepared compound 6a reach the highest. This is because different oxidants have different degrees of fitness for the substrate, which in turn affects the reaction conversion rate.

[0194] Therefore, in the fifth-step reaction for synthesizing compound 6a, the preferred oxidant is Dess-Martin periodinane, and the prepared compound 6a has the highest yield and isotope abundance at this time.

[0195] II. Synthesis of Deuterium-Labeled Sphingosine and Ceramide

[0196] 1. Second Step: In the second-step reaction, the process of the reaction between compound 8a and the labeled fatty aldehyde to form compound 9a was studied. The reaction was carried out according to the optimal method of Example 1, with the difference being the adoption of different reaction conditions, and the yields of the reactions were determined. The selected different reaction conditions and the corresponding results are shown in Table 6 below.

[0197] Table 6. Effects of Different Reaction Conditions on the Yield of Compound 9a

[0198]

[0199] According to the test results, there is a significant correlation between different reaction conditions and the yield of compound 9a. The results show that when the reaction conditions are selected as the combination of inorganic lithium salt reagents and bases, the finally prepared compound 9a has a good yield. And when the combination of lithium chloride and triethylamine is preferred, the yield of the compound 9a prepared at this time is the best. This may be because lithium chloride is an inorganic lithium salt reagent and triethylamine is an organic base reagent. When the two act synergistically, the reaction efficiency and selectivity can be significantly improved. Lithium chloride can accurately activate the reaction site of compound 8a through ion complexation, effectively reducing the activation energy of the target reaction; triethylamine, as an organic base, can accurately extract the protons generated in the reaction, maintaining the stability of the alkaline environment in the reaction system and ensuring the continuous forward progress of the reaction. And limiting the appropriate molar ratio of the two (1:1.2 - 1.5) is beneficial to strictly control the reaction rate and degree, avoiding side reactions caused by excessive base amount or incomplete reaction due to insufficient base amount, thereby greatly improving the product yield and isotope abundance.

[0200] Therefore, in the process of synthesizing compound 9a, the preferred reaction conditions are the combination of inorganic lithium salt reagents and organic base reagents. And when the combination of lithium chloride and triethylamine is preferred, the yield of the compound 9a prepared at this time reaches the best.

[0201] 2. The third step: In the third-step reaction, the reaction of compound 9a to compound 10a was studied. The reaction was carried out according to the best method of Example 1, with the difference being the optimization and screening of different reducing agents. The yield and isotope abundance of compound 10a were measured. The selected reducing agents and the corresponding results are shown in Table 7.

[0202] Table 7. Effects of different reducing agents on the synthesized compound 10a

[0203]

[0204] According to the test results, in the process of synthesizing compound 10a, the selection of the reducing agent is significantly correlated with the yield of compound 10a. The results show that when different reducing agents are selected, there are obvious differences in the yield of the finally prepared compound 10a. And when the preferred reducing agent is lithium tri-tert-butoxyaluminum hydride, the yield of the compound 10a prepared at this time reaches the highest. This may be because lithium tri-tert-butoxyaluminum hydride has a high steric hindrance effect and has good stereoselective induction during the reduction process, thus efficiently converting the target configuration product.

[0205] Therefore, in the process of synthesizing compound 10a in the third-step reaction, the preferred reducing agent is lithium tri-tert-butoxyaluminum hydride, and the compound 10a prepared at this time has the highest yield.

[0206] 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. An alkyl chain terminal deuterium labeled fatty aldehyde compound, characterized in that: The alkyl chain terminal deuterium-labeled fatty aldehyde compound has the following chemical structure: ； Wherein, R1 is H; n is 10.

2. The method for preparing an alkyl chain terminal deuterium-labeled fatty aldehyde compound according to claim 1, characterized in that: The alkyl chain terminal deuterium-labeled fatty aldehyde compound is prepared using aliphatic methyl ketone compounds as raw materials by using a DH exchange method; the synthetic route is as follows: ； Wherein, R2 is any one or more combinations of -Bn, -TBDS, and -MOM; The invention comprises the following steps: step (1) hydroxyl protection: taking an aliphatic methyl ketone compound as a raw material, reacting it with a protecting group reagent to prepare compound 2; step (2) DH exchange: compound 2 is subjected to DH exchange under the action of a base and a deuterium source to prepare compound 3; the base is any one of potassium carbonate, potassium tert-butoxide, sodium methoxide and sodium bicarbonate; the deuterium source of DH exchange is heavy water; step (3) compound 3 is reacted with p-toluenesulfonyl hydrazide under the action of a reducing agent to convert it into compound 4, the reducing agent is sodium cyanoborodeuteride, and the preparation is carried out by a "one-pot method"; in step (4), compound 4 is deprotected to prepare compound 5; step (5) oxidation reaction: compound 5 is oxidized by an oxidant to generate compound 6, in step (5), the oxidant is any one or more of pyridinium chlorochromate, Dess-Martin oxidant, tetramethylpiperidinium oxide, dimethyl sulfoxide and sulfur trioxide-pyridine complex; compound 6 is a fatty aldehyde compound labeled with deuterium at the end of the alkyl chain.

3. The preparation method according to claim 2, characterized in that: In the step (1), the protecting group reagent includes any one or more combinations of BnBr, TBDSCl, and MOMCl.

4. The preparation method according to claim 2, characterized in that: In the step (4), compound 4 is deprotected under certain conditions to obtain compound 5; when R2 is -Bn, the "certain conditions" are the use of a catalyst, and the catalyst is any one or more combinations of Pd / C and H2; when R2 is -TBDS or MOM, the "certain conditions" are the use of any one or more combinations of fluorine reagents or acid reagents.

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