Synthesis method of deuterium-labeled long-chain fatty acid acyl carnitine

Abstract

The invention discloses a synthesis method of deuterium-labeled long-chain fatty acid acyl carnitine, and belongs to the technical field of medicinal chemistry. (R)-4-chloro-3-hydroxy-ethyl butyrate is used as a raw material, and is placed in a benzyl alcohol solvent to prepare (R)-4-chloro-3-hydroxy-benzyl butyrate; the preparation method comprises the following steps: reacting (R)-4-chloro-3-hydroxy-benzyl butyrate with dimethylamine hydrochloride D6 to prepare (R)-4-(dimethylamino-D6)-3-hydroxy-benzyl butyrate; the preparation method comprises the following steps: carrying out quaternization reaction on (R)-4-(dimethylamino-D6)-3-hydroxy-benzyl butyrate in methyl iodide to prepare an L-carnitine benzyl ester-D6 iodide; the L-carnitine benzyl ester-D6 iodide is prepared into L-carnitine benzyl ester-D6 hydrochloride under the condition of silver oxide and hydrochloric acid; condensing the L-carnitine benzyl ester-D6 hydrochloride and long-chain fatty acid to obtain acyl carnitine benzyl ester; and debenzylating the acyl carnitine benzyl ester under the conditions of palladium, carbon and hydrogen to synthesize the deuterium-labeled long-chain fatty acid acyl carnitine. According to the synthesis method provided by the invention, various long-chain fatty acid acyl carnitine labeled by deuterium is realized, a method for mass spectrum quantitative analysis of acyl carnitine is simplified, and the disease screening efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly relates to a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine. Background Art

[0002] Carnitine is an amino acid derivative containing a quaternary ammonium cation, first discovered in meat extracts in 1905. Endogenous carnitine in the human body mainly exists in the forms of free carnitine and short, medium, and long-chain conjugated acyl carnitines. Acyl carnitine is a metabolite of fatty acids, participates in the energy metabolism of various cells, and has long been used as a diagnostic marker for abnormal fatty acid metabolism. In recent years, acyl carnitine has shown potential as a biomarker in the research of other different types of metabolic disorders and related diseases. Especially in the detection of cardiovascular diseases, diabetes, depression, neurological diseases, and certain cancers, it has become an important metabolic index.

[0003] There are a wide variety of fatty acids in the human body, including carbon chains of different lengths, functional group modifications at different positions, and varying numbers of unsaturated bonds, which makes the structure of acyl carnitine complex and diverse. Therefore, comprehensive quantitative analysis of acyl carnitine is crucial for understanding the pathological mechanisms of various diseases, differential diagnosis, and treatment research.

[0004] The use of tandem mass spectrometry technology to determine the levels of free carnitine and acyl carnitine in patients has been widely applied in clinical screening for various disease types. By measuring the absolute concentrations of different types of acyl carnitines in patients, multiple fatty acid metabolism diseases can be screened simultaneously, and positive results can be further confirmed through biochemical experiments or gene sequencing.

[0005] Since the mass spectrometry quantitative analysis of complex biological samples usually requires stable isotope standards, acyl carnitine containing stable isotope labels is particularly important in disease screening. Therefore, developing a technology capable of synthesizing deuterium-labeled long-chain fatty acyl carnitine will greatly simplify the method design for mass spectrometry quantitative analysis of acyl carnitine. However, the existing chemical synthesis methods for deuterium-labeled long-chain fatty acyl carnitine currently cannot achieve this goal. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine to solve the problems in the background art.

[0007] To achieve the above purpose, the present invention provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine, including the following steps:

[0008] S1. Using (R)-ethyl 4-chloro-3-hydroxybutyrate as a raw material, preparing (R)-benzyl 4-chloro-3-hydroxybutyrate in a benzyl alcohol solvent;

[0009] (R)-4-Chloro-3-hydroxy-butanoic acid benzyl ester and dimethylamine hydrochloride-D prepared from S2 and S1 6 React to obtain (R)-4-(dimethylamino-D 6 )-3-hydroxy-butanoic acid benzyl ester;

[0010] (R)-4-(dimethylamino-D prepared from S3 and S2 6 )-3-hydroxy-butanoic acid benzyl ester undergoes quaternization reaction in iodomethane to obtain levocarnitine benzyl ester-D 6 iodide;

[0011] Levocarnitine benzyl ester-D prepared from S4 and S3 6 iodide is prepared into levocarnitine benzyl ester-D under the conditions of silver oxide and hydrochloric acid 6 hydrochloride;

[0012] Levocarnitine benzyl ester-D prepared from S5 and S4 6 hydrochloride is condensed with long-chain fatty acid to obtain acylcarnitine benzyl ester;

[0013] The acylcarnitine benzyl ester prepared from S6 and S5 is debenzylated under the conditions of palladium-carbon and hydrogen to synthesize deuterium-labeled long-chain fatty acid acylcarnitine;

[0014] The synthetic route is as follows:

[0015]

[0016] Preferably, the specific steps of S1 are as follows: Add (R)-4-chloro-3-hydroxy-butanoic acid ethyl ester and benzyl alcohol to a three-necked flask, dropwise add concentrated hydrochloric acid under ice bath conditions, reflux and react overnight at 140 °C. After the reaction is completed, add ether, store overnight at -20 °C, pour out the ether layer, and distill off the solvent under reduced pressure to obtain (R)-4-chloro-3-hydroxy-butanoic acid benzyl ester.

[0017] Preferably, the specific steps of S2 are as follows: Under the protection of argon and ice-salt bath conditions, add anhydrous potassium carbonate and anhydrous tetrahydrofuran to a three-necked flask, and then add dimethylamine hydrochloride-D 6 , stir well; then dissolve the (R)-4-chloro-3-hydroxy-butanoic acid benzyl ester prepared in S1 in anhydrous tetrahydrofuran, and drop it into the above three-necked flask for reaction overnight. After the reaction is completed, spin-dry the solvent, add water and extract with DCM, combine the organic phases, then spin-dry the solvent, and finally perform column chromatography to obtain (R)-4-(dimethylamino-D 6 )-3-hydroxy-butanoic acid benzyl ester.

[0018] Preferably, the specific steps of S3 are as follows: Add the (R)-4-(dimethylamino-D prepared in S2 6) -3-hydroxybutyric acid benzyl ester was dissolved in acetone, and methyl iodide was added and reacted overnight. After the reaction was completed, the solvent was concentrated by distillation under reduced pressure, and purified by silica gel column chromatography to obtain L-carnitine benzyl ester-D 6 iodide.

[0019] Preferably, the specific steps of S4 are: Dissolve the L-carnitine benzyl ester-D 6 iodide obtained in S3 in MeOH / H 2 O, add silver oxide and react. After the reaction is completed, filter to remove insoluble substances. The filter residue is washed with MeOH / H 2 O and the solvent is evaporated by rotary evaporation. The residue is dissolved in concentrated hydrochloric acid and freeze-dried to obtain L-carnitine benzyl ester-D 6 hydrochloride.

[0020] Preferably, the specific steps of S5 are: Dissolve the long-chain fatty acid in a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, add 4-dimethylaminopyridine and the L-carnitine benzyl ester-D 6 hydrochloride obtained in step S4, then add N,N'-diisopropylcarbodiimide and react with stirring overnight. After the reaction is completed, remove the solvent, and separate by column chromatography to obtain acyl carnitine benzyl ester.

[0021] Preferably, in S5, the long-chain fatty acid is any one of dodecanoic acid (lauric acid, C12:0), tridecanoic acid (C13:0), tetradecanoic acid (myristic acid, C14:0), cis-9-tetradecenoic acid (myristoleic acid, C14:1), cis-2,4-tetradecadienoic acid (C14:2), 3-hydroxytetradecanoic acid (3-hydroxymyristic acid, C14:OH), pentadecanoic acid (C15:0), cis-10-pentadecenoic acid (C15:1), hexadecanoic acid (palmitic acid, C16:0), cis-9-hexadecenoic acid (palmitoleic acid, C16:1), heptadecanoic acid (C17:0), cis-10-heptadecenoic acid (C17:1), octadecanoic acid (stearic acid, C18:0), cis-9-octadecenoic acid (oleic acid, C18:1), cis,cis-9,13-octadecadienoic acid (linoleic acid, C18:2), 3-hydroxy-octadecanoic acid (C18:OH), nonadecanoic acid (C19:0), eicosanoic acid (arachidic acid, C20:0), cis-11-eicosenoic acid (C20:1), cis-11,14-eicosadienoic acid (C20:2), cis-8,11,14-eicosatrienoic acid (C20:3), cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid, C20:4), heneicosanoic acid (C21:0), docosanoic acid (behenic acid, C22:0), cis-13-docosenoic acid (erucic acid, C22:1), cis-13,16-docosadienoic acid (C22:2), cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6), tricosanoic acid (23:0), tetracosanoic acid (24:0), cis-15-tetracosenoic acid (nervonic acid, C24:1), pentacosanoic acid (C25:0), hexacosanoic acid (C26:0).

[0022] Preferably, the specific steps of S6 are as follows: Dissolve the acyl carnitine benzyl ester prepared in step S5 in MeOH, add 10% palladium on carbon, introduce hydrogen for reaction, filter off the palladium on carbon after the reaction is completed, wash the filter residue with MeOH, and then freeze-dry to obtain deuterium-labeled long-chain fatty acid acyl carnitine.

[0023] Therefore, the synthesis method of deuterium-labeled long-chain fatty acid acyl carnitine provided by the present invention realizes the deuterium labeling of various long-chain fatty acid acyl carnitines, greatly simplifies the method for mass spectrometry quantitative analysis of acyl carnitine, and improves the efficiency of disease screening.

[0024] The technical solutions of the present invention will be further described in detail below through examples. Detailed Embodiments

[0025] The technical solutions of the present invention will be further described below through embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application and belong to the protection scope of the present invention.

[0026] As used herein, the mention of "embodiment" means that the specific features, structures, or characteristics described in connection with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" that appears in various positions in the specification does not necessarily refer to the same embodiment, nor is it particularly limited to the independence or relevance with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0027] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the technical field to which this application belongs; the use of the relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0028] Unless otherwise specified in the present invention, the reagents, instruments, equipment, etc. used are all the reagents, instruments, and equipment commonly used by those skilled in the art in this field and can be obtained on the market. For those not specifically indicating specific conditions, conventional conditions or conditions recommended by the manufacturer are adopted.

[0029] In the following embodiments, the structures of the compounds are determined by nuclear magnetic resonance (NMR) and techniques.

[0030] The measuring instrument for nuclear magnetic resonance (NMR) is a Bruker 400 MHz nuclear magnetic resonance spectrometer, and the solvents used are deuterated methanol (CD 3 OD) and deuterated chloroform (CDCl 3 ), and the internal standard substance is tetramethylsilane (TMS).

[0031] The meanings represented by the abbreviations in the following nuclear magnetic resonance (NMR) data of the embodiments are as follows:

[0032] s: singlet, D: doublet, t: triplet, q: quartet, DD: double doublet, m: multiplet, br: broad peak, J: coupling constant, Hz: Hertz, δ: chemical shift.

[0033] Example 1

[0034] This embodiment provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine, comprising the following steps:

[0035] S1. Preparation of compound II [(R)-4-chloro-3-hydroxy-butanoic acid benzyl ester]

[0036] Structural formula of compound II:

[0037] Compound I [(R)-4-chloro-3-hydroxy-butanoic acid ethyl ester] (16.6 g, 100.0 mmol, 1.1 eq) and benzyl alcohol (155.8 mL, 1.5 mol, 15.0 eq) were added to a 500 mL three-necked flask to obtain a colorless suspension. Under ice bath conditions, concentrated hydrochloric acid (10.0 mL) was slowly added dropwise. The mixture was refluxed at 140 °C overnight, and the reaction progress was monitored by TLC. After the reaction was completed, 300 mL of ether was added and stored overnight at -20 °C. The next day, the ether layer was poured out, and the solvent was removed by distillation under reduced pressure to obtain a brown liquid II (9.4 g, yield 45%).

[0038] S2. Preparation of compound III [(R)-4-(dimethylamino-D 6 )-3-hydroxy-butanoic acid benzyl ester]

[0039] Structural formula of compound III:

[0040] Under argon protection and ice-salt bath conditions, anhydrous potassium carbonate (4.2 g, 30 mmol, 3.0 eq) was added to a 50 mL three-necked flask, and then anhydrous tetrahydrofuran (20 mL) was added. Then, dimethylamine hydrochloride-D 6 (1.74 g, 20 mmol, 2.0 eq) was slowly added and stirred well for 30 minutes. Then, (R)-4-chloro-3-hydroxy-butanoic acid benzyl ester (2.28 g, 10 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (5 mL) and placed in a dropping funnel. The ice-salt bath was maintained and the tetrahydrofuran solution of (R)-4-chloro-3-hydroxy-butanoic acid benzyl ester was slowly added dropwise (1 drop / second), and the reaction was carried out overnight, and the temperature was always maintained between -5 °C and 5 °C. After the overnight reaction, it was naturally restored to room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated to dryness, water (20 mL) was added, and extraction was carried out with DCM (3 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. Finally, column chromatography was carried out using neutral alumina to obtain a colorless oil III (1.51 g, yield 62%).

[0041] 1 HNMR(400MHz,CDCl 3): δ = 7.38–7.28 (m, 5H), 5.15 (s, 2H), 4.15–4.08 (m, 1H), 2.56–2.43 (m, 2H), 2.38–2.32 (m, 1H), 2.25–2.21 (m, 1H).

[0042] S3. Preparation of Compound IV [L-Carnitine Benzyl Ester-D 6 Iodide]

[0043] Structural formula of Compound IV:

[0044] Dissolve (R)-4-(Dimethylamino-D 6 )-3-hydroxybutyric acid benzyl ester (4.91 g, 20.24 mmol, 1.0 eq) in acetone (25 mL), add methyl iodide (1.88 mL, 30.36 mmol, 1.5 eq) and react overnight. After the reaction is completed, concentrate the solvent by distillation under reduced pressure, and purify and separate by silica gel column chromatography to obtain white solid IV (3.79 g, yield 49%).

[0045] 1 1H NMR (400 MHz, D 2 2O): δ = 7.44–7.33 (m, 5H), 4.61 (s, 2H), 4.55–4.49 (m, 1H), 3.38–3.37 (m, 2H), 3.17 (s, 3H), 2.46–2.35 (m, 2H).

[0046] S4. Preparation of Compound V [L-Carnitine Benzyl Ester-D 6 Hydrochloride]

[0047] Structural formula of Compound V:

[0048] At room temperature, dissolve L-carnitine benzyl ester-D 6 iodide (3.85 g, 10.0 mmol, 1.0 eq) in MeOH / H 2 2O (v / v = 1 / 6, 29 mL), add an equivalent amount of silver oxide (2.3 g, 10.0 mmol, 1.0 eq). Monitor the reaction progress by TLC. After the reaction is completed, filter off the insoluble matter using diatomaceous earth, wash the filter residue with MeOH / H 2 2O (v / v = 1 / 6, 30 mL), and then evaporate the solvent to dryness. Dissolve the residue in concentrated hydrochloric acid (10 mL) and freeze-dry to obtain white solid V.

[0049] 1 1H NMR (400 MHz, CD 3OD): δ = 7.42–7.28 (m, 5H), 5.24–5.12 (m, 2H), 4.62 - 4.59 (m, 1H), 3.52–3.40 (m, 2H), 3.22 (s, 3H), 2.64 (d, J = 7.5 Hz, 2H).

[0050] S5. Preparation of Compound VI [Dodecanoyl L-Carnitine Benzyl Ester - D 6 Hydrochloride] (VI-C12:0)

[0051] Structural formula of Compound VI:

[0052] Under anhydrous and dry conditions, dodecanoic acid (150.0 mg, 0.75 mmol, 1.5 eq) was dissolved in a mixed solvent of dry N,N-dimethylformamide (1.0 mL) and tetrahydrofuran (0.15 mL). L-Carnitine benzyl ester - D 6 Hydrochloride (146.5 mg, 0.5 mmol, 1.0 eq) and 4-dimethylaminopyridine (12.2 mg, 0.1 mmol, 0.2 eq) were added. Subsequently, N,N'-diisopropylcarbodiimide (126.2 mg, 1.0 mmol, 2.0 eq) was added. The reaction was stirred overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed, and column chromatography was used to separate to obtain white solid VI-C12:0 (83.4 mg, yield 34%).

[0053] 1 HNMR (400 MHz, CD 3 OD): δ = 7.42–7.29 (m, 5H), 5.66 (q, J = 6.8 Hz, 1H), 5.16 (s, 2H), 3.86 (dd, J = 14.4, 8.5 Hz, 1H), 3.69 (dd, J = 14.5 1.5 Hz, 1H), 3.19 (s, 3H), 2.84 (d, 6.1 Hz, 2H), 2.31 (td, J = 7.5, 6.2 Hz, 2H), 1.65–1.48 (m, 2H), 1.30 (s, 16H), 0.91 (m, t, J = 6.6 Hz, 3H).

[0054] S6. Preparation of Compound VII [Dodecanoyl L-Carnitine - D 6 Hydrochloride] (VII-C12:0)

[0055] Structural formula of Compound VII:

[0056] Under anhydrous and dry conditions, dodecanoyl L-carnitine benzyl ester - D 6The hydrochloride salt (49.2 mg, 0.1 mmol, 1.0 eq) was dissolved in MeOH (1.0 mL), 10% palladium on carbon (4.9 mg) was added, and the reaction was carried out under hydrogen. The reaction was monitored by TLC. After the reaction was completed, the palladium on carbon was removed by filtration through diatomaceous earth, and the residue was washed with MeOH (20 mL) and then freeze-dried to obtain white solid VII-C12:0 (32.7 mg, yield 82%).

[0057] 1 HNMR(400MHz,CD 3 OD): δ = 5.62(q, J = 7.0Hz, 1H), 3.87(dd, J = 14.4, 8.5Hz, 1H), 3.71(dd, J = 14.4, 1.3Hz, 1H), 3.21(s, 3H), 2.76(dd, J = 6.3, 3.8Hz, 2H), 2.39(t, J = 7.5Hz, 2H), 1.62(q, J = 7.2Hz, 2H), 1.29(s, 16H), 0.94–0.86(m, 3H).

[0058] Example 2:

[0059] This example provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine. Steps S1 - S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0060] S5. Preparation of compound VI [cis-2,4-tetradecadienoyl-L-carnitine benzyl ester-D 6 hydrochloride] (VII-C14:2)

[0061] Structural formula of compound VI:

[0062]

[0063] Under anhydrous and dry conditions, cis-2,4-tetradecenoic acid (336.1 mg, 1.5 mmol, 1.5 eq) was dissolved in dry N,N-dimethylformamide (2.0 mL) and tetrahydrofuran (0.15 mL), L-carnitine benzyl ester-D 6 hydrochloride (293.8 mg, 1.0 mmol, 1.0 eq) and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol, 0.2 eq) were added, and then N,N'-diisopropylcarbodiimide (252.2 mg, 2.0 mmol, 2.0 eq) was added and the reaction was carried out overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed, and column chromatography was used for separation to obtain white solid VI-C14:2 (131.2 mg, yield 32%).

[0064] 1HNMR (400 MHz, CD 3 OD): δ = 7.42–7.28 (m, 6H), 6.33–6.20 (m, 2H), 5.88 (d, J = 15.2 Hz, 1H), 5.74–5.69 (m, 1H), 5.16 (s, 2H), 3.97–3.91 (m, 1H), 3.77–3.73 (m, 1H), 3.23–3.15 (s, 3H), 2.91–2.79 (m, 2H), 2.25–2.21 (m, 2H), 1.47 (q, J = 7.2, 14.4 Hz, 2H), 1.32–1.30 (m, 12H), 0.92 (t, J = 6.8 Hz, 3H).

[0065] S6. Preparation of Compound VII [cis-2,4-Tetradecadienoyl L-Carnitine-D 6 Hydrochloride] (VII-C14:2)

[0066] Structural formula of Compound VII:

[0067] Under anhydrous and dry conditions, dissolve cis-2,4-tetradecadienoyl L-carnitine benzyl ester-D 6 Hydrochloride (50.0 mg, 0.1 mmol, 1.0 eq) in MeOH (1.0 mL), add 10% palladium on carbon (5.0 mg), react at -20 °C under hydrogen atmosphere, and monitor the reaction by TLC. After the reaction is completed, filter off the palladium on carbon using diatomaceous earth, wash the residue with MeOH (20 mL), and then freeze-dry to obtain white solid VII-C14:2 (30.7 mg, yield 75%).

[0068] 1 HNMR (400 MHz, CD 3 OD): δ = 7.42–7.35 (m, 1H), 6.32–6.19 (m, 2H), 5.87 (d, J = 15.2 Hz, 1H), 5.72–5.67 (m, 1H), 3.96–3.90 (m, 1H), 3.76–3.72 (m, 1H), 3.21 (s, 3H), 2.84–2.73 (m, 2H), 2.23–2.18 (m, 2H), 1.44 (q, J = 7.2, 14.4 Hz, 2H), 1.32–1.29 (m, 12H), 0.90 (t, J = 6.8 Hz, 3H).

[0069] Example 3

[0070] This example provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine. Steps S1 - S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0071] S5. Preparation of compound VI [3-hydroxyoctadecanoyl-L-carnitine benzyl ester-D 6 hydrochloride] (VI-C18:OH)

[0072] Structural formula of compound VI:

[0073] In a round-bottom flask, dissolve 3-hydroxyoctadecanoic acid (29.9 mg, 0.1 mmol, 1.0 eq) in 1.0 mL of anhydrous THF. Under stirring, successively add DMAP (3.7 mg, 0.03 mmol, 0.3 eq), L-carnitine benzyl ester-D 6 hydrochloride (32.3 mg, 0.11 mmol, 1.1 eq) and condensing agent DCC (61.9 mg, 0.3 mmol, 3.0 eq). React overnight. After the reaction is completed (monitored by TLC), filter out the insoluble solid. The filtrate is rotary-evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography to obtain the product as a white solid VI-C18:OH (12.2 mg, yield 40.8%).

[0074] 1 HNMR(400MHz,CD 3 OD):δ=7.40–7.31(m,5H),5.69–5.64(m,1H),5.18–5.13(m,2H),3.75–3.70(m,2H),3.62(s,2H),3.23–3.15(m,3H),2.91–2.79(m,2H),2.37–2.32(m,2H),1.60–1.52(m,2H),1.28(s,24H),0.89(t,J=6.6Hz,3H).

[0075] S6. Preparation of compound VII [3-hydroxyoctadecanoyl carnitine hydrochloride] (VII-C18:OH)

[0076] Structural formula of compound VII:

[0077] In a round-bottom flask, take 3-hydroxyoctadecanoyl-L-carnitine benzyl ester-D 6The hydrochloride salt (287.5 mg, 0.5 mmol, 1.0 eq) was dissolved in 6.0 mL of methanol, and then 10% palladium on carbon (57.5 mg) was added. Under hydrogen gas, the mixture was stirred at room temperature for 2 h. After the reaction was completed (monitored by TLC), after the reaction was completed, the palladium on carbon was removed by filtration through diatomaceous earth. The residue was washed with MeOH (20 mL) and then freeze-dried to obtain white solid VII-C18:OH (210.0 mg, yield 86.5%).

[0078] 1 HNMR(400MHz,CD 3 OD): δ = 5.72–5.58 (m, 1H), 4.02–4.00 (m, 1H), 3.87–3.85 (m, 1H), 3.73–3.69 (m, 1H), 3.24–3.20 (m, 3H), 2.92–2.65 (m, 2H), 2.65–2.51 (m, 1H), 2.44–2.40 (m, 1H), 1.50–1.46 (m, 2H), 1.48–1.29 (m, 26H), 0.90 (t, J = 6.7 Hz, 3H).

[0079] Example 4

[0080] This example provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine. Steps S1-S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0081] S5. Preparation of compound VI [eicosanoyl-L-carnitine benzyl ester-D 6 hydrochloride] (VI-C20:0)

[0082] Structural formula of compound VI:

[0083] Under anhydrous and dry conditions, eicosanoic acid (0.39 g, 1.26 mmol, 1.50 eq) was dissolved in dry N,N-dimethylformamide (1.60 mL) and tetrahydrofuran (0.30 mL). L-Carnitine benzyl ester-D 6 hydrochloride (246.1 mg, 0.84 mmol, 1.00 eq) and 4-dimethylaminopyridine (20.80 mg, 0.17 mmol, 0.2 eq) were added. Subsequently, N,N'-diisopropylcarbodiimide (0.26 mL, 1.68 mmol, 2.0 eq) was added and the reaction was carried out overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed and column chromatography was performed with silica gel powder to obtain off-white solid VI-C20:0 (148.2 mg, yield 30%).

[0084] 1 HNMR(400MHz,CD3 OD): δ = 7.42–7.29 (m, 5H), 5.66 (q, J = 6.8 Hz, 1H), 5.16 (s, 2H), 3.86 (dd, J = 14.4, 8.5 Hz, 1H), 3.69 (dd, J = 14.5 1.5 Hz, 1H), 3.18 (s, 3H), 2.84 (d, 6.1 Hz, 2H), 2.31 (td, J = 7.5, 6.2 Hz, 2H), 1.65–1.48 (m, 2H), 1.29 (s, 32H), 0.90 (m, t, J = 6.6 Hz, 3H).

[0085] S6. Preparation of Compound VII [Eicosanoyl-L-carnitine-D 6 Hydrochloride] (VII-C20:0)

[0086] Structural formula of Compound VII:

[0087] Under anhydrous and dry conditions, dissolve eicosanoyl-L-carnitine benzyl ester-D 6 Hydrochloride (176.4 mg, 0.3 mmol, 1.0 eq) in MeOH (3.0 mL), add 10% palladium on carbon (8.8 mg, 5 mol%), and react under hydrogen conditions. Monitor the reaction by TLC. After the reaction is completed, filter off the palladium on carbon using diatomaceous earth, wash the residue with MeOH (20 mL), and then freeze-dry to obtain white solid VII-C20:0 (118.2 mg, yield 79%).

[0088] 1 HNMR (400 MHz, CD 3 OD): δ = 5.62 (q, J = 7.0 Hz, 1H), 3.87 (dd, J = 14.4, 8.5 Hz, 1H), 3.71 (dd, J = 14.4, 1.3 Hz, 1H), 3.21 (s, 3H), 2.76 (dd, J = 6.3, 3.8 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 1.62 (q, J = 7.2 Hz, 2H), 1.29 (s, 32H), 0.94–0.86 (m, 3H).

[0089] Example 5

[0090] This example provides a method for synthesizing deuterium-labeled long-chain fatty acid acyl carnitine. Steps S1 - S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0091] S5. Preparation of Compound VI [Docosanoyl-L-carnitine benzyl ester-D 6 Hydrochloride] (VI-C22:0)

[0092] Structural formula of Compound VI:

[0093] Under anhydrous and dry conditions, dissolve docosanoic acid (1.02 g, 3 mmol, 1.5 eq) in dry N,N-dimethylformamide (2.5 mL) and tetrahydrofuran (0.5 mL), add L-carnitine benzyl ester-D 6 hydrochloride (0.56 g, 2.0 mmol, 1.0 eq) and 4-dimethylaminopyridine (50.0 mg, 0.4 mmol, 0.2 eq), and then add N,N'-diisopropylcarbodiimide (0.46 mL, 3.0 mmol, 1.5 eq) and react overnight. Monitor the reaction by TLC. After the reaction is completed, remove the solvent and perform column chromatography with silica gel powder to obtain off-white solid VI-C22:0 (460 mg, yield 37.3%).

[0094] 1 HNMR (400 MHz, CD 3 OD): δ = 7.42–7.29 (m, 5H), 5.66 (d, J = 7.5 Hz, 1H), 5.16 (s, 2H), 3.87 (dd, J = 14.4, 8.5 Hz, 1H), 3.70 (dd, J = 14.4, 1.4 Hz, 1H), 3.18 (s, 3H), 2.84 (d, J = 6.1 Hz, 2H), 2.38–2.22 (m, 2H), 1.56 (q, J = 7.2 Hz, 2H), 1.24 (s, 36H), 0.90 (t, J = 6.7 Hz, 3H).

[0095] S6. Preparation of Compound VII [docosanoyl-L-carnitine-D 6 hydrochloride] (VII-C22:0)

[0096] Structural formula of Compound VII:

[0097] At room temperature, dissolve docosanoyl-L-carnitine benzyl ester-D 6 hydrochloride (1.0 g, 1.62 mmol, 1.0 eq) in MeOH (12 mL), add 10% Pd / C (50 mg), evacuate, and then introduce hydrogen and react for 2 - 4 hours. After the reaction is completed, filter off the palladium carbon using diatomaceous earth, wash the residue with MeOH (50 mL), and then freeze-dry to obtain white solid VII-C22:0 (356 mg, yield 43%).

[0098] 1 HNMR (400 MHz, CD 3OD): δ = 5.63 (q, J = 7.0 Hz, 1H), 3.87 (dd, J = 14.4, 8.4 Hz, 1H), 3.71 (d, J = 14.4 Hz, 1H), 3.21 (s, 3H), 2.80–2.68 (m, 2H), 2.39 (t, J = 7.5 Hz, 2H), 1.62 (q, J = 7.3 Hz, 2H), 1.29 (s, 36H), 0.90 (t, J = 6.7 Hz, 3H).

[0099] Example 6

[0100] This example provides a method for synthesizing deuterium-labeled long-chain fatty acyl carnitine. Steps S1 - S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0101] S5. Preparation of compound VI [tetracosanoyl-L-carnitine benzyl ester-D 6 hydrochloride] (VI-C24:0)

[0102] Structural formula of compound VI:

[0103] Under anhydrous and dry conditions, tetracosanoic acid (1.09 g, 3 mmol, 1.5 eq) was dissolved in dry N,N-dimethylformamide (2.5 mL) and tetrahydrofuran (0.5 mL). L-carnitine benzyl ester-D 6 hydrochloride (0.56 g, 2.0 mmol, 1.0 eq) and 4-dimethylaminopyridine (50.0 mg, 0.4 mmol, 0.2 eq) were added. Subsequently, N,N'-diisopropylcarbodiimide (0.46 mL, 3.0 mmol, 1.5 eq) was added and the reaction was carried out overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed and column chromatography was performed with silica gel powder to obtain a grayish-white solid VI-C24:0 (405 mg, yield 31%).

[0104] 1 HNMR (400 MHz, CD 3 OD): δ = 7.48–7.21 (m, 5H), 5.78–5.58 (m, 1H), 5.16 (s, 2H), 3.87 (dd, J = 14.4, 8.5 Hz, 1H), 3.70 (dd, J = 14.5, 1.5 Hz, 1H), 3.18 (s, 3H), 2.31 (hept, J = 8.0 Hz, 2H), 1.75–1.52 (m, 2H), 1.28 (s, 40H), 0.90 (t, J = 6.6 Hz, 3H).

[0105] S6. Compound VII [tetracosanoyl-L-carnitine-D 6Preparation of hydrochloride (VII-C24:0)

[0106] Structural formula of compound VII:

[0107] At room temperature, dissolve tetracosanoyl-L-carnitine benzyl ester-D 6 hydrochloride (1.0 g, 1.7 mmol, 1.0 eq) in MeOH (12 mL), add Pd / C (50 mg), evacuate, and then introduce hydrogen gas to react for 2 - 4 hours. After the reaction, filter off Pd / C using diatomaceous earth, wash the residue with MeOH, and then evaporate the solvent by rotary evaporation. After the reaction, filter off palladium carbon using diatomaceous earth, wash the residue with MeOH (50 mL), and then freeze-dry to obtain white solid VII-C24:0 (180 mg, yield 19%).

[0108] 1 1H NMR (400 MHz, CD 3 OD): δ = 5.62 (q, J = 7.1 Hz, 1H), 3.87 (dd, J = 14.4, 8.5 Hz, 1H), 3.71 (d, J = 14.4 Hz, 1H), 3.20 (s, 3H), 2.83–2.67 (m, 2H), 2.39 (t, J = 7.5 Hz, 2H), 1.81–1.51 (m, 2H), 1.29 (s, 40H), 0.90 (t, J = 6.5 Hz, 3H).

[0109] Example 7

[0110] This example provides a method for synthesizing deuterium-labeled long-chain fatty acid acyl carnitine. Steps S1 - S4 are the same as those in Example 1, except that the type of long-chain fatty acid is changed. Specifically, the change is as follows:

[0111] S5. Preparation of compound VI [hexacosanyl-L-carnitine benzyl ester-D 6 hydrochloride (VI-C26:0)

[0112] Structural formula of compound VI:

[0113] Under anhydrous and dry conditions, dissolve hexacosanoic acid (1.2 g, 3 mmol, 1.5 eq) in dry N,N-dimethylformamide (2.5 mL) and tetrahydrofuran (0.5 mL), add L-carnitine benzyl ester-D 6The hydrochloride salt (0.56 g, 2.0 mmol, 1.0 eq) and 4-dimethylaminopyridine (50.0 mg, 0.4 mmol, 0.2 eq) were added, and then N,N'-diisopropylcarbodiimide (0.46 mL, 3.0 mmol, 1.5 eq) was added and the reaction was carried out overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed, and column chromatography was performed using silica gel powder to obtain a grayish-white solid VI-C26:0 (454.3 mg, yield 33%).

[0114] 1 HNMR(400MHz,CD 3 OD):δ7.42–7.31(m,5H),5.66(d,J=7.2Hz,1H),5.17(d,J=1.3Hz,2H),3.87(dd,J=14.4,8.5Hz,1H),3.69(d,J=14.4Hz,1H),3.18(s,3H),2.84(m,2H),2.38–2.27(m,2H),1.65–1.51(m,2H),1.29(s,44H),0.94–0.86(m,3H).

[0115] S6, Preparation of Compound VII [Hexadecanoyl L-carnitine-D 6 Hydrochloride] (VII-C26:0)

[0116] Structural formula of Compound VII:

[0117] Under anhydrous and dry conditions, hexadecanoyl L-carnitine benzyl ester-D 6 Hydrochloride (910.0 mg, 1.44 mmol, 1.0 eq) was dissolved in MeOH (12.0 mL), 10% palladium on carbon (45.5 mg, 5%) was added, and hydrogen was introduced for reaction. The reaction was monitored by TLC (silica gel plate, D:M = 2:1, Rf = 0.2). After the reaction was completed, the palladium on carbon was removed by filtration using diatomaceous earth, and the filter residue was washed with MeOH (50 mL) and then freeze-dried to obtain a white solid VII-C26:0 (367 mg, yield 44%).

[0118] 1 HNMR(400MHz,CD 3 OD):δ=5.62(d,J=7.2Hz,1H),3.93–3.79(m,1H),3.70(d,J=13.9Hz,1H),3.20(s,3H),2.82–2.68(m,2H),2.39(t,J=7.4Hz,2H),1.63(s,2H),1.29(s,44H),0.91(d,J=6.4Hz,3H).

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine, characterized in that: The following steps are involved: S1, using (R)-4-chloro-3-hydroxy-butyric acid ethyl ester as a raw material, placing it in benzyl alcohol solvent to prepare (R)-4-chloro-3-hydroxy-butyric acid benzyl ester; The (R)-4-chloro-3-hydroxy-butyric acid benzyl ester prepared in S2 and S1 is reacted with dimethylamine hydrochloride-D6 to prepare (R)-4-(dimethylamino-D6)-3-hydroxy-butyric acid benzyl ester; The (R)-4-(dimethylamino-D6)-3-hydroxy-butyric acid benzyl ester prepared in S3 and S2 is subjected to quaternization reaction in methyl iodide to prepare L-carnitine benzyl ester-D6 iodide; The L-carnitine benzyl ester-D6 hydrochloride is prepared from the L-carnitine benzyl ester-D6 iodide prepared in S4 and S3 under the conditions of silver oxide and hydrochloric acid; S5, L-carnitine benzyl ester-D6 hydrochloride prepared in S4 is condensed with long-chain fatty acid to prepare acylcarnitine benzyl ester; The acylcarnitine benzyl ester prepared in S6 and S5 is debenzylated under palladium carbon hydrogen conditions to synthesize deuterium-labeled long-chain fatty acid acylcarnitine; The synthetic route is as follows:

2. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S1 are: add (R)-4-chloro-3-hydroxy-butyric acid ethyl ester and benzyl alcohol into a three-necked flask, add concentrated hydrochloric acid dropwise under ice bath condition, reflux at 140°C for overnight reaction, add ether after the reaction is completed, store at -20°C for overnight, pour out the ether layer and remove the solvent by vacuum distillation to obtain (R)-4-chloro-3-hydroxy-butyric acid benzyl ester.

3. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S2 are: under argon protection and ice-salt bath conditions, anhydrous potassium carbonate and anhydrous tetrahydrofuran are added to a three-necked flask, and then dimethylamine hydrochloride-D6 is added and stirred thoroughly; then the (R)-4-chloro-3-hydroxy-butyric acid benzyl ester prepared in S1 is dissolved in anhydrous tetrahydrofuran, and the mixture is added dropwise to the above three-necked flask to react overnight. After the reaction is completed, the solvent is dried up, water is added and extracted with DCM, the organic phases are combined, the solvent is dried up, and finally column chromatography is performed to obtain (R)-4-(dimethylamino-D6)-3-hydroxy-butyric acid benzyl ester.

4. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S3 are: dissolving (R)-4-(dimethylamino-D6)-3-hydroxybutyric acid benzyl ester obtained in S2 in acetone, adding iodomethane to react overnight, and after the reaction is completed, concentrating the solvent by vacuum distillation, and purifying and separating by silica gel column to obtain L-carnitine benzyl ester-D6 iodide.

5. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S4 are: dissolving the L-carnitine benzyl ester-D6 iodide prepared in S3 in MeOH / H2O, adding silver oxide to react, filtering to remove insoluble matter after the reaction is completed, washing the filter residue with MeOH / H2O and then drying the solvent, dissolving the residue in concentrated hydrochloric acid, and freeze-drying to obtain L-carnitine benzyl ester-D6 hydrochloride.

6. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S5 are: dissolving a long-chain fatty acid in a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, adding 4-dimethylaminopyridine and L-carnitine benzyl ester-D6 hydrochloride obtained in step S4, then adding N,N'-diisopropylcarbodiimide to react and stir overnight, removing the solvent after the reaction is completed, and separating by column chromatography to obtain acylcarnitine benzyl ester.

7. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: In S5, the long-chain fatty acids are dodecanoic acid, tridecanoic acid, tetradecanoic acid, cis-9-tetradecenoic acid, cis-2,4-tetradecadienoic acid, 3-hydroxytetradecanoic acid, pentadecanoic acid, cis-10-pentadecaenoic acid, hexadecanoic acid, cis-9-hexadecenoic acid, heptadecanoic acid, cis-10-heptadecaenoic acid, octadecanoic acid, cis-9-octadecenoic acid, cis-9,13-octadecadienoic acid, 3-hydroxy-octadecanoic acid, nonadecanoic acid, eicosanoic acid, cis-11-eicosanoic acid, any one of cis-11,14-eicosadienoic acid, cis-8,11,14-eicosatrienoic acid, cis-5,8,11,14-eicosatetraenoic acid, heneicosanoic acid, docosanoic acid, cis-13-docosaenoic acid, cis-13,16-docosadienoic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, tricosanoic acid, tetracosanoic acid, cis-15-tetracosenoic acid, pentacosanoic acid, and hexacosanoic acid.

8. The method for synthesizing deuterium-labeled long-chain fatty acid acylcarnitine according to claim 1, characterized in that: The specific steps of S6 are: dissolving the acylcarnitine benzyl ester obtained in step S5 in MeOH, adding 10% palladium carbon, introducing hydrogen to react, filtering to remove the palladium carbon after the reaction, washing the filter residue with MeOH and freeze-drying to obtain deuterium-labeled long-chain fatty acid acylcarnitine.

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