A method for efficiently preparing fatty diacid derivatives and its application

Abstract

The present invention provides a method for efficiently preparing fatty diacid derivatives and its application, and relates to the field of biomedicine technology. The method comprises the following steps: (1) dissolving hexadecane mono-tert-butyl ester in solvent 1, adding 2-succinimidyl-1,1,3,3-tetramethylurea tetrafluoroborate, keeping warm and stirring, and then adding L-glutamic acid-1-tert-butyl ester to keep warm and react, thereby obtaining product 1; (2) dissolving product 1 in solvent 2, adding 2-succinimidyl-1,1,3,3-tetramethylurea tetrafluoroborate to keep warm and react, thereby obtaining product 2; (3) dissolving product 2 in solvent 3, adding a mixed solution of isopropanol and n-heptane, filtering, and drying, thereby obtaining; the method uses hexadecane dioic acid mono-tert-butyl ester as a raw material, and undergoes fatty acid carboxyl activation and glutamic acid condensation, glutamic acid carboxyl activation, and de-tert-butyl protection reaction to obtain fatty diacid derivatives. The method is simple, low in cost, and the final product has high purity and yield, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for efficiently preparing fatty diacid derivatives and application thereof. Background Art

[0002] Long-chain fatty diacids and their derivatives are often used in a series of high-value-added special chemicals such as synthetic drugs, special materials, and spices, and have an important impact on the extension of drug efficacy and drug metabolism dynamics in some known drug molecules, especially in the research of long-acting insulin analogs. As an important drug of long-acting insulin analogs, glargine insulin has a wide market in the world, but glargine insulin is difficult to stably control blood sugar concentration, and its pharmacokinetic curve is also unstable, which poses certain safety risks.

[0003] In recent years, the search for alternative drugs to glargine insulin has gradually become a hot topic in the field of diabetes treatment. Patent CN1829738A discloses a new type of insulin derivative, specifically a long-acting insulin analogue with a fatty diacid side chain modified by glutamic acid - degludec insulin; Patent CN102065884A discloses a PEGylated lispro insulin compound, specifically a polyethylene glycol-conjugated insulin analogue; Patent WO2011 / 122921 discloses an insulin conjugate coupled to an immunoglobulin Fc fragment by PEG. Among the studies of many long-acting insulin analogues, degludec insulin has the longest half-life and a stable pharmacokinetic curve, and is very likely to become the trump card drug after glargine insulin. During use, degludec insulin self-aggregates to form multi-hexamer through fatty diacid deprotection modifiers and forms a storage reservoir with the injection site, exerting a stable and lasting blood sugar lowering effect. Thereafter, as the multi-hexamer slowly dissociates to release monomers that enter the blood circulation through the capillaries, the added fatty diacid deprotection modifier reversibly binds to plasma proteins, further slowing down its diffusion rate to target tissues and blood circulation, thereby achieving a long-term blood sugar lowering effect.

[0004] The preparation and modification reaction of long-chain fatty diacid derivatives have become an important link in the preparation process of insulin analogs. Patent CN105001140A discloses the use of fatty diacids and thionyl chloride to react to obtain acyl chlorides, followed by the reaction of acyl chlorides with tert-butyl alcohol to obtain monoacyl chloride mono-tert-butyl ester intermediates, and finally hydrolysis to obtain the target product. This method has poor selectivity, difficult purification and low yield, and is not suitable for industrial amplification. The prior art also discloses the use of 4-dimethylaminopyridine to catalyze the condensation of dicyclohexylcarbodiimide dodecanedioic acid and tert-butyl alcohol to prepare dodecane mono-tert-butyl ester. When this method is used to prepare hexadecanedioic acid mono-tert-butyl ester, the byproduct hexadecanedioic acid N-acylurea is high in proportion and difficult to purify, which is also not suitable for factory amplification.

[0005] Since the known methods cannot meet the requirements in the industrial and biomedical fields in terms of selectivity, cost, yield, etc., there is still a need for a method for efficiently preparing fatty diacid derivatives that is suitable for industrial scale-up. Summary of the invention

[0006] Based on the deficiencies in the prior art, the present invention aims to provide a method for efficiently preparing fatty diacid derivatives. The method uses hexadecane mono-tert-butyl ester as a raw material, activates the carboxyl group of fatty acid, condenses with glutamic acid, activates the carboxyl group of glutamic acid, and performs a de-tert-butyl protection reaction to finally obtain hexadecane dioyl-L-Glu (OSu). The preparation method of the present invention has low cost, and the purity and yield of the final product are high, which is suitable for industrial production.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for efficiently preparing fatty diacid derivatives comprises the following steps:

[0009] (1) dissolving hexadecane mono-tert-butyl ester in solvent 1, adding 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, stirring at room temperature, and then adding L-glutamic acid-1-tert-butyl ester, reacting at room temperature, to obtain tert-butyl hexadecane dioyl-L-Glu-OtBu;

[0010]

[0011] (2) tert-butyl hexadecanoyl-L-Glu-OtBu is dissolved in solvent 2, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is added and the mixture is heated to react, thereby obtaining tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu;

[0012]

[0013] (3) dissolving tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu in solvent 3, stirring at room temperature, adding a mixed solution of isopropanol and n-heptane under ice bath conditions, filtering and drying to obtain hexadecanoyl-L-Glu (OSu);

[0014] .

[0015] The solvent 1 in the above step (1) is dichloromethane.

[0016] Diisopropylethylamine needs to be added at the same time as the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and L-glutamic acid-1-tert-butyl ester described in the above step (1).

[0017] The molar ratio of hexadecane mono-tert-butyl ester, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and L-glutamic acid-1-tert-butyl ester in the above step (1) is 1-1.5:1-1.5:1-1.5.

[0018] The molar ratio of the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate to diisopropylethylamine is 1-1.5:1-5.

[0019] The time of the heat preservation and stirring in the above step (1) is 0.5-5h; the time of the heat preservation reaction is 1-20h; and the heat preservation temperature is 2-8°C.

[0020] The solid-liquid ratio of hexadecane mono-tert-butyl ester and solvent 1 in the above step (1) is 1-2 g:5-60 mL.

[0021] The above step (1) also includes a purification step, specifically: washing the product after the reaction with 10 mL 1M HCl, water, and saturated brine in sequence, drying over anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain tert-butylhexadecandioyl-L-Glu-OtBu.

[0022] The solvent 2 described in the above step (2) is ethyl acetate.

[0023] Diisopropylethylamine needs to be added at the same time as the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate described in step (2), and the molar ratio of the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate to diisopropylethylamine is 1-1.5:1-5.

[0024] The heat preservation and stirring time in the above step (2) is 1-10 hours, and the heat preservation temperature is 2-8°C.

[0025] The solid-liquid ratio of tert-butylhexadecanoyl-L-Glu-OtBu and solvent 2 in the above step (2) is 1-2 g:10-60 mL.

[0026] The molar ratio of tert-butylhexadecanoyl-L-Glu-OtBu and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate in the above step (2) is 1-1.5:1-2.

[0027] The above step (2) also includes a purification step, specifically: the product after the reaction is completed is washed with 20 mL 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and then filtered. The solvent is evaporated under reduced pressure to obtain an oily substance, a mixed solution of ethyl acetate: petroleum ether = 1:3 is added, and the solid precipitate is collected by filtration, and dried under reduced pressure to a constant weight to obtain tert-butylhexadecanoyl-L-Glu (OSu) -OtBu.

[0028] The solvent 3 in the above step (3) is trifluoroacetic acid;

[0029] The solid-liquid ratio of tert-butylhexadecanoyl-L-Glu(OSu)-OtBu and solvent 3 in the above step (3) is 1-2 g: 3-30 mL.

[0030] The room temperature in the above step (3) is 25° C., and the stirring time is 1-10 h, preferably 2 h.

[0031] The volume ratio of isopropanol to n-heptane in the above step (3) is 1-10:1-30, preferably 1:3.

[0032] The fatty diacid derivatives prepared by the above preparation method can be used to prepare insulin analogs, that is, the present invention also provides the use of the fatty diacid derivatives prepared by the above preparation method in the preparation of insulin analogs.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The preparation method provided by the present invention is simple to operate, has controllable quality, and is suitable for industrial scale-up production.

[0035] (2) The preparation method provided by the present invention uses hexadecane mono-tert-butyl ester as a raw material, activates the carboxyl group of fatty acid, condenses with glutamic acid, activates the carboxyl group of glutamic acid, and undergoes a de-tert-butyl protection reaction to finally obtain hexadecane dioyl-L-Glu (OSu). The preparation method of the present invention has low cost, and the final product has high purity (greater than 95%) and yield (greater than 55%).

[0036] (3) The subsequent operation of the product of the preparation method provided by the present invention is simple and feasible, the product quality is controllable, and it can be directly used to prepare high-purity insulin analogs through protein modification technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a liquid chromatogram of tert-butylhexadecandioyl-L-Glu(OSu)-OtBu prepared by the preparation method of Example 1 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0039] The reaction route is:

[0040]

[0041] Example 1

[0042] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0043]

[0044] Dissolve hexadecane mono-tert-butyl ester (1.7 g, 4.96 mmol) in dichloromethane (10 mL), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (1.5 g, 4.98 mmol) and diisopropylethylamine (1.2 mL, 7.26 mmol) at 5 ° C, and stir for 2 h. Add L-glutamic acid-1-tert-butyl ester (1.11 g, 5.46 mmol) and diisopropylethylamine (0.7 g, 5.42 mmol), and react for 5 h. Wash with 10 mL 1M HCl, water, and saturated brine in sequence, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 2.5 g, with a yield of 95.5%, and a purity of 96.4% measured by HPLC.

[0045] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0046]

[0047] Tert-butyl hexadecanoyl-L-Glu-OtBu (2.5 g, 4.74 mmol) was dissolved in ethyl acetate (20 mL), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (1.5 g, 4.83 mmol) and diisopropylethylamine (0.92 g, 7.12 mmol) were added at 5°C, and the mixture was stirred for 4 h. The mixture was washed with 20 mL 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain an oily substance. A mixed solution of ethyl acetate: petroleum ether = 1:3 (4 mL) was added, and the solid precipitate was collected by filtration. The solid precipitate was dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu) -OtBu, with a weight of 2.4 g and a yield of 81.1%. The purity of the obtained product tert-butyl hexadecanoyl-L-Glu (OSu) -OtBu was detected by liquid chromatography. The detection spectrum is attached. Figure 1 The purity was 99.56% as measured by HPLC.

[0048] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0049]

[0050] Tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (2 g, 3.19 mmol) was dissolved in trifluoroacetic acid (3 mL) and stirred at room temperature (25 ° C) for 2 h. Isopropanol (6 mL) and n-heptane (18 mL) were added under ice bath conditions. The solid precipitate was collected by suction filtration and dried under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 1.1 g, with a yield of 67.0% and a purity of 96.4% as measured by HPLC.

[0051] Example 2

[0052] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0053]

[0054] Dissolve hexadecane mono-tert-butyl ester (10.0 g, 29.20 mmol) in dichloromethane (100 mL), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (9.23 g, 30.66 mmol) and diisopropylethylamine (5.66 g, 43.80 mmol) at 10 ° C, and stir for 2 h. Add L-glutamic acid-1-tert-butyl ester (6.23 g, 30.65 mmol) and diisopropylethylamine (4.02 g, 31.10 mmol), and react for 5 h. Wash with 100 mL 1M HCl, water, and saturated brine in turn, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 17.5 g, with a yield of 100% (including solvent), and a purity of 99.0% as measured by HPLC.

[0055] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0056]

[0057] Tert-butyl hexadecanoyl-L-Glu-OtBu (17.5 g, 33.20 mmol) was dissolved in ethyl acetate (175 mL), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (10.5 g, 34.88 mmol) and diisopropylethylamine (6.28 g, 48.60 mmol) were added at 10 °C, and the mixture was stirred for 4 h. The mixture was washed with 175 mL 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain an oily substance. A mixed solution of ethyl acetate: petroleum ether = 1:3 (68 mL) was added, and the solid precipitate was collected by filtration. The solid precipitate was dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu, with a weight of 15.2 g, a yield of 83.4%, and a purity of 99.2% as measured by HPLC.

[0058] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0059]

[0060] Tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (15.2 g, 24.34 mmol) was dissolved in trifluoroacetic acid (23 mL) and stirred at room temperature (25 ° C) for 2 h. Most of the trifluoroacetic acid was concentrated, and isopropanol (45 mL) and n-heptane (135 mL) were added to the concentrate under ice bath conditions. The solid precipitate was collected by suction filtration and dried under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 11.6 g, with a yield of 93.0%, and a purity of 97.07% measured by HPLC.

[0061] Example 3

[0062] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0063]

[0064] Dissolve hexadecane mono-tert-butyl ester (10.0 g, 29.20 mmol) in dichloromethane (100 mL), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (9.23 g, 30.66 mmol) and diisopropylethylamine (4.91 g, 37.99 mmol) at 5 ° C, and stir for 2 h. Add L-glutamic acid-1-tert-butyl ester (6.23 g, 30.65 mmol) and diisopropylethylamine (4.15 g, 32.11 mmol), and react for 5 h. Wash with 100 mL 1M HCl, water, and saturated brine in turn, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 18.13 g, with a yield of 100%, and a purity of 99.84% as measured by HPLC.

[0065] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0066]

[0067] Tert-butyl hexadecanoyl-L-Glu-OtBu (15.4 g, 29.20 mmol) was dissolved in ethyl acetate (150 mL), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (9.23 g, 30.66 mmol) and diisopropylethylamine (4.91 g, 37.99 mmol) were added at 5°C, and the mixture was stirred for 4 h. The mixture was washed with 150 mL 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain an oily substance. A mixed solution of ethyl acetate: petroleum ether = 1:3 (60 mL) was added, and the solid precipitate was collected by filtration. The solid precipitate was dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu, which weighed 14.42 g, had a yield of 79.10%, and a purity of 99.95% as measured by HPLC.

[0068] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0069]

[0070] Dissolve tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (14.35 g, 22.98 mmol) in trifluoroacetic acid (42 mL) and stir at room temperature for 2 h. Concentrate most of the trifluoroacetic acid, and add isopropanol (42 mL) and n-heptane (126 mL) to the remaining yellow-white concentrate under ice bath conditions. Collect the solid precipitate by suction filtration and dry it under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 11.72 g, with a yield of 99.54% and a purity of 98.31% as measured by HPLC.

[0071] Example 4

[0072] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0073]

[0074] Dissolve hexadecane mono-tert-butyl ester (30.0 g, 87.60 mmol) in dichloromethane (300 mL), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (27.69 g, 91.98 mmol) and diisopropylethylamine (14.73 g, 113.97 mmol) at 5 ° C, and stir for 2 h. Add L-glutamic acid-1-tert-butyl ester (19.58 g, 96.36 mmol) and diisopropylethylamine (12.45 g, 96.33 mmol), and react for 5 h. Wash with 300 mL 1M HCl, water, and saturated brine in turn, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 55.4 g, with a yield of 100%, and a purity of 99.83% as measured by HPLC.

[0075] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0076]

[0077] Tert-butyl hexadecanoyl-L-Glu-OtBu (55.4 g, 87.60 mmol) was dissolved in ethyl acetate (460 mL), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (27.69 g, 91.98 mmol) and diisopropylethylamine (14.73 g, 113.97 mmol) were added at 5°C, and the mixture was stirred for 6 h. The mixture was washed with 460 mL 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain an oily substance. A mixed solution of ethyl acetate: petroleum ether = 1:4 (225 mL) was added, and the solid precipitate was collected by filtration. The solid precipitate was dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu) -OtBu, with a weight of 39.4 g, a yield of 72.04%, and a purity of 99.0% as measured by HPLC.

[0078] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0079]

[0080] Tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (39.8 g, 63.74 mmol) was dissolved in trifluoroacetic acid (120 mL) and stirred at room temperature (25 ° C) for 2 h. Most of the trifluoroacetic acid was concentrated, and the remaining yellow-white concentrate was added with isopropanol (120 mL) and n-heptane (360 mL) under ice bath conditions. The solid precipitate was collected by suction filtration and dried under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 30.7 g, with a yield of 94.01% and a purity of 99.56% measured by HPLC.

[0081] Example 5

[0082] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0083]

[0084] Dissolve hexadecane mono-tert-butyl ester (100.0 g, 0.292 mol) in dichloromethane (1000 mL), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (92.36 g, 0.307 mol) and diisopropylethylamine (49.10 g, 0.380 mol) at 5 ° C, and stir for 2 hours. Add L-glutamic acid-1-tert-butyl ester (65.28 g, 0.322 mol) and diisopropylethylamine (41.50 g, 0.321 mol), and keep warm overnight. Wash with 1L 1M HCl, water, and saturated brine in turn, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 189.36 g, with a yield of 100%, and a purity of 99.68% measured by HPLC.

[0085] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0086]

[0087] Tert-butyl hexadecanoyl-L-Glu-OtBu (154.0 g, 0.29 mol) was dissolved in ethyl acetate (1.5 L), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (92.30 g, 0.307 mol) and diisopropylethylamine (49.10 g, 0.380 mol) were added at 5 ° C, and the mixture was kept warm overnight. The mixture was washed with 1.5 L 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to an oily substance, and a mixed solution of ethyl acetate: petroleum ether = 1:4 (1 L) was added. The solid precipitate was collected by filtration and dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu) -OtBu, with a weight of 171.1 g, a yield of 93.85%, and a purity of 99.6% as measured by HPLC.

[0088] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0089]

[0090] Tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (150.0 g, 0.240 mol) was dissolved in trifluoroacetic acid (450 mL) and stirred at room temperature (25 ° C) for 3 h. Most of the trifluoroacetic acid was concentrated, and the remaining yellow-white concentrate was added with isopropanol (450 mL) and n-heptane (1350 mL) under ice bath conditions. The solid precipitate was collected by suction filtration and dried under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 120.0 g, with a yield of 97.50%, and a purity of 99.15% measured by HPLC.

[0091] Example 6

[0092] 1. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0093]

[0094] Dissolve hexadecane mono-tert-butyl ester (14.7kg, 42.92mol) in dichloromethane (195kg), add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (14.21Kg, 47.20mol) and diisopropylethylamine (7.20kg, 55.71mol) at 5℃, and stir for 3h. Add L-glutamic acid-1-tert-butyl ester (9.61kg, 47.30mol) and diisopropylethylamine (6.10kg, 47.20mol), and react overnight. Wash with 150L 1MHCl, water, and saturated brine in turn, dry with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 27.34kg, with a yield of 100%, and a purity of 99.84% as measured by HPLC.

[0095] 2. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0096]

[0097] Tert-butyl hexadecanoyl-L-Glu-OtBu (27.34 kg, 42.92 mol) was dissolved in ethyl acetate (213 kg), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (14.21 kg, 47.20 mol) and diisopropylethylamine (7.20 kg, 55.71 mol) were added at 5°C, and the mixture was stirred for 5 hours. The mixture was washed with 230L 1M HCl, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain an oily substance. A mixed solution of ethyl acetate: petroleum ether = 1:4 (141 kg) was added, and the solid precipitate was collected by centrifugation and dried under reduced pressure to constant weight to obtain tert-butyl hexadecanoyl-L-Glu (OSu) -OtBu, weighing 24.52 kg, with a yield of 91.50%, and a purity of 99.1% as measured by HPLC.

[0098] 3. Preparation of Hexadecanoyl-L-Glu (OSu)

[0099]

[0100] Tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu (24.0 kg, 38.44 mol) was dissolved in trifluoroacetic acid (110.52 kg) and stirred at room temperature (25 ° C) for 3 hours. Most of the trifluoroacetic acid was concentrated, and the remaining yellow-white concentrated liquid was added with isopropanol (72 L) and n-heptane (216 L) after the temperature dropped below 20 ° C. The solid precipitate was collected by centrifugation and dried under reduced pressure to constant weight to obtain hexadecanoyl-L-Glu (OSu), weighing 19.0 kg, with a yield of 96.49% and a purity of 96.72% measured by HPLC.

[0101] Comparative Example 1

[0102] The preparation was carried out according to the method disclosed in patent CN105001140A, and the route is as follows:

[0103]

[0104] The specific steps are:

[0105] 1. Preparation of mono-tert-butyl hexadecanedioate

[0106] Suspend hexadecanedioic acid (10.0 g, 34.9 mmol) in thionyl chloride (50 mL) and heat at 80 ° C for 3 h. Evaporate the excess thionyl chloride under reduced pressure, add dichloromethane (100 mL) to dissolve, add tert-butyl alcohol (3.67 mL, 38.40 mmol) and pyridine (2.95 mL, 36.66 mmol), add pyridine within 2 h, and keep at 25 ° C for 3 h. Evaporate the solvent under reduced pressure, add 300 mL of DMF / water mixed solvent (DMF and water are mixed in a volume ratio of 1:2) precooled to 0-4 ° C, and filter and collect the precipitate. The mixture was dried under vacuum to constant weight, and dichloromethane (300 mL) was added to resuspend the mixture. The filtrate was collected and the solvent was evaporated under reduced pressure. Petroleum field ether (200 mL) was added, and the filtrate was collected and evaporated to dryness under reduced pressure. Heptane (40 mL) was added to recrystallize the mixture, and the precipitate was collected by filtration. The TLC spot plate was sampled and showed (iodine development) that it was rather messy without obvious main spots. It was speculated that there were unreacted raw materials, and some target mono-tert-butyl esters and di-tert-butyl esters were by-products, which were difficult to separate and purify.

[0107] 2. Preparation of hexadecanedioic acid mono-tert-butyl ester succinimide

[0108] Dissolve hexadecanedioic acid tert-butyl ester (10.3g, 30.1mmol) in dichloromethane (200ml), add N-hydroxysuccinimide (3.64g, 31.6mmol) and dicyclohexylcarbodiimide (6.84g, 33.1mmol) at -20℃, continue to react for 2h at -20℃, return to room temperature (25℃) and stir overnight. Filter under reduced pressure, evaporate the solvent under reduced pressure, add isopropanol (90mL) for recrystallization, filter and collect the filter cake, wash the filter cake with n-heptane (30mL), collect the filter cake (i.e. hexadecanedioic acid succinimidyl tert-butyl ester), and dry under reduced pressure to constant weight, weight 10.5g, yield: 77%. TLC spot plate shows one spot, the product is pure, and continue to the next step.

[0109] 3. Preparation of tert-butyl hexadecanoyl-L-Glu-OtBu

[0110] Take hexadecanedioic acid succinimidyl tert-butyl ester (10.2g, 23.2mmol) and dissolve it in N,N-dimethyl

[0111] Formamide (200 mL), add L-glutamic acid a-tert-butyl ester (5.18 g, 25.5 mmol), N, N-diisopropylethylamine (5.94 mL, 34.7 mmol) in sequence, and stir at room temperature overnight. The solvent N, N-dimethylformamide and residual N, N-diisopropylethylamine were evaporated under reduced pressure, and the residual oil was dissolved in ethyl acetate (200 mL). Washed with 0.2M HCl, purified water and saturated brine in sequence, dried over anhydrous sodium sulfate and filtered, and the solvent was evaporated under reduced pressure to obtain a viscous oil (i.e., tert-butyl hexadecane dioyl-L-Glu-OtBu), weighing 12 g, with a yield of 98%, and TLC showed (iodine display) pure.

[0112] 4. Preparation of tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu

[0113] Tert-butyl hexadecane dioyl-L-Glu-OtBu (11.63 g, 22.04 mmol) was dissolved in dichloromethane (220 mL) under an ice bath. N-hydroxysuccinimide (2.66 g, 24.14 mmol) and dicyclohexylcarbodiimide (5.0 g, 24.24 mmol) were added at -20 ° C, and the reaction was continued for 2 h at -20 ° C, and the mixture was returned to room temperature and stirred overnight. The mixture was filtered and the solvent was evaporated under reduced pressure to obtain an oily substance. n-heptane (100 mL) was added, and the solid precipitate was collected by filtration and dried under reduced pressure to constant weight to obtain tert-butyl hexadecane dioyl-L-Glu (OSu) -OtBu, weighing 13.6 g, with a yield of 97%. TLC showed that the obtained product was pure, but the baseline was inferred to contain dicyclohexyl urea, which could not be filtered out.

[0114] 5. Preparation of Hexadecanoyl-L-Glu (OSu)

[0115] Tert-butyl hexadecane dioyl-L-Glu(OSu)-OtBu (13.2 g, 21.12 mmol) was dissolved in trifluoroacetic acid (66 mL). The mixture was stirred at room temperature for 2 h, and anhydrous ether (130 mL) and n-heptane (390 mL) were added successively under ice bath conditions. The solid precipitate was collected by suction filtration and dried under reduced pressure to constant weight to obtain hexadecane dioyl-L-Glu(OSu), with a weight of 9.5 g, a yield of 90%, and a purity of 93% as measured by HPLC.

[0116] The difference from the present invention is that comparative example 1 uses solvents such as DCC, which cannot be completely removed in post-treatment, and the product in step (2) in this method needs to be separated and purified for use in step (3), while the present invention does not use reagents such as DCC that are difficult to filter out in post-treatment, has small residues, is easy to operate, and has a relatively mild temperature, a higher yield, and is more suitable for industrialization. In addition, step (2) and step (3) are two steps in a row, without separation and purification in the middle, without using DMF, and the temperature is relatively mild, with a higher yield and less industrial waste liquid.

[0117] In summary, the present invention does not use ether as a low-boiling-point reagent, is easy to operate, has high purity, higher yield, and is more suitable for industrialization.

[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for efficiently preparing fatty diacid derivatives, characterized in that: The steps include: (1) dissolving hexadecane mono-tert-butyl ester in solvent 1, adding 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, stirring at room temperature, and then adding L-glutamic acid-1-tert-butyl ester, reacting at room temperature, to obtain tert-butyl hexadecane dioyl-L-Glu-OtBu; The solvent 1 is dichloromethane; the solid-liquid ratio of hexadecane mono-tert-butyl ester to solvent 1 is 1-2g:5-60mL; Diisopropylethylamine needs to be added at the same time as the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and L-glutamic acid-1-tert-butyl ester are added; The molar ratio of hexadecane mono-tert-butyl ester, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and L-glutamic acid-1-tert-butyl ester is 1-1.5:1-1.5:1-1.5; (2) tert-butyl hexadecanoyl-L-Glu-OtBu is dissolved in solvent 2, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is added and the mixture is heated to react, thereby obtaining tert-butyl hexadecanoyl-L-Glu(OSu)-OtBu; Diisopropylethylamine needs to be added at the same time as the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is added, and the molar ratio of the 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate to diisopropylethylamine is 1-1.5:1-5; The molar ratio of tert-butyl hexadecanoyl-L-Glu-OtBu and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is 1-1.5:1-2; (3) dissolving tert-butyl hexadecanoyl-L-Glu (OSu)-OtBu in solvent 3, stirring at room temperature, adding a mixed solution of isopropanol and n-heptane under ice bath conditions, filtering and drying to obtain hexadecanoyl-L-Glu (OSu); 。 2. The preparation method according to claim 1, characterized in that: The solvent 2 described in step (2) is ethyl acetate; the solid-liquid ratio of the tert-butylhexadecanoyl-L-Glu-OtBu to the solvent 2 is 1-2 g:10-60 mL.

3. The preparation method according to claim 1, characterized in that: The solvent 3 in step (3) is trifluoroacetic acid; the solid-liquid ratio of the tert-butylhexadecanoyl-L-Glu(OSu)-OtBu to the solvent 3 is 1-2 g:3-30 mL.

4. The preparation method according to claim 1, characterized in that: The volume ratio of isopropanol to n-heptane in step (3) is 1-10:1-30.

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