Preparation method of diillain acetate

Abstract

The invention relates to the technical field of medicine preparation, and provides a preparation method of diillain acetate, which comprises the following steps: taking Boc-D-Phe-D-Phe-D-Leu and D-Lys (Boc)-Ome as starting materials, condensing, hydrolyzing, condensing with alpha-Boc-Phe-Ome, deprotecting, hydrolyzing, salifying and the like to obtain the diillain acetate. The preparation method provided by the invention overcomes the problems that the impurity separation is difficult and the purity is difficult to break through 80% in the traditional preparation method, and the preparation method is mild in condition, simple to operate, high in safety and suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, and in particular to a method for preparing difacillin acetate. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Difelikefalin is the first KOR agonist approved by the FDA. It is used to treat chronic kidney disease-associated pruritus (CKD-aP) and was first launched in the United States in 2021. The original research company is Cara Therap.

[0004] The chemical name is 4-amino-1-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)piperidine-4-carboxylic acid acetate, and its structural formula is as follows Figure 1 Molecular formula: C 36 H 53 N 7 O 6 xAcOH (1.0≤x≤2.0), molecular weight: 679.84 (free base), CAS number: 1024828-77-0.

[0005] At present, CN101627049B reports a method for solid phase synthesis of difalin, which obtains a resin-bound peptide by sequentially combining with amino acids on a resin, and then completely deprotects the peptide by a peptide cutting agent to obtain a crude target product. Finally, the crude target product is purified by high performance liquid chromatography and freeze-dried to obtain a purified target product. However, in the manufacture using solid phase peptide synthesis, after the pentapeptide protection body is synthesized, it is removed from the resin and completely deprotected, and then refined using a high pressure preparative liquid phase. In this refining, a large high pressure preparative liquid phase equipment is required, the batch size is small, and the refining time is long.

[0006] In addition, CN106459150B reports a method for preparing difacillin acetate by a liquid phase method, which improves the purity of the reaction product by refining, breaking through the limit that the purity of the traditional refining is difficult to exceed 80%. However, in this method, the deprotection agent reaction requires the use of a Pd / C hydrogenation reaction, which has a high risk in the industrial production process. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing difalan acetate in view of the defects of the existing difalan preparation method, so as to solve the problem that the industrial production process of difalan liquid phase preparation has high risks and complex operations.

[0008] The technical solution of the present invention is as follows:

[0009] A liquid phase preparation method of phytoplankton comprises the following steps:

[0010] Step (1): Boc-D-Phe-D-Phe-D-Leu and D-Lys(Boc)-Ome are condensed under the action of a condensing agent;

[0011] Step (2): Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome is hydrolyzed under the action of a base;

[0012] Step (3): Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) and α-Boc-Pic-Ome are condensed in the presence of a condensing agent;

[0013] Step (4): R 1 -D-Phe-D-Phe-D-Leu-D-Lys(R 1 )-α-R 1 -Pic-Ome is deprotected under the action of a deprotecting agent;

[0014] Step (5): D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome is hydrolyzed under the action of a base.

[0015] According to a preferred embodiment, in step (4), R 1 The N-protecting groups shown include alkoxycarbonyl-type N-protecting groups, acyl-type N-protecting groups, alkyl-type N-protecting groups or sulfonamide-type N-protecting groups.

[0016] Preferably, in step (4), R 1 The N-protecting group shown is one or more of tert-butyloxycarbonyl (Boc), acetyl (Ac), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), trityl (Trt), methoxymethyl (MOM), preferably Boc.

[0017] According to a preferred embodiment, the concentration of the deprotecting agent in step (4) is 5% to 100%; the deprotecting agent includes an acidic deprotecting agent or a basic deprotecting agent, preferably HCl, H 2 SO 4 , trifluoroacetic acid, acetic acid, p-toluenesulfonic acid, and unsaturated fatty acid (TFA) or more.

[0018] Preferably, step (4) is specifically carried out in the presence of a deprotecting agent, in a deprotecting solvent, at a temperature of 0 to 40° C., for a reaction time of 1 to 48 hours;

[0019] The deprotection solvent is one or more of dichloromethane (DCM), 1,4-dioxane (1,4-Dioxane), ethyl acetate (EtOAc), methanol (MeOH), acetonitrile (MeCN), tetrahydrofuran, and ethanol (EtOH).

[0020] According to a preferred embodiment, the condensing agent used in step (1) and step (3) includes carbodiimides, onium salts, and organophosphorus condensing agents, preferably N,N,-dicyclohexylcarbodiimide (DCC), carbonyldiimidazole, N,N'-diisopropylcarbodiimide, 1-hydroxy-benzotriazole (HOBt), 0-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole One or more of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinyl uronium hexafluorophosphate (COMU) or 1-propylphosphonic acid cyclic anhydride (T3P).

[0021] Preferably, the amount of the condensing agent used in step (1) and step (3) is 0.5 to 5.0 equivalents, preferably 0.8 to 2.0 equivalents.

[0022] Preferably, the condensation reaction in step (1) and step (3) is carried out in a condensation reaction solvent under the action of a condensation agent, and the condensation reaction conditions are: a condensation temperature of 0 to 80° C., and a condensation time of 1 to 48 hours;

[0023] The condensation reaction solvent includes toluene, xylene, ethyl acetate, isopropyl acetate, butyl acetate, chloroform, dimethyl sulfoxide, N,N-dimethylformamide (DMF), acetonitrile, N,N-dimethylacetamide (DMAC), N-methylpyrrolidone (NMP) or dimethyl sulfoxide (DMSO).

[0024] According to a preferred embodiment, the amount of base used in steps (2) and (5) is 0.8 to 5.0 equivalents, preferably 1.0 to 3.0 equivalents;

[0025] The base includes a monobasic base, a dibasic base or a ternary base; preferably one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide and aluminum hydroxide.

[0026] Preferably, step (2) and step (5) are specifically carried out in the presence of a base in tetrahydrofuran, water or an alcohol solvent at 0 to 40° C. for 1 to 48 hours.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. A method for preparing difalin acetate, such as Figure 1 The salt of compound (1) can be easily purified by crystallization or the like. In addition, the salt of compound (1) has a sufficiently high purity even without purification, which is greater than the 80% purity limit of conventional liquid phase preparation.

[0029] 2. A method for preparing difacillin acetate. Compared with the solid phase synthesis method, the compound (1) produced by the method can achieve large-scale synthesis and reduce the unit production cost. In addition, the intermediate compound can be separated during the preparation process, and then the impurities such as non-enantiomers and defective peptides can be removed by extraction, crystallization and other simple operations without the need for expensive equipment. Finally, a high-purity target compound with a purity of 99% as determined by HPLC can be obtained.

[0030] 3. A method for preparing difacillin acetate, compared with other liquid phase synthesis methods, avoids the use of protecting groups such as Cbz, can effectively avoid the less safe hydrogenation reaction, can effectively reduce the demand for industrial production equipment, and greatly reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the structural formula of difalin acetate;

[0032] Figure 2 This is a schematic diagram of the preparation process of difalin acetate in this application;

[0033] Figure 3 Shown is the LC-MS detection chart of compound (1) prepared in Example 1;

[0034] Figure 4 The HPLC chart of compound (1) prepared in Example 1 is shown. DETAILED DESCRIPTION

[0035] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0037] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0038] The structural formula of D-Phe-D-Phe-D-Leu-D-Lys-Pic is as follows Figure 1 As shown, using Figure 2 The synthetic route shown specifically comprises the following steps:

[0039] Step (1): Synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome (4), Boc-D-Phe-D-Phe-D-Leu and D-Lys(Boc)-Ome are condensed under the action of a condensing agent:

[0040] Example 1: Add Boc-D-Phe-D-Phe-D-Leu (2): 30.0 g (57.07 mmol, 1.00 eq), D-Lys (Boc) -Ome (3): 17.8 g (59.98 mmol, 1.05 eq), EDC·HCl: 16.4 g (85.55 mmol, 1.50 eq), HOBt: 7.9 g (58.47 mmol, 1.03 eq), and DCM 300 mL to a reaction bottle and stir to dissolve. Add DIPEA (N, N-diisopropylethylamine): 11.1 g (85.89 mmol, 1.50 eq) under ice-water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, add 7% NaHCO 3 Solution: 300 mL Stir for 30 minutes, separate the organic layer. Use 1N HCl solution: 300 mL, 7% NaHCO 3 Solution: 300 mL, 10% NaCl solution: 300 mL to wash the organic layer. Add n-heptane: 900 mL to the organic phase under stirring for crystallization, filter, and obtain a white solid: 39.1 g, namely Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4), with a yield of 89.2% and a purity of 98.3% as determined by HPLC.

[0041] Example 2: Add Boc-D-Phe-D-Phe-D-Leu (2): 30.0 g (57.07 mmol, 1.00 eq), D-Lys (Boc) -Ome (3): 17.8 g (59.98 mmol, 1.05 eq), EDC·HCl: 16.4 g (85.55 mmol, 1.50 eq), HATU 23.87 g (62.78 mmol, 1.10 eq), DCM: 300 mL to a reaction flask and stir to dissolve. Add DIPEA (N, N-diisopropylethylamine): 11.1 g (85.89 mmol, 1.50 eq) under ice water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, add 7% NaHCO 3 Solution: 300 mL Stir for 30 minutes, separate the organic layer. Use 1N HCl solution: 300 mL, 7% NaHCO 3 Solution: 300 mL, 10% NaCl solution: 300 mL to wash the organic layer. Add n-heptane: 900 mL to the organic phase under stirring for crystallization, filter, and obtain a white solid: 41.1 g, namely Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4), with a yield of 93.9% and a purity of 96.3% as determined by HPLC.

[0042] Example 3: Add Boc-D-Phe-D-Phe-D-Leu (2): 30.0 g (57.07 mmol, 1.00 eq), D-Lys (Boc) -Ome (3): 17.8 g (59.98 mmol, 1.05 eq), EDC·HCl: 16.4 g (85.55 mmol, 1.50 eq), PYBOP: 31.39 g (59.92 mmol, 1.05 eq), DCM: 300 mL to a reaction flask and stir to dissolve. Add DIPEA (N, N-diisopropylethylamine): 11.1 g (85.89 mmol, 1.50 eq) under ice water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, add 7% NaHCO 3 Solution: 300 mL Stir for 30 minutes, separate the organic layer. Use 1N HCl solution: 300 mL, 7% NaHCO 3 Solution: 300 mL, 10% NaCl solution: 300 mL to wash the organic layer. Add n-heptane: 900 mL to the organic phase under stirring for crystallization, filter and obtain a white solid: 37.1 g, namely Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4), with a yield of 89.2% and a purity of 84.6% as determined by HPLC.

[0043] Step (2): Synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5), Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome is hydrolyzed under the action of a base:

[0044] Example 4: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4): 30.0g (39.06mmol, 1.00eq), THF: 300mL, water: 90mL to the reaction bottle and stir to dissolve, add lithium hydroxide monohydrate: 2.0g (48.05mmol, 1.23eq) under ice water bath, warm to room temperature after addition and react for 5 hours. After the reaction is completed, add 3N HCl to adjust the pH to 2-5, then extract twice with EtOAc (ethyl acetate): 300mL, and wash the organic layer with 10% NaCl solution: 300mL. Add n-heptane: 450mL to the organic phase under stirring for crystallization, filter, and obtain a white solid: 22.8g, which is

[0045] Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5), yield 77.4%, purity detected by HPLC: 98.7%.

[0046] Example 5: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4): 30.0g (39.06mmol, 1.00eq), THF: 300mL, water: 90mL to the reaction bottle and stir to dissolve, add potassium hydroxide: 2.6g (46.87mmol, 1.20eq) under ice water bath, warm to room temperature after addition and react for 5 hours. After the reaction is completed, add 3N HCl solution to adjust the pH to 2-5, then extract twice with EtOAc (ethyl acetate): 300mL, and wash the organic layer with 10% NaCl solution: 300mL. Add n-heptane: 450mL to the organic phase under stirring for crystallization, filter, and obtain a white solid: 23.6g, which is

[0047] Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5), yield 80.2%, purity determined by HPLC: 96.4%.

[0048] Example 6: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome(4): 30.0g (39.06mmol, 1.00eq), THF: 300mL, water: 90mL to a reaction flask and stir to dissolve, add sodium hydroxide: 1.9g (46.9mmol, 1.20eq) under an ice-water bath, and warm to room temperature to react for 5 hours after the addition. After the reaction is completed, add 3N HCl to adjust the pH to 2-5, then extract twice with EtOAc (ethyl acetate): 300mL, and wash the organic layer with 10% NaCl solution: 300mL. Add n-heptane: 450mL to the organic phase under stirring for crystallization, filter, and obtain a white solid: 21.6g, which is Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5), with a yield of 73.5% and a purity of 94.3% as determined by HPLC.

[0049] Step (3): Synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome (7), Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc) and α-Boc-Pic-Ome are condensed in the presence of a condensing agent:

[0050] Example 7: Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5):13.0g(17.2mmol, 1.00eq), α-Boc-Pic-Ome(6)5.3g(18.0mmol, 1.05eq), EDC·HCl:4.9g(25.8mmol, 1.5eq), HOBt:2.8g(20.6mmol, 1.2eq), DCM 130mL were added to the reaction bottle and stirred to dissolve, DIPEA:6.7g(51.6mmol, 3.0eq) was added under ice water bath, and the temperature was raised to room temperature for reaction for 5 hours. After the reaction was completed, 7% NaHCO 3 Solution: 130 mL was stirred for 30 minutes, and the organic layer was separated. 130 mL of 1N HCl solution and 7% NaHCO 3 Solution: 130 mL, 10% NaCl solution: 130 mL to wash the organic layer. Add n-heptane (30 v / w) to the organic phase under stirring for crystallization, filter, and obtain a white solid: 8.9 g, which is Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7), with a yield of 52.1% and a purity of 95.3% as determined by HPLC.

[0051] Example 8: Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5): 13.0 g (17.2 mmol, 1.00 eq), α-Boc-Pic-Ome(6): 5.3 g (18.0 mmol, 1.05 eq), COMU 8.1 g (18.9 mmol, 1.1 eq), HOBt: 2.8 g (20.64 mmol, 1.2 eq), and DCM 130 mL were added to the reaction bottle and stirred to dissolve. DIPEA: 6.7 g (51.6 mmol, 3.0 eq) was added under ice-water bath. After the addition, the temperature was raised to room temperature and reacted for 5 hours. After the reaction was completed, 7% NaHCO 3 :130mL, stirred for 30 minutes, and the organic layer was separated. 1N HCl solution: 130mL, 7% NaHCO 3 Solution: 130mL, 10% NaCl solution: 130mL to wash the organic layer. Add n-heptane (30v / w) to the organic phase under stirring for crystallization, filter, and obtain a white solid: 13.7g, which is Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7), with a yield of 79.9% and a purity of 99.3% as determined by HPLC.

[0052] Example 9: Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)(5): 13.0 g (17.2 mmol, 1.00 eq), α-Boc-Pic-Ome(6): 5.3 g (18.0 mmol, 1.05 eq), EDC·HCl: 4.9 g (25.8 mmol, 1.5 eq), PyBoP: 10.7 g (20.6 mmol, 1.2 eq), and DCM 130 mL were added to the reaction bottle and stirred to dissolve. DIPEA: 6.7 g (51.6 mmol, 3.0 eq) was added under ice-water bath. After the addition, the temperature was raised to room temperature and reacted for 5 hours. After the reaction was completed, 7% NaHCO 3 Solution: 130 mL Stir for 30 minutes, separate the organic layer. Use 1N HCl solution: 130 mL, 7% NaHCO 3 Solution: 130mL, 10% NaCl solution: 130mL to wash the organic layer. Add n-heptane (30v / w) to the organic phase under stirring for crystallization, filter, and obtain a white solid: 9.6g, which is Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7), with a yield of 56.1% and a purity of 93.3% as determined by HPLC.

[0053] Step (4): Synthesis of D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome (8), R 1 -D-Phe-D-Phe-D-Leu-D-Lys(R 1 )-α-R 1 -Pic-Ome is deprotected by a deprotecting agent:

[0054] Example 10: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7): 9.5 g (9.6 mmol, 1.00 eq) and DCM: 50 mL to a reaction bottle and stir to dissolve. Add TFA: 10 mL under an ice-water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, add methyl tert-butyl ether: 300 mL and crystallize for 1 hour. Filter and dry the filter cake to obtain a white solid: 6.5 g, which is D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome(8), with a yield of 84.3% and a HPLC purity of 98.3%.

[0055] Example 11: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7): 9.5 g (9.6 mmol, 1.00 eq) and DCM: 50 mL to a reaction flask and stir to dissolve. Add acetic acid: 20 mL under an ice-water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, add methyl tert-butyl ether: 300 mL and crystallize for 1 hour. Filter and dry the filter cake to obtain a white solid: 6.3 g, which is D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome(8), with a yield of 81.5% and a HPLC purity of 86.7%.

[0056] Example 12: Add Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-α-Boc-Pic-Ome(7): 3.2g (mol, eq) and EtOH: 15mL to a reaction flask and stir to dissolve. Add 3N HCl / 1,4-Dioxane: 5mL under an ice-water bath. After the addition, warm to room temperature and react for 5 hours. After the reaction is completed, concentrate to 10mL, add methyl tert-butyl ether: 700mL, and crystallize for 1 hour. Filter and dry the filter cake to obtain a white solid; 1.8g is D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome(8), the yield is 79.6%, and the purity detected by HPLC is 99.2%.

[0057] Step (5): Synthesis of D-Phe-D-Phe-D-Leu-D-Lys-Pic (1), hydrolysis of D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome under the action of alkali:

[0058] Example 13: D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome (8): 4.8 g (5.9 mmol, 1.00 eq), MeOH: 15 mL, and water: 15 mL were added to a reaction bottle and stirred to dissolve. Lithium hydroxide monohydrate: 0.4 g (8.9 mmol, 1.5 eq) was added under an ice-water bath. After the addition, the temperature was raised to room temperature and reacted for 5 hours. After the reaction was completed, methanol was concentrated to remove methanol, acetic acid: 2.5 g (41.6 mmol, 7.0 eq) was added, and after stirring for 1 hour, MeCN: 75 mL was added and crystallized for 1 hour. Filter and dry the filter cake to obtain a white solid: 3.9 g, which is D-Phe-D-Phe-D-Leu-D-Lys-Pic (1). The yield was 72.6%, and the purity was 99.4% as determined by HPLC. Example 14: D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome (8): 1.8 g (2.2 mmol, 1.00 eq), MeOH 15 mL, and water: 15 mL were added to a reaction flask and stirred to dissolve. Sodium hydroxide: 0.1 g (2.7 mmol, 1.2 eq) was added under an ice-water bath. After the addition, the temperature was raised to room temperature and reacted for 5 hours. After the reaction was completed, methanol was concentrated to remove methanol, acetic acid: 0.9 g (15.0 mmol, 7.0 eq) was added, and the reaction was stirred for 1 hour. MeCN: 25 mL was added and crystallized for 1 hour. Filter and dry the filter cake to obtain a white solid: 1.2 g, which is D-Phe-D-Phe-D-Leu-D-Lys-Pic (1). The yield was 77.3% and the purity was 99.1% as determined by HPLC. The parameters for LCMS and HPLC purity detection of the prepared product are as follows:

[0059] The detection parameters for LCMS detection are:

[0060] Column: Waters X Bridge C18: 50mm*4.6mm*3.5um;

[0061] Mobile phase: A: water (0.01 mol / L NH 4 HCO 3 )B:ACN

[0062] Gradient: B from 5% to 95% for 1.6 min, hold at 95% for 1.4 min;

[0063] Flow rate: 2.0 ml / min;

[0064] Column temperature: 40°C

[0065] The LC-MS diagram of Example 13 is as follows: Figure 3 As shown, MS (M+H +): The detected molecular weight is 680.3. The predicted molecular weight is 680.41, and the detected result is consistent with the prediction.

[0066] The HPLC detection spectrum of Example 13 is as follows Figure 4 shown.

[0067] In summary, combined with the data of the examples, it can be seen that the substance prepared in the present application is D-Phe-D-Phe-D-Leu-D-Lys-Pic (1). The purity of the preparation of the present application is as high as 99.4%. Compared with the prior art, it not only breaks through the defect that the purity of the traditional refining is difficult to be higher than 80%, but also overcomes the problem that the deprotection agent reaction used in the prior art to improve the purity requires the use of Pd / C hydrogenation reaction, which is high risk and complicated in the industrial production process. The technical solution of the present application is simple to operate, highly safe, and high in purity, and is very suitable for large-scale industrial production.

[0068] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for preparing difacillin acetate, characterized in that: The following steps are involved: Step (1): Boc-D-Phe-D-Phe-D-Leu and D-Lys(Boc)-Ome are condensed under the action of a condensing agent; Step (2): Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Ome is hydrolyzed under the action of a base; Step (3): Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) and α-Boc-Pic-Ome are condensed in the presence of a condensing agent; Step (4): R1-D-Phe-D-Phe-D-Leu-D-Lys(R1)-α-R1-Pic-Ome is deprotected under the action of a deprotecting agent; Step (5): D-Phe-D-Phe-D-Leu-D-Lys-Pic-Ome is hydrolyzed under the action of a base.

2. The method for preparing difacillin acetate according to claim 1, characterized in that: The N-protecting group represented by R1 in step (4) includes an alkoxycarbonyl N-protecting group, an acyl N-protecting group, an alkyl N-protecting group or a sulfonamide N-protecting group.

3. The method for preparing difacillin acetate according to claim 2, characterized in that: The N-protecting group represented by R1 in step (4) is one or more of tert-butoxycarbonyl, acetyl, 2-(trimethylsilyl)ethoxycarbonyl, trityl, and methoxymethyl.

4. The method for preparing difacillin acetate according to claim 1, characterized in that: The condensing agent used in step (1) and step (3) includes carbodiimides, onium salts, and organic phosphorus condensing agents, preferably one or more of N,N,-dicyclohexylcarbodiimide, carbonyldiimidazole, N,N'-diisopropylcarbodiimide, 1-hydroxy-benzotriazole, 0-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 0-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or benzotriazol-1-yloxytris(dimethylamino)phosphine hexafluorophosphate.

5. The method for preparing difacillin acetate according to claim 4, characterized in that: The amount of the condensing agent used in step (1) and step (3) is 0.5 to 5.0 equivalents, preferably 0.8 to 2.0 equivalents.

6. The method for preparing difacillin acetate according to claim 1, characterized in that: The condensation reaction under the action of the condensation agent in step (1) and step (3) is carried out in a condensation reaction solvent, and the condensation reaction conditions are: the condensation temperature is 0 to 80° C., and the condensation time is 1 to 48 hours; The condensation reaction solvent includes toluene, xylene, ethyl acetate, isopropyl acetate, butyl acetate, chloroform, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, N,N-dimethylacetamide, N-methylpyrrolidone or dimethyl sulfoxide.

7. The method for preparing difacillin acetate according to claim 1, characterized in that: The amount of base used in step (2) and step (5) is 0.8 to 5.0 equivalents, preferably 1.0 to 3.0 equivalents; The base includes a monobasic base, a dibasic base or a ternary base; preferably one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide and aluminum hydroxide.

8. The method for preparing difacillin acetate according to claim 7, characterized in that: Step (2) and step (5) are specifically carried out in the presence of a base in tetrahydrofuran, water or an alcoholic solvent at 0 to 40° C. for a reaction of 1 to 48 hours.

9. The method for preparing difacillin acetate according to claim 1, characterized in that: The concentration of the deprotecting agent in step (4) is 5% to 100%; The deprotecting agent includes an acidic deprotecting agent or a basic deprotecting agent, preferably one or more of HCl, H2SO4, trifluoroacetic acid, acetic acid, and p-toluenesulfonic acid.

10. The method for preparing difacillin acetate according to claim 9, characterized in that: Step (4) is specifically to react in the presence of a deprotecting agent at a temperature of 0 to 40° C. for 1 to 48 hours; The deprotection solvent is one or more of dichloromethane, 1,4-dioxane, ethyl acetate, methanol, acetonitrile, and tetrahydrofuran.

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