A preparation method of tyrosine-serine pseudo-dipeptide

Abstract

The invention discloses a method for preparing a tyrosine serine pseudo dipeptide, and relates to the technical field of chemical synthesis. The preparation method includes condensing Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride under the conditions of a first solvent, a condensing agent and a base, and the condensing agent is HOBT and TBTU to obtain a first intermediate; the first intermediate and 2-dimethoxypropylene are subjected to a closed-loop reaction under the conditions of 4-methylbenzenesulfonic acid pyridine and a second solvent to obtain a second intermediate; the second intermediate and palladium carbon are reacted in a third solvent to obtain a target product, tyrosine serine pseudo dipeptide. The preparation method of the present invention has mild synthesis conditions, is green and environmentally friendly, is simple to operate, is easy to mass produce, and can obtain a product of higher purity at the same time, with a purity of ≥98.5%, a single impurity of less than 0.2%, and an isomer purity of less than 0.1%.

Description

Technical Field

[0001] The invention relates to the technical field of chemical synthesis, and in particular to a method for preparing a tyrosine-serine pseudo-dipeptide. Background Art

[0002] Drug peptides are small molecule proteins composed of amino acids that can regulate physiological functions or treat diseases in organisms. They have been widely used in the field of drug development because of their advantages such as high efficiency, low side effects and broad spectrum. Compared with traditional chemotherapy drugs, peptide drugs have the characteristics of small molecular weight, sensitivity to tumors, low resistance and low cost, which has led to active research and development of peptide drugs at home and abroad. In addition, peptides are also the main active components of tumor antigens, and the development of peptide vaccines will also bring hope to cancer patients.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a tyrosine-serine pseudo-dipeptide.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a method for preparing a tyrosine-serine pseudo-dipeptide, comprising:

[0007] S1. Conducting a condensation reaction between Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride in the presence of a first solvent, a condensing agent and a base, wherein the condensing agent is HOBT and TBTU, to obtain a first intermediate; the structural formula of the Fmoc-O-tert-butyl-L-tyrosine is , the structural formula of the L-serine benzyl ester hydrochloride is: , the structural formula of the first intermediate is ;

[0008] S2, subjecting the first intermediate and 2-dimethoxypropylene to a ring-closing reaction in the presence of 4-methylbenzenesulfonate pyridine and a second solvent to obtain a second intermediate; the structural formula of the second intermediate is ;

[0009] S3, reacting the second intermediate and palladium carbon in a third solvent to obtain a target product - tyrosine serine pseudo-dipeptide, the structural formula of the tyrosine serine pseudo-dipeptide is: .

[0010] In an optional embodiment, step S1 includes at least one of features (1) to (5):

[0011] Feature (1): The molar ratio of the Fmoc-O-tert-butyl-L-tyrosine, the L-serine benzyl ester hydrochloride, the HOBT, the TBTU and the base is 1:(1-2):(1-2):(1.1-2.5):(2-4);

[0012] Feature (2): The mass ratio of the Fmoc-O-tert-butyl-L-tyrosine to the first solvent is 1:(7-8);

[0013] Feature (3): The first solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and ethyl acetate;

[0014] Feature (4): The base is N,N-diisopropylethylamine;

[0015] Feature (5): The operating steps of the condensation reaction include: adding the Fmoc-O-tert-butyl-L-tyrosine to the first solvent, cooling to 0-5°C, then adding the HOBT, the TBTU and the base in sequence, maintaining the temperature at 0-5°C for 20-30 minutes, and finally adding the L-serine benzyl ester hydrochloride and naturally heating to room temperature for reaction.

[0016] In an optional embodiment, step S2 includes at least one of features (6) to (9):

[0017] Feature (6): The molar ratio of the first intermediate, the 2-dimethoxypropylene and the 4-methylbenzenesulfonate pyridine is 1:(2-8):(0.01-0.1);

[0018] Feature (7): The mass ratio of the first intermediate to the second solvent is 1:(5-7);

[0019] Feature (8): the second solvent is at least one of toluene, tetrahydrofuran and DMF;

[0020] Feature (9): The reaction temperature of the ring-closing reaction is 60-110°C.

[0021] In an optional embodiment, step S3 includes at least one of features (10) to (12):

[0022] Feature (10): The molar ratio of the second intermediate to the palladium carbon is 1:(0.2-0.3);

[0023] Feature (11): The mass ratio of the second intermediate to the third solvent is 1:(5-7);

[0024] Feature (12): The third solvent is ethyl acetate.

[0025] In an optional embodiment, the method of adding palladium carbon includes: first adding 40-60% of the total amount of palladium carbon to the second intermediate, then replacing it with nitrogen 2-4 times, replacing it with hydrogen 2-4 times, reacting for 1-3 hours, adding the remaining amount of palladium carbon, replacing it with hydrogen 2-4 times, and continuing the reaction for 1-3 hours.

[0026] In an optional embodiment, in step S1, after the condensation reaction is completed and before the first intermediate is obtained, it also includes extracting the first reaction system with water and EA, combining the organic phases, acid washing the organic phase until the pH of the aqueous phase is 1, then adding sodium bicarbonate solution to wash until the pH of the aqueous phase is 8, then washing with saturated brine, concentrating, then adding petroleum ether and ethyl acetate for crystallization, and continuing to add petroleum ether and ethyl acetate for slurrying after filtration, filtering the slurry, and drying.

[0027] In an optional embodiment, in step S2, after the ring-closing reaction is completed and before the second intermediate is obtained, the second reaction system is further cooled and vacuum concentrated to remove the second solvent, followed by filtration to obtain a solid product and a filtrate, the solid product is rinsed with petroleum ether to remove the remaining second solvent, and the solid product is then eluted with petroleum ether and ethyl acetate. After the elution is completed, the eluate is concentrated to remove the petroleum ether and ethyl acetate.

[0028] In an optional embodiment, the filtration is performed using a filter cylinder, the filter cylinder is provided with a filter screen with a size of 1000-1400 mesh, and the filter cylinder is also filled with silica gel with a size of 100-200 mesh.

[0029] In an optional embodiment, in step S3, before obtaining the target product, the third reaction system is further concentrated to remove the third solvent, followed by washing with sodium bicarbonate solution until the aqueous phase has a pH of 7-8, then washing with saturated brine, concentrating, and then adding methyl tert-butyl ether and n-heptane for crystallization, and after filtration, methyl tert-butyl ether and n-heptane are continued to be added for slurrying, and the slurry is filtered and dried.

[0030] In an optional embodiment, during crystallization and slurrying, the total amount of the methyl tert-butyl ether and the n-heptane added is 2-4 times the mass of the concentrate after concentration, and the mass ratio of the methyl tert-butyl ether to the n-heptane is 1:(4-6).

[0031] The present invention has the following beneficial effects:

[0032] The preparation method of the tyrosine serine pseudo dipeptide provided by the present invention uses Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride as raw materials, and uses cheap HOBT and TBTU as condensing agents, which can improve the condensation effect and ensure the yield and purity of the first intermediate. When preparing the second intermediate, 2-dimethoxypropylene is used as an auxiliary material, which is easy to react with the specific site of the hydroxyl group on the first intermediate. 4-methylbenzenesulfonic acid pyridine is used as a catalyst to improve the yield and stereoselectivity of the second intermediate, thereby improving the purity of the second intermediate. Finally, the second intermediate reacts with palladium carbon, and the hydrogenation reducibility and selectivity of palladium carbon are utilized to efficiently prepare a high-purity target product. The preparation method of the present invention has mild synthesis conditions, is green and environmentally friendly, is simple to operate, is easy to mass-produce, and can obtain a product of higher purity at the same time, with a purity of ≥98.5% and a single impurity of less than 0.2%. The optical purity can control the purity of (4S)-3-[(2S)-3-[4-(tert-butyloxy)phenyl]-2-[[fluorenylmethoxycarbonyl]amino]-1-oxopropyl]-2,2-dimethyl-4-oxazolidinecarboxylic acid isomers to be less than 0.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 The HPLC chromatogram of the tyrosine-serine pseudo-dipeptide provided in Example 1 of the present invention;

[0035] Figure 2 This is the hydrogen spectrum of the tyrosine-serine pseudo-dipeptide provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] The present invention provides a method for preparing a tyrosine-serine pseudo-dipeptide, which comprises:

[0038] S1. Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride are subjected to a condensation reaction in the presence of a first solvent, a condensing agent and a base, wherein the condensing agent is HOBT and TBTU, to obtain a first intermediate. The structural formula of Fmoc-O-tert-butyl-L-tyrosine is , the structural formula of L-serine benzyl ester hydrochloride is: , the structural formula of the first intermediate is .

[0039] In the present invention, Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride are used as raw materials for condensation reaction, the conditions of the condensation reaction are mild, green and environmentally friendly, and the whole operation process is simple. At the same time, the condensation agent is limited to a mixture of HOBT and TBTU. The selection of the condensation agent can affect the yield and purity of the first intermediate, and also has a certain influence on the purity of the target product.

[0040] The molar ratio of Fmoc-O-tert-butyl-L-tyrosine, L-serine benzyl ester hydrochloride, HOBT, TBTU and base is 1:(1-2):(1-2):(1.1-2.5):(2-4); the mass ratio of Fmoc-O-tert-butyl-L-tyrosine to the first solvent is 1:(7-8).

[0041] The first solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and ethyl acetate; and the base is N,N-diisopropylethylamine (DIPEA).

[0042] Specifically, the operating steps of the condensation reaction include: adding Fmoc-O-tert-butyl-L-tyrosine to the first solvent, cooling to 0-5°C, then adding HOBT, TBTU and a base in sequence, maintaining the temperature at 0-5°C for 20-30 minutes, and finally adding L-serine benzyl hydrochloride, and naturally heating to room temperature for reaction.

[0043] In the present invention, by first adding Fmoc-O-tert-butyl-L-tyrosine to the first solvent, the full dispersion of Fmoc-O-tert-butyl-L-tyrosine can be achieved, and the subsequent cooling operation is conducive to maintaining the stability of Fmoc-O-tert-butyl-L-tyrosine, and then HOBT and TBTU are added together as condensing agents, wherein HOBT refers to 1-hydroxybenzotriazole, TBTU refers to 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, and HOBT and TBTU are both used as polypeptide condensing agents. However, the present invention has found that the yield and purity of HOBT and TBTU are poor when used alone, and the compound use can significantly improve the yield and purity. The base selected in the present invention is N, N-diisopropylethylamine, which is different from other alkaline reagents (sodium hydroxide, etc.) because the reaction conditions are mild and there will not be too many side reactions. Finally, L-serine benzyl ester hydrochloride is added to start the condensation reaction. The conditions for the condensation reaction of Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride are mild. Under the condensation action of HOBT and TBTU, it is easy to prepare a first intermediate with high yield and high purity, which is conducive to ensuring the purity of the subsequent target product.

[0044] After the condensation reaction is completed, before obtaining the first intermediate, the process also includes: after the TLC spot plate shows that the raw material Fmoc-O-tert-butyl-L-tyrosine has reacted, extracting the first reaction system with water and disodium ethylenediaminetetraacetic acid (EA), combining the organic phases, acid-washing the organic phase to a water phase pH of 1, then adding a sodium bicarbonate solution to wash the water phase to a pH of 8, then washing with saturated brine, concentrating, then adding petroleum ether and ethyl acetate for crystallization, and then continuing to add petroleum ether and ethyl acetate for pulping after suction filtration, suction filtration of the slurry, and drying. In the present invention, after the condensation reaction is completed, the first intermediate can be purified by extraction, washing, crystallization and recrystallization to ensure the purity of the first intermediate.

[0045] S2, the first intermediate and 2-dimethoxypropylene are subjected to a ring-closing reaction in the presence of 4-methylbenzenesulfonate pyridine and a second solvent to obtain a second intermediate, the structural formula of which is .

[0046] In the present invention, 2-dimethoxypropylene is used as an auxiliary material and 4-methylbenzenesulfonate pyridine (PPTS) is used as a catalyst, wherein 2-dimethoxypropylene has a methoxy group and a double bond, and is easy to undergo a ring-closing reaction with a specific site of a hydroxyl group on the first intermediate to obtain a second intermediate. 4-Methylbenzenesulfonate pyridine is usually used as a mild and efficient catalyst for forming and cracking acetals. In the present invention, it is innovatively used as a catalyst for the ring-closing reaction, which can improve the yield and stereoselectivity of the second intermediate, thereby improving the purity of the second intermediate. Other stereoselective catalysts, such as left-rotating camphorsulfonic acid or rac camphorsulfonic acid, are not suitable for the ring-closing reaction of the present invention. When other catalysts are used for catalysis, the yield and purity of the obtained second intermediate are significantly reduced, which is not conducive to the preparation of the subsequent target product.

[0047] In the present invention, the molar ratio of the first intermediate, 2-dimethoxypropylene and 4-methylbenzenesulfonate pyridine is 1: (2-8): (0.01-0.1); the mass ratio of the first intermediate and the second solvent is 1: (5-7); the second solvent is at least one of toluene, tetrahydrofuran and DMF; and the reaction temperature of the ring-closing reaction is 60-110°C.

[0048] After the ring-closing reaction is completed and before the second intermediate is obtained, the process also includes: when the TLC spot plate shows that the reaction of the first intermediate of the raw material is complete, the second reaction system is cooled and vacuum concentrated to remove the second solvent, followed by suction filtration, wherein the suction filtration is performed using a filter cylinder, the filter cylinder is provided with a filter screen with a size of 1000-1400 mesh, and the filter cylinder is also filled with 100-200 mesh silica gel. A solid product and a filtrate are obtained, the solid product is rinsed with petroleum ether to remove the remaining second solvent, and then the solid product is eluted with petroleum ether and ethyl acetate (the mass ratio of petroleum ether to ethyl acetate is 4-6:1). After the elution is completed, the eluent is concentrated to remove the petroleum ether and ethyl acetate.

[0049] Since conventional purification requires column purification, the present invention adopts the method of suction filtration to achieve purification, avoiding the need for column brushing and making purification simpler.

[0050] S3, reacting the second intermediate and palladium carbon in a third solvent to obtain a target product, the structural formula of the target product is: .

[0051] The molar ratio of the second intermediate to palladium carbon is 1:(0.2-0.3); the mass ratio of the second intermediate to the third solvent is 1:(5-7); and the third solvent is ethyl acetate.

[0052] The palladium carbon in the present invention is palladium carbon with a mass percentage of 5%, and the method for adding palladium carbon includes: first adding 40-60% of the total amount of palladium carbon to the second intermediate, then replacing with nitrogen 2-4 times, replacing with hydrogen 2-4 times, reacting for 1-3 hours, adding the remaining amount of palladium carbon, replacing with hydrogen 2-4 times, and continuing to react for 1-3 hours.

[0053] The present invention has found that the dosage of 5% palladium carbon affects the yield. When the dosage of palladium carbon is too much or too little, its yield is significantly reduced. In addition, in the present invention, the feeding method of palladium carbon also adopts a method of feeding a part of the material first, and then supplementing the material after a period of reaction. This feeding method can ensure purity and yield. If the whole amount of palladium carbon is added at one time, part of the raw materials will be decomposed into impurities, which will also affect the yield.

[0054] Before obtaining the target product, the TLC spot plate shows that the reaction of the second intermediate of the raw material is complete. The third reaction system is concentrated to remove the third solvent, and then washed with sodium bicarbonate solution until the pH of the aqueous phase is 7-8, and then washed with saturated brine, concentrated, and then methyl tert-butyl ether and n-heptane are added for crystallization, and methyl tert-butyl ether and n-heptane are added for slurrying after suction filtration, and the slurry is suction filtered and dried.

[0055] During crystallization and pulping, the total amount of methyl tert-butyl ether and n-heptane added is 2-4 times the mass of the concentrate after concentration, and the mass ratio of methyl tert-butyl ether to n-heptane is 1:(4-6).

[0056] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0057] Example 1

[0058] S1, prepare the first intermediate, the reaction formula is as follows:

[0059]

[0060] (1) Add Fmoc-O-tert-butyl-L-tyrosine (20 kg) to N,N-dimethylformamide (DMF) (150 kg) and cool to 0-5°C; add HOBT (6.2 kg) (1.05 eq), TBTU (16.8 kg) (1.2 eq), DIPEA (14.1 kg) (2.5 eq) in sequence, keep the temperature at 0-5°C for 30 mins, and finally add L-serine benzyl ester hydrochloride (11.1 kg) (1.05 eq) and naturally raise the temperature to room temperature for reaction.

[0061] (2) TLC spot plate shows that the raw material Fmoc-O-tert-butyl-L-tyrosine has been reacted. Water (360 kg) and EA (180 kg x 2) are added for extraction. The organic phases are combined and washed with 0.5N hydrochloric acid (180 kg x 2). The pH value of the aqueous phase is measured to be 1. Saturated sodium bicarbonate solution (200 kg) is added for washing. The pH value of the aqueous phase is measured to be 8. The organic phase is washed with saturated brine (200 kg). The organic phase is concentrated to a small volume, ethyl acetate is crystallized, and filtered. The filtered solid is placed in an oven and dried at 55°C.

[0062] (3) 26.7 kg of the first intermediate as a yellow-brown solid was obtained, with a yield of 96.4%.

[0063] S2, prepare the second intermediate, the reaction formula is as follows:

[0064]

[0065] (1) The first intermediate obtained in Example 1 was added to toluene (162 kg), 4-methylbenzenesulfonate pyridine (1 kg) (0.05 eq) was added, and dimethoxypropylene (11.8 kg) (4 eq) was added, and the temperature was raised to 100° C. for reaction.

[0066] (2) When the TLC plate shows that the reaction of the first intermediate of the raw material is complete, the temperature is lowered to 55°C and the toluene is concentrated in vacuo. After the concentration is complete (a small amount of toluene remains), the toluene is rinsed clean with petroleum ether in a filter cylinder (1200 mesh) (160 kg silica gel 100 mesh-200 mesh), and the product is eluted with a mixture (ethyl acetate: methanol = 20:1), and the ethyl acetate and methanol are concentrated.

[0067] (3) Finally, 24 kg of the second intermediate was obtained with a yield of 85%.

[0068] S3, prepare the target product, the reaction formula is as follows:

[0069]

[0070] (1) The second intermediate was added to ethyl acetate (150 kg), Pd / C5% (2.4 kg) (0.1 eq) was added, nitrogen was replaced 3 times, hydrogen was replaced 3 times, the reaction was continued for 2 h, Pd / C5% (2.4 kg) was added, hydrogen was replaced 3 times, and the reaction was continued for 2 h.

[0071] (2) TLC spot plate showed that the reaction of the second intermediate of the raw material was complete. Ethyl acetate was concentrated to 114 kg, washed with 5% sodium bicarbonate (100 kg), the pH of the aqueous phase was measured to be 7-8, washed twice with saturated brine (125 kg), concentrated to a small volume (60 kg), 145 kg of methyl tert-butyl ether was added, and n-heptane (135 kg) was added for crystallization, and filtered and slurried (n-heptane: methyl tert-butyl ether = 5:1) (135 kg: 45 kg).

[0072] (3) Finally, 18.7 kg of the target product - tyrosine serine pseudo-dipeptide was obtained. The chemical name of tyrosine serine pseudo-dipeptide is (4S)-3-[(2S)-3-[4-(tert-butyloxy)phenyl]-2-[[fluorenylmethoxycarbonyl]amino]-1-oxopropyl]-2,2-dimethyl-4-oxazolidinecarboxylic acid, and its English name is Fmoc-Tyr(tBu)-Ser(psi(Me,Me)pro)-OH. The yield is 93%, HPLC = 98.8351%.

[0073] from Figure 1 It can be seen that the area percentage report is recorded as follows:

[0074]

[0075] Target product 1 H NMR spectrum Figure 2 As shown, 1 H NMR (400 MHz, DMSO) δH (ppm) = 1.13 (s, 9H), 1.37 (s, 3H), 1.55 (s, 3H), 2.84 (d, J = 2.88-2.82 Hz, 2H), 3.82 (s, 1H), 4.10 (m, J = 4.10-4.05 Hz, 5H), 4.35 (s, 1H), 6.76 (s, 2H), 7.14 (s, 2H), 7.35 (m, J = 7.40-7.33 Hz, 5H), 7.72 (s, 2H), 7.86 (s, 2H), active hydrogen is not shown.

[0076] Example 2

[0077] S1, prepare the first intermediate, the reaction formula is as follows:

[0078]

[0079] (1) Add Fmoc-O-tert-butyl-L-tyrosine (20 kg) to N,N-dimethylformamide (DMF) (140 kg) and cool to 0-5°C; add HOBT (5.88 kg) (1 eq), TBTU (14 kg) (1.1 eq), DIPEA (11.3 kg) (2 eq) in sequence, keep the temperature at 0-5°C for 30 mins, and finally add L-serine benzyl ester hydrochloride (10.1 kg) (1 eq) and naturally raise the temperature to room temperature for reaction.

[0080] (2) TLC spot plate shows that the raw material Fmoc-O-tert-butyl-L-tyrosine has been reacted. Water (360 kg) and EA (180 kg x 2) are added for extraction. The organic phases are combined and washed with 0.5N hydrochloric acid (180 kg x 2). The pH value of the aqueous phase is measured to be 1. Saturated sodium bicarbonate solution (200 kg) is added for washing. The pH value of the aqueous phase is measured to be 8. The organic phase is washed with saturated brine (200 kg). The organic phase is concentrated to a small volume, ethyl acetate is crystallized, and filtered. The filtered solid is placed in an oven and dried at 55°C.

[0081] (3) 26.0 kg of the first intermediate as a yellow-brown solid was obtained with a yield of 93.8%.

[0082] S2, prepare the second intermediate, the reaction formula is as follows:

[0083]

[0084] (1) The first intermediate obtained in Example 1 was added to toluene (160 kg), 4-methylbenzenesulfonate pyridine (0.26 kg) (0.01 eq) was added, and 2-dimethoxypropylene (5.9 kg) (2 eq) was added, and the temperature was raised to 100° C. for reaction.

[0085] (2) When the TLC plate shows that the reaction of the first intermediate of the raw material is complete, the temperature is lowered to 55°C and the toluene is concentrated in vacuo. After the concentration is complete (a small amount of toluene remains), the toluene is rinsed clean with petroleum ether in a filter cylinder (1200 mesh) (160 kg silica gel 100 mesh-200 mesh), and the product is eluted with a mixture (ethyl acetate: methanol = 20:1), and the ethyl acetate and methanol are concentrated.

[0086] (3) Finally, 24 kg of the second intermediate was obtained with a yield of 85%.

[0087] S3, prepare the target product, the reaction formula is as follows:

[0088]

[0089] (1) The second intermediate was added to ethyl acetate (150 kg), Pd / C5% (4.8 kg) (0.20 eq) was added, nitrogen was replaced 3 times, hydrogen was replaced 3 times, the reaction was continued for 2 h, Pd / C5% (4.8 kg) was added, hydrogen was replaced 3 times, and the reaction was continued for 2 h.

[0090] (2) TLC spot plate showed that the reaction of the second intermediate of the raw material was complete. Ethyl acetate was concentrated to 114 kg, washed with 5% sodium bicarbonate (100 kg), the pH of the aqueous phase was measured to be 7-8, washed twice with saturated brine (125 kg), concentrated to a small volume (60 kg), 145 kg of methyl tert-butyl ether was added, and n-heptane (135 kg) was added for crystallization, and filtered and slurried (n-heptane: methyl tert-butyl ether = 5:1) (135 kg: 45 kg).

[0091] (3) Finally, 18.7 kg of the target product (4S)-3-[(2S)-3-[4-(tert-butyloxy)phenyl]-2-[[fluorenylmethoxycarbonyl]amino]-1-oxopropyl]-2,2-dimethyl-4-oxazolidinecarboxylic acid was obtained with a yield of 93% and HPLC=98.8567%.

[0092] Target product 1 H NMR spectrum data are: 1 H NMR (400MHz, DMSO) δH (ppm)=1.13(s,9H),1.37(s,3H),1.55(s,3H),2.84(d,J=2.88-2.82 Hz,2H),3.82(s,1H), 4.10(m,J=4.10-4.05 Hz,5H),4.35(s,1H),6.76(s,2H),7.14(s,2H),7.35(m,J=7.40-7.33 Hz,5H),7.72(s,2H),7.86(s,2H).

[0093] Example 3

[0094] S1, prepare the first intermediate, the reaction formula is as follows:

[0095]

[0096] (1) Add Fmoc-O-tert-butyl-L-tyrosine (20 kg) to N,N-dimethylformamide (DMF) (160 kg) and cool to 0-5°C; add HOBT (11.8 kg) (2 eq), TBTU (35 kg) (2.5 eq), DIPEA (22.5 kg) (4 eq) in sequence, keep the temperature at 0-5°C for 30 mins, and finally add L-serine benzyl ester hydrochloride (20.2 kg) (2 eq) and naturally raise the temperature to room temperature for reaction.

[0097] (2) TLC spot plate shows that the raw material Fmoc-O-tert-butyl-L-tyrosine has been reacted. Water (360 kg) and EA (180 kg x 2) are added for extraction. The organic phases are combined and washed with 0.5N hydrochloric acid (180 kg x 2). The pH value of the aqueous phase is measured to be 1. Saturated sodium bicarbonate solution (200 kg) is added for washing. The pH value of the aqueous phase is measured to be 8. The organic phase is washed with saturated brine (200 kg). The organic phase is concentrated to a small volume, ethyl acetate is crystallized, and filtered. The filtered solid is placed in an oven and dried at 55°C.

[0098] (3) 26.4 kg of the first intermediate as a yellow-brown solid was obtained, with a yield of 95.3%.

[0099] S2, prepare the second intermediate, the reaction formula is as follows:

[0100]

[0101] (1) The first intermediate obtained in Example 1 was added to toluene (162 kg), 4-methylbenzenesulfonate pyridine (2.7 kg) (0.1 eq) was added, and 2-dimethoxypropylene (27.3 kg) (8 eq) was added, and the temperature was raised to 100° C. for reaction.

[0102] (2) When the TLC plate shows that the reaction of the first intermediate of the raw material is complete, the temperature is lowered to 55°C and the toluene is concentrated in vacuo. After the concentration is complete (a small amount of toluene remains), the toluene is rinsed clean with petroleum ether in a filter cylinder (1200 mesh) (160 kg silica gel 100 mesh-200 mesh), and the product is eluted with a mixture (ethyl acetate: methanol = 20:1), and the ethyl acetate and methanol are concentrated.

[0103] (3) Finally, 24 kg of the second intermediate was obtained with a yield of 85%.

[0104] S3, prepare the target product, the reaction formula is as follows:

[0105]

[0106] (1) Add the second intermediate to ethyl acetate (kg), add Pd / C5% (7.2kg) (0.3eq), replace the atmosphere with nitrogen 3 times, replace the atmosphere with hydrogen 3 times, react for 2h, add more Pd / C5% (7.2kg), replace the atmosphere with hydrogen 3 times, and continue the reaction for 2h.

[0107] (2) TLC spot plate showed that the reaction of the second intermediate of the raw material was complete. Ethyl acetate was concentrated to 114 kg, washed with 5% sodium bicarbonate (100 kg), the pH of the aqueous phase was measured to be 7-8, washed twice with saturated brine (125 kg), concentrated to a small volume (60 kg), 145 kg of methyl tert-butyl ether was added, and n-heptane (135 kg) was added for crystallization, and filtered and slurried (n-heptane: methyl tert-butyl ether = 5:1) (135 kg: 45 kg).

[0108] (3) Finally, 18.5 kg of the target product (4S)-3-[(2S)-3-[4-(tert-butyloxy)phenyl]-2-[[fluorenylmethoxycarbonyl]amino]-1-oxopropyl]-2,2-dimethyl-4-oxazolidinecarboxylic acid was obtained with a yield of 93% and HPLC=98.632%.

[0109] Target product 1 H NMR spectrum data are: 1 H NMR (400MHz, DMSO) δH (ppm)=1.13(s,9H),1.37(s,3H),1.55(s,3H),2.84(d,J=2.88-2.82 Hz,2H),3.82(s,1H), 4.10(m,J=4.10-4.05 Hz,5H),4.35(s,1H),6.76(s,2H),7.14(s,2H),7.35(m,J=7.40-7.33 Hz,5H),7.72(s,2H),7.86(s,2H).

[0110] Experimental Example 1

[0111] On the basis of step S1 of Example 1, Fmoc-O-tert-butyl-L-tyrosine, L-serine benzyl ester hydrochloride, DIPEA, and DMF were used as standard substrates. The condensing agent in the reaction was studied without changing the temperature and reaction time. HOBT was used as a ligand and TBTU was used as a condensing agent. The condensing agent equivalents in different comparative examples should be consistent with those in Example 1. The results are shown in the following table:

[0112]

[0113] As can be seen from the above table, Example 1 of the present invention selects specific HOBT and TBTU for mixing, which can significantly improve the yield and purity of the first intermediate, and on the basis of high purity, has no effect on the purity of the target product, and can ensure the purity of the target product.

[0114] Experimental Example 2

[0115] (1) Based on step S2 of Example 1, the catalyst in the reaction was studied with the first intermediate, toluene, 2-dimethoxypropylene and the temperature unchanged. The results are shown in the following table:

[0116]

[0117] It can be seen from the above table that when 4-methylbenzenesulfonate pyridine is selected as the catalyst in the present invention, the yield and purity of the second intermediate are significantly better than those of other catalysts.

[0118] (2) Based on step S2 of Example 1, the auxiliary materials in the reaction were studied with the first intermediate, toluene, and 4-methylbenzenesulfonate pyridine at a constant temperature. The results are shown in the following table:

[0119]

[0120] As can be seen from the above table, the present invention uses 2-dimethoxypropylene as an auxiliary material, which contains both a methoxy group and a double bond. It can not only improve the yield of the second intermediate, but also react efficiently with the first intermediate, and the residual amount of the raw material is 0.

[0121] (3) Based on step S2 of Example 1, the first intermediate, 2-dimethoxypropylene, 4-methylbenzenesulfonate pyridine, and the temperature were kept constant to study the solvent in the reaction. The results are shown in the following table:

[0122]

[0123] As can be seen from the above table, the choice of solvent in the present invention also has a significant effect on the yield of the final second intermediate. The yield of the solvents within the scope of this application can reach more than 65%, among which toluene has the best effect as the solvent, while when methanol is used as the solvent, the yield of the second intermediate is only 20%, which is significantly lower than the examples of this application.

[0124] Experimental Example 3

[0125] On the basis of step S3 of Example 1, with the second intermediate and the solvent unchanged, the equivalent of palladium carbon added in the reaction was studied at room temperature and pressure. The results are shown in the following table:

[0126]

[0127] As can be seen from the above table, the amount of palladium carbon added in the present invention also has a significant effect on the yield of the target product. When the amount of palladium carbon added is too much or too little, the yield is significantly reduced.

[0128] In summary, the preparation method of the tyrosine serine pseudo dipeptide provided by the present invention is to use Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride as raw materials, and to use cheap HOBT and TBTU as condensing agents, which can improve the condensation effect and ensure the yield and purity of the first intermediate. When preparing the second intermediate, 2-dimethoxypropylene is used as an auxiliary material, which is easy to react with the specific site of the hydroxyl group on the first intermediate. 4-methylbenzenesulfonic acid pyridine is used as a catalyst to improve the yield and stereoselectivity of the second intermediate, thereby improving the purity of the second intermediate. Finally, the second intermediate is reacted with palladium carbon, and the hydrogenation reducibility and selectivity of palladium carbon are utilized to efficiently prepare a high-purity target product. The preparation method of the present invention has mild synthesis conditions, is green and environmentally friendly, is simple to operate, is easy to mass-produce, and can obtain a product of higher purity at the same time, with a purity of ≥98.5% and a single impurity of less than 0.2%. The optical purity can control the purity of (4S)-3-[(2S)-3-[4-(tert-butyloxy)phenyl]-2-[[fluorenylmethoxycarbonyl]amino]-1-oxopropyl]-2,2-dimethyl-4-oxazolidinecarboxylic acid isomers to be less than 0.1%.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a tyrosine-serine pseudo-dipeptide, characterized in that: It includes: S1. Conducting a condensation reaction between Fmoc-O-tert-butyl-L-tyrosine and L-serine benzyl ester hydrochloride in the presence of a first solvent, a condensing agent and a base, wherein the condensing agent is HOBT and TBTU, to obtain a first intermediate; the structural formula of the Fmoc-O-tert-butyl-L-tyrosine is , the structural formula of the L-serine benzyl ester hydrochloride is: , the structural formula of the first intermediate is ; S2, subjecting the first intermediate and 2-dimethoxypropylene to a ring-closing reaction in the presence of 4-methylbenzenesulfonate pyridine and a second solvent to obtain a second intermediate; the structural formula of the second intermediate is , the second solvent is at least one of toluene, tetrahydrofuran and DMF; S3, reacting the second intermediate and palladium carbon in a third solvent to obtain a target product - tyrosine serine pseudo-dipeptide, the structural formula of the tyrosine serine pseudo-dipeptide is: .

2. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: Step S1 includes at least one of features (1) to (5): Feature (1): The molar ratio of the Fmoc-O-tert-butyl-L-tyrosine, the L-serine benzyl ester hydrochloride, the HOBT, the TBTU and the base is 1:(1-2):(1-2):(1.1-2.5):(2-4); Feature (2): The mass ratio of the Fmoc-O-tert-butyl-L-tyrosine to the first solvent is 1:(7-8); Feature (3): The first solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran and ethyl acetate; Feature (4): The base is N,N-diisopropylethylamine; Feature (5): The operating steps of the condensation reaction include: adding the Fmoc-O-tert-butyl-L-tyrosine to the first solvent, cooling to 0-5°C, then adding the HOBT, the TBTU and the base in sequence, maintaining the temperature at 0-5°C for 20-30 minutes, and finally adding the L-serine benzyl ester hydrochloride and naturally heating to room temperature for reaction.

3. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: Step S2 includes at least one of features (6) to (8): Feature (6): The molar ratio of the first intermediate, the 2-dimethoxypropylene and the 4-methylbenzenesulfonate pyridine is 1:(2-8):(0.01-0.1); Feature (7): The mass ratio of the first intermediate to the second solvent is 1:(5-7); Feature (8): The reaction temperature of the ring-closing reaction is 60-110°C.

4. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: Step S3 includes at least one of features (9) to (11): Feature (9): The molar ratio of the second intermediate to the palladium carbon is 1:(0.2-0.3); Feature (10): The mass ratio of the second intermediate to the third solvent is 1:(5-7); Feature (11): The third solvent is ethyl acetate.

5. The method for preparing the tyrosine-serine pseudo-dipeptide according to claim 1 or 4, characterized in that: The method for adding palladium carbon comprises: first adding 40-60% of the total amount of palladium carbon to the second intermediate, then replacing with nitrogen 2-4 times, replacing with hydrogen 2-4 times, reacting for 1-3 hours, adding the remaining amount of palladium carbon, replacing with hydrogen 2-4 times, and continuing to react for 1-3 hours.

6. The method for preparing the tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: In step S1, after the condensation reaction is completed and before the first intermediate is obtained, the first reaction system is extracted with water and EA, the organic phases are combined, the organic phases are acid-washed until the pH of the aqueous phase is 1, and then a sodium bicarbonate solution is added to wash until the pH of the aqueous phase is 8, and then washed with saturated brine, concentrated, and then petroleum ether and ethyl acetate are added for crystallization, and petroleum ether and ethyl acetate are continued to be added for slurrying after filtration, and the slurry is filtered and dried.

7. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: In step S2, after the ring-closing reaction is completed and before the second intermediate is obtained, the second reaction system is cooled and vacuum concentrated to remove the second solvent, followed by filtration to obtain a solid product and a filtrate, the solid product is rinsed with petroleum ether to remove the remaining second solvent, and the solid product is then eluted with petroleum ether and ethyl acetate. After the elution is completed, the eluent is concentrated to remove the petroleum ether and ethyl acetate.

8. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 7, characterized in that: The filtration is performed by using a filter cylinder, wherein the filter cylinder is provided with a filter screen with a size of 1000-1400 meshes, and the filter cylinder is also filled with silica gel with a size of 100-200 meshes.

9. The method for preparing tyrosine-serine pseudo-dipeptide according to claim 1, characterized in that: In step S3, before obtaining the target product, the third reaction system is also concentrated to remove the third solvent, followed by washing with sodium bicarbonate solution until the aqueous phase has a pH of 7-8, then washing with saturated brine, concentrating, then adding methyl tert-butyl ether and n-heptane for crystallization, and after filtration, continuing to add methyl tert-butyl ether and n-heptane for slurrying, filtering the slurry, and drying.

10. The method for preparing the tyrosine-serine pseudo-dipeptide according to claim 9, characterized in that: During crystallization and pulping, the total amount of the methyl tert-butyl ether and the n-heptane added is 2-4 times the mass of the concentrated substance after concentration, and the mass ratio of the methyl tert-butyl ether to the n-heptane is 1:(4-6).

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