CN109810169B - Liquid phase preparation method of Reltecimod - Google Patents

Liquid phase preparation method of Reltecimod Download PDF

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CN109810169B
CN109810169B CN201711158124.2A CN201711158124A CN109810169B CN 109810169 B CN109810169 B CN 109810169B CN 201711158124 A CN201711158124 A CN 201711158124A CN 109810169 B CN109810169 B CN 109810169B
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陈友金
宓鹏程
陶安进
袁建成
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Hybio Pharmaceutical Co Ltd
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Abstract

The invention relates to a liquid phase preparation method of Reltecimod, which comprises the following steps: step 1), three fully protected polypeptide fragments, R1-Ser (R) were synthesized4)‑Pro‑OH，R2-Met-Leu-Val-OH and R3‑Ala‑Tyr(R5)‑Asp(R6) -OH; step 2) coupling of R2-Met-Leu-Val-OH and R3‑Ala‑Tyr(R5)‑Asp(R6) OH to obtain R2-Met-Leu-Val-Ala-Tyr (R)5) -Asp (R6) -OH; step 3) coupling of R1-Ser (R)4) Pro and R2-Met-Leu-Val-Ala-Tyr (R)5)‑Asp(R6) -OH to obtain a fully protected polypeptide; step 4), removing a protecting group to obtain a crude product; and 5) purifying.

Description

Liquid phase preparation method of Reltecimod
Technical Field
The invention relates to the field of medicines, in particular to a preparation method of Reltecimod.
Background
Reltecimod is a drug under development by Atox Bio for the treatment of severe necrotic soft tissue infections caused by mesophilic bacteria. The Reltecimod is octapeptide composed of natural amino acids, and has the chemical name of 2- ((5S,11S,14S,17S) -5- (1- ((S) -2-amino-3-hydroxyproamino) pyrrolidine-2-carb oxyamido) -17- (4-hydroxybenzyl) -8-isobutyl-11-isoproxyl-14-methyl-6, 9,12, 15-t-etraoxo-2-thia-7, 10,13, 16-tetraazaoctadecarbamido) succininic acid and the molecular formula of C40H62N8O13S, molecular weight 895.03, its structure is as follows:
Figure BDA0001474883210000011
the transmission of carnivorous bacteria is rapid, often causing significant tissue damage and systemic disease, resulting in multiple organ dysfunction, failure and death. There is currently no standard treatment for carnivorous bacteria other than antibiotics, and relatec will provide a significant non-antibiotic treatment.
The prior published literature has no report on a synthesis method of Reltecimod, so that a process capable of realizing industrial production is necessary.
The peptide drugs are generally synthesized by a solid-phase synthesis method, which needs to adopt a solid carrier and ensures complete reaction by excessive feeding, and has higher cost and low economic value.
Disclosure of Invention
In order to solve the above technical problems, an aspect of the present invention provides a method for preparing a meltecimod, including the steps of:
step 1), three fully protected polypeptide fragments, R, were synthesized1-Ser(R4)-Pro-OH，R2-Met-Leu-Val-OH and R3-Ala-Tyr(R5)-Asp(R6)-OH；
Step 2) coupling of R2-Met-Leu-Val-OH and R3-Ala-Tyr(R5)-Asp(R6) -OH to obtain R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH；
Step 3) coupling of R1-Ser(R4) -Pro and R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) -OH to obtain R1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH；
Step 4), removing a protecting group to obtain a crude product;
step 5) purifying to obtain a pure Reltecimod product;
wherein, the steps R1, R2 and R3 are all amino protecting groups, R4 is a serine side chain protecting group, R5 is a tyrosine side chain protecting group, and R6 is an aspartic acid side chain protecting group.
In the technical scheme of the invention, R is synthesized1-Ser(R4) The method of-Pro-OH is that R is1-Ser(R4) Activation of the carboxyl group by-OH, followed by reaction with NH2The coupling is carried out in liquid phase with-Pro-OH.
In the technical scheme of the invention, R is synthesized2The method of-Met-Leu-Val-OH is that R is7-Leu-OH, then activated with NH2Liquid phase coupling of-Val-OH to obtain R7-Leu-Val-OH, removal of amino protecting group R7 to obtain NH2-Leu-Val-OH; activating R2-Met-OH by carboxyl, and then reacting with NH2Liquid phase coupling of-Leu-Val-OH to give R2-Met-Leu-Val-OH。
In the technical scheme of the invention, R is synthesized3-Ala-Tyr(R5)-Asp(R6) The method of-OH is to react R8-Tyr(R5) OH is activated by carboxyl groups and then reacted with NH2-Asp(R6) Liquid phase coupling of-OH to give R8-Tyr(R5)-Asp(R6) -OH, removal of the amino protecting group R8To obtain NH2-Tyr(R5)-Asp(R6) -OH; activating R3-Ala-OH with carboxyl, and reacting with NH2-Tyr (R)5)-Asp(R6) Liquid phase coupling of-OH to give R3-Ala-Tyr(R5)-Asp(R6)-OH。
In the technical scheme of the invention, R is synthesized2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) The process of-OH is R3-Ala-Tyr(R5)-Asp(R6) OH-removal of the amino protecting group R3Obtaining NH2-Ala-Tyr (R)5)-Asp(R6) -OH; activating the carboxyl group of R2-Met-Leu-Val-OH, and reacting with NH2-Ala-Tyr (R)5)-Asp(R6) Liquid phase coupling of-OH to give R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH。
In the technical scheme of the invention, R is synthesized1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) The process of-OH is R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) OH-removal of the amino protecting group R2To obtain NH2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) -OH; r is to be1-Ser(R4) Carboxy activation by Pro-OH followed by NH2-Met-Leu-Val-Ala-Tyr (R)5)-Asp(R6) Liquid phase coupling of-OH to give R1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH。
In the present invention, the conditions for removing the protecting group in step 4) are 50% piperidine/DCM and TFA.
In the technical scheme of the invention, R1, R2, R3, R7 and R8 are respectively and independently selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyl methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc) and trimethylsiloxycarbonyl (Teoc);
R4selected from tBu, Trt, R5Selected from tBu, R6Selected from tBu.
In the technical scheme of the invention, the carboxyl activation is carried out on a compound raw material with carboxyl by using a condensing agent, wherein the condensing agent is selected from DCC, HOBt, HOAt, EDC, HONB, HOSu, DIC and … ….
In the technical scheme of the invention, the liquid phase coupling is to couple a compound activated by carboxyl under alkaline conditions and a compound with an amino group under alkaline conditions.
In the technical scheme of the invention, the method for removing the amino protecting group is to treat 20% piperidine/DCM or 50% piperidine/DCM or treat TFA.
In the technical scheme of the invention, the method for purifying Reltecimod is to purify in a liquid chromatography mode or in a pure crystallization mode.
In step 5) of the present invention, purification was performed by HPLC under conditions of mobile phase a of 0.1% TFA and phase B of acetonitrile, gradient: 25% B-45% B.
In a specific technical scheme of the invention, the preparation method comprises the following steps:
step 1): synthesizing three full-protection fragments of Fmoc-Ser (tBu) -Pro-OH, Fmoc-Met-Leu-Val-OH and Fmoc-Ala-Tyr (tBu) -Asp (OtBu) -OH;
step 2): Fmoc-Met-Leu-Val-OH and Fmoc-Ala-Tyr (tBu) -Asp (OtBu) -OH are connected to obtain Fmoc-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH;
step 3): connecting Fmoc-Ser (tBu) -Pro-OH and Fmoc-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH to obtain Fmoc-Ser (tBu) -Pro-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH;
and 4, step 4: deprotection is carried out to obtain a Reltecimod crude product;
and 5: and (5) preparing by HPLC to obtain a pure Reltecimod product.
Figure BDA0001474883210000031
Figure BDA0001474883210000041
The method adopts a liquid phase method to synthesize 1 dipeptide fragment and 23 peptide fragments, then sequentially connects and deprotects the fragments according to the sequence from C end to N end to obtain a crude product, and the crude product is prepared by HPLC to obtain a pure product with the purity of more than 99 percent and the single impurity of less than 0.1 percent.
Advantageous effects
The invention adopts the fragments with the C-terminal amino acid with larger rigidity to synthesize, controls the fragment quality and a specific connection sequence, so that the target product has no single amino acid default and interpolated impurities and almost no isomerized impurities, and the obtained crude product can be prepared into a high-purity product only by one-step simple HPLC.
Drawings
FIG. 1 is a mass spectrum of a pure product obtained in example 12.
FIG. 2 is a HPLC chromatogram of a pure product obtained in example 12.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only, and are not intended to limit the scope of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Example 1: Fmoc-Ser (tBu) -Pro-OH Synthesis
Dissolving Fmoc-Ser (tBu) -OH (383.4g, 1mol) and HONB (197.1g, 1.1mol) in 2L DCM, slowly dropwise adding DIC (154.1g, 1.2mol) at 0-5 ℃, naturally heating the reaction solution to room temperature after dropwise adding, and then, heating the reaction solution to room temperatureStirring and reacting for more than 3 h. After the reaction mixture was warmed to room temperature, the reaction was checked every 1 hour by TLC (developing reagent: PE: EA: AcOH ═ 2:1:0.03), and when TLC showed completion of Fmoc-Ser (tBu) -OH reaction, stirring was stopped. And filtering the reaction solution, concentrating under reduced pressure to obtain a white solid, adding 2 liters of EA into the solid, stirring and washing for 0.5h, and slightly dissolving the solid. And standing the suspension at 0 +/-2 ℃ for 20-24 h. The product is filtered under reduced pressure, Et2O wash (544 ml. times.2) and vacuum drying gave 470.1g of Fmoc-Ser (tBu) -ONB as a white solid in yield: 81.6 percent and the purity is 99.54 percent.
H-Pro-OH (172.7g, 1.5mol) was dissolved in a mixed solvent of 4.5L acetonitrile-water (2:1), and the above Fmoc-Ser (tBu) -ONB (470.1g, 0.82mol) was added, and Fmoc-Ser (tBu) -ONB was dispersed in the solvent and slightly soluble. Stirring vigorously at room temperature, slowly adding DIPEA (129.2g, 1mol) dropwise, continuously reacting Fmoc-Ser (tBu) -ONB during the dropwise adding process until the solid disappears, and then stirring at room temperature for more than 2 h. After disappearance of the solid, TLC (developing reagent: PE: EA: AcOH ═ 2:1:0.03) was checked once every 1 hour, and when TLC showed completion of Fmoc-Ser (tBu) -ONB reaction, stirring was stopped. Acetonitrile in the reaction system was spin-dried, 100ml of a 10% aqueous solution of sodium carbonate was added to the residual solution, extraction was performed with ethyl acetate (1L. times.3), the aqueous layer was acidified to pH3-4 with saturated citric acid, extraction was performed with ethyl acetate (1L. times.2), the organic phases were combined, washed with saturated brine (500 ml. times.2), and dried over anhydrous sodium sulfate. Filtering, vacuum concentrating the filtrate, adding 2L Et to the residue2And O, heating to 40 ℃ for dissolution, standing to room temperature, and crystallizing for 20-24 hours at 2-8 ℃. Filtration, washing of the residue with frozen anhydrous ether for 2 times, vacuum drying to give 247g of white solid, yield: 51.5 percent and the purity is 99.67 percent.
Example 2: Boc-Leu-Val-OH Synthesis
Boc-Leu-OH (231.3g, 1mol) and HONB (197.1g, 1.1mol) were dissolved in 1L THF, DCC (227.0g, 1.1mol) was added under ice bath conditions, stirred at room temperature for 4h, TLC showed complete reaction of Boc-Leu-OH, filtered to remove solids, and the filtrate was used.
Dissolve Na with 2L deionized water2CO3(318g, 3mol), H-Val-OH (117.8g, 1mol), slowly adding the newly prepared filtrate dropwise, stirring at room temperature for 18H, TLC showing Boc-Leu-ONB reaction is complete, and concentrating the reaction solution in vacuumThe THF was condensed off, the residue was diluted with 2L of deionized water, EA extracted (1L × 2), the aqueous layer was acidified to pH3-4 with 1mol/L hydrochloric acid solution, EA extracted, the EA layers were combined, flash dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and the residue was washed with THF: EA: PE (1:2:10, V/V) was recrystallized. The crystals were collected by filtration and dried in vacuo to give 218.7g of Boc-Leu-Val-OH, yield: 66.2 percent and the purity is 99.59 percent.
Example 3: Fmoc-Met-Leu-Val-OH Synthesis
Boc-Leu-Val-OH (165.2g, 0.5mol) was dissolved in 1L trifluoroacetic acid and stirred at room temperature for 1H, TLC showed complete Boc-Leu-Val-OH reaction, TFA was concentrated off in vacuo and an oily H-Leu-Val-OH was obtained for use.
Fmoc-Met-OH (185.7g, 0.5mol) and HONB (98.6g, 0.55mol) were dissolved in 0.5L acetonitrile, DCC (227.0g, 1.1mol) was added under ice bath conditions, stirring was carried out at room temperature for 2h, TLC showed completion of Fmoc-Met-OH reaction, DCU was removed by filtration, and the resulting Fmoc-Met-ONB filtrate was used as it was.
Dissolve the H-Leu-Val-OH oil, saturated Na, with 0.5L deionized water2CO3And adjusting the pH value of the solution to 8.0-8.5, slowly dropwise adding the Fmoc-Met-ONB solution, continuously stirring at room temperature for 4 hours after dropwise adding is finished, and indicating that the Fmoc-Met-ONB reaction is complete by TLC. Concentrating THF in the solution in vacuum, supplementing 0.5L of distilled water to the residual aqueous solution, extracting with EA (0.5L multiplied by 2), adjusting the pH of the aqueous phase to 3-4 with saturated citric acid solution, extracting with EA (0.5L multiplied by 2), combining organic phases, washing with 10% citric acid (0.5L multiplied by 2), washing with saturated saline (0.5L multiplied by 2), and adding anhydrous Na2SO4Dried, concentrated in vacuo, DCM/Et2Crystallization of O gave 207.2g of a white solid in 71.0% yield and 99.75% purity.
Example 4: Fmoc-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Tyr (tBu) -OH (459.5g, 1mol) and HONB (197.1g, 1.1mol) were dissolved in THF (1L), DCC (227.0g, 1.1mol) was added under ice bath conditions, the reaction was stirred for 2h, TLC showed complete reaction of the starting materials, filtered and the filtrate was used.
H-Asp (OtBu) -OH (208.1g, 1.1mol) and Na2CO3(212g, 2mol) in 1L distilled water, adding the filtrate dropwise under stirring at room temperature, and continuing to addThe reaction was carried out at room temperature for 6h, and TLC showed complete reaction of the starting materials. Concentrating THF in the solution in vacuum, supplementing 1L of distilled water to the residual aqueous solution, EA extracting (1L multiplied by 2), adjusting the pH of the aqueous phase to 3-4 by using saturated citric acid solution, EA extracting (1L multiplied by 2), combining organic phases, washing by 10% citric acid (1L multiplied by 2), washing by using saturated saline (1L multiplied by 2), and anhydrous Na2SO4Drying, vacuum concentration, EA/n-hexane crystallization gave 425.1g of white solid, yield 67.4%, purity 99.47%.
Example 5: synthesis of H-Tyr (tBu) -Asp (OtBu) -OH
Fmoc-Tyr (tBu) -Asp (OtBu) -OH (315.4g, 0.5mol) was dissolved in 1L 20% piperidine/DMF solution and stirred at room temperature for 0.5h, TLC indicated the starting material reaction. The reaction was added to 5L 10% citric acid ice water, stirred for 5min, the solid collected by filtration, washed with water (1L × 2), and dried to give 192.0g of a white solid, yield: 94.0 percent and the purity is 99.54 percent.
Example 6 Fmoc-Ala-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Ala-OH (124.5g, 0.4mol) and HONB (78.8g, 0.44mol) were dissolved in THF (0.5L), DCC (90.8g, 0.44mol) was added under ice bath conditions, the reaction was stirred for 2h, TLC showed complete reaction of starting material, filtered and the filtrate was ready for use.
H-Tyr (tBu) -Asp (OtBu) -OH (163.4g, 0.4mol) and Na2CO3(106g, 1mol) was dissolved in 1L of distilled water, the filtrate was added dropwise with stirring at room temperature, and after completion of the addition, the reaction was continued at room temperature for 4 hours, and TLC showed completion of the reaction of the starting materials. Concentrating THF in the solution in vacuum, supplementing 1L of distilled water to the residual aqueous solution, EA extracting (1L multiplied by 2), adjusting the pH of the aqueous phase to 3-4 by using saturated citric acid solution, EA extracting (1L multiplied by 2), combining organic phases, washing by 10% citric acid (1L multiplied by 2), washing by using saturated saline (1L multiplied by 2), and anhydrous Na2SO4Drying, vacuum concentration and EA crystallization gave 227.9g of a white solid with a yield of 81.2% and a purity of 99.59%.
Example 7: H-Ala-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Ala-Tyr (tBu) -Asp (OtBu) -OH (140.4g, 0.2mol) was dissolved in 50% piperidine/DCM (300ml) and stirred at room temperature for 0.5h, TLC indicated complete reaction of starting material. The reaction solution was slowly added to 2L of frozen anhydrous ether, the solid was collected by filtration, washed with frozen anhydrous ether (200 ml. times.3), and dried to obtain 95.2g of a white solid with a yield of 95.1% and a purity of 99.48%.
Example 8: Fmoc-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Met-Leu-Val-OH (116.7g, 0.2mol) and HONB (39.4g, 0.22mol) were dissolved in THF (200ml), DCC (45.4g, 0.22mol) was added under ice bath conditions, the reaction was stirred for 2h, TLC showed complete reaction of the starting material, filtered and the filtrate was used.
H-Ala-Tyr (tBu) -Asp (OtBu) -OH (95.9g, 0.2mol) and Na2CO3(42.4g, 0.4mol) was dissolved in 200ml of distilled water, the filtrate was added dropwise with stirring at room temperature, and after completion of the addition, the reaction was continued at room temperature for 12 hours, and TLC showed completion of the reaction of the starting materials. Vacuum concentrating to remove THF, adding 200ml distilled water to obtain residual water solution, EA extracting (200ml × 3), adjusting pH of water phase to 3-4 with saturated citric acid solution, EA extracting (200L × 2), mixing organic phases, washing with water (200ml × 3), washing with saturated saline (200ml × 3), and removing anhydrous Na2SO4Drying, vacuum concentrating, DMF/H2Crystallization of O gave 142.6g of a white solid in 68.2% yield and 99.42% purity.
Example 9: H-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH (104.5g, 100mmol) was added to 500ml of 20% piperidine/DMF and stirred at room temperature for 0.5h, TLC showed complete reaction of starting material. The reaction mixture was slowly added to 2.5L of distilled water, stirred for 5min, filtered, washed with water (500 ml. times.3), and dried to obtain 78.3g of a white solid, yield 95.1%, purity 99.46%.
Example 10: Fmoc-Ser (tBu) -Pro-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH Synthesis
Fmoc-Ser (tBu) — Pro-OH (24.0g, 50mmol) and HOSu (6.33g, 55mmol) were dissolved in THF (50ml), DCC (11.3g, 55mmol) was added under ice bath conditions, the reaction was stirred at room temperature for 4h, TLC showed that the starting material was substantially reacted completely, filtered and the filtrate was ready for use.
H-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH (41.2g, 50mmol) and NaHCO3(16.8g, 200mmol) was dissolved in 100ml of deionized waterAnd 50ml of tetrahydrofuran, adding the filtrate while stirring, stirring at room temperature for 24 hours, and indicating that the raw materials are completely reacted by TLC. The solvent was concentrated off in vacuo, EA (50ml) was added to the residue, stirred for 0.5h, filtered, EA washed (50ml × 2), 10% citric acid washed (50ml × 3), water washed (50ml × 3) ether washed (50ml × 3) and dried to give 35.0g of a white solid, yield 57.6%, purity 98.9%.
Example 11: synthesis of crude Reltecimod
To a round-bottomed flask containing Fmoc-Ser (tBu) -Pro-Met-Leu-Val-Ala-Tyr (tBu) -Asp (OtBu) -OH (12.2g, 10mmol) was added a solution of 50% piperidine/DCM (100ml) and stirred at room temperature for 0.5h, HPLC showed complete reaction of starting material. The reaction solution was added to Et2O (1L), stirring for 0.5h, filtering, Et2O wash (200 ml. times.2) and dry to give a white solid.
A mixed solution of TFA (95ml) and water (5ml) was added to a round bottom flask containing the above solid, stirred at room temperature for 3h and HPLC showed complete reaction of the starting materials. The reaction solution was added to Et2In O (1L), the solid was collected by centrifugation, Et2O wash (200 ml. times.3) and vacuum dry to give 8.63g of a white solid in 98.0% yield and 95.7% purity.
Example 12: HPLC preparation of pure Reltecimod
Sample treatment: 2g of Reltecimod crude product is taken, 25% acetonitrile/water solution (200ml) is added, ultrasonic treatment is carried out to completely dissolve a sample, then a filter membrane is used for filtration, and filtrate is collected for standby.
HPLC purification conditions: the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, and the diameter and the length of the column are 50mm multiplied by 250 mm. Mobile phase a phase was 0.1% TFA and B phase acetonitrile, flow rate 80ml/min, gradient: 25% B-45% B, detection wavelength, 280 nm.
And (3) purification process: and (4) after the chromatographic column is balanced for 5min, loading, performing gradient purification, monitoring and collecting target peak fractions. The desired peak fraction was concentrated to 30ml under reduced pressure and lyophilized. Freeze-drying to obtain 1.68g of white powdery solid pure product. The purity is 99.82%, and the single impurities are all less than 0.1%. Yield 84.0%, MS:896.35(M + 1).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A preparation method of Reltecimod comprises the following steps:
step 1), three fully protected polypeptide fragments, R, were synthesized1-Ser(R4)-Pro-OH，R2-Met-Leu-Val-OH and R3-Ala-Tyr(R5)-Asp(R6)-OH；
Step 2) coupling of R2-Met-Leu-Val-OH and R3-Ala-Tyr(R5)-Asp(R6) -OH to obtain R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH；
Step 3) coupling of R1-Ser(R4) -Pro and R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) -OH to obtain R1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH；
Step 4), removing a protecting group to obtain a crude product;
step 5) purifying to obtain a pure Reltecimod product;
wherein, step R1、R2And R3Are each an amino protecting group, R4Is a serine side chain protecting group, R5Is a tyrosine side chain protecting group, R6Is an aspartic acid side chain protecting group;
synthesis of R1-Ser(R4) The method of-Pro-OH is that R is1-Ser(R4) Activation of the carboxyl group by-OH, followed by reaction with NH2-Pro-OH in liquid phase;
synthesis of R2The method of-Met-Leu-Val-OH is that R is7-Leu-OH carboxyl activation followed by NH2Liquid phase coupling of-Val-OH to give R7-Leu-Val-OH, deamino-protectionProtecting group R7To obtain NH2-Leu-Val-OH; r is to be2Activation of the carboxyl group by-Met-OH followed by NH2Liquid phase coupling of-Leu-Val-OH to give R2-Met-Leu-Val-OH；
Synthesis of R3-Ala-Tyr(R5)-Asp(R6) The method of-OH is to react R8-Tyr(R5) OH is activated by carboxyl groups and then reacted with NH2-Asp(R6) Liquid phase coupling of-OH to give R8-Tyr(R5)-Asp(R6) -OH, removal of the amino protecting group R8To obtain NH2-Tyr(R5)-Asp(R6) -OH; r is to be3Activation of the carboxyl group by Ala-OH followed by NH2-Tyr(R5)-Asp(R6) Liquid phase coupling of-OH to give R3-Ala-Tyr(R5)-Asp(R6)-OH。
2. The method of claim 1, wherein R is synthesized2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) The process of-OH is R3-Ala-Tyr(R5)-Asp(R6) OH-removal of the amino protecting group R3To obtain NH2-Ala-Tyr(R5)-Asp(R6) -OH; r is to be2Activation of the carboxyl group by-Met-Leu-Val-OH followed by reaction with NH2-Ala-Tyr (R)5)-Asp(R6) Liquid phase coupling of-OH to give R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH。
3. The method of claim 1, wherein R is synthesized1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) The process of-OH is R2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) OH-removal of the amino protecting group R2To obtain NH2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) -OH; r is to be1-Ser(R4) Activation of the carboxyl group by-Pro-OH followed by reaction with NH2-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6) Liquid phase coupling of-OH to give R1-Ser(R4)-Pro-Met-Leu-Val-Ala-Tyr(R5)-Asp(R6)-OH。
4. The method of claim 1, wherein the deprotection in step 4) is performed by reacting 50% piperidine/DCM followed by TFA.
5. The method of claim 1, wherein the carboxyl group activation is carried out by subjecting a compound having a carboxyl group to carboxyl group activation using a condensing agent selected from the group consisting of DCC, HOBt, HOAt, EDC, HONB, HOSu and DIC.
6. The method of claim 1, wherein the liquid phase coupling is performed under basic conditions by coupling a compound having an activated carboxyl group with a compound having an amino group under basic conditions.
7. The method of manufacturing Reltecimod according to claim 1, wherein the step 5) of purifying the Reltecimod is a purification by liquid chromatography or a purification by pure crystallization.
8. The method of claim 7, wherein the Reltecimod is purified by HPLC under conditions of mobile phase A of 0.1% TFA and phase B of acetonitrile, and gradient: 25% B-45% B.
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