阅读说明：本技术 醋酸戈那瑞林的制备方法 (Preparation method of gonadorelin acetate ) 是由 王林鹏 安宁 张嫣 于 2020-11-23 设计创作，主要内容包括：本发明涉及医药技术领域,尤其是涉及一种醋酸戈那瑞林的制备方法。醋酸戈那瑞林的制备方法,包括如下步骤：(a)以为起始原料,以Boc-氨基酸衍生物和pGlu为单体,逐步接肽处理,得多肽树脂(b)将所述多肽树脂进行氨解脱树脂后,再进行纯化,然后转醋酸盐处理,得到醋酸戈那瑞林；每个接肽处理的步骤中,所述单体、第一缩合剂、第二缩合剂和的摩尔比为(1.5～3)﹕(1.72～3.45)﹕(1.72～3.45)﹕1。本发明采用Boc法固相多肽合成醋酸戈那瑞林,在减少单体和缩合剂用量的情况下,兼顾降低原辅料成本和保证甚至提高产物收率。(The invention relates to the technical field of medicines, in particular to a preparation method of gonadorelin acetate. The preparation method of the gonadorelin acetate comprises the following steps: (a) to be provided with Taking Boc-amino acid derivatives and pGlu as monomers as starting materials, gradually carrying out peptide grafting treatment to obtain polypeptide resin (b), carrying out ammonolysis on the polypeptide resin to remove resin, then carrying out purification, and then carrying out transacetate treatment to obtain gonadorelin acetate; in each step of peptide-joining treatment, the monomer, the first peptideA mixture, a second condensing agent and)

1. The preparation method of gonadorelin acetate is characterized by comprising the following steps:

(a) to be provided withUsing Boc-amino acid derivative and pGlu as monomers as starting materials, and gradually carrying out peptide grafting treatment to obtain polypeptide resin

(b) Carrying out ammonolysis on the polypeptide resin, purifying, and then carrying out transacetate treatment to obtain gonadorelin acetate;

the condensing agent used in the peptide-joining treatment includes a first condensing agent selected from at least one of HOBT and HOAT and a second condensing agent including DCCI;

in each step of the peptide-joining treatment, the monomer, the first condensing agent, the second condensing agent andthe molar ratio of (1.5 to 3): 1.72 to 3.45.

2. The method according to claim 1, wherein in each step of the peptide-binding treatment, the monomer, the first condensing agent, the second condensing agent, and the second condensing agent are addedThe molar ratio of the components is 1.5: 2: 1;

preferably, the first condensing agent is HOAT and the second condensing agent is DCCI.

3. The method for producing gonadorelin acetate according to claim 1 or 2, wherein each step of the peptide-grafting treatment comprises: removing protecting groups, neutralizing free amino terminal, coupling corresponding monomers and washing after coupling; the washing treatment after coupling comprises: with DMF and CH₂Cl₂Carrying out the washing treatment;

preferably, the washing treatment after coupling comprises: washing with DMF for 2-3 times and using CH₂Cl₂Washing for 2-4 times.

4. The method for producing gonadorelin acetate according to claim 3, wherein the washing treatment after coupling of N-BOC-O-t-butyl-L-serine and Boc-L-tryptophan comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

5. The method for producing gonadorelin acetate according to claim 3, wherein the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing for 3 times;

preferably, the washing treatment after coupling to pGlu comprises: by sequential use of CH₂Cl₂Washing 3 times with DMF 3 times, CH₂Cl₂Washing for 1 time;

preferably, the washing treatment after coupling Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine comprises: sequentially washing with DMF for 2 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

6. The method for preparing gonadorelin acetate according to claim 1, wherein the ammonolysis method comprises:

(b1) the polypeptide resin is put in an alcohol solvent, shaken for 18-30 h under the closed condition of the action of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtrate is collected;

(b2) concentrating the filtrate to be dry, adding an alcohol solvent for dissolving, concentrating to be dry, repeating the operation of dissolving and concentrating to be dry for at least three times to obtain a foamy concentrate I;

(b3) dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid water solution, concentrating to be dry, adding methanol for dissolution, concentrating to be dry, repeating the operation of adding methanol for dissolution, and concentrating to be dry for at least three times to obtain a foamy concentrate II; wherein the volume ratio of the methanol to the acetone is 1: 4-5;

(b4) dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using diethyl ether for 2 times, and drying to obtain a gonadorelin crude product; wherein the volume ratio of the methanol to the acetone is 1: 1.5-2;

preferably, in step (b3), the ratio of concentrate I, methanol and acetone is 1 g: 5 mL: 20-25 mL;

preferably, in step (b4), the ratio of concentrate II to methanol to acetone is 1 g: 10mL (15-20) mL.

7. The method of claim 6, wherein the ratio of methanol to acetone in the step (b3) is 1: 5 by volume; in step (b4), the volume ratio of methanol to acetone is 1: 2;

alternatively, in step (b3), the volume ratio of methanol to acetone is 1: 4; in step (b4), the volume ratio of methanol to acetone was 1: 1.5.

8. The method of claim 6, wherein the ratio of concentrate I, methanol and acetone in step (b3) is 1 g/5 mL/20 mL; in step (b4), the ratio of concentrate II, methanol and acetone was 1 g: 10 mL: 15 mL.

9. The method for producing gonadorelin acetate according to claim 1, characterized in that the acetate is converted by a reverse phase HPLC method;

preferably, the method for transacetate comprises the following steps: diluting the purified gonadorelin intermediate with water, adsorbing the diluted gonadorelin intermediate onto an HPLC (high performance liquid chromatography) preparation column after equilibrium treatment, washing the intermediate by using an ammonium acetate buffer solution, washing the intermediate by using acetonitrile aqueous solution with the volume fraction of 3%, then eluting the intermediate by using acetonitrile aqueous solution with the volume fraction of 80%, and collecting eluent with an absorption value;

preferably, the washing volume of the ammonium acetate buffer solution is 5-5.5 times of the column volume; the washing volume of the acetonitrile water solution with the volume fraction of 3% is 10-11 times of the column volume.

10. The method for producing gonadorelin acetate according to claim 9, wherein the ammonium acetate buffer is an ammonium acetate buffer with a pH of 5.0 and a concentration of 0.1 mol/L;

preferably, the method further comprises the step of carrying out freeze-drying treatment on the substance after the transacetate treatment.

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of gonadorelin acetate.

Background

Gonadorelin, chemically known as 5' -oxidized prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide, has the following structural formula:

gonadorelin is an artificially synthesized gonadotropin releasing hormone, belongs to a peptide compound and is decapeptide. Gonadorelin can be used for diagnosing hypothalamic-pituitary-gonadal dysfunction, treating amenorrhea and hypogonadotropic hormone hyposecretion and infertility caused by multi-follicular ovary, and promoting ovulation to treat infertility caused by hypothalamic amenorrhea, primary ovarian dysfunction, etc.

In the existing methods for synthesizing gonadorelin, Chinese patent application CN105646671A discloses a method for gradient elution and purification by combining an SPE column and a reversed-phase chromatographic column to obtain high-purity gonadorelin, but a trifluoroacetic acid aqueous solution system and a phosphoric acid aqueous solution system are adopted in the method, trifluoroacetic acid is strong acid and is difficult to volatilize and prepare, phosphoric acid is difficult to remove in a subsequent organic solvent removal process, and the risk of solvent residue in the raw material medicine is increased; in the method, 0.05-0.3% acetic acid aqueous solution is adopted for acetate conversion, the acetic acid content is low, the salt conversion is not thorough, most acetic acid exists in a free state, the combination is not firm, and the acetic acid is easy to remove in the later rotary evaporation process, so that the acetic acid content of the raw material medicine is low, and the result is difficult to control and repeat. Chinese patent application CN107176975A discloses a method for synthesizing gonadorelin by a solid phase method, which has complex post-process, needs at least 3 times of freeze-drying operation, has high freeze-drying cost and long production period, needs more highly corrosive TFA, HF or TFE, and has difficulty in treating the generated waste liquid. In the step of synthesizing the crude gonadorelin peptide product by the gonadorelin peptide resin, ether is adopted for precipitation, the formed particles are fine, and centrifugation and filtration are difficult to perform.

In the prior art, in the process for synthesizing other micromolecular polypeptides similar to gonadorelin, the problems of relatively high consumption of amino acid monomers and condensing agents in the peptide splicing process, complex washing process after peptide splicing and large consumption of solvents exist, so that the raw material cost is high, and the industrial development of gonadorelin is limited to a certain extent.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method of gonadorelin acetate, which aims to solve the technical problems of high cost of raw materials for preparing the gonadorelin acetate and the like in the prior art.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the preparation method of the gonadorelin acetate comprises the following steps:

(a) to be provided withUsing Boc-amino acid derivative and pGlu as monomers as starting materials, and gradually carrying out peptide grafting treatment to obtain polypeptide resin

(b) Carrying out ammonolysis on the polypeptide resin, purifying, and then carrying out transacetate treatment to obtain gonadorelin acetate;

the condensing agent used in the peptide-joining treatment includes a first condensing agent selected from at least one of HOBT and HOAT and a second condensing agent including DCCI;

in each step of the peptide-joining treatment, the monomer, the first condensing agent, the second condensing agent andthe molar ratio of (1.5-3) to (1.72-3.45) to 1.

According to the invention, the Gorenelin acetate is synthesized by adopting Boc method solid-phase polypeptide, and the cost of raw and auxiliary materials is reduced, and the product yield is ensured and even improved under the condition of reducing the dosage of the monomer and the condensing agent.

In the preferred embodiment of the present inventionIn an embodiment, the monomer, the first condensing agent, the second condensing agent, and the third condensing agent are added to the mixture in each step of the peptide-bonding treatmentThe molar ratio of (A) to (B) is 1.5: 2: 1.

In a preferred embodiment of the present invention, the first condensing agent is HOAT. The condensing agent combination method using HOAT and DCCI is preferred.

In a specific embodiment of the invention, said Boc-amino acid derivatives comprise Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, Boc-L-tryptophan and N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine.

In practice, said Boc-amino acid derivative may also be a hydrochloride and/or a monohydrate of the respective Boc-amino acid.

In a specific embodiment of the invention, each step of the peptide-joining treatment comprises: removing protecting groups, neutralizing free amino terminal, coupling corresponding monomers and washing after coupling;

the washing treatment after coupling comprises: with DMF and CH₂Cl₂The washing treatment is performed.

In a preferred embodiment of the invention, the post-coupling washing treatment comprises: washing with DMF for 2-3 times and using CH₂Cl₂Washing for 2-4 times.

In the washing treatment conditions after coupling of the present invention, DMF and CH are used₂Cl₂The resin after coupling is washed without ethanol, so that the problems of resin shrinkage, small space, large steric hindrance and the like caused by ethanol washing are solved, washing of other reagents is not facilitated, the required washing procedures are increased, and the process is complicated. Particularly, the influence is particularly remarkable for amino acid derivative monomers having side chains.

Furthermore, by optimizing the washing conditions, the washing effectiveness can be ensured under fewer washing times, the process is simplified, and meanwhile, the yield and the purity are ensured and even improved.

In a preferred embodiment of the invention, the washing treatment after coupling of N-BOC-O-tert-butyl-L-serine and Boc-L-tryptophan respectively comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

In a preferred embodiment of the invention, the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing was carried out 3 times.

In a preferred embodiment of the invention, the washing treatment after coupling of pGlu comprises: by sequential use of CH₂Cl₂Washing 3 times with DMF 3 times, CH₂Cl₂Washing is carried out for 1 time.

In a preferred embodiment of the present invention, the washing process after coupling Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine comprises respectively: sequentially washing with DMF for 2 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

In a specific embodiment of the present invention, the method for deprotecting comprises: and (3) putting the material to be deprotected in an organic solvent containing HCl/iPrOH, stirring for 40-60 min, then draining, and washing.

In a specific embodiment of the present invention, in the deprotection method, the organic solvent is dichloromethane in the peptide grafting treatment to respectively graft Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, and Boc-L-tryptophan. Preferably, the volume ratio of the HCl/iPrOH to the dichloromethane is 1: 1. Furthermore, the concentration of HCl in the HCl/iPrOH is 9-10 mol/L.

In a specific embodiment of the present invention, in the deprotection method, the organic solvent includes dichloromethane and mercaptoethanol in the peptide-grafting treatment to N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine and pGlu, respectively. Preferably, the volume ratio of the HCl/iPrOH, the dichloromethane and the mercaptoethanol is 5: 4: 1. Furthermore, the concentration of HCl in the HCl/iPrOH is 9-10 mol/L.

In the tool of the inventionIn an embodiment, the washing treatment during the deprotection process comprises: by CH₂Cl₂And DMF. Further, CH is adopted in sequence₂Cl₂Washing for 3 times, draining, washing for 1 time by adopting DMF, and draining.

In a specific embodiment of the invention, the method of neutralizing the free amino terminus comprises: washing with dichloromethane solution containing triethylamine for 3 times, draining, and washing. Furthermore, in the dichloromethane solution containing triethylamine, the volume ratio of triethylamine to dichloromethane is 5: 95. Further, the washing treatment comprises: washing with DMF for 1 time, draining, and adding CH₂Cl₂Washing for 5-6 times until the solution is neutral, and draining.

In a particular embodiment of the invention, the method of coupling the corresponding monomers comprises: respectively dissolving a monomer and a condensing agent by using an organic solvent, mixing and activating for 5-30 min at the temperature of 0-5 ℃, then carrying out solid-liquid separation, mixing the liquid and the neutralized precursor peptide-resin, reacting for 12-24 h at the temperature of 5-10 ℃, and then pumping out the solvent.

In a specific embodiment of the invention, the method of ammonolysis comprises:

(b1) the polypeptide resin is put in an alcohol solvent, shaken for 18-30 h under the closed condition of the action of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtrate is collected;

(b2) concentrating the filtrate to be dry, adding an alcohol solvent for dissolving, concentrating to be dry, repeating the operation of dissolving and concentrating to be dry for at least three times to obtain a foamy concentrate I;

(b3) dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid water solution, concentrating to be dry, adding methanol for dissolution, concentrating to be dry, repeating the operation of adding methanol for dissolution, and concentrating to be dry for at least three times to obtain a foamy concentrate II; wherein the volume ratio of the methanol to the acetone is 1: 4-5; preferably, the ratio of the concentrate I, the methanol and the acetone is 1g to 5mL to (20-25) mL;

(b4) dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using ether for 2 times, and drying to obtain a gonadorelin crude product; wherein the volume ratio of the methanol to the acetone is 1: 1.5-2; preferably, the ratio of the concentrate II, the methanol and the acetone is 1g to 10mL to (15-20) mL.

Wherein the proportion of the concentrate I, the methanol and the acetone is that the dosage of the methanol is 5mL and the dosage of the acetone is 20-25 mL relative to 1g of the concentrate I; the proportion of the concentrate II, the methanol and the acetone is that the dosage of the methanol is 10mL and the dosage of the acetone is 15-20 mL relative to 1g of the concentrate II.

In a preferred embodiment of the present invention, the volume ratio of methanol to acetone in step (b3) is 1: 5, and the volume ratio of methanol to acetone in step (b4) is 1: 2; alternatively, in step (b3), the volume ratio of methanol to acetone is 1: 4, and in step (b4), the volume ratio of methanol to acetone is 1: 1.5.

In a preferred embodiment of the invention, the ratio of concentrate I, methanol and acetone is 1 g: 5 mL: 20 mL; the ratio of the concentrate II, methanol and acetone is 1 g: 10 mL: 15 mL.

In a specific embodiment of the invention, the method of purification comprises: CM purification and HPLC purification.

The invention reduces the impurity content by adopting a certain purification method and pre-purification, thereby reducing the impurity loading capacity in the chromatographic column, prolonging the use times of the chromatographic column such as a C18 column and simultaneously improving the product purity.

In a specific embodiment of the invention, the acetate is converted using a reverse phase HPLC method. Further, the stationary phase of the trans-acetate in the reversed phase HPLC method is octadecylsilane chemically bonded silica.

In a specific embodiment of the invention, the method of transacetate comprises: and diluting the purified gonadorelin intermediate with water, adsorbing the diluted gonadorelin intermediate onto an HPLC (high performance liquid chromatography) preparation column after equilibrium treatment, washing the intermediate by using an ammonium acetate buffer solution, washing the intermediate by using acetonitrile aqueous solution with the volume fraction of 3%, eluting the intermediate by using acetonitrile aqueous solution with the volume fraction of 80%, and collecting eluent with an absorption value.

In a specific embodiment of the present invention, the washing volume of the ammonium acetate buffer solution is 5 to 5.5 times of the column volume, preferably 5 times of the column volume; the washing volume of the 3% acetonitrile aqueous solution is 10-11 times of the column volume, preferably 10 times of the column volume.

Further, the HPLC preparative column was subjected to the equilibration treatment with 3% volume fraction acetonitrile in water.

Further, the ammonium acetate buffer solution is an ammonium acetate buffer solution with pH of 5.0 and concentration of 0.1 mol/L.

In a specific embodiment of the invention, the eluent is concentrated to dryness, then dissolved in water and concentrated to dryness, and repeated for at least 3 times to obtain concentrate III.

In the step of transacetate, acetonitrile solution with lower concentration (such as volume fraction of 3%) is adopted during balancing and washing, so that the adsorption force of target peptide on a column during sample loading can be enhanced, and loss caused by flow-through can be avoided during washing, so that the sample loading amount in a single time can be greatly increased, and the acetonitrile solution with higher concentration (such as volume fraction of 80%) is adopted during subsequent elution, thereby ensuring the elution effect. Therefore, the step of the invention reduces the sample loading times, not only ensures the sufficient salt transfer effect, but also avoids the loss caused by incomplete elution.

In the embodiment of the invention, the method further comprises the step of carrying out freeze-drying treatment on the substance after the transacetate treatment.

In practice, the freeze-drying process comprises:

pre-freezing: the temperature of a heat conducting oil inlet is less than-40 ℃, and the heat is preserved for about 2 hours;

sublimation: the temperature rise speed of the heat conducting oil is about 10 ℃/h until the oil temperature rises to 38 ℃, and the vacuum degree of the process control front box is set to be not more than 20 Pa;

and when the temperature of the heat conducting oil reaches about 38 ℃, keeping the temperature for about 18 hours until the drying end point.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the invention, through optimization of the amino acid monomer, the condensing agent, the washing process and the like, the-OH falling is reduced, the generation of side reactions is reduced, the reaction conversion rate is improved, the use amount of the amino acid monomer and the condensing agent is reduced, and the cost of raw and auxiliary materials is greatly reduced; meanwhile, the usage amount of the organic solvent and the generation of organic waste liquid are reduced by about 40 percent, and the purchase cost of the organic solvent and the treatment cost of the waste liquid are reduced;

(2) in the preparation method of the gonadorelin acetate, the washing process conditions after the coupling step of the docking peptide are improved, and the product yield is ensured and even improved under the condition of simplifying the washing process; meanwhile, the purity of the product is greatly improved by optimizing the solvent dosage in the ammonolysis step; in addition, the method further explores the conditions of the salt-removing process, and improves the sample loading amount while ensuring the yield.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following detailed description, but those skilled in the art will understand that the following described examples are some, not all, of the examples of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the present invention, the expression "N times volume" of a certain solvent is used, N refers to a positive number or a numerical range, and for example, in the specific examples, the "5 times volume" of methanol, the "5 times volume" of acetic acid aqueous solution with a volume fraction of 50%, the "10 times volume" of methanol, the "15 times volume" of acetone, the "20 times volume" of acetone, the "25 times volume" of acetone, the "50 times volume" of acetone, the "1-2 times volume" of water for injection, and the like, the representative meaning is: the amount of a certain solvent employed is N X mL (mL stands for volume unit) relative to the mass Xg (g stands for mass unit) of the corresponding solid substance.

The following description will be made by taking as an example "the foamy concentrate I is weighed, dissolved in 5 volumes of methanol, precipitated in 20 volumes of acetone, filtered, and the filter cake is dissolved in 5 volumes of an aqueous solution of acetic acid having a volume fraction of 50%" described in step (11) of example 1: the dosage of methanol is 5mL and the dosage of acetone is 20mL relative to 1g of the concentrate I; the amount of the 50% volume aqueous acetic acid solution used was 5mL per 1g of the cake.

The preparation method of the gonadorelin acetate comprises the following steps:

(a) to be provided withUsing Boc-amino acid derivative and pGlu as monomers as starting materials, and gradually carrying out peptide grafting treatment to obtain polypeptide resin

(b) Carrying out ammonolysis on the polypeptide resin, purifying, and then carrying out transacetate treatment to obtain gonadorelin acetate;

the condensing agent used in the peptide-joining treatment includes a first condensing agent selected from at least one of HOBT and HOAT and a second condensing agent including DCCI;

in each step of the peptide-joining treatment, the monomer, the first condensing agent, the second condensing agent andthe molar ratio of (1.5-3) to (1.72-3.45) to 1.

Is/are as followsRefers to polystyrene resin.

According to the invention, the Gorenelin acetate is synthesized by adopting Boc method solid-phase polypeptide, and the cost of raw and auxiliary materials is reduced, and the product yield is ensured and even improved under the condition of reducing the dosage of the monomer and the condensing agent.

In actual operation, pressIn the order of (1) in the starting materialsThe amino acid monomer structures containing Pro, Arg, Leu, Gly, Tyr, Ser, Trp, His and pGlu are sequentially connected on the polypeptide, and the raw material of each peptide connection step is precursor peptide-resin obtained after the previous step of peptide connection.

As in the different embodiments, the monomer, the first condensing agent, the second condensing agent, and the third condensing agent are added to each of the steps of the peptide bonding processThe molar ratio of (B) may be 1.5: 1.72: 1, 1.5: 12: 2: 1, 3: 3.45: 1, etc.

In a preferred embodiment of the present invention, in each step of the peptide-bonding treatment, the monomer, the first condensing agent, the second condensing agent andthe molar ratio of (A) to (B) is 1.5: 2: 1.

Wherein the content of the first and second substances,the number of moles of (b) is calculated as the number of moles of Boc-Gly bound on the resin.

As in the different embodiments, the condensing agent may be HOBT and DCCI, or HOAT and DCCI. In a preferred embodiment of the present invention, the first condensing agent is HOAT. Preferably, a condensing agent combination of HOAT and DCCI is used.

In a specific embodiment of the invention, said Boc-amino acid derivatives comprise Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, Boc-L-tryptophan and N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine.

In practice, said Boc-amino acid derivative may also be a hydrochloride and/or a monohydrate of the respective Boc-amino acid. For example, Boc-L-arginine may be Boc-L-arginine hydrochloride (Boc-Arg-HCl), and Boc-L-arginine hydrochloride monohydrate (Boc-Arg-HCl. H)₂O), Boc-L-leucine can also be Boc-L-leucine monohydrate (Boc-Leu. H)₂O)。

In a specific embodiment of the invention, each step of the peptide-joining treatment comprises: removing protecting groups, neutralizing free amino terminal, coupling corresponding monomers and washing after coupling;

the washing treatment after coupling comprises: with DMF and CH₂Cl₂The washing treatment is performed.

Wherein the coupling of the corresponding monomers is referred to asIn the order of (1) in the starting materialsAnd amino acid monomer structures containing Pro, Arg, Leu, Gly, Tyr, Ser, Trp, His and pGlu are sequentially connected on the surface of the substrate.

In a preferred embodiment of the invention, the post-coupling washing treatment comprises: washing with DMF for 2-3 times and using CH₂Cl₂Washing for 2-4 times.

In the washing treatment conditions after coupling of the present invention, DMF and CH are used₂Cl₂The resin after coupling is washed without ethanol, so that the problems of resin shrinkage, small space, large steric hindrance and the like caused by ethanol washing are solved, washing of other reagents is not facilitated, the required washing procedures are increased, and the process is complicated. Particularly, the influence is particularly remarkable for amino acid derivative monomers having side chains.

In a specific embodiment of the present invention, the amount of the solvent used in each washing in the peptide-connecting treatment step is 2000 to 2200mL/300 mmol. Specifically, the amount of the solvent used for each washing is 2000-2200 mL per 300mmol of the starting material.

Furthermore, by optimizing the washing conditions, the washing effectiveness can be ensured under fewer washing times, the process is simplified, and meanwhile, the yield and the purity are ensured and even improved.

In a preferred embodiment of the invention, the washing treatment after coupling of N-BOC-O-tert-butyl-L-serine and Boc-L-tryptophan respectively comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

In a preferred embodiment of the invention, the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: sequentially washing with DMF for 3 times, and washing with CH₂Cl₂Washing was carried out 3 times.

In a preferred embodiment of the invention, the washing treatment after coupling of pGlu comprises: by sequential use of CH₂Cl₂Washing 3 times with DMF 3 times, CH₂Cl₂Washing is carried out for 1 time.

In a preferred embodiment of the present invention, the washing process after coupling Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine comprises respectively: sequentially washing with DMF for 2 times, and washing with CH₂Cl₂Washing is carried out for 2 times.

In a specific embodiment of the present invention, the method for deprotecting comprises: and (3) putting the material to be deprotected in an organic solvent containing HCl/iPrOH, stirring for 40-60 min, then draining, and washing. Wherein, the material to be deprotected refers to precursor peptide-resin material obtained after the previous step of peptide grafting.

In a specific embodiment of the present invention, in the deprotection method, the organic solvent is dichloromethane in the peptide grafting treatment to respectively graft Boc-L-proline, Boc-L-arginine, Boc-L-leucine, Boc-glycine, Boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, and Boc-L-tryptophan. Preferably, the volume ratio of the HCl/iPrOH to the dichloromethane is 1: 1. Furthermore, the concentration of HCl in the HCl/iPrOH is 9-10 mol/L.

In a specific embodiment of the present invention, in the deprotection method, the organic solvent includes dichloromethane and mercaptoethanol in the peptide-grafting treatment to N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine and pGlu, respectively. Preferably, the volume ratio of the HCl/iPrOH, the dichloromethane and the mercaptoethanol is 5: 4: 1. Furthermore, the concentration of HCl in the HCl/iPrOH is 9-10 mol/L.

In a specific embodiment of the present invention, the washing treatment during the deprotection process comprises: by CH₂Cl₂And DMF. Further, CH is adopted in sequence₂Cl₂Washing for 3 times, draining, washing for 1 time by adopting DMF, and draining.

In a specific embodiment of the invention, the method of neutralizing the free amino terminus comprises: washing with dichloromethane solution containing triethylamine for 3 times, draining, and washing. Furthermore, in the dichloromethane solution containing triethylamine, the volume ratio of triethylamine to dichloromethane is 5: 95. Further, the washing treatment comprises: washing with DMF for 1 time, draining, and adding CH₂Cl₂Washing for 5-6 times until the solution is neutral, and draining.

In a particular embodiment of the invention, the method of coupling the corresponding monomers comprises: respectively dissolving a monomer and a condensing agent by using an organic solvent, mixing and activating for 5-30 min at the temperature of 0-5 ℃, then carrying out solid-liquid separation, mixing the liquid and the neutralized precursor peptide-resin, reacting for 12-24 h at the temperature of 5-10 ℃, and then pumping out the solvent. Further, the organic solvent includes dichloromethane.

In a particular embodiment of the invention, the starting materialsThe preparation method comprises the following steps:

(1) Boc-Gly and Cs₂CO₃Reacting in the solution to obtain Boc-Gly-Cs;

(2) Boc-Gly-Cs and chloromethyl resinsAfter reaction in solution, post-treatment to obtain

Further, in step (1), Boc-Gly and Cs₂CO₃The mass ratio of (1) to (1.15-1.25); in step (2), Boc-Gly-Cs and chloromethyl resinThe molar ratio of (1-1.5) to 1. Chloromethyl resinThe number of moles of (A) is calculated from the amount of substitution of chlorine.

In a specific embodiment of the invention, the method of ammonolysis comprises:

(b1) the polypeptide resin is put in an alcohol solvent, shaken for 18-30 h under the closed condition of the action of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtrate is collected;

(b2) concentrating the filtrate to be dry, adding an alcohol solvent for dissolving, concentrating to be dry, repeating the operation of dissolving and concentrating to be dry for at least three times to obtain a foamy concentrate I;

(b3) dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid water solution, concentrating to be dry, adding methanol for dissolution, concentrating to be dry, repeating the operation of adding methanol for dissolution, and concentrating to be dry for at least three times to obtain a foamy concentrate II; wherein the volume ratio of the methanol to the acetone is 1: 4-5; preferably, the ratio of the concentrate I, the methanol and the acetone is 1g to 5mL to (20-25) mL;

(b4) dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using ether for 2 times, and drying to obtain a gonadorelin crude product; wherein the volume ratio of the methanol to the acetone is 1: 1.5-2; preferably, the ratio of the concentrate II, the methanol and the acetone is 1g to 10mL to (15-20) mL.

In a specific embodiment of the present invention, the alcoholic solvent includes methanol.

In a preferred embodiment of the present invention, the volume ratio of methanol to acetone in step (b3) is 1: 5, and the volume ratio of methanol to acetone in step (b4) is 1: 2; alternatively, in step (b3), the volume ratio of methanol to acetone is 1: 4; in step (b4), the volume ratio of methanol to acetone is 1: 1.5.

In a preferred embodiment of the invention, the ratio of concentrate I, methanol and acetone is 1 g: 5 mL: 20 mL; the ratio of the concentrate II, methanol and acetone is 1 g: 10 mL: 15 mL.

In practice, the concentration conditions may be: concentrating under reduced pressure at 40-45 deg.C. Furthermore, the vacuum degree of the reduced pressure concentration is-0.08 MPa to-0.1 MPa.

The invention can obviously improve the purity and content of the crude product while ensuring the yield by further optimizing the proportion of the methanol and the acetone which are adopted for dissolving and precipitating in the ammonolysis process.

In a specific embodiment of the invention, the method of purification comprises: CM purification and HPLC purification.

In practice, the method of CM purification comprises: adopting CM-Sephadex C25 chromatographic column, balancing, loading, washing with washing solution until the light absorption value is less than 0.2, eluting with eluent, determining the light absorption value of the eluate at 280nm, and determining the OD of the light absorption value₂₈₀When the temperature rises to be more than or equal to 0.4, the elution effluent liquid starts to be collected, and when the temperature drops to be less than or equal to 0.4, the collection is stopped; wherein the washing solution is 0.05mol/L sodium acetate aqueous solution with pH 6.0, and the eluent is 0.5mol/L sodium acetate aqueous solution with pH 6.0. Further, the balancing process includes: the solution was washed to neutrality with purified water and then equilibrated with 0.05mol/L sodium acetate solution (buffer) having a pH of 6.0 until the effluent pH and conductivity values were substantially the same as those of the buffer. Further, dissolving the gonadorelin crude product by using 0.05mol/L sodium acetate water solution with the pH value of 6.0, passing through a 0.45 mu m membrane, collecting a sample solution and loading the sample solution; wherein the concentration of the gonadorelin crude product in the sample solution is about 1g/100 mL; the loading amount was 0.05mmol/mL of column.

In practice, the HPLC purification method comprises: passing the elution effluent collected by CM purification through a 0.45 mu m membrane, loading the elution effluent on a preparative HPLC column after equilibrium treatment, performing gradient elution, and collecting and combining elution effluent with gonadorelin intermediate purity being more than or equal to 97%; wherein, the mobile phase A of the gradient elution is an aqueous solution containing acetic acid with the volume fraction of 0.5% and acetonitrile with the volume fraction of 5%, and the mobile phase B is an aqueous solution containing acetic acid with the volume fraction of 0.5% and acetonitrile with the volume fraction of 80%, so as to ensure the separation and purification.

In a specific embodiment of the invention, the acetate is converted using a reverse phase HPLC method. Further, the stationary phase of the trans-acetate in the reversed phase HPLC method is octadecylsilane chemically bonded silica.

In a specific embodiment of the invention, the method of transacetate comprises: diluting the purified gonadorelin intermediate with water, adsorbing the diluted gonadorelin intermediate onto an HPLC (high performance liquid chromatography) preparation column after equilibrium treatment, washing the intermediate by using an ammonium acetate buffer solution, washing the intermediate by using acetonitrile aqueous solution with the volume fraction of 3%, eluting the intermediate by using acetonitrile aqueous solution with the volume fraction of 80%, and collecting eluent with an absorption value.

In a specific embodiment of the present invention, the washing volume of the ammonium acetate buffer solution is 5 to 5.5 times of the column volume, preferably 5 times of the column volume; the washing volume of the 3% acetonitrile aqueous solution is 10-11 times of the column volume, preferably 10 times of the column volume.

Further, the HPLC preparative column was subjected to the equilibration treatment with 3% volume fraction acetonitrile in water.

Further, the ammonium acetate buffer solution is an ammonium acetate buffer solution with pH of 5.0 and concentration of 0.1 mol/L.

The invention adopts ammonium acetate to convert acetate, utilizes the competitive combination mode of ammonium acetate to convert all the acetate into diacetate, and improves the combination fastness.

In a specific embodiment of the invention, the eluent is concentrated to dryness, then dissolved in water and concentrated to dryness, and repeated for at least 3 times to obtain concentrate III.

In the embodiment of the invention, the method further comprises the step of carrying out freeze-drying treatment on the substance after the transacetate treatment.

In practice, the freeze-drying process comprises:

pre-freezing: the temperature of a heat conducting oil inlet is less than-40 ℃, and the heat is preserved for about 2 hours;

sublimation: the temperature rise speed of the heat conducting oil is about 10 ℃/h until the oil temperature rises to 38 ℃, and the vacuum degree of the process control front box is set to be not more than 20 Pa;

and when the temperature of the heat conducting oil reaches about 38 ℃, keeping the temperature for about 18 hours until the drying end point.

Example 1

The embodiment provides a preparation method of gonadorelin acetate, which comprises the following steps:

(1)preparation of

Weighing 86.7g of Boc-Gly and placing the Boc-Gly in a container, and adding about 300mL of methanol for dissolving to obtain a methanol solution of the Boc-Gly; 104.7g of Cs were weighed₂CO₃Placing the mixture into another container, adding about 300mL of water to dissolve the mixture to obtain Cs₂CO₃An aqueous solution of (a); mixing Cs₂CO₃The aqueous solution is slowly added into a methanol solution of Boc-Gly, and the mixture is gently shaken by hands until the mixture is fully reacted to obtain a colorless and transparent Boc-Gly-Cs solution with the pH value of 7.0-7.5.

Decompressing, concentrating and steaming the Boc-Gly-Cs solution obtained in the step one in a water bath at the temperature of 40-50 ℃ to remove the solvent to obtain a solid; adding about 150mL of methanol into the solid, evaporating to dryness under reduced pressure, and repeating twice (the step of evaporating to dryness after adding methanol) to obtain a solid substance; placing the solid substance in P₂O₅And (4) drying in a dryer in vacuum to constant weight to obtain a Boc-Gly-Cs dried product.

③ according toCalculating the chlorine substitution amount of (A), weighing 330mmol of chloromethyl polystyrene resin(cas # 55844-94-5) in a 3L three-necked flask; dissolving the Boc-Gly-Cs dried product obtained in the step (II) into about 2100mL of DMF to obtain a DMF solution of Boc-Gly-Cs; adding a DMF solution of Boc-Gly-Cs into the three-necked flask; placing the three-necked bottle in a water bath kettle at 50 ℃, stirring at constant temperature and reacting for 48 hours; after the reaction was complete, the reaction was filtered, and the solid resin was washed 3 times with DMF (about 750mL each time) and washed with purified water to Cl free^-Reaction (AgNO)₃And (3) reagent detection: adding silver nitrate into the washed purified water, slightly shaking to obtain colorless and transparent solution, washing with ethanol for 4 times (300 mL each time), and washing with methanol for 2 times (300 mL each time); putting the washed resin into an oven to be dried at 40-50 ℃, and then putting the resin into P₂O₅Vacuum drying in a drier to constant weight to obtainAnd (5) drying the product.

(2) Linker dipeptides

Protecting a protecting group: weighing 300mmol of Boc-Gly according to mole number of Boc-Gly bonded on the resinDried product, put into a 10L reaction kettle, CH₂Cl₂Washing for 2-3 times, and draining. Adding 9-10 mol/L HCl/iPrOH to about 780mL and CH₂Cl₂About 780mL, stirring for 40min, draining, and adding CH to the solid₂Cl₂Washing for 3 times, and draining, and washing for 1 time by adopting DMF.

(ii) neutralizing the free amino terminus: adding triethylamine/CH with volume ratio of 5: 95 into the material obtained in the step (i)₂Cl₂Washing for 3 times, and draining; washing with DMF for 1 time, draining, and adding CH₂Cl₂Washing for 5-6 times until the solution is neutral, and draining.

③ coupling of the monomer Boc-Pro-OH: weighing 97g (450mmol) of Boc-Pro-OH, 81g (600mmol) of HOAT and 124g (600mmol) of DCCI, dissolving the Boc-Pro-OH, the HOAT and the DCCI respectively by adopting DMF, and then reacting for 5-30 min after uniformly mixing at 0-5 ℃; and after the reaction is finished, filtering and collecting liquid, adding the liquid into the resin material treated in the step II, sealing the bottle mouth, reacting for 18 hours at the temperature of 5-10 ℃, and draining the solvent to obtain the coupled resin material.

And (4) washing treatment after coupling: washing the coupled resin material obtained in the third step with DMF for 2 times and CH₂Cl₂Washing for 2 times, and draining to obtain

(3) Tripeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared

Step three, the coupling monomer is Boc-Arg-HCl & H₂O, weighing Boc-Arg-HCl & H₂148g (450mmol) of O, 81g (600mmol) of HOAT and 124g (600mmol) of DCCI; the monomer is not limited thereto, and 140g (450mmol) of Boc-Arg-HCl may be weighed;

step four, the final product is obtained

(4) Tetetrapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared

Step three, the coupling monomer is Boc-Leu.H₂O, weighing Boc-Leu. H₂O112g (450mmol), HOAT 81g (600mmol), DCCI 124g (600 mmol); the monomer is not limited thereto, but Boc-Leu 105g (450mmol) may be weighed;

step four, the final product is obtained

(5) Linker pentapeptide

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared

Step three, weighing 79g (450mmol) of Boc-Gly, 81g (600mmol) of HOAT and 124g (600mmol) of DCCI, wherein the coupling monomer is Boc-Gly;

step four, the final product is obtained

(6) Joining hexapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared

Step three, weighing Boc-Tyr 127g (450mmol), HOAT 81g (600mmol) and DCCI 124g (600mmol) as coupling monomers;

step four, the final product is obtained

(7) Joined heptapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared

Step three, weighing Boc-Ser (tBu) -OH 118g (450mmol), HOAT 81g (600mmol) and DCCI 124g (600mmol) as coupling monomers;

step four, washing the coupled resin material obtained in the step three by DMF for 3 timesBy using CH₂Cl₂Washing for 2 times, and draining to obtain

(8) Joined octapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared And the stirring time of the deprotection reaction is 60 min;

step three, weighing 137g (450mmol), 81g (600mmol) of HOAT and 124g (600mmol) of DCCI, wherein the coupling monomer is Boc-Trp;

step four, washing the coupled resin material obtained in the step three by DMF (dimethyl formamide) for 3 times in sequence, and adopting CH (methyl chloride)₂Cl₂Washing for 2 times, and draining to obtain

(9) Joined nonapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all preparedAnd the deprotection system is as follows: about 780mL of 9-10 mol/L HCl/iPrOH, CH₂Cl₂About 624mL and 156mL mercaptoethanol, and the stirring time of the deprotection reaction is 60 min;

step three, weighing 184g (450mmol), 81g (600mmol) of HOAT and 124g (600mmol) of DCCI, wherein the coupling monomer is Boc-His (tos);

step four, washing the coupled resin material obtained in the step three by DMF (dimethyl formamide) for 3 times in sequence, and adopting CH (methyl chloride)₂Cl₂Washing for 3 times, and draining to obtain

(10) Joined decapeptides

Reference is made to the method for conjugating dipeptides, with the difference that:

the raw materials for removing the protecting group in the step I are all prepared And the deprotection system is as follows: about 780mL of 9-10 mol/L HCl/iPrOH, CH₂Cl₂About 624mL and 156mL mercaptoethanol, and the stirring time of the deprotection reaction is 60 min;

step three, taking coupling monomer pGlu, and weighing pGlu 58g (450mmol), HOAT 81g (600mmol) and DCCI 124g (600 mmol);

step IV, sequentially adopting CH for the coupled resin material obtained in the step III₂Cl₂Washing 3 times with DMF, 3 times with CH₂Cl₂After washing for 1 time, draining. Drying the solid material in an oven at 40-50 ℃ for more than 0.5h, and then placing the dried solid material in P₂O₅Drying in drying oven for more than 12h to obtain polypeptide resin

(11) Ammonolysis

Firstly, the polypeptide resin prepared in the step (10)Placing in a cracking bottle, adding 3000mL of methanol, adding 4500mL of anhydrous liquid ammonia, sealing, shaking at room temperature for 24h, opening the cracking bottle under cooling condition, filtering, washing solid resin material with methanol for 4 times, and mixing the filtrate and wash solution;

concentrating the filtrate at 40-45 deg.c under reduced pressure to dry (vacuum degree of-0.08 MPa-0.1 MPa), dissolving in methanol, concentrating under reduced pressure to dry (40-45 deg.c, vacuum degree of-0.08 MPa-0.1 MPa), repeating the steps for three times to obtain foamed concentrate I;

thirdly, weighing the foamed concentrate I, dissolving the foamed concentrate I by 5 times of volume of methanol, precipitating the foamed concentrate I by 20 times of volume of acetone, filtering, dissolving a filter cake by using an acetic acid aqueous solution with the volume fraction of 50 percent by 5 times of volume, concentrating the filter cake at 40-45 ℃ under reduced pressure (the vacuum degree is-0.08 MPa-0.1 MPa) until the filter cake is dry, adding methanol for dissolving, concentrating the filter cake at 40-45 ℃ under reduced pressure (the vacuum degree is-0.08 MPa-0.1 MPa) until the filter cake is dry, and repeating the steps for at least three times until the foamed concentrate II is obtained.

Fourthly, weighing the foamy concentrate II, dissolving the foamy concentrate II by 10 times of methanol, precipitating the foamy concentrate II by 15 times of acetone, filtering, washing a filter cake for 1 time by acetone and 2 times by diethyl ether, and draining; and (4) drying the solid obtained by pumping in vacuum to obtain the gonadorelin crude product. And (c) recycling the filtrate generated by filtering in the step (c) and the step (c), and repeating the step (c) and the step (c) to obtain the gonadorelin crude product for recycling.

(12) Purification of CM

Selecting a column: the filler is a pretreatment chromatographic column of CM-Sephadex C25; a suitable volume of the pretreatment column is selected according to the loading amount of 0.05mmol/mL, for example, about 2000mL of the pretreatment column is selected when 100mmol of the gonadorelin crude product is charged.

② chromatographic column balancing: washing with purified water to neutrality, and balancing with 0.05mol/L sodium acetate buffer solution with pH of 6.0 until the effluent pH value and conductivity value are substantially the same as those of the buffer solution.

Loading and processing: dissolving the gonadorelin crude product obtained in the step (11) by using 0.05mol/L sodium acetate buffer solution with the pH value of 6.0, passing through a 0.45 mu m membrane, and then loading on a chromatographic column; after the sample loading, washing with a washing solution (0.05 mol/L sodium acetate buffer solution with pH 6.0) until the absorbance is less than 0.2, eluting with an eluent (0.5 mol/L sodium acetate aqueous solution with pH 6.0), measuring the absorbance of the eluate at 280nm, and determining the absorbance OD (OD) when the absorbance is less than₂₈₀When the temperature rises to be more than or equal to 0.4, the elution effluent liquid starts to be collected, and when the temperature drops to be less than or equal to 0.4, the collection is stopped.

(13) HPLC purification

Column balancing: the preparative HPLC column (4L reverse phase C18 liquid chromatography column) was equilibrated with mobile phase A at a flow rate of 400mL/min to baseline stability for future use.

Sample loading: and (4) passing the elution effluent collected in the step (12) through a 0.45-micron membrane, and carrying out balance treatment on the elution effluent in the step (i).

③ gradient elution: gradient elution is carried out by adopting a mobile phase A and a mobile phase B; the mobile phase A is an aqueous solution containing 0.5% by volume of acetic acid and 5% by volume of acetonitrile, and the mobile phase B is an aqueous solution containing 0.5% by volume of acetic acid and 80% by volume of acetonitrile.

And fourthly, collecting: and (3) collecting main peak solutions in sections, detecting by an HPLC method, determining that the part with the purity of more than or equal to 97.0% is qualified, recovering the unqualified part, purifying by HPLC again, combining all the parts with the purity of more than or equal to 97.0%, and uniformly mixing to obtain the gonadorelin intermediate.

(14) Salt conversion-concentration

Column balancing: preparative HPLC columns (4L reverse phase C18 liquid chromatography column) were equilibrated with 3% volume fraction acetonitrile in water until the baseline stabilized for use.

Diluting the gonadorelin intermediate obtained in the step (13) by 1-2 times of volume of injection water, adsorbing the diluted gonadorelin intermediate to the HPLC preparation column after the balance treatment in the step (I), washing the gonadorelin intermediate by 5 times of column volume by using ammonium acetate buffer solution with pH of 5.0 and 0.1mol/L, washing the gonadorelin intermediate by using acetonitrile aqueous solution with the volume fraction of 3% for 10 times of column volume, then eluting the gonadorelin intermediate by using acetonitrile aqueous solution with the volume fraction of 80%, and collecting eluent with an absorption value; concentrating the collected eluent under reduced pressure (vacuum degree of-0.08 MPa-0.1 MPa) at 40-45 ℃ to dryness, then adding a proper amount of water for injection to dissolve the eluent, concentrating the eluent again to dryness, and repeating the concentration for at least 3 times to obtain the concentrate.

(15) And (3) freeze-drying treatment: dissolving the concentrate obtained in step (14) with an appropriate amount of water for injection, filtering through two series-connected 0.22 μm filters, and filtering into a sterilized container under partial A-stage laminar flow. Finally controlling the concentration of the filtered collection liquid to be 8% -10%; pouring the filtered and collected liquid into a sterilized freeze-drying tray, freeze-drying in a sterilized freeze-drying box, and taking out of the box to obtain sterile gonadorelin acetate raw material medicine (pharmaceutical grade);

the freeze-drying process conditions are as follows: pre-freezing-heat conducting oil inlet temperature is less than-40 ℃, and heat preservation is carried out for about 2 hours; the temperature rising speed of the sublimation-heat conduction oil is about 10 ℃/h until the oil temperature rises to 38 ℃, and the vacuum degree of the process control front box is not more than 20 Pa; and when the temperature of the heat conducting oil reaches about 38 ℃, keeping the temperature for about 18h to the end point of drying.

Example 2

This example refers to the preparation of example 1, with the only difference that: in the process of coupling a dipeptide to a decapeptide, the respective coupling monomers, HOAT, DCCI and starting materialsThe molar ratio of the dried products is different; the method comprises the following specific steps:

starting materialsDried product300mmol；

And (3) dipeptide grafting: coupling monomers Boc-Pro-OH 193.5g (900mmol), HOAT 139.7g (1035mmol), DCCI 213.0g (1035 mmol);

tripeptide grafting: coupling monomer Boc-Arg-HCl. H₂O 295.5g(900mmol)、HOAT 139.7g(1035mmol)、DCCI 213.0g(1035mmol)；

Grafting tetrapeptide: coupling of monomeric Boc-Leu. H₂O 225.0g(900mmol)、HOAT 139.7g(1035mmol)、DCCI 213.0g(1035mmol)；

Connecting pentapeptide: coupling monomers Boc-Gly 157.5g (900mmol), HOAT 139.7g (1035mmol), DCCI 213.0g (1035 mmol);

grafting hexapeptide: 253.5g (900mmol) of coupling monomers Boc-Tyr, 139.7g (1035mmol) of HOAT and 213.0g (1035mmol) of DCCI;

grafting a heptapeptide: coupling monomers Boc-Ser (tBu) -OH 235.5g (900mmol), HOAT 139.7g (1035mmol), DCCI 213.0g (1035 mmol);

and (3) adding octapeptide: 274.5g (900mmol) of coupling monomer Boc-Trp, 139.7g (1035mmol) of HOAT and 213.0g (1035mmol) of DCCI;

connecting nonapeptide: coupling monomers Boc-His (tos)369.0g (900mmol), HOAT 139.7g (1035mmol), DCCI 213.0g (1035 mmol);

connecting decapeptide: coupling monomers pGlu 115.5g (900mmol), HOAT 139.7g (1035mmol) and DCCI 213.0g (1035 mmol).

Example 3

This example refers to the preparation of example 1, with the only difference that: in the process of coupling a dipeptide to a decapeptide, the respective coupling monomers, HOAT, DCCI and starting materialsThe molar ratio of the dried products is different; the method comprises the following specific steps:

starting materialsDried product300mmol；

And (3) dipeptide grafting: coupling monomers Boc-Pro-OH 97g (450mmol), HOAT 70g (517mmol), DCCI 107g (517 mmol);

tripeptide grafting: coupling monomer Boc-Arg-HCl. H₂O 148g(450mmol)、HOAT 70g(517mmol)、DCCI 107g(517mmol)；

Grafting tetrapeptide: coupling of monomeric Boc-Leu. H₂O 112g(450mmol)、HOAT 70g(517mmol)、DCCI 107g(517mmol)；

Connecting pentapeptide: coupling monomers Boc-Gly 79g (450mmol), HOAT 70g (517mmol) and DCCI 107g (517 mmol);

grafting hexapeptide: coupling monomers Boc-Tyr 127g (900mmol), HOAT 70g (517mmol), DCCI 107g (517 mmol);

grafting a heptapeptide: coupling monomers Boc-Ser (tBu) -OH 118g (450mmol), HOAT 70g (517mmol), DCCI 107g (517 mmol);

and (3) adding octapeptide: coupling monomers Boc-Trp 137g (450mmol), HOAT 70g (517mmol), DCCI 107g (517 mmol);

connecting nonapeptide: coupling of monomeric Boc-His (tos)184g (450mmol), HOAT 70g (517mmol), DCCI 107g (517 mmol);

connecting decapeptide: coupling monomers pGlu 58g (450mmol), HOAT 70g (517mmol) and DCCI 107g (517 mmol).

Example 4

This example refers to the preparation of example 2, with the only difference that:

the condensing agent is HOBT and DCCI, and each peptide connecting step is carried out according to monomer, HOBT, DCCI and DCCIThe molar ratio of the raw materials is 3: 3.45: 1;

the washing conditions after coupling in each peptide-joining treatment were different, specifically as follows:

the washing treatments after coupling of dipeptide to nonapeptide were all: by sequential use of CH₂Cl₂Washing 3 times, washing 4 times with absolute ethanol, washing 2 times with DMF, CH₂Cl₂Washing for 3 times, and draining;

the washing treatments after coupling of the decapeptide were: by sequential use of CH₂Cl₂Washing 3 times, 4 times with absolute ethanol, 2 times with DMF, CH₂Cl₂Washing for 2-3 times, washing for 4-5 times with methanol, and draining.

Example 5

This example refers to the preparation of example 1, with the only difference that:

in the step (11), the ammonolysis step (c) is carried out by dissolving 5 times of methanol and 25 times of acetone, and the rest operations are the same;

in the step (11), the same procedure is used for dissolving 10 times of methanol and precipitating 20 times of acetone in the step (11).

Example 6

This example refers to the preparation of example 1, with the only difference that:

in the step (11), the ammonolysis step (c) is carried out by dissolving 5 times of methanol and 50 times of acetone, and the rest operations are the same;

in the step (11), the same procedure is used except that in the step (11), methanol is dissolved in an amount of 10 times the volume of the mixture, and acetone is precipitated in an amount of 50 times the volume of the mixture.

Example 7

This example refers to the preparation of example 1, with the only difference that:

(14) salt conversion-concentration

Column balancing: preparative HPLC columns (4L reverse phase C18 liquid chromatography column) were equilibrated with an aqueous solution containing 0.5% by volume acetic acid and 5% by volume acetonitrile until the baseline stabilized for use.

Diluting the gonadorelin intermediate obtained in the step (13) with 1-2 times volume of injection water, adsorbing the diluted gonadorelin intermediate to an HPLC (high performance liquid chromatography) preparation column subjected to balance treatment in the step (I), washing the gonadorelin intermediate by using an ammonium acetate buffer solution with pH of 5.0 and 0.1mol/L, washing the gonadorelin intermediate by using an aqueous solution containing 0.5% by volume of acetic acid and 5% by volume of acetonitrile, eluting the gonadorelin intermediate by using an aqueous solution containing 0.5% by volume of acetic acid and 40% by volume of acetonitrile, and collecting an eluent with an absorption value; concentrating the collected eluent under reduced pressure (vacuum degree of-0.08 MPa-0.1 MPa) at 40-45 ℃ to dryness, then adding a proper amount of water for injection to dissolve the eluent, concentrating the eluent again to dryness, and repeating the concentration for at least 3 times to obtain the concentrate.

Example 8

This example refers to the preparation of example 1, with the only difference that:

the condensing agent is HOAT and DCCI, and each peptide connecting step is carried out according to monomer, HOBT, DCCI and DCCIThe molar ratio of the raw materials is 1.5: 2: 1;

the washing conditions after coupling in each peptide-joining treatment were different, specifically as follows:

the washing treatments after coupling of dipeptide to nonapeptide were all: by sequential use of CH₂Cl₂Washing 3 times, washing 4 times with absolute ethanol, washing 2 times with DMF, CH₂Cl₂Washing for 3 times, and draining;

the washing treatments after coupling of the decapeptide were: by sequential use of CH₂Cl₂Washing 3 times, 4 times with absolute ethanol, 2 times with DMF, CH₂Cl₂Washing for 2-3 times, washing for 4-5 times with methanol, and draining.

Experimental example 1

In order to compare and illustrate the process differences of the preparation method of gonadorelin acetate, the yield, purity and the like of the preparation method of gonadorelin acetate of different examples are tested from various aspects. The test results are shown in tables 1 to 3.

TABLE 1 Effect of the amount of condensing agent and monomer in the respective peptide-grafting step on the polypeptide resin obtained after decapeptide grafting

As can be seen from the peptide grafting process conditions of examples 1 to 4, when HOBT is used as the first condensing agent and the washing treatment conditions are not optimized, the amounts of the coupling monomer, the first condensing agent and the second condensing agent are large, the cost is high, and the yield of the polypeptide resin is low [ i.e., (800g/900g) × 100% ═ 88.9%](ii) a The invention discovers through a large number of creative experiments that when HOAT is selected as the first condensing agent, the use amount of the monomer can be reduced, reaction impurities are reduced, and on the basis, the specific washing treatment conditions of the invention are further adopted, so that the reaction efficiency is improved, the yield of the polypeptide resin can be obviously improved, and the use amounts of the coupling monomer, the first condensing agent and the second condensing agent are reduced to 1mol per unitThe yield was 96.3% or more [ i.e., (867g/900 g). times.100%: 96.3%; or more ] using only 1.5mol of the coupling monomer, 2mol of the first condensing agent, and 2mol of the second condensing agent]. Therefore, by adopting the specific condensing agent and the washing treatment conditions after coupling, the yield of the polypeptide resin can be improved under the condition of obviously reducing the consumption of the coupling monomer and the condensing agent, the cost of raw and auxiliary materials and the cost of a solvent used for washing are greatly reduced, and the process is simplified.

TABLE 2 influence of the particular process of the ammonolysis step on the purity, content and yield of the gonadorelin crude product

Wherein, the purity of the crude product is: detecting the crude product by HPLC, wherein the ratio of the peak area of the target peptide to the total peak area (the sum of all the peaks in the HPLC) in the obtained HPLC map is the purity of the crude product (in the HPLC detection process, not all substances are detected and appear peaks); the purity of the crude product mainly reflects the relationship between the target peptide and related impurities (such as other peptide chains of non-target peptides and the like);

crude product content: the ratio of the mass of the target peptide in the crude product to the total mass of the crude product; the content of the crude product mainly reflects the content of the target peptide in the crude product.

As can be seen from the ammonolysis process conditions of example 1, example 5 and example 6, the specific ammonolysis process dissolution precipitation conditions are adopted, so that the yield is ensured, the purity of the crude product is remarkably improved, and the use amount of organic solvents (methanol and acetone) is relatively less.

TABLE 3 Effect of the specific Process for the salt-transfer-concentration step on the Single Loading

Numbering	Single sample application amount
		Example 1	About 90g
Example 7	About 30g

From the process conditions of the trans-salting-concentrating process of examples 1 and 7, it can be seen that the present invention can avoid the loss due to the flow-through during the loading and washing processes, increase the loading amount, and improve the treatment efficiency by improving the process conditions of the trans-salting-concentrating step.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.