CN109187804B

CN109187804B - Method for preparing octreotide acetate

Info

Publication number: CN109187804B
Application number: CN201811215470.4A
Authority: CN
Inventors: 王敬; 江威; 姚波; 张莲莲; 余茹; 吕吉; 赵东明
Original assignee: CHENGDU TIANTAISHAN PHARMACEUTICAL CO LTD
Current assignee: CHENGDU TIANTAISHAN PHARMACEUTICAL CO LTD
Priority date: 2018-10-18
Filing date: 2018-10-18
Publication date: 2021-12-24
Anticipated expiration: 2038-10-18
Also published as: CN109187804A

Abstract

The invention relates to a method for preparing octreotide acetate. Specifically, one aspect of the present invention relates to a method for preparing octreotide acetate, comprising the steps of: using chloromethyl resin as a starting material, preparing cesium salt from Boc-Thr (tBu) -OH, sequentially connecting amino acids with protective groups according to a solid-phase synthesis method to obtain protected octapeptide resin, sequentially removing the Boc-protective groups by HCl/isopropanol, and carrying out a peptide-connecting reaction by using a condensing agent; reducing by using palladium carbon/hydrogen, and simultaneously cutting off a peptide chain to obtain reduced octreotide; under the acidic condition, the reduced octreotide reacts with an oxidant and an auxiliary agent to generate a crude octreotide product with disulfide bond cyclization; separating and purifying the crude octreotide product by a C18 column to obtain a refined octreotide acetate product. Also relates to octreotide acetate injection pharmaceutical compositions and a quality detection method thereof. The method of the present invention exhibits excellent technical effects such as high production efficiency, high yield, and less impurities, compared with the existing methods.

Description

Method for preparing octreotide acetate

Technical Field

The invention belongs to the technical field of medicines, and particularly discloses a preparation method of octreotide acetate and an injection pharmaceutical composition of the octreotide acetate. The octreotide acetate and the octreotide acetate injection pharmaceutical composition prepared by the invention have excellent properties, for example, the method for preparing octreotide acetate can enable the content of oxidative degradation impurities in the product to be lower, and the preparation efficiency of the method is remarkably better.

Background

Octreotide Acetate (Octreotide Acetate) is a synthetic polypeptide consisting of eight amino acids, with the chemical name: D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N- [ (1R,2R) -2-hydroxy-1- (hydroxymethyl) propyl ] -L-cysteinamide ring (2 → 7) -disulfide acetate, whose english chemical name is: l-cysteine amide, D-phenylallyl-L-phenylallyl-D-tryptophyl-L-lysyl-L-threonyl-N- [2-hydroxy-1- (hydroxymethyl) propyl ] -, cyclic (2 → 7) -disulphide; [ R- (R, R) ] acetate salt, CAS: 83150-76-9, the chemical structural formula, molecular formula and molecular weight are respectively:

or, the chemical structural formula is:

since the isolation of Somatostatin (ST) from the hypothalamus of sheep by Brazeau et al in 1973, researchers have conducted extensive studies on the production, distribution and biological activity of ST and determined its primary structure.

With the development of radioimmunoassay and immunocytochemistry, ST family hormones were found to be widely distributed in vivo. Such as central nervous system, hypothalamus, peripheral nervous system, islet D cells, epithelial glandular epithelium of gastrointestinal tract, axonal cells, etc. In the anterior pituitary, ST inhibits the release of growth hormone and thyroid stimulating hormone. In the pancreas, ST inhibits secretion of pancreatic islets and glucagon. ST has a wide range of effects on the gastrointestinal system, inhibiting the release of hormones such as gastrin, secretin, vasoactive intestinal peptide and other hormones, inhibiting the secretion of gastric acid, pepsin, reducing visceral blood flow and intestinal activity, reducing carbohydrate absorption, increasing the absorption of water and electrolytes by the large intestine. ST also has a cytoprotective effect. And are therefore believed to be useful in the treatment of certain gastrointestinal and surgical disorders. However, the half-life period of ST in vivo is very short, only 2-3min, continuous intravenous injection is needed, ST has no organ specificity, excessive secretion of growth hormone and the like is easily caused after drug withdrawal, and long-term application of ST can cause intestinal malabsorption and glucose tolerance reduction. The clinical use of ST has been limited.

In 1982, Sandoz pharmaceutical factory, Switzerland (now Novartis Co.) synthesized a new generation of long-acting somatostatin analogue Octreotide acetate (Octreotide, Sandostatin, SMS201-995, hereinafter abbreviated as Octreotide). It is a peptide chain similar to ST with 6 amino acids removed. It can be combined with ST receptors widely existing in central nervous system, pituitary and pancreatic beta cells to produce biological effect, and has stronger ability of inhibiting secretion of Growth Hormone (GH), insulin, glucagon and gastric acid than ST, and higher specificity. Meanwhile, 1, 4-position L-amino acid is respectively replaced by corresponding D-amino acid, 8-position is amino L alcohol, so that octreotide is not easy to be rapidly hydrolyzed by protease, the half-life period in vivo is prolonged, t1/2 is 80-160min, and continuous intravenous injection is not needed when the octreotide is used.

Therefore, octreotide has the following characteristics in addition to the inhibitory function of ST analogs:

(1) the inhibitory effect on endocrine is stronger, and the inhibitory effect on growth hormone, insulin and insulin is 45 times, 11 times and 1.3 times of ST respectively.

(2) The half-life period is long: the half-life of subcutaneous injection is 2 h.

(3) After stopping taking the medicine, rebound high secretion does not occur.

Therefore, octreotide has been used for the treatment of various diseases in recent years with relatively reduced side effects, and has been widely used for the treatment of diseases such as digestive tract tumors, upper gastrointestinal hemorrhage, acute pancreatitis, metastatic carcinoid, acromegaly, and the like.

Octreotide was first marketed in New Zealand in 1988 in the form of injections and in 1989 in the United states approved by the FDA. The octreotide acetate injection can be clinically used for intravenous injection, subcutaneous injection and intravenous drip (the drip administration time can reach more than 12 hours), the storage mode is 2-8 ℃, the octreotide acetate injection can be stored in the dark and can be stored for 14 days at room temperature (20-30 ℃), and therefore, the stability of the octreotide acetate solution is an important measurement standard for the quality of the octreotide acetate solution.

There are several publications disclosing methods for the preparation of octreotide acetate. For example,

CN103965291A (patent application No. 201410228795.1) discloses a method for preparing octreotide and octreotide acetate. The preparation method of the octreotide comprises the following steps: performing reversed-phase purification and reversed-phase desalting on the octreotide crude product solution by high performance liquid reversed-phase chromatography in sequence; the filler of the high performance liquid phase reverse phase chromatography is styrene-divinylbenzene (PS-DVB) copolymer. The invention combines reversed-phase purification and reversed-phase desalination, designs the latest application of the polymer filler styrene-divinylbenzene, and can prepare octreotide and octreotide acetate in large batch.

CN103965291A (patent application No. 201010165270.X) discloses a method for preparing octreotide by solid phase synthesis, comprising the following steps: (1) connecting the fluorenylmethyloxycarbonyl-O-tert-butylthrenol to a resin serving as a carrier to obtain the fluorenylmethyloxycarbonyl-O-tert-butylthrenol resin; (2) according to the Fmoc/tBu solid-phase polypeptide synthesis method, according to the sequence of octreotide, protective amino acids are coupled to the resin obtained in the step (1) one by one according to the sequence from C end to N end to obtain octreotide octapeptide resin; (3) cleaving octapeptide from the octreotide octapeptide resin using a cleavage agent to obtain linear crude peptide; (4) and (3) oxidizing the linear crude peptide obtained in the step (3) to form a disulfide bond, and purifying by preparative high performance liquid chromatography to obtain the octreotide, wherein the method comprises the following steps: in the step (4), the oxidation reaction is carried out in the presence of an oxidant H2O2 and the pH value is 7.0-7.5, wherein the feeding molar ratio of H2O2 to the linear crude peptide is 7-9: 1. According to the invention, the linear crude peptide is oxidized by hydrogen peroxide to obtain octreotide, the oxidation reaction is completed in 1-3 hours, compared with the traditional air oxidation method, the reaction time is greatly shortened, the yield of octreotide products is greatly improved, and the method is suitable for large-scale production of octreotide and salts thereof.

CN103102390A (patent application No. 201110352635.4) discloses a method for preparing octreotide, which is characterized by comprising the following steps: step 1: obtaining a dipeptide with an amino acid sequence of D-Phe-Cys; synthesizing hexapeptide resin by using resin as a carrier according to a solid phase method; the amino acid residue sequence in the hexapeptide resin is Phe-D-Trp-Lys-Thr-Cys-Thr-ol; under alkaline conditions, taking the dipeptide and the hexapeptide resin to carry out condensation reaction, and cutting the peptide to prepare linear octapeptide; the amino acid residue sequence of the linear octapeptide is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol; step 2: dissolving the linear octapeptide in an excessive N, N-dimethylformamide aqueous solution, adjusting the pH value to 6.8-8.0, adding an excessive oxidant, cyclizing at 18-30 ℃ for 2-3 h, adjusting the pH value to 3.5-5.0, and purifying to obtain octreotide with a structure shown in a formula I; wherein Thr-ol is threoninol, Cys is cysteine, Thr is threonine, Lys is lysine, D-Trp is D-form tryptophan, Phe is phenylalanine, and D-Phe is D-form phenylalanine. It is believed that the invention improves the yield and purity of octreotide.

CN1355173A (patent application No. 00134258.4) discloses a process for preparing octreotide acetate, which comprises: a) reacting the tripeptide R1-D-Phe-Cys (R2) -Phe-OH of the formula with the pentapeptide R3-D-Trp-Lys (R4) -Thr (R5) -Cys (R2) -NH-CH (CH2-OR6) -CH (OR5) -CH3 of the formula to give the octapeptide R1-D-Phe-Cys (R2) -Phe-D-Trp-Lys (R4) -Thr (R5) -Cys (R2) -NH-CH (CH2-OR6) -CH (OR5) -CH3 of the tripeptides, pentapeptides and octapeptides mentioned above, R1 ═ BOC; r2 ═ Bzl, 4-Me-Bzl, or 4-MeO-Bzl; r3 ═ Fmoc; r4 ═ Z or 2-Cl-Z; r5 ═ Bzl; r6 ═ CH3-, CH3CH2-, CH3CH2CH2-, or CH3CH2CH 2-. b) Removing all side chain protecting groups on the octapeptide obtained in the step a) by using anhydrous hydrogen fluoride to obtain reduced octreotide with the following formula, D-Phe-Cys-Phe-D-Try-Lys-Thr-Cys-Thr-Ol; c) stirring the aqueous solution of the octapeptide obtained in the step b) in the air for natural oxidation to obtain octreotide, and regulating the pH value of the octreotide aqueous solution to 7.0-8.0 by using an alkaline aqueous solution after purification; d) injecting octreotide solution with the pH value of 7.0-8.0 in c) into a nonpolar column, eluting with acetonitrile aqueous solution, mixing eluates, adding a proper amount of glacial acetic acid to obtain octreotide acetate, and if necessary, freeze-drying the octreotide acetate to obtain octreotide acetate dry powder.

CN1490330A (patent application No. 02137521.6) discloses a process for producing octreotide acetate, which is characterized in that: putting Boc-Thr (Ac) -Wang resin into a reaction vessel, adding mixed liquor of TFA and dichloromethane for reaction, adding triethylamine and mixed liquor of dichloromethane for reaction after washing, adding Fmoc-Cys (Trt) -OH, TBTU and HOBt, dissolving by using a peptide connecting reagent, detecting amino positive by using a Kan method, detecting amino negative by using a peptide connecting reagent after the Kan method detects amino positive, adding a head sealing reagent, a cap removing reagent, washing, adding Fmoc-Thr (tBu) -OH, TBTU, HOBt, adding oc-Lys (Boc) -OH, TBTU, HOBt, dissolving, adding Fmoc-D-Trp (Boc) -OH, TBTU, HOBt, dissolving by using a peptide connecting reagent, adding Fmoc-Phe-OH, TBTU and HOBt, adding the reaction vessel, washing and draining, adding Fmoc-Cys (Trt) -OH, TBTU, HOBt, Fmoc-Phe-OH, TBTU and HOBt, drying to obtain octapeptide resin, adding side chain removing reagent, ethanol, sodium borohydride and ethanol solution into the octapeptide resin, washing with ethanol, adding acetic acid and dropwise adding NHOH to a pH value of 7.8, adjusting the pH value to 5.5 with glacial acetic acid, purifying the filtrate in batches, and freeze-drying to obtain a white loose blocky finished product. The method has the advantages that sodium borohydride is used for reducing and replacing expensive L-Thr (ol), so that the cost for producing and synthesizing octreotide acetate can be greatly reduced.

CN1569890A (patent application No. 200410010833.2) discloses a solid phase synthesis method of octreotide acetate, which comprises the following steps: synthesizing octapeptide; preparing octapeptide into an aqueous solution, and naturally oxidizing the aqueous solution in the air to prepare octreotide; adding glacial acetic acid into octreotide aqueous solution, and freeze-drying to obtain octreotide acetate freeze-dried powder; characterized in that the process for synthesizing the octapeptide comprises the following steps: the method comprises the steps of reacting fluorenylmethyloxycarbonyl-threitol with p-carboxybenzaldehyde to generate fluorenylmethyloxycarbonyl-threitol p-carboxybenzaldehyde, bonding the fluorenylmethyloxycarbonyl-threitol p-carboxybenzaldehyde on a polymer resin carrier, sequentially bonding the fluorenylmethyloxycarbonyl-threitol p-carboxybenzaldehyde with protective amino acid residues to obtain octapeptide resin, and cutting the octapeptide from the resin. The synthesis process is simple, easy to operate, short in process time, small in reaction ratio, raw material-saving, low in cost, high in yield and purity, less in side reaction and by-product, convenient to purify and beneficial to mass production.

CN1810829A (patent application No. 200510002874.1) discloses a method for preparing octreotide acetate, which comprises the following steps: 1) synthesizing a linear octapeptide with an amino acid residue sequence of D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr by using a solid phase synthesis method; 2) oxidizing the linear octapeptide to obtain oxidized octreotide; 3) and (3) performing salt conversion and purification on the oxidized octreotide in an acetate solution to obtain octreotide acetate. According to the preparation method of octreotide acetate, DMF, HOBT/HBTU and DIEA are all adopted for peptide-joining reaction, so that the preparation method has the advantages of simplicity and convenience in operation and few side reactions, the minimum protection strategy can be achieved by the method, and a large amount of synthesis can be carried out. The method has the advantages of low cost and high yield, and has wide application prospect in the industrial production of octreotide acetate.

CN1923849A (patent application No. 200510029221.2) discloses a preparation method of octreotide by solid phase polypeptide synthesis, which is characterized by comprising the following steps: taking 2-chloro-trityl resin, trityl resin as resin and 4-methyl trityl resin or 4-methoxy trityl resin as starting materials, sequentially connecting amino acids with protecting groups according to a solid-phase synthesis method to obtain protected octapeptide resin, sequentially removing Fmoc-protecting groups, carrying out a peptide-joining reaction by using TBTU or HBTU and HOBT as condensing agents to obtain protected reduced octapeptide resin, synchronously removing side chain protecting groups and cutting peptides to obtain reduced octreotide, oxidizing with air under the condition of pH7-11 to obtain crude octreotide, separating and purifying by using a C18 (or C8) column, and freeze-drying to obtain the refined octreotide. The method has the advantages of large-scale production capacity, stable process, convenient source of raw and auxiliary materials, short production period, high yield, stable quality, low production cost, high peptide receiving yield, avoidance of using highly toxic reagents such as hydrogen fluoride and the like, less pollution of three wastes and adoption of a C18 (or C8) column for separation and purification.

CN103351426A (patent application No. 201310333598.1) discloses a method for synthesizing polypeptide of octreotide acetate, which is characterized by comprising the following steps: using chloromethyl resin as a starting material, preparing Boc-Thr (tBu) -OH into cesium salt, and sequentially connecting amino acids with protective groups according to a solid-phase synthesis method, wherein the method sequentially comprises the following steps: Boc-Cys (Trt) -OH, Boc-Thr (tBu) -OH, Boc-Lys (Fmoc) -OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Cys (Trt) -OH and Fmoc-D-Phe-OH to obtain a protected octapeptide resin, removing the Boc-protecting group by HCl/isopropanol in sequence, carrying out peptide splicing reaction by DIC and HOBT, reducing by palladium carbon/hydrogen, cutting off peptide chains to obtain reduced octreotide, introducing air to form disulfide bonds under the condition of pH7.8-9 to obtain crude octreotide, and separating and purifying by a C18 column by adopting a unique mobile phase to obtain the refined octreotide acetate. According to the method, threoninol and Fmoc-threoninol are not adopted, so that the production cost is extremely low, the method has large-scale production capacity, stable process, convenient raw and auxiliary material sources, short production period, high peptide yield and stable quality, and avoids using highly toxic reagents such as hydrogen fluoride, trifluoroacetic acid and the like, so that the three-waste pollution is less.

CN106543269A (patent application No. 201610938844.X) discloses a synthesis method for industrial production of octreotide, which is characterized in that: the synthesis method sequentially comprises the following steps: the method comprises the following steps of firstly, taking aminomethyl Resin as initial Resin, coupling raw material Fmoc-Thr-x onto the Resin by utilizing condensation reagents HOBT and DIC to generate Fmoc-Thr-x-AM Resin, wherein the substitution degree of the aminomethyl Resin is 0.55-0.65 mmol/g, and the molar weight of the Fmoc-Thr-x, the molar weight of the HOBT and the molar weight of the DIC are 2-3 times of the molar weight of octreotide to be synthesized; then sequentially coupling amino acids Cys, Thr, Lys, D-Trp, Phe, Cys and D-Phe to Fmoc-Thr-x-AM Resin one by one to obtain octreotide peptide Resin; the chemical structural formula of Fmoc-Thr-x is shown as follows; secondly, adding a lysis solution into the octreotide resin, and uniformly stirring for lysis for 2-3 h, wherein the ratio of the octreotide resin to the lysis solution is that 10ml of lysis solution is added into 1g of octreotide resin; after cracking, filtering, slowly adding the filtrate into pre-frozen anhydrous ether, settling for 20-40 min, and centrifuging to obtain crude octreotide linear peptide; dissolving the octreotide linear crude peptide into a TFA/water mixed solution, and then putting the solution into a water bath or a heating jacket at 35-38 ℃ to heat for 2-3 hours to ensure that the peak type of the octreotide linear crude peptide in an HPLC (high performance liquid chromatography) spectrum is single, wherein the ratio of the octreotide linear crude peptide to the TFA/water mixed solution is that 300ml of TFA/water mixed solution is added into every 1g of the octreotide linear crude peptide, and the volume percentage content of TFA in the TFA/water mixed solution is 5 per thousand; and fourthly, adding DMSO into the heated TFA/water mixed solution dissolved with the octreotide linear crude peptide in the third step, oxidizing for 1-1.5 h, wherein the volume of the added DMSO accounts for 5-8% of the total volume of the TFA/water mixed solution dissolved with the octreotide linear crude peptide, detecting a cyclization end point by HPLC, and purifying and separating to obtain an octreotide refined product.

CN106866788A (patent application No. 201710259689.3) discloses a method for preparing octreotide acetate, which comprises the following steps: using chloromethyl resin as a starting material, preparing cesium salt from Boc-Thr (tBu) -OH, sequentially connecting amino acids with protective groups according to a solid-phase synthesis method to obtain protected octapeptide resin, sequentially removing the Boc-protective groups by HCl/isopropanol, and carrying out a peptide-connecting reaction by using a condensing agent; reducing by using palladium carbon/hydrogen, and simultaneously cutting off a peptide chain to obtain reduced octreotide; introducing air under the condition of pH7.8-9 to form disulfide bond into ring to obtain crude octreotide product; separating and purifying the crude octreotide product by a C18 column to obtain a refined octreotide acetate product. The operation steps of the disulfide bond cyclization of the invention are as follows: dissolving the reduced octreotide crude product in water, dropwise adding 2mol/L ammonia water under stirring until the pH value is 7.8-9, introducing air at room temperature for 24-36 hours to form disulfide bonds into rings, adding glacial acetic acid, adding activated carbon, and filtering to obtain the octreotide crude product. However, the cyclization reaction time is too long, the production efficiency is low, and a large variety of impurities may be easily introduced.

Therefore, it is still highly desirable for those skilled in the art to provide a highly efficient production method.

Disclosure of Invention

The present invention aims to provide a method for preparing octreotide acetate and to overcome the problems associated with the prior art. Another object of the present invention is to provide a method for preparing octreotide acetate pharmaceutical composition. It has been surprisingly found that one or more technical effects can be obtained using the octreotide acetate and pharmaceutical compositions thereof of the present invention prepared using the methods and formulations of the present invention, and the present invention has been completed based on such findings.

To this end, the present invention provides, in a first aspect, a process for preparing octreotide acetate, comprising the steps of:

(a) using chloromethyl resin as a starting material, preparing Boc-Thr (tBu) -OH into cesium salt, and sequentially connecting amino acids with protective groups according to a solid-phase synthesis method, wherein the method sequentially comprises the following steps: Boc-Cys (Trt) -OH, Boc-Thr (tBu) -OH, Boc-Lys (Fmoc) -OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Cys (Trt) -OH, Fmoc-D-Phe-OH to obtain a protected octapeptide resin, in which the Boc-protecting group is removed by HCl/isopropanol in sequence, and DIC and HOBT are used as condensing agents to perform a peptide-joining reaction;

(b) reducing by using palladium carbon/hydrogen, and simultaneously cutting off a peptide chain to obtain reduced octreotide;

(c) introducing air under the condition of pH7.8-9 to form disulfide bond into ring to obtain crude octreotide product;

(d) separating and purifying the crude octreotide product by a C18 column to obtain a refined octreotide acetate product.

The method according to any one of the embodiments of the first aspect of the present invention, wherein in the step (c), the operation of disulfide cyclization comprises: dissolving the reduced octreotide crude product in water, dropwise adding 2mol/L ammonia water under stirring until the pH value is 7.8-9, introducing air at room temperature for 24-36 hours to form disulfide bonds into rings, adding glacial acetic acid, adding activated carbon, and filtering to obtain the octreotide crude product.

The process according to any one of the embodiments of the first aspect of the present invention, wherein the air used is previously treated successively with a saturated calcium hydroxide solution and a 2M sulfuric acid solution in the course of carrying out the disulfide bond cyclization.

The method according to any one of the embodiments of the first aspect of the present invention, wherein in the step (c), the operation of disulfide cyclization comprises: dissolving reduced octreotide with 4-6 times of 50% glacial acetic acid, diluting with purified water until the content of the glacial acetic acid is 25%, stirring uniformly, and filtering to obtain a filtrate; to the filtrate was added dropwise saturated I under stirring₂(iodine 1.5-2.0 mol times of reduced octreotide)/glacial acetic acid solution, stirring for 30min, and adding water solution saturated with sodium pyrosulfite and sodium bicarbonate (wherein sodium pyrosulfiteThe amount of the reduced octreotide is 0.02-0.03 mol times of the reduced octreotide) until the reddish brown disappears; concentrating under reduced pressure to obtain octreotide with disulfide bond forming ring. It has been surprisingly found that the addition of the adjuvants sodium metabisulphite and sodium bicarbonate during the disulfide cyclisation of reduced octreotide with iodine under acidic conditions leads to a significant reduction in the content of oxidatively degrading impurities in the product. And compared with the original product obtained by adopting air oxidation in an ammonia water environment, the product is not deteriorated or even better in various technical indexes, particularly, the reaction time is obviously shortened, and the production efficiency is obviously improved.

The method according to any one of the embodiments of the first aspect of the present invention, wherein amino acids having protecting groups are sequentially linked to obtain a protected octapeptide resin, wherein the Boc-protecting groups are sequentially removed, comprises the steps of:

(1) preparation of Boc-Thr (tBu) -OCs

Dissolving Boc-Thr (tBu) -OH in methanol and water, dissolving cesium carbonate in water to clarify, adding the Boc-Thr (tBu) -OH solution with stirring and pH7.5, and concentrating under reduced pressure to dryness to obtain a syrupy residue Boc-Thr (tBu) -OCs; [ in one embodiment of this step, the number of moles of cesium carbonate is 1 to 5 times that of Boc-Thr (tBu) -OH ]

(2) Preparation of Boc-Thr (tBu) -O-resin

Soaking chloromethyl resin in DMF to swell the resin, and adding the syrupy residue Boc-Thr (tBu) -OCs and DMF for reaction; filtering, washing the resin twice with DMF, washing with purified water, washing with absolute ethyl alcohol, and drying under reduced pressure to obtain Boc-Thr (tBu) -O-resin [ in one embodiment of the step, the mole number of Boc-Thr (tBu) -OCs is 1-3 times of that of the resin; in one embodiment, the reaction temperature of chloromethyl resin with Boc-Thr (tBu) -OCs: 40-65 ℃, reaction time: 36-72 hours ];

(3) preparation of Boc-Cys (Trt) -Thr (tBu) -O-resin:

adding 2mol/L HCl/isopropanol solution into the Boc-Thr (tBu) -O-resin in the step (2) to react with DCM, draining, washing twice with DCM, anhydrous methanol and DCM respectively, draining, adding 10% of DCM solution of triethylamine (volume ratio) to react, draining and washing with DCM; adding Boc-Cys (Trt) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF, and draining to obtain [ in one embodiment, removing Boc-group adopts 2mol/L HCl/isopropanol solution, HCl concentration is 1 mol/L-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(4) preparation of Boc-Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding a 2mol/L HCl/isopropanol solution into the Boc-Cys (Trt) -Thr (tBu) -O-resin in the step (3) to react with DCM, draining, washing twice with DCM, absolute methanol and DCM respectively, draining, adding a 10% DCM solution of triethylamine to react, draining, and washing with DCM; adding Boc-Thr (tBu) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF for three times respectively, and draining to obtain [ in one embodiment, removing Boc-group adopts 2mol/L HCl/isopropanol solution, HCl concentration is 1 mol/L-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(5) preparation of Boc-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding 2mol/L HCl/isopropanol solution into Boc-Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (4) to react with DCM, draining, washing with DCM, anhydrous methanol and DCM respectively, draining, adding 10% triethylamine solution into DCM to react, draining and washing with DCM; adding Boc-Lys (Fmoc) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF respectively, and draining to obtain [ in one embodiment, removing Boc-group adopts 2mol/L HCl/isopropanol solution, the HCl concentration is 1 mol/L-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(6) preparation of Boc-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding 2mol/L HCl/isopropanol solution into Boc-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (5) to react with DCM, draining, washing twice with DCM, anhydrous methanol and DCM respectively, adding 10% triethylamine to react with DCM, draining and washing with DCM; adding Boc-D-Trp-OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF for three times respectively, and draining to obtain [ in one embodiment, removing Boc-group adopts 2mol/L HCl/isopropanol solution, HCl concentration is 1 mol/L-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(7) preparation of Boc-Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Boc-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (6), reacting, draining, washing with DCM, anhydrous methanol and DCM respectively, draining, adding a 10% DCM solution of triethylamine for reacting, draining and washing with DCM; adding Boc-Phe-OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF respectively, and draining to obtain [ in one embodiment, removing Boc-group adopts a mixed solution of 2mol/L HCl/isopropanol solution and ethanedithiol, and HCl concentration is 1 mol/L-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(8) preparation of Boc-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys ((tBu) -Thr (tBu) -O-resin

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Boc-Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin of the step (7), reacting, draining, washing with DCM, anhydrous methanol and DCM, draining, adding a 10% DCM solution of triethylamine, draining, and washing with DCM; adding Boc-Cys (Trt) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing the DMF, absolute methanol and DMF, and draining to obtain [ in one embodiment, a mixed solution of 2 mol/LHCl/isopropanol solution and ethanedithiol is adopted for removing Boc-group, and the HCl concentration is 1-9 mol/L; the dosage of the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, such as 2.5-5.5 times;

(9) preparation of Fmoc-D-Phe-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into Boc-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys ((tBu) -Thr) (tBu) -O-resin in the step (8), reacting, draining, washing with DCM, anhydrous methanol and DCM, draining, adding a DCM solution of 10% triethylamine for reacting, draining, washing with DCM, adding Fmoc-D-Phe-OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF, and draining to obtain [ in one embodiment, removing Boc-group adopts a mixed solution of 2mol/L HCl/isopropanol solution and ethanedithiol, the HCl concentration is 1 mol/L-9 mol/L, and the 2mol/L HCl/isopropanol is 2-8 times of the weight of the resin, for example, 2.5 to 5.5 times;

(10) preparation of Fmoc-D-Phe-Cys (SH) -Phe-D-Trp-Lys (Fmoc) -Thr-Cys (SH) -Thr-O-resin

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Fmoc-D-Phe-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (9), reacting, draining, washing by DCM, anhydrous methanol and DCM, and draining; washing with anhydrous methanol for ten times, pumping, and drying in a vacuum drier to obtain octapeptide resin (in one embodiment, removing Boc-group is performed with a mixed solution of 2mol/L HCl/isopropanol solution and ethanedithiol, HCl concentration is 1 mol/L-9 mol/L; the amount of 2mol/L HCl/isopropanol is 2-8 times, for example 2.5-5.5 times, of the weight of the resin.

The process according to any one of the embodiments of the first aspect of the present invention, wherein the reduction with palladium on carbon and hydrogen is carried out while the peptide chain is cleaved to obtain reduced octreotide (i.e., D-Phe-Cys (SH) -Phe-D-Trp-Lys-Thr-Cys (SH) -Thr-ol), comprises the steps of:

adding anhydrous methanol and 5% palladium/carbon into the octapeptide resin, introducing hydrogen, sealing, stirring, filtering, removing carbon powder and resin, washing with anhydrous methanol, concentrating under reduced pressure to obtain an oily substance, adding an anhydrous methanol solution of piperidine, reacting, concentrating under reduced pressure to obtain an oily substance, dissolving DMF, adding anhydrous diethyl ether for precipitation, filtering, and collecting the precipitate to obtain a crude product of reduced octreotide [ in one embodiment, the amount of 5% palladium/carbon is 1/10-5/10 (mass ratio) of the resin ].

The process according to any one of the embodiments of the first aspect of the present invention, wherein the crude product is separated and purified by a C18 column, comprises the steps of: the filtrate was purified in portions over a C18 column, mobile phase: 0.25-0.5mol/L potassium acetate: acetonitrile 7-8: 3-2; the flow rate is: 600-; the detection wavelength is as follows: 280 nm; and combining sample peaks, desalting, and freeze-drying to obtain the octreotide acetate finished product.

Further, the second aspect of the present invention relates to an octreotide acetate injection pharmaceutical composition, which comprises: 0.1g of octreotide acetate counted by octreotide, 45g of mannitol, 3.4g of lactic acid, a proper amount of sodium bicarbonate for adjusting the pH value to 3.7-4.7 and a proper amount of water for injection added to 1000 ml. The injection can be 1ml per bottle.

The octreotide acetate injection pharmaceutical composition according to any embodiment of the second aspect of the present invention, wherein the octreotide acetate is prepared by the method according to any embodiment of the first aspect of the present invention.

The octreotide acetate injection pharmaceutical composition according to any embodiment of the second aspect of the present invention is prepared by a method comprising the following steps:

taking mannitol in a prescription amount and lactic acid in a prescription amount of 2/3 and 700ml of water for injection, stirring for dissolving, adding 0.1% of activated carbon, heating to 80 ℃, stirring for 30 minutes, performing cyclic decarburization through a titanium rod, cooling filtrate to 30 ℃, adding sodium bicarbonate to adjust the pH to 3.7-4.7, such as 4.0, and keeping for later use;

taking octreotide acetate and the balance of lactic acid according to the prescription, adding 100ml of water for injection at 30 ℃, adding 0.1% of activated carbon, stirring for 30 minutes, performing cyclic decarburization through a titanium rod, and adding sodium bicarbonate into the filtrate to adjust the pH value to 3.7-4.7, such as 4.0;

combining the two filtrates, adjusting pH to 3.7-4.7 such as 4.0 with sodium bicarbonate if necessary, and adding water for injection to 1000 ml; pumping into sterile room by peristaltic pump, filtering with 0.22 μm microporous membrane to clarify and sterilize, bottling in glass bottle at a volume of 1ml per bottle, and sealing to obtain octreotide acetate injection pharmaceutical composition.

Further, the third aspect of the present invention provides a method for determining impurities, such as oxidative degradation impurities, in octreotide acetate or a composition thereof, the method comprising the operations of:

dissolving and/or diluting octreotide acetate or its composition in water to obtain solution containing octreotide acetate 0.5mg per 1ml as test solution; precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a control solution;

measuring according to high performance liquid chromatography (specification of 0512 in the four-part general regulation of the Chinese pharmacopoeia 2015 edition), and using octadecylsilane chemically bonded silica as a filling agent; using a tetramethylammonium hydroxide solution (20 ml of 10% tetramethylammonium hydroxide solution, 1ml of triethylamine and 880ml of water, adjusting the pH value to 5.4-acetonitrile (900: 100) by using a 10% phosphoric acid solution as a mobile phase A, and using a tetramethylammonium hydroxide solution (20 ml of 10% tetramethylammonium hydroxide solution, 1ml of triethylamine and 380ml of water and adjusting the pH value to 5.4-acetonitrile (400: 600) by using a 10% phosphoric acid solution as a mobile phase B); the flow rate was 1ml per minute; the detection wavelength is 210 nm; gradient elution was performed as follows: the gradient of the mobile phase A76% -the mobile phase B24% is changed to the mobile phase A63% -the mobile phase B37% from 0 minute to 30 minutes, the gradient of the mobile phase A63% -the mobile phase B37% is changed to the mobile phase A40% -the mobile phase B60% from 30 minutes to 40 minutes, the gradient of the mobile phase A40% -the mobile phase B60% is changed to the mobile phase A76% -the mobile phase B24% from 40 minutes to 41 minutes, and the gradient of the mobile phase A76% -the mobile phase B24% is maintained from 41 minutes to 55 minutes;

taking about 10mg of octreotide acetate reference substance, placing the reference substance in a 10ml measuring flask, adding 1ml of 30% hydrogen peroxide solution, placing for 1 hour, adding water to dilute to a scale, shaking up, filtering, taking 20 mul of subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, wherein the number of theoretical plates is not less than 3000 calculated according to the octreotide peak (the retention time is about 18 minutes), and the separation degree of the octreotide peak and the oxidative degradation product peak with the relative retention time of about 1.45 is not less than 3.0;

precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, recording chromatogram of the test solution, reading peak area of each chromatogram peak, comparing with main peak area of the control solution to calculate content of each impurity, and calculating content of RRT1.45 impurity if the impurity exists.

The method according to the third aspect of the present invention, wherein the octreotide acetate is octreotide acetate prepared by the method according to any one of the first aspect of the present invention.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, chloromethyl resin is used as a solid phase carrier, the first amino acid Boc-Thr (tBu) -OH is prepared into cesium salt, DIC and HOBT are used as condensing agents, protected amino acids are connected one by one, and the last peptide chain is Fmoc-D-Phe-OH to obtain protected octapeptide resin, no threonine or Fmoc-threonine is used, so that the production cost is extremely low, the method has large-scale production capacity, the process is stable, the raw and auxiliary materials are convenient to obtain, the production period is short, the peptide yield is high, the quality is stable, the Boc-protecting group is removed by HCl/isopropanol, palladium/carbon is used for hydrogenation reduction, the peptide chain is cut down and cut, the condition is mild, highly toxic reagents such as hydrogen fluoride and trifluoroacetic acid are avoided, and the three-waste pollution is less.

In addition, compared with the prior art, the invention has the beneficial effects that: the process of the present invention is carried out in a more efficient manner when carrying out the epoxidation and the amount of oxidatively degrading impurities due to over-oxidation during the reaction is also significantly reduced.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it.

In the embodiments of the invention, the amounts of the materials to be prepared are schematic and may be scaled up or increased in number of operations. In the embodiments of the present invention, the raw materials used are all commercially available, for example, part of the raw materials and their sources are as follows:

Boc-Thr (tBu) -OH, Gill Biochemical (Shanghai)

Boc-Cys (Trt) -OH, Gill Biochemical (Shanghai)

Boc-Thr (tBu) -OH, Gill Biochemical (Shanghai)

Boc-Lys (Fmoc) -OH, Gill Biochemical (Shanghai)

Boc-D-Trp-OH, Gill Biochemical (Shanghai) Co

Boc-Phe-OH, Gill Biochemical (Shanghai)

Fmoc-D-Phe-OH, Gill Biochemical (Shanghai) Co

HOBt (N-Hydroxybenzotriazole), Gill Biochemical (Shanghai) Inc

The 2mol/L HCl/isopropanol (hydrogen chloride/isopropanol) solution used hereinafter was prepared as follows: dropping industrial concentrated sulfuric acid into edible crude salt (dried) to generate HCl gas, introducing the HCl gas into an isopropanol absorption bottle (weighing gross weight and net weight) filled with 1000ml (about 1050 g) of isopropanol through two sulfuric acid washing gas bottles, stopping when the weight of the isopropanol absorption bottle is increased to about 1123 g, filling the isopropanol absorption bottle into a reagent bottle, tightly covering an inner plug, and placing the reagent bottle into a refrigerator.

Example 1: preparation of octreotide acetate

One, synthetic peptide chain

(1) Preparation of Boc-Thr (tBu) -OCs

Boc-Thr (tBu) -OH (FW: 275.3, 110mmol)30.3g was dissolved in 250ml methanol and 250ml water at pH 2.5. Another 24g cesium carbonate (147mmol) was dissolved in 100ml water until clarified, and the above Boc-Thr (tBu) -OH solution was slowly added with stirring with generation of carbon dioxide gas. The pH of the solution was 7.5. Dissolving in water at 40-50 deg.C, and concentrating under reduced pressure to dryness to obtain syrup-like residue Boc-Thr (tBu) -OCs (54.2 g).

(2) Preparation of Boc-Thr (tBu) -O-resin

100 g (100 meshes, 200 meshes, 1.0mmol/g, 100mmol) of chloromethyl resin was taken out, soaked with DMF for 30 minutes to fully swell the resin, 107g of syrupy residue obtained in the step (1) and 700ml of DMF were added, and reacted at 50-55 ℃ for 48 hours.

Filtration, resin washing with DMF two times, purified water washing 20 times, absolute ethanol washing 6 times. Drying under reduced pressure at 50-55 ℃ to obtain 124.4g of Boc-Thr (tBu) -O-resin.

(3) Preparation of Boc-Cys (Trt) -Thr (tBu) -O-resin

The resin from the previous step was reacted with 350ml of a 2mol/L HCl/isopropanol solution and 350ml of DCM at 25 ℃ for 30 minutes. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. 700ml of 10% triethylamine in DCM was added and reacted for 5 minutes at 25 ℃ and then the reaction mixture was dried by suction and washed 7 times with DCM.

Boc-Cys (Trt) -OH (MW: 463.6, 200mmol)92.7g, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, DMF600ml were added and the mixture was reacted at 25 ℃ for 3 h. And (5) pumping to dry. Washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dry to obtain the final product.

(4) Preparation of Boc-Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

350ml of 2mol/L HCl/isopropanol solution (2mol/L HCl/isopropanol is 3.5 times of the weight of the resin) and 350ml of DCM are added to the resin obtained in the previous step, and the mixture is reacted for 30 minutes at 25 ℃. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. Add 10% triethylamine 700ml in DCM, react at 25 ℃ for 5 min, pump dry and wash 7 times with DCM.

55.1g (FW: 275.3, 200mmol) Boc-Thr (tBu) -OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, DMF600ml were added and the mixture was reacted at 25 ℃ for 3 hours. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness to obtain the finished product.

(5) Preparation of Fmoc-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

350ml of 2mol/L HCl/isopropanol solution (2mol/L HCl/isopropanol is 4 times of the weight of the resin) and 350ml of DCM are added to the resin obtained in the last step, and the mixture is reacted for 30 minutes at 25 ℃. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. 10% triethylamine 700ml in DCM was added and the reaction was carried out for 5 minutes at 25 ℃ and then the reaction mixture was dried by suction and washed 7 times with DCM.

93.7g (FW: 468.6, 200mmol) Boc-Lys (Fmoc) -OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, 600ml DMF were added and the mixture was reacted at 25 ℃ for l hours. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness to obtain the finished product.

350ml of 2mol/L HCl/isopropanol solution (2mol/L HCl/isopropanol is 5.5 times of the weight of the resin) and 350ml of DCM are added to the resin obtained in the previous step, and the mixture is reacted for 30 minutes at 25 ℃. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. 10% triethylamine 700ml in DCM was added and the reaction was carried out for 5 minutes at 25 ℃ and then the reaction mixture was dried by suction and washed 7 times with DCM.

60.9g (FW: 304.4, 200mmol) Boc-D-Trp-OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, 600ml DMF were added and the mixture was reacted at 25 ℃ for l h. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness to obtain the finished product.

(7) Preparation of Boc-Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-lipid

315ml of 2mol/L HCl/isopropanol (2mol/L HCl/isopropanol is used in an amount which is 3 times of the weight of the resin) and 315ml of DCM and 70ml of ethanedithiol are added into the resin obtained in the previous step, and the mixture is reacted for 30 minutes at 25 ℃. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. 10% triethylamine 700ml in DCM was added and the reaction was carried out for 5 minutes at 25 ℃ and then the reaction mixture was dried by suction and washed 7 times with DCM.

53.1g (FW: 265.3, 200mmol) Boc-Phe-OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, 600ml DMF were added and the mixture was reacted at 25 ℃ for l h. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness to obtain the finished product.

315ml of 2mol/L HCl/isopropanol (2mol/L HCl/isopropanol is used in an amount which is 3.5 times of the weight of the resin) and 315ml of DCM and 70ml of ethanedithiol are added to the resin obtained in the previous step, and the mixture is reacted at 25 ℃ for 30 minutes. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining. 10% triethylamine 700ml in DCM was added and the reaction was carried out for 5 minutes at 25 ℃ and then the reaction mixture was dried by suction and washed 7 times with DCM.

92.7g (FW: 463.6, 200mmol) Boc-Cys (Trt) -OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, 600ml DMF were added and the mixture was reacted at 25 ℃ for l h. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness.

53.1g (FW: 265.3, 200mmol) Fmoc-D-Phe-OH, DIC (MW: 126.2, 200mmol)25.2g, HOBT (MW: 153, 200mmol)30.6g, 600ml DMF were added and the mixture was reacted at 25 ℃ for l h. And (4) pumping to dryness, washing DMF, anhydrous methanol and DMF for three times respectively, and pumping to dryness.

630ml of 2mol/L HCl/isopropanol (2mol/L HCl/isopropanol is used in an amount of 4.5 times the weight of the resin) mixed with 70ml of ethanedithiol was added to the resin obtained in the above step, and the mixture was stirred at 25 ℃ for 30 minutes. And (4) draining, washing with DCM, absolute methanol and DCM twice respectively, and draining.

And then washed ten times with anhydrous methanol. After being pumped to dryness, the mixture is put into a vacuum drier for drying and weighing to obtain about 190.7g of octapeptide resin, and the total yield of the peptide is about 84 percent.

(11) Preparation of reduced octreotide D-Phe-Cys (SH) -Phe-D-Trp-Lys-Thr-Cys (SH) -Thr-ol

190g of octapeptide resin is taken, absolute methanol is added, 10 g of 5 percent palladium/carbon is added, hydrogen is introduced, and the mixture is sealed and stirred for more than 4 hours at 25 ℃. Filtering, removing carbon powder and resin, washing three times with anhydrous methanol, and concentrating under reduced pressure to obtain oily substance. 300ml of a 20% solution of piperidine in dry methanol (vol/vol) was added and the reaction was carried out at 25 ℃ for 30 minutes. Concentrated to an oil under reduced pressure. DMF is dissolved, absolute ether is added for precipitation, and 92g of reduced octreotide crude product is obtained by filtering and collecting the precipitate.

Second, disulfide bond cyclization

Dissolving 90g of reduced octreotide crude product in 90L of water, slowly dripping 2M ammonia water to pH7.8 under stirring, introducing air into the solution at room temperature to perform disulfide bond cyclization reaction for about 24-36 hours (the cyclization reaction is found to have no obvious influence on the yield of octreotide acetate finished products within the range of 24-36 hours, the molar yield of the finished products is within the range of 31-33%), adding glacial acetic acid to adjust to pH5.5, adding activated carbon to stir for 30 minutes, and filtering.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 31.2-32.7% (in mole percentage, the yield range of about 24-36 hours of cyclization reaction time in two parts of disulfide bond cyclization and separation and purification operations, such as disulfide bond cyclization, is referred to below).

Example 2: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 1.

Second, disulfide bond cyclization

Dissolving 90g of crude reduced octreotide in 90L of water, slowly adding 2M ammonia water dropwise under stirring until the pH value is 7.8, introducing air into the solution at room temperature (the used air sequentially passes through a saturated calcium hydroxide solution and a 2M sulfuric acid solution in advance) to perform a disulfide bond cyclization reaction for about 24-36 hours, adding glacial acetic acid to adjust the pH value to 5.5, adding activated carbon, stirring for 30 minutes, and filtering.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduction octreotide to the octreotide acetate finished product is 57.4-59.7% (by mol percent).

Example 3: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 1.

Second, disulfide bond cyclization

Dissolving 90g of crude reduced octreotide in 90L of water, slowly adding 2M ammonia water dropwise under stirring until the pH value is 7.8, introducing air into the solution at room temperature (the air is sequentially added with 2M sulfuric acid solution and saturated calcium hydroxide solution in advance) to perform disulfide bond cyclization reaction for about 24-36 hours, adding glacial acetic acid to adjust the pH value to 5.5, adding activated carbon, stirring for 30 minutes, and filtering.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 30.2-31.4% (by mol percent).

Example 4: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 1.

Second, disulfide bond cyclization

90g of crude reduced octreotide was dissolved in 90L of water, 2M ammonia water was slowly dropped under stirring to pH7.8, air was introduced into the solution at room temperature (the air used passed through 2M sulfuric acid solution) to cause disulfide bond cyclization for about 24 to 36 hours, glacial acetic acid was added to adjust pH5.5, activated carbon was added and stirred for 30 minutes, and filtration was carried out.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 25.6-26.9% (by mol percent).

Example 5: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 1.

Second, disulfide bond cyclization

90g of crude reduced octreotide was dissolved in 90L of water, 2M ammonia water was slowly dropped under stirring to pH7.8, air was introduced into the solution at room temperature (the air used passed through a saturated calcium hydroxide solution) to cause disulfide bond cyclization for about 24 to 36 hours, glacial acetic acid was added to adjust pH5.5, activated carbon was added and stirred for 30 minutes, and filtration was carried out.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; and combining sample peaks, desalting, freeze-drying, wherein a white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 29.3-29.8% (in mol percent).

Example 11: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 2.

Second, disulfide bond cyclization

Dissolving 90g of reduced octreotide with 5 times of 50% glacial acetic acid, diluting with purified water to 25% glacial acetic acid, stirring, and filtering to obtain filtrate; to the filtrate was added dropwise saturated I under stirring₂(iodine with amount of 1.75 mol times of reduced octreotide)/glacial acetic acid solution until the solution is reddish brown, stirring for 30min, adding water solution saturated with sodium pyrosulfite and sodium bicarbonate (wherein the amount of sodium pyrosulfite is 0.025 mol times of reduced octreotide) until reddish brown disappears; concentrating under reduced pressure to obtain octreotide with disulfide bond forming ring.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 63.7 percent (calculated by mole percent).

Example 12: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 2.

Second, disulfide bond cyclization

Dissolving 90g of reduced octreotide with 50% glacial acetic acid in an amount which is 4 times that of the reduced octreotide, diluting the solution with purified water until the solution contains 25% glacial acetic acid, stirring the solution uniformly and filtering the solution to obtain filtrate; to the filtrate was added dropwise saturated I under stirring₂(iodine in an amount of 2.0 times by mole of reduced octreotide)/glacial acetic acid solutionStirring for 30min, and adding water solution saturated with sodium pyrosulfite and sodium bicarbonate (wherein the amount of sodium pyrosulfite is 0.03 mol times of reduced octreotide) until the reddish brown disappears; concentrating under reduced pressure to obtain octreotide with disulfide bond forming ring.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 61.9 percent (calculated by mole percent).

Example 13: preparation of octreotide acetate

One, synthetic peptide chain

The same as in example 2.

Second, disulfide bond cyclization

Dissolving 90g of reduced octreotide with 50% glacial acetic acid in an amount which is 6 times that of the reduced octreotide, diluting the solution with purified water until the solution contains 25% glacial acetic acid, stirring the solution uniformly and filtering the solution to obtain filtrate; to the filtrate was added dropwise saturated I under stirring₂(iodine with amount of 1.5 mol times of reduced octreotide)/glacial acetic acid solution, stirring for 30min, adding water solution saturated with sodium pyrosulfite and sodium bicarbonate (wherein sodium pyrosulfite with amount of 0.02 mol times of reduced octreotide) until the reddish brown disappears; concentrating under reduced pressure to obtain octreotide with disulfide bond forming ring.

Thirdly, separation and purification

All filtrates obtained during disulfide cyclisation were purified in portions on a C18 column, mobile phase: 0.25mol/L potassium acetate: acetonitrile (7.2: 2.8); the flow rate is: 800 ml/min; and tracking and collecting the required effluent liquid by using a liquid chromatograph, wherein the detection wavelength is as follows: 280 nm; after the sample peaks are combined, desalting and freeze-drying are carried out, the white loose block is an octreotide acetate finished product, and the yield from reduced octreotide to the octreotide acetate finished product is 64.4 percent (calculated by mole percent).

The disulfide cyclization reaction of examples 11 to 13 can be completed within 2 hours.

The determination of oxidative degradation impurities in octreotide acetate crude or refined products or compositions is carried out by the following [ HPLC method ]:

[ HPLC method ] -is used for treating diabetes

Dissolving and/or diluting a proper amount of octreotide acetate crude product or refined product or composition with water to obtain a solution containing about 0.5mg of octreotide acetate per 1ml as a test solution; precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a control solution;

measuring by high performance liquid chromatography (0512 in accordance with the national pharmacopoeia 2015 edition of the general rules of four departments), and using octadecylsilane chemically bonded silica as a filling agent; using a tetramethylammonium hydroxide solution (20 ml of 10% tetramethylammonium hydroxide solution, 1ml of triethylamine and 880ml of water, adjusting the pH value to 5.4-acetonitrile (900: 100) by using a 10% phosphoric acid solution as a mobile phase A, and using a tetramethylammonium hydroxide solution (20 ml of 10% tetramethylammonium hydroxide solution, 1ml of triethylamine and 380ml of water and adjusting the pH value to 5.4-acetonitrile (400: 600) by using a 10% phosphoric acid solution as a mobile phase B); the flow rate was 1ml per minute; the detection wavelength is 210 nm; gradient elution was performed as follows: the gradient of the mobile phase A76% -the mobile phase B24% is changed to the mobile phase A63% -the mobile phase B37% from 0 minute to 30 minutes, the gradient of the mobile phase A63% -the mobile phase B37% is changed to the mobile phase A40% -the mobile phase B60% from 30 minutes to 40 minutes, the gradient of the mobile phase A40% -the mobile phase B60% is changed to the mobile phase A76% -the mobile phase B24% from 40 minutes to 41 minutes, and the gradient of the mobile phase A76% -the mobile phase B24% is maintained from 41 minutes to 55 minutes;

taking about 10mg of octreotide acetate reference substance, placing the octreotide acetate reference substance in a 10ml measuring flask, adding 1ml of 30% hydrogen peroxide solution, placing for 1 hour, adding water to dilute to a scale, shaking up, filtering, taking 20 mul of subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, wherein the number of theoretical plates is not less than 3000 calculated according to the octreotide peak (retention time is about 18 minutes), and the separation degree of the octreotide peak and an oxidative degradation product (which can also be called RRT1.45 impurity in the invention) peak with the relative retention time of about 1.45 is not less than 3.0; (the RRT1.45 impurity shows a significant increase with the time of retention of octreotide in a hydrogen peroxide solution, and the RRT1.45 impurity is distinguished from 21 impurities described in the plum alizarin document (plum alizarin et al, J. Pharmacology, 2017, 37(3): 492); further, if triethylamine is not added to the mobile phase, the RRT1.45 impurity overlaps with the impurity 13 at a retention time of about 28 minutes although the retention time of other typical impurities is not substantially changed with reference to the plum alizarin document method, and the peak time of the RRT1.45 impurity advances to RRT1.45 at about 26 minutes after the addition of a small amount of triethylamine, whereby the amount of the oxidized impurity can be accurately determined);

precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, recording chromatogram of the test solution, reading peak area of each chromatogram peak, reading peak area of RRT1.45 impurity if RRT1.45 impurity, namely oxidation degradation product exists, and comparing with main peak area of the control solution to calculate impurity content.

In the various raw materials prepared by the invention, especially in the process of converting reduced octreotide acetate into octreotide acetate by cyclization, an excessive oxidant is needed for reaction, and the excessive oxidant can further oxidize the octreotide acetate subjected to oxidative cyclization to form oxidative degradation impurities, which can be called RRT1.45 impurities in the invention, and the determination and the quantification can be carried out by an HPLC method.

Through determination, the content of the RRT1.45 impurity in the octreotide acetate (crude product) obtained through the disulfide bond cyclization in the step two of the above embodiments 1 to 5 is within the range of 0.143 to 0.165%, the content of the RRT1.1 impurity in the octreotide acetate (refined product) obtained through the separation and purification refining process in the step three is within the range of 0.117 to 0.128%, and the reduction of the RRT1.45 impurity in the purification process of the octreotide acetate from the crude product to the refined product in each embodiment is within the range of 17 to 22%; for example, the impurity content of RRT1.45 in the product of step two in example 1 is 0.154%, the impurity content of RRT1.45 in the product subjected to step three is 0.122%, and the impurity content of RRT1.45 in the purification process of the product of example 1 from crude product to fine product is reduced by about 20.8%.

In the invention, the content of RRT1.45 impurities in octreotide acetate (crude product) obtained by cyclization through disulfide bonds in the second step of the previous embodiments 11 to 13 is in the range of 0.011 to 0.016 percent, and the reduction of the RRT1.45 impurities in the purification process from the crude product to the fine product in the third step is in the range of 18 to 22 percent; for example, the content of the RRT1.45 impurity in the product obtained in the second step of the example 11 is 0.013%, and the reduction of the RRT1.45 impurity in the purification process of the product obtained in the second step of the example 11 from the crude product to the refined product is about 19.4%.

The results show that the reduction of the RRT1.45 impurity in the purification process from crude product to fine product in the third step is basically the same under various test conditions, while the content of the oxidation degradation RRT1.45 impurity in the purification process is different when the method in the ring formation process of disulfide bond in the second step is different.

In a complementary example (which may be referred to herein as example 14), reference is made to example 11, except that in step two, no sodium bicarbonate, or no sodium metabisulphite, is used; the yields of two refined products from reduced octreotide to octreotide acetate finished products are 41.3 percent and 38.7 percent (calculated by mol percent); the results of the final product such as the specific rotation degree, the amino acid ratio, the acetic acid content, related substances, the content measurement and the like are basically the same as those of the product in the example 1; in this example 14, the content of RRT1.45 impurities in octreotide acetate (crude product) obtained by cyclization through disulfide bond in step two is in the range of 0.136-0.143%, and the reduction of RRT1.45 impurities in the purification process from crude product to refined product in step three is in the range of 17-21%. The above results show that it has been surprisingly found that the use of iodine as oxidant and the addition of an auxiliary agent comprising a combination of sodium metabisulfite and sodium bicarbonate during the formation of cyclized octreotide from reduced octreotide acetate significantly reduces the formation of oxidative degradation impurities during the oxidation reaction by a factor of up to about 12, and yields significantly higher than the prior art; however, the effect of using sodium metabisulfite or sodium bicarbonate alone as a reaction aid was not acceptable.

In addition, for the reaction of iodine in the octreotide acetate synthesis step two related to the invention, iodine, sodium metabisulfite and/or sodium bicarbonate in the obtained octreotide acetate crude product and refined product can not be detected, which indicates that all reactants are completely removed in the final product.

In the following composition examples, octreotide acetate obtained in example 2 was used as a drug substance unless otherwise described.

Composition example 1: preparation of octreotide acetate injection pharmaceutical composition

Prescription of octreotide acetate injection:

0.1g of octreotide acetate in terms of octreotide,

45g of mannitol,

3.4g of lactic acid,

Adjusting the pH value of sodium bicarbonate to 3.7-4.7,

The right amount of water for injection is added to 1000 ml.

The preparation method comprises the following steps:

taking mannitol in a prescription amount and lactic acid in a prescription amount of 2/3 and adding 700ml of water for injection, stirring for dissolving, adding 0.1% of activated carbon, heating to 80 ℃, stirring for 30 minutes, performing cyclic decarburization through a titanium rod, cooling the filtrate to 30 ℃, adding sodium bicarbonate to adjust the pH value to 4.0 for later use;

taking octreotide acetate and the rest lactic acid according to the prescription amount, adding 100ml of water for injection at 30 ℃, adding 0.1 percent of active carbon, stirring for 30 minutes, circularly decarburizing by a titanium rod, and adding sodium bicarbonate into the filtrate to adjust the pH value to 4.0;

mixing the two filtrates, adjusting pH to 4.0 with sodium bicarbonate if necessary, and adding water for injection to 1000 ml; pumping into sterile room by peristaltic pump, filtering with 0.22 μm microporous membrane to clarify and sterilize, bottling in glass bottle at a volume of 1ml per bottle, and sealing to obtain octreotide acetate injection pharmaceutical composition.

In the supplementary test of the present composition example 1, it was found that the results of the injection solution obtained in test example 2 were the same as those of the injection solution of composition example 1 described above when the pH was adjusted to the whole range of 3.7 to 4.7, for example, to 3.7, 4.2 and 4.6 with an appropriate amount of sodium bicarbonate.

Composition example 2: preparation of octreotide acetate injection pharmaceutical composition

Prescription of octreotide acetate injection:

0.1g of octreotide acetate in terms of octreotide,

45g of mannitol,

3.4g of lactic acid,

Adjusting the pH value of sodium bicarbonate to 3.7-4.7,

The right amount of water for injection is added to 1000 ml.

The preparation method comprises the following steps:

taking 700ml of mannitol and lactic acid in the amount of a prescription, adding water for injection, stirring for dissolving, adding 0.1% of activated carbon, heating to 80 ℃, stirring for 30 minutes, performing cyclic decarburization through a titanium rod, cooling the filtrate to 30 ℃, adding sodium bicarbonate to adjust the pH value to 4.0 for later use;

adding 100ml of water for injection at 30 ℃ into octreotide acetate of the prescription amount, adding 0.1% of active carbon, stirring for 30 minutes, circularly decarburizing by a titanium rod, and adding sodium bicarbonate into the filtrate to adjust the pH value to 4.0;

Composition example 3: preparation of octreotide acetate injection pharmaceutical composition (#700BE6)

Prescription of octreotide acetate injection:

0.1g of octreotide acetate in terms of octreotide,

45g of mannitol,

3.4g of lactic acid,

Adjusting the pH value of sodium bicarbonate to 3.7-4.7,

The right amount of water for injection is added to 1000 ml.

The preparation method comprises the following steps:

taking 800ml of mannitol and lactic acid in the amount of a prescription and injection water, stirring and dissolving, adding 0.1% of activated carbon, heating to 80 ℃, stirring for 30 minutes, performing cyclic decarburization through a titanium rod, cooling the filtrate to 15 ℃, adding octreotide acetate in the amount of the prescription, stirring and dissolving, adding sodium bicarbonate to adjust the pH value to 4.6, and adding the injection water to 1000 ml. Pumping into sterile room by peristaltic pump, filtering with 0.22 μm microporous membrane to clarify and sterilize, bottling in glass bottle at a volume of 1ml per bottle, and sealing to obtain octreotide acetate injection pharmaceutical composition.

In the supplementary test of the present composition example 3, it was found that the results of the injection solution obtained in test example 2 were the same as those of the above composition example 3 injection solution when the pH was adjusted to the whole range of 3.7 to 4.7, for example, to 3.7 and 4.0 with an appropriate amount of sodium bicarbonate.

Composition example 4: preparation of octreotide acetate injection pharmaceutical composition

Referring to the formulation and preparation method of the composition examples 1-3, respectively, three batches of injection were obtained with the octreotide acetate obtained in example 11 as the raw material.

Detection example 1: quality detection of octreotide acetate

According to the method for checking related substances in octreotide acetate, the method for measuring the content of the octreotide acetate and other detection indexes, which are recorded in the second part of the 2015 edition of Chinese pharmacopoeia, the finished octreotide acetate products obtained in the examples 1-5 and the examples 11-13 of the invention are measured. Through determination, 8 batches of octreotide acetate finished products obtained in examples 1-5 and examples 11-13:

the specific rotation degrees are all in the range of-69.2 degrees to-73.6 degrees, and all samples are not obviously different and all accord with standard regulations;

the relative ratio of the amino acids is in the following range: 1.93-2.14 parts of cysteine, 0.88-1.08 parts of threonine, 1.90-2.11 parts of phenylalanine and 0.94-1.05 parts of lysine, wherein threonine can be detected, and all samples have no obvious difference and meet the standard regulation;

the acetic acid content is in the range of 7.5-9.6%, and all samples have no obvious difference and meet the standard regulation;

the inspection results of related substances show that the maximum single impurities are all within the range of 0.15-0.29%, the total impurities are all within the range of 0.63-0.74%, and all samples have no obvious difference and all accord with standard regulations;

the content measurement result shows that the content of octreotide acetate in terms of octreotide (C49H66N10O10S2) is within the range of 97.2-100.8% in terms of anhydrous and acetic acid-free substances, and the samples have no obvious difference and meet the standard regulation.

Detection example 2: property examination of the injection pharmaceutical composition:

the 6 batches of injections obtained in composition examples 1 to 4 were tested according to the test methods of octreotide acetate injection item collected on page 1545 of the second part of the 2015 pharmacopoeia, and the results showed that all injections uniformly met the standard specification. And the clarity of all injections (checked according to methods under the items of clarity and color of the solution of octreotide acetate crude drug collected on page 1544 of the second part of the 2015, China pharmacopoeia) is uniform and accords with the specification, and all injections are colorless clear liquids.

The 6 batches of injections obtained in composition examples 1 to 4 were allowed to stand at a temperature of 4 ℃ for 7 days and then at a temperature of 28 ℃ for 7 days, thus completing one cycle; the four cycles are carried out for 56 days, and the injection on the 0 th day and the 56 th day is detected according to various detection methods under the item of octreotide acetate injection collected on the 1545 page of the second part of the 2015 version in Chinese pharmacopoeia to investigate the physical stability of the drug under the high-low temperature alternation condition. The results show that the content of all injections and related substances meet the standard specification on 0 day and 56 days, and other indexes except clarity also meet the standard specification on 0 day and 56 days. However, the clarity of the injections in 56 days changed differently, that is, the clarity of the injections in 56 days obtained in composition example 1 and composition example 4 with reference to composition example 1 was defined as colorless clear liquid; in contrast, the clarity of the 4 injections obtained in composition examples 2 and 3 and composition example 4 with reference to composition examples 2 to 3 did not meet the standard specification in 56 days, and all appeared white turbid substances. This indicates that the injections prepared by different methods exhibit different physical stabilities even with the same formulation.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. A method for preparing octreotide acetate, comprising the steps of:

using chloromethyl resin as a starting material, preparing Boc-Thr (tBu) -OH into cesium salt, and sequentially connecting amino acids with protective groups according to a solid-phase synthesis method, wherein the method sequentially comprises the following steps: Boc-Cys (Trt) -OH, Boc-Thr (tBu) -OH, Boc-Lys (Fmoc) -OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Cys (Trt) -OH, Fmoc-D-Phe-OH to obtain a protected octapeptide resin, in which the Boc-protecting group is removed by HCl/isopropanol in sequence, and DIC and HOBT are used as condensing agents to perform a peptide-joining reaction;

reducing by using palladium carbon/hydrogen, and simultaneously cutting off a peptide chain to obtain reduced octreotide;

dissolving reduced octreotide with 4-6 times of 50% glacial acetic acid, diluting with purified water until the content of the glacial acetic acid is 25%, stirring uniformly, and filtering to obtain a filtrate; dropwise adding saturated iodine/glacial acetic acid solution into the filtrate under stirring until the solution is reddish brown, continuously stirring for 30min, and adding water solution saturated with sodium metabisulfite and sodium bicarbonate until the reddish brown disappears; concentrating under reduced pressure to obtain octreotide crude product with disulfide bond forming ring; wherein the amount of iodine is 1.5-2.0 molar times of the reduced octreotide, and the amount of sodium pyrosulfite is 0.02-0.03 molar times of the reduced octreotide;

separating and purifying the crude octreotide product by a C18 column to obtain a refined octreotide acetate product.

2. The process according to claim 1, wherein the sequential attachment of amino acids having protecting groups to obtain a protected octapeptide resin, during which the Boc-protecting groups are sequentially removed, comprises the steps of:

(1) preparation of Boc-Thr (tBu) -OCs

Dissolving Boc-Thr (tBu) -OH in methanol and water, dissolving cesium carbonate in water to clarify, adding Boc-Thr (tBu) -OH solution with stirring and pH7.5, and concentrating under reduced pressure to dry to obtain syrupy residue Boc-Thr (tBu) -OCs;

(2) preparation of Boc-Thr (tBu) -O-resin

Soaking chloromethyl resin in DMF to swell the resin, and adding the syrupy residue Boc-Thr (tBu) -OCs and DMF for reaction; filtering, washing the resin twice with DMF, washing with purified water, washing with absolute ethyl alcohol, and drying under reduced pressure to obtain Boc-Thr (tBu) -O-resin;

(3) preparation of Boc-Cys (Trt) -Thr (tBu) -O-resin:

adding 2mol/L HCl/isopropanol solution into the Boc-Thr (tBu) -O-resin in the step (2) to react with DCM, draining, washing twice with DCM, anhydrous methanol and DCM respectively, draining, adding 10% of triethylamine DCM solution to react, draining, and washing with DCM; adding Boc-Cys (Trt) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF, and draining to obtain the final product;

(4) preparation of Boc-Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding a 2mol/L HCl/isopropanol solution into the Boc-Cys (Trt) -Thr (tBu) -O-resin in the step (3) to react with DCM, draining, washing twice with DCM, absolute methanol and DCM respectively, draining, adding a 10% DCM solution of triethylamine to react, draining, and washing with DCM; adding Boc-Thr (tBu) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF for three times respectively, and draining to obtain the final product;

(5) preparation of Boc-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin

Adding 2mol/L HCl/isopropanol solution into Boc-Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (4) to react with DCM, draining, washing with DCM, anhydrous methanol and DCM respectively, draining, adding 10% triethylamine solution into DCM to react, draining and washing with DCM; adding Boc-Lys (Fmoc) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF respectively, and draining to obtain the final product;

Adding 2mol/L HCl/isopropanol solution into Boc-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (5) to react with DCM, draining, washing twice with DCM, anhydrous methanol and DCM respectively, adding 10% triethylamine to react with DCM, draining and washing with DCM; adding Boc-D-Trp-OH, DIC, HOBT and DMF, reacting the mixture, draining, washing with DMF, anhydrous methanol and DMF for three times, and draining to obtain the final product;

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Boc-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (6), reacting, draining, washing with DCM, anhydrous methanol and DCM respectively, draining, adding a 10% DCM solution of triethylamine for reacting, draining and washing with DCM; adding Boc-Phe-OH, DIC, HOBT and DMF, reacting, draining, washing with DMF, anhydrous methanol and DMF, and draining;

(8) preparation of Boc-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (tBu) -Thr (tBu) -O-resin

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Boc-Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin of the step (7), reacting, draining, washing with DCM, anhydrous methanol and DCM, draining, adding a 10% DCM solution of triethylamine, draining, and washing with DCM; adding Boc-Cys (Trt) -OH, DIC, HOBT and DMF, reacting the mixture, draining, washing DMF, anhydrous methanol and DMF, and draining to obtain the final product;

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Boc-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (tBu) -Thr (tBu) -O-resin in the step (8), reacting, draining, washing by DCM, absolute methanol and DCM, draining, adding a 10% DCM solution of triethylamine, draining, and washing by DCM; adding Fmoc-D-Phe-OH, DIC, HOBT and DMF, reacting the mixture, draining, washing the DMF, anhydrous methanol and DMF, and draining to obtain the product;

Adding a mixed solution of 2mol/L HCl/isopropanol and DCM and ethanedithiol into the Fmoc-D-Phe-Cys (Trt) -Phe-D-Trp-Lys (Fmoc) -Thr (tBu) -Cys (Trt) -Thr (tBu) -O-resin in the step (9), reacting, draining, washing by DCM, anhydrous methanol and DCM, and draining; and washing the octapeptide resin by using anhydrous methanol for ten times, draining, and drying in a vacuum drier to obtain the octapeptide resin.

3. The method according to claim 2, wherein in the step (1), the mole number of cesium carbonate is 1 to 5 times that of Boc-Thr (tBu) -OH.

4. The method according to claim 2, wherein in the step (3) to the step (10), 2mol/L HCl/isopropanol is used in an amount of 2 to 8 times the weight of the resin.

5. The process according to claim 2, wherein the reduction of the peptide chain with palladium on carbon and hydrogen is carried out simultaneously with cleaving off the peptide chain to obtain reduced octreotide (D-Phe-Cys (SH) -Phe-D-Trp-Lys-Thr-Cys (SH) -Thr-ol), which process comprises the steps of:

adding anhydrous methanol and 5% palladium/carbon into the octapeptide resin, introducing hydrogen, sealing, stirring, filtering, removing carbon powder and resin, washing with anhydrous methanol, concentrating under reduced pressure to obtain an oily substance, adding an anhydrous methanol solution of hexahydropyridine, reacting, concentrating under reduced pressure to obtain an oily substance, dissolving DMF, adding anhydrous ether for precipitation, filtering, and collecting the precipitate to obtain a crude product of reduced octreotide.

6. The method of claim 5, wherein the 5% palladium/carbon is used in a mass ratio of 1/10 to 5/10% of the resin.

7. The method of claim 2, wherein the crude product is separated and purified by a C18 column comprising the steps of: the filtrate was purified in portions over a C18 column, mobile phase: 0.25-0.5mol/L potassium acetate: acetonitrile = 7-8: 3-2; the flow rate is: 600-; the detection wavelength is as follows: 280 nm; and combining sample peaks, desalting, and freeze-drying to obtain the octreotide acetate finished product.

8. A method for determining oxidative degradation impurities in octreotide acetate prepared by the method of any one of claims 1 to 7, comprising the operations of:

dissolving and/or diluting octreotide acetate in water to obtain solution containing octreotide acetate 0.5mg per 1ml as test solution; precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a control solution;

measuring according to the specification of high performance liquid chromatography of the national pharmacopoeia 2015 edition of rules of four parts 0512, and using octadecylsilane chemically bonded silica as a filling agent; mixing tetramethylammonium hydroxide solution-acetonitrile in a volume ratio of 900: 100 as a mobile phase A, and mixing tetramethylammonium hydroxide solution-acetonitrile in a volume ratio of 400: 600 as mobile phase B; the flow rate was 1ml per minute; the detection wavelength is 210 nm; gradient elution was performed as follows: the gradient of the mobile phase A76% -the mobile phase B24% is changed to the mobile phase A63% -the mobile phase B37% from 0 minute to 30 minutes, the gradient of the mobile phase A63% -the mobile phase B37% is changed to the mobile phase A40% -the mobile phase B60% from 30 minutes to 40 minutes, the gradient of the mobile phase A40% -the mobile phase B60% is changed to the mobile phase A76% -the mobile phase B24% from 40 minutes to 41 minutes, and the gradient of the mobile phase A76% -the mobile phase B24% is maintained from 41 minutes to 55 minutes; the preparation method of the tetramethylammonium hydroxide solution in the mobile phase A comprises the following steps: taking 20ml of 10% tetramethyl ammonium hydroxide solution and 1ml of triethylamine, adding 880ml of water, and adjusting the pH value to 5.4 by using 10% phosphoric acid solution; the preparation method of the tetramethylammonium hydroxide solution in the mobile phase B comprises the following steps: taking 20ml of 10% tetramethylammonium hydroxide solution and 1ml of triethylamine, adding 380ml of water, and adjusting the pH value to 5.4 by using 10% phosphoric acid solution;

taking about 10mg of an octreotide acetate reference substance, putting the octreotide acetate reference substance into a 10ml measuring flask, adding 1ml of 30% hydrogen peroxide solution, standing for 1 hour, adding water to dilute to a scale, shaking up, filtering, taking 20 mu l of subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, wherein the number of theoretical plates is not less than 3000 calculated according to the octreotide peak, and the separation degree of the octreotide peak and an oxidative degradation product peak with the relative retention time of about 1.45 is not less than 3.0;

precisely measuring 20 mu l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, recording the chromatogram of the test solution, reading the peak area of each chromatogram peak, comparing with the main peak area of the control solution to calculate the content of each impurity, and calculating the content of each impurity if RRT1.45 impurity exists.