Oxytocin derivatives and methods for their preparation

Abstract

The present application relates to a kind of intermediate compounds of novel oxytocin analogues, novel oxytocin analogues and preparation method, pharmaceutical composition and purposes thereof.The intermediate compound has the structure shown in formula (IV);Oxytocin analogues are compounds with the structure shown in formula (I);The preparation method of compound includes: the thiol alkene reaction of intermediate compound shown in formula (IV) is carried out so that the compound shown in formula (I);Pharmaceutical composition includes the compound shown in formula (I).The compound of the present application can effectively promote breast lactation and prevent or treat induced labor hemorrhage and postpartum hemorrhage, and has longer half-life and lower EC 50 value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular, the present application relates to a oxytocin derivative and a preparation method thereof, more particularly, the present application relates to an intermediate compound, a compound and a preparation method thereof, a pharmaceutical composition and use. BACKGROUND

[0002] Oxytocin is a polypeptide hormone secreted by hypothalamus in mammals, which contains nine amino acid residues and a pair of disulfide bonds. It is involved in many physiological processes, such as neurotransmission in central nervous system, contraction of uterine and mammary smooth muscle, breast tumor growth, and autocrine and paracrine of ovary. The current oxytocin and its analog products have the disadvantages of short half-life or EC 50 value too large or even no effect.

[0003] Therefore, there is an urgent need for a new oxytocin analog product. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides an intermediate compound for synthesizing a new oxytocin analog, the new oxytocin analog and a preparation method thereof, a pharmaceutical composition and use, which has a longer half-life and a lower EC 50 value.

[0005] The present application is based on the following findings of the inventors:

[0006] Oxytocin is mainly used for labor induction and postpartum hemorrhage control in clinical practice, but it has the disadvantage of short half-life (2-5 minutes). Disulfide bond plays a crucial role in maintaining the rigidity of oxytocin structure and physiological activity, but disulfide bond itself is easily affected by reducing substances in the body (such as free thiol), which directly affects the half-life of oxytocin. Therefore, modification of disulfide bond is one of the important modification directions of oxytocin. According to the literature, the disulfide bond of oxytocin is replaced by C-C bond through olefin metathesis reaction, and the half-life of the modified compound (i.e. oxytocin analog) is greatly increased (5-60 times), but the EC 50 value of the oxytocin analog containing C-C bond is increased by three orders of magnitude, or even has no effect. In addition, replacing S-S bond with sulfide bond is a popular modification strategy for disulfide bond. The sulfide bond is close in biochemical and geometric parameters such as dihedral angle and covalent radius, so the sulfide bond has higher stability. According to the literature, one S atom of the disulfide bond of oxytocin is usually replaced by C atom, and the obtained oxytocin analog contains sulfide bond, but the results show that the above modification can increase the half-life of the oxytocin analog, but has an impact on the activity of the oxytocin analog, greatly increasing the EC50 Values.

[0007] However, the inventors of the present application have found through a large number of experiments that, by replacing the side chain -SH of one of the cysteines in oxytocin with -R4-CH=CH2 (e.g. R4 is nothing or a C1-C5 alkylene), the alkenyl group can be connected to the thiol of the other cysteine under thiol-ene reaction to form a thioether bond, and the -S-S- bond of the original oxytocin can be modified into -R1-CH2-S- (e.g. R1 is a C1-C6 alkylene), the modified compound (i.e. an oxytocin analogue) has a longer half-life, while still being able to maintain the original activity, and the EC 50 value of the compound is comparable to or even lower than the EC 50 value of oxytocin.

[0008] Based on this, in one aspect of the present application, the present application provides a compound, which is a compound shown in formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt of the compound shown in formula (I),

[0009] wherein X is -S-CH2-R1-, and R1 is a C1-C6 alkylene.

[0010] The inventors have obtained the above-mentioned compound through a large number of experiments, and the compound is a novel oxytocin analogue, which has a longer half-life and a lower EC 50 value, and can effectively promote breast milk ejection and prevent or treat induced labor and postpartum hemorrhage.

[0011] In another aspect of the present application, the present application provides an intermediate compound. According to an embodiment of the present application, the intermediate compound has a structure shown in formula (IV),

[0012] wherein R2 is -SH, R3 is -R4-CH=CH2, or R3 is -SH, R2 is -R4-CH=CH2; and R4 is nothing or a C1-C5 alkylene.

[0013] The intermediate compound of the present application can be used for synthesizing the aforementioned oxytocin analogue shown in formula (I).

[0014] In another aspect of the present application, the present application provides a method for preparing the compound shown in formula (I). According to an embodiment of the present application, the method comprises: subjecting an intermediate compound shown in formula (IV) to thiol-ene reaction, so as to obtain the compound shown in formula (I);

[0015]

[0016] wherein X is -S-CH2-R1-; R1 is C1-C6 alkylene; R2 is -SH, R3 is -R4-CH=CH2, or R3 is -SH, R2 is -R4-CH=CH2; R4 is null or C1-C5 alkylene.

[0017] The inventors have found, through a large number of experiments, that the oxytocin analogue shown in formula (I) can be effectively synthesized by the above method, and the oxytocin analogue can effectively promote mammary gland milk ejection and prevent or treat induced labor and postpartum hemorrhage, and the preparation method is simple.

[0018] In another aspect of the present application, a pharmaceutical composition is provided. According to an embodiment of the present application, the composition comprises the aforementioned compound or the compound prepared according to the aforementioned method. The pharmaceutical composition of the present application has a longer half-life and a lower EC 50 value, and can effectively promote mammary gland milk ejection and prevent or treat induced labor and postpartum hemorrhage.

[0019] In another aspect of the present application, the use of the aforementioned compound, the compound prepared according to the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of a medicament for promoting mammary gland milk ejection and preventing or treating induced labor and postpartum hemorrhage is provided.

[0020] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0022] Figure 1 A preparation process schematic diagram of the oxytocin analogue in Example 1 of the present application;

[0023] Figure 2 An HPLC analysis diagram of the oxytocin analogue in Example 1 of the present application;

[0024] Figure 3 An ESI-MS mass spectrum diagram of the oxytocin analogue in Example 1 of the present application;

[0025] Figure 4 EC 50 values of the oxytocin analogue and oxytocin in Example 2 of the present application. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not to be construed as limiting the present application.

[0027] It should be noted that the terms "first", "second", etc. are used herein only for descriptive purposes and do not necessarily connote relative importance or an ordering. Thus, a feature defined with "first", "second", etc. can include one or more of the features. Further, in the description of the application, the meaning of "a", "an", and "the" includes plural references unless the context clearly dictates otherwise.

[0028] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Within the range values, endpoints are provided as a separate embodiment. Other values within the range are neither precluded or essential.

[0029] So that the application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document connotate the meanings that would be given to them by one of ordinary skill in the art of the present application.

[0030] In this document, the terms "comprises", "comprising", "includes", "including" or "having" are used in their open-ended, non-limiting sense, i.e. they mean that the stated features, items, components, elements or steps are included, but not excluded.

[0031] In this document, the terms "optionally", "optional" or "may" are used as antonyms for "definitely" or "must". They mean that the subsequently described event or circumstance can, but need not, occur, and that the description includes the case where the event or circumstance occurs, as well as the case where it does not.

[0032] In this document, the term "alkylene" refers to a straight-chain or branched saturated divalent alkane radical. It includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), isopropylene (-CH2(CH3)CH2-).

[0033] In this document, the term "C1-C n " refers to an alkyl group having a finite number of carbon atoms of 1 to n, for example 1, 2, 3, 4, 5, or 6. It is further understood that the term "C1-C6" is to be interpreted in the same way as "C1-C6 " and "C1-C6 ", i.e. to include any sub-range that falls within the main reference range. Further, it is understood that the term "C1-C6 " is to be interpreted in the same way as "C1-C6 ", i.e. to include any sub-range that falls within the main reference range. In particular, C1-C2, C1-C3.

[0034] In the present context, the compounds of the present application can be preferred compounds. The term "preferred compounds" refers to compounds that result in more desirable biological activity, for example, stereoisomers, tautomers, or pharmaceutically acceptable salts of the compounds. Exemplarily, isolated, purified or partially purified stereoisomers of the compounds of the present application are also included within the scope of the present application. Purification and isolation of such materials can be achieved by standard techniques known in the art.

[0035] It is noted that the compounds of the present application can also include all possible stereoisomers of the compounds of the present application, either as single stereoisomers, or as any mixture in all proportions in the said stereoisomers, for example, any mixture of (R) or (S) isomers, or (E) or (Z) isomers. Isolation of single stereoisomers of the compounds of the present application, for example, isolation of single enantiomers or single diastereomers, can be achieved by any suitable method illustrated in the art, for example, by chromatography, especially chiral chromatography.

[0036] It is noted that the compounds of the present application can also include all possible tautomers of the compounds of the present application, either as single tautomers, or as any mixture in all proportions in the said tautomers.

[0037] In the present context, the term "pharmaceutically acceptable salts" refers to salts, or inner salts thereof, of a compound which are non-toxic with the compound or its stereoisomers, with an acid and / or a base, which are relatively non-toxic, inorganic and / or organic, and also to zwitterionic salts, and also to quaternary ammonium salts, for example alkylammonium salts. These salts can be obtained directly by the final isolation and purification of the compounds. They can also be obtained by mixing an appropriate amount of an acid or a base with the above-mentioned compounds, or their stereoisomers. These salts can form a precipitate in solution, which is collected by filtration, or they are recovered after evaporation of the solvent, or they are obtained by lyophilization after the reaction in an aqueous medium. The salts mentioned in the present application can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluorides, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates or trifluoroacetates of the compounds. For example, see S. M. Berge et al. "Pharmaceutical Salts" J. Pharm. Sci. 1977, 66, 1-19.

[0038] In the present context, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the excipient that constitutes one or more accessory ingredients. Ordinarily, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid excipient, a finely divided solid excipient, or both.

[0039] In the present context, the term "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved or approvable by a regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0040] In the present context, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" can include any solvent, solid diluent, diluent liquid excipient, or other liquid excipient, etc., suitable for the particular target dosage form. The use of any of these excipients is contemplated insofar as they are not incompatible with the compounds of the present application, for example, they do not produce any adverse biological effects or interact in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition.

[0041] For further pharmaceutically acceptable excipients mentioned herein and their processes, reference is made to the extensive literature on this subject, in particular to the Handbook of Pharmaceutical Excipients, 3rdEdition, edited by Arthur H. Kibbe, American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and to the Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, edited by H.P. Fiedler, 4thEdition, edited by Cantor, Aulendorf and earlier editions.

[0042] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The fusion proteins or pharmaceutical compositions of the present application can be administered by any common route of administration, as long as it reaches the intended tissue. Various modes of administration are contemplated, including intravenous injection, intramuscular injection, subcutaneous injection, etc., but the present application is not limited to these exemplified modes of administration. Preferably, the compositions of the present application are administered by intravenous injection.

[0043] As used herein, the term "treatment" refers to any action providing a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any administration of a drug or compound to a subject for therapeutic, curative, palliative, ameliorative, improving, alleviating or inhibiting a disease in a subject, including, but not limited to, administration of a drug containing a compound described herein to a subject in need thereof.

[0044] The present application provides an intermediate compound, a compound, a preparation method of the compound, a pharmaceutical composition and use thereof, which will be described in detail as follows.

[0045] Compound

[0046] In one aspect of the present application, the present application provides a compound, which is a compound shown in formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt of the compound shown in formula (I).

[0047] wherein X is -S-CH2-R1-, and R1 is C1-C6 alkylene.

[0048] It has been found that replacing one S atom of the disulfide bond of the original compound (oxytocin) with a C atom, so that a thioether bond replaces the disulfide bond to form a new compound (i.e., an oxytocin analog), the compound has a longer half-life, but its activity is significantly reduced, greatly improving the EC 50 value. Based on this, the inventors have found through experiments that the number of atoms at the connection of the thioether bond affects the activity of the compound, and thus the inventors have obtained the above-mentioned compound through a large number of experiments, which has a longer half-life and a lower EC 50 value, can effectively promote breast milk ejection and prevent or treat induced labor and postpartum hemorrhage.

[0049] According to an embodiment of the present application, R1 is methylene, ethylene, propylene or isopropylene. The inventors have found through experiments that replacing the side chain -SH of cysteine with an alkenyl structure, the number of atoms of the alkenyl structure can affect the activity of the compound, and when R1 is methylene, ethylene, propylene or isopropylene, the activity of the obtained compound is better, and R1 is preferably methylene.

[0050] According to an embodiment of the present application, the compound has a structure shown in formula (II) or formula (III),

[0051]

[0052] The inventors have found that the oxytocin analogue, which is obtained by replacing one S atom of oxytocin with ethylene, has a longer half-life and substantially maintains the activity of oxytocin, and can even further improve the activity of oxytocin.

[0053] Intermediate compound

[0054] In yet another aspect of the present application, an intermediate compound is provided. According to embodiments of the present application, the intermediate compound has a structure as shown in formula (IV),

[0055] wherein R2 and R3 are each independently selected from -R4-CH=CH2 or -SH, and one of R2 and R3 must be -SH, and R4 is nothing or a C1-C5 alkylene.

[0056] The intermediate compound of the present application can be used to synthesize the aforementioned compound, so that the synthesized compound has a longer half-life and a lower EC 50 value, and can effectively promote breast milk ejection and prevent or treat induced labor bleeding and postpartum hemorrhage.

[0057] According to embodiments of the present application, R2 is -SH, R3 is -CH=CH2, or R3 is -SH, R2 is -CH=CH2.

[0058] Method for preparing the compound shown in formula (I)

[0059] In another aspect of the present application, a method for preparing the compound shown in formula (I) is provided. According to embodiments of the present application, the method comprises: performing a thiol-ene reaction on an intermediate compound shown in formula (IV) so as to obtain the compound shown in formula (I);

[0060]

[0061] wherein X is -S-CH2-R1-, R1 is a C1-C6 alkylene; R2 and R3 are each independently selected from -R4-CH=CH2 or -SH, and one of R2 and R3 must be -SH, and R4 is nothing or a C1-C5 alkylene.

[0062] The inventors have found, through a large number of experiments, that the aforementioned method can effectively synthesize the compound shown in formula (I), which can effectively promote breast milk ejection and prevent or treat induced labor and postpartum hemorrhage, and the preparation method is simple.

[0063] According to embodiments of the present application, R1 is a methylene, ethylene, propylene or isopropylene, and is preferably a methylene.

[0064] According to an embodiment of the present application, R2 is -SH, and R3 is -CH=CH2, or R3 is -SH, and R2 is -CH=CH2.

[0065] According to an embodiment of the present application, the intermediate compound shown in formula (IV) is obtained by a solid phase synthesis method.

[0066] According to an embodiment of the present application, the solid phase synthesis method is selected from Fmoc solid phase synthesis method.

[0067] According to an embodiment of the present application, the thiol-ene reaction comprises: performing light irradiation treatment on the intermediate compound shown in formula (IV) in the presence of guanidine hydrochloride and a photoinitiator.

[0068] According to an embodiment of the present application, the light irradiation treatment is performed under the light irradiation conditions of light wavelength of 365 nm, light intensity of (5-8) x 10 5 μJ / cm 2 The above light irradiation treatment conditions are obtained by the inventors through a large number of experiments, and thus the compound shown in formula (I) can be effectively synthesized.

[0069] According to an embodiment of the present application, the photoinitiator is selected from 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (i.e. photoinitiator 2959). The above photoinitiator is screened by the inventors through a large number of experiments, and thus the compound shown in formula (I) can be effectively synthesized. The inventors have found through experiments that when the compound shown in formula (I) is synthesized by using other photoinitiators such as p-dimethylaminopyridine (DMAP), initiator VA-044, sodium anthraquinone-2-sulfonate and photoinitiator 819, it is found that the above photoinitiators all form dimers through disulfide bonds or do not react directly, and thus the compound shown in formula (I) cannot be obtained.

[0070] According to an embodiment of the present application, the molar ratio of the photoinitiator to the intermediate compound shown in formula (IV) is (0.2-1):1. The above ratio is obtained by the inventors through a large number of experiments, and thus the compound shown in formula (I) can be effectively synthesized.

[0071] According to an embodiment of the present application, the final concentration of the intermediate compound shown in formula (IV) in the mixture of guanidine hydrochloride and the intermediate compound shown in formula (IV) is 3-5 mM.

[0072] According to an embodiment of the present application, the intermediate compound shown in formula (IV) is subjected to a purification treatment before the thiol-ene reaction is performed.

[0073] According to an embodiment of the present application, the purification treatment is performed by high performance liquid chromatography purification.

[0074] According to embodiments of the present application, the intermediate compound of formula (IV) obtained from the purification process is further subjected to a freeze-drying process.

[0075] It is understood by those skilled in the art that the features and advantages described above for the compound of formula (I) and the intermediate compound of formula (IV) also apply to the method for preparing the compound of formula (I), which will not be repeated here.

[0076] Pharmaceutical composition

[0077] In another aspect of the present application, a pharmaceutical composition is provided. According to embodiments of the present application, the pharmaceutical composition comprises: the aforementioned compound or the compound prepared according to the aforementioned method. The pharmaceutical composition of the present application is effective in promoting breast milk expression and preventing or treating induced labor bleeding and postpartum hemorrhage, and has a longer half-life and a lower EC 50 value.

[0078] According to embodiments of the present application, the composition further comprises: a pharmaceutically acceptable excipient. Any solvent, solid excipient, diluent or other liquid excipient, and the like, suitable for the particular intended dosage form is included. Except insofar as any conventional excipient is incompatible with the compound of the present application, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application. These pharmaceutical compositions can be prepared into various forms. For example, liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (e.g., injection solutions and infusion solutions). A typical pharmaceutical composition is an injection solution. The pharmaceutical composition can be administered by intravenous injection.

[0079] It is understood by those skilled in the art that the features and advantages described above for the compound of formula (I) and the method for preparing the compound of formula (I) also apply to the pharmaceutical composition, which will not be repeated here.

[0080] Method

[0081] In another aspect of the present application, a method for promoting breast milk expression is provided. According to embodiments of the present application, the method comprises: administering to a subject a pharmaceutically acceptable amount of the aforementioned compound of formula (I) or the aforementioned pharmaceutical composition. According to embodiments of the present application, the method is effective in promoting breast milk expression.

[0082] According to embodiments of the present application, the administration route of the method comprises intravenous injection.

[0083] It is understood by those skilled in the art that the features and advantages described above for the compound of formula (I), the method for preparing the compound of formula (I) and the pharmaceutical composition are also applicable to the method for promoting breast milk ejection, and thus will not be repeated here.

[0084] In another aspect of the present application, the present application provides a method for preventing or treating labor hemorrhage and postpartum hemorrhage. According to an embodiment of the present application, the method comprises: administering to a subject a pharmaceutically acceptable amount of the aforementioned compound of formula (I) or the aforementioned pharmaceutical composition. According to an embodiment of the present application, the method is effective in preventing or treating labor hemorrhage and postpartum hemorrhage.

[0085] According to an embodiment of the present application, the administration route of the method comprises intravenous injection.

[0086] It is understood by those skilled in the art that the features and advantages described above for the compound of formula (I), the method for preparing the compound of formula (I) and the pharmaceutical composition are also applicable to the method for preventing or treating labor hemorrhage and postpartum hemorrhage, and thus will not be repeated here.

[0087] Use

[0088] In another aspect of the present application, the present application provides a use of the aforementioned compound of formula (I), the compound prepared according to the aforementioned method or the aforementioned pharmaceutical composition in the preparation of a medicament for promoting breast milk ejection and preventing or treating labor hemorrhage and postpartum hemorrhage.

[0089] It is understood by those skilled in the art that the features and advantages described above for the compound of formula (I), the method for preparing the compound of formula (I) and the pharmaceutical composition are also applicable to the use, and thus will not be repeated here.

[0090] The schemes of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0091] Example 1: Preparation of a compound (i.e. oxytocin analogue)

[0092] I. Process for synthesizing intermediate compounds in Fmoc solid phase synthesis

[0093] The amino acid sequence of the intermediate compound to be prepared is: allyl glycine-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-CONH2.

[0094] Take 1300 mg of aminolysis resin (Rink amide AM resin, purchased from Tianjin Nankai Hesheng Technology Co., Ltd.) with a degree of substitution of 0.31 mmol / g (0.4 mmol) and place it in a polypeptide synthesis tube. Add 5 mL of N,N-dimethylformamide (DMF) and 5 mL of dichloromethane (DCM) and let it stand at room temperature for 30 min to swell the aminolysis resin. Add 10 mL of a 20% piperidine solution (20% piperidine solution is 100 mL of piperidine dissolved in 400 mL of DMF, with additional addition of 2-oxyma cyanohydrin (Oxyma) to a final concentration of 0.1 M) to the polypeptide synthesis tube to remove the Fmoc protecting group, and react at 33°C for two times, with reaction times of 5 min and 10 min respectively. Weigh 475 mg (1.6 mmol) of Fmoc-Gly-OH (glycine) and 628 mg (1.52 mmol) of condensing agent HCTU (i.e. 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate) into a 50 mL centrifuge tube, dissolve in 10 mL of DMF, then add 528 μL (3.2 mmol) of N,N-diisopropylethylamine (DIEA), and transfer the above obtained mixed solution to the polypeptide synthesis tube. Perform the amino acid coupling reaction under the condition of 33°C oscillation, with a reaction time of 1 h. The remaining amino acid sequence is Leu, Pro, Cys, Asn, Gin, Ile, Tyr and allyl glycine in order. Perform Fmoc deprotection and amino acid coupling for each amino acid in order according to the sequence of the remaining amino acid sequence. The coupling process weighs the Fmoc-protected amino acid, the condensing agent HCTU and the DIEA according to the addition amount of 4 equiv of the amino acid resin.

[0095] After the completion of solid-phase synthesis, 10 mL of cleavage reagent (prepared from 10 mL of trifluoroacetyl (TFA), 500 μL of H2O, 500 mg of phenol and 250 μL of Tips (triisopropylsilane, cas: 6485-79-6)) is added to the above-mentioned amino acid coupling reaction completed aminolysis resin, and then oscillated at 26°C for 2.5 h. Filter and collect the filtrate, and then use nitrogen to blow out at room temperature to obtain a cleavage solution. Concentrate the cleavage solution to less than 5 mL. Add an appropriate amount of ethyl ether to precipitate and centrifuge to collect the precipitate. Repeat the ethyl ether washing process once to obtain a crude peptide. Perform semi-preparative separation on the crude peptide using HPLC and then freeze-dry to obtain 128 mg of pure peptide, with a total yield of 32%.

[0096] Ⅱ. Photo-induced thiol-ene reaction of the compound (oxytocin analogue)

[0097] Take 50 mg of pure peptide and dissolve in 13 mL of guanidine hydrochloride solution with a concentration of 6 M (the final concentration of pure peptide is 3.85 mM), PH = 5. Add 5.6 mg of photoinitiator 2959 (the amount of photoinitiator 2959 added is 0.5 equiv of pure peptide) to the solution. After ultrasonic dissolution, place it in a UVP CL-1000L ultraviolet crosslinking instrument, and carry out a light irradiation reaction for 1 h under the condition of a light wavelength of 365 nm and a light intensity of 7.2 x 10 5 μJ / cm 2 . After the light irradiation reaction, the obtained solution is filtered by using a 0.22 μm biological membrane, and then is subjected to semi-preparative separation by using high performance liquid chromatography (HPLC) and is freeze-dried to obtain 15 mg of oxytocin analog, with a yield of 30%. The oxytocin analog is detected by using HPLC and ESI-MS. The preparation step of the oxytocin analog is shown in Figure 1 , and the detection result is shown in Figures 2-3 , wherein the letters in the circle in Figure 1 are the abbreviations of amino acids.

[0098] Example 2: Analysis of calcium flow experiment activity of oxytocin analog

[0099] 1. Preparation of detection solution:

[0100] 1) 250 mM probenecid (propenecid, Sigma-Aldrich, item # P8761). Probenecid has the effect of inhibiting the efflux of Ca 2+ fluorescent dye from cells, so as to ensure that the dye remains in the cells.

[0101] 2) Preparation of Flex buffer (also known as "Flex buffer"): 10 x HBSS, final concentration 1 x; 1 M Hepes, final concentration 20 nM; 1 M MgSO4, final concentration 1 mM; 1 M Na2CO3, final concentration 3.3 mM; 1 M CaCl2, final concentration 1.3 mM; 10% (w / v) BSA, final concentration 0.1%; 250 mM probenecid, final concentration 2.5 mM; adjust pH to 7.4 with 10 M HCl (about 90 μL).

[0102] 3) 20% (w / v) pluronic acid: dissolve 20 mg of pluronic acid (trade name Pluronic F-127, Sigma-Aldrich) in 100 μL of dimethyl sulfoxide (DMSO) and store at 4°C for later use. Pluronic acid is a surfactant that can prevent the aggregation of calcium ion fluorescent probe (Fluo-4 AM) dye in solution and help it enter the cells, and also can reduce the damage to cells caused by repeated replacement of culture medium.

[0103] 4) 1 mM Fluo-4 AM solution (about 8 mL needed for each 96-well plate), freshly prepared: 8 mL Flex buffer added with 8 mL 1 mM Fluo-4 AM solution and 4 mL 20% pluronic acid.

[0104] 2, HEK293T cell preparation:

[0105] 1) When the HEK293T cells in the dish reached 60% to 70% confluence, transfection was performed, and 7 pg of plasmid expressing oxytocin receptor (OTR, purchased from Jin Siree Biotechnology) was transfected.

[0106] 2) After 24 hours of transfection, the cells were transferred, i.e. the black 96-well plate was coated with 100 mL / well poly-lysine, then the cells were blown evenly with culture medium and counted, the cell density was adjusted with culture medium, and 60,000 cells / 100 mL / well were plated into the 96-well plate.

[0107] 3, Experimental process:

[0108] 1) Remove the cell culture medium from the 96-well plate containing HEK293T cells, add 150 mL Flex buffer to wash the cells, and then add 75 mL Fluo-4 AM solution to each well, incubate at 37°C in a 5% carbon dioxide cell incubator for 1 hour.

[0109] 2) The modified oxytocin analog was dissolved in Flex buffer to obtain a drug solution with a final concentration of 0.2 mM. Then it was distributed into the drug-added well plate, 50 mL of drug solution was added to each well of the cell, and a total of three wells. At the same time, the original oxytocin (amino acid sequence: Cys-Tyr-Ile-Gly-Asp-Cys-Pro-Leu-Gly, two Cys connected by disulfide bond) was treated in the same way and added to the three wells containing cells in the drug-added well plate as a control.

[0110] 3) After the Fluo-4 AM incubation of the cells was completed, the cells were washed with 150 mL Flex buffer, then 50 mL Flex buffer was added, and the cell plate, drug-added plate and gun head were placed into the Flexstation (Molecular Devices) machine, the real-time temperature in the machine was 37°C. After 20 seconds, 50 mL of two-fold concentrated drug solution was added by automatic sampler. The fluorescence group of the dye was excited by 485 nm fluorescence, and the emission light at 525 nm was recorded. Within 1 minute, the fluorescence value was recorded every 1.5 seconds, see Figure 4 .

[0111] After the experimental data were recorded, the half maximal effective concentration (EC50) of the oxytocin analog and oxytocin were obtained by GraphPad Prism processing, which were 56 nM and 68 nM, respectively. It was confirmed that the modified oxytocin had similar or slightly higher biological activity than the prototype. 50 ) respectively. It was confirmed that the modified oxytocin had similar or slightly higher biological activity than the prototype.

[0112] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0113] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A compound, which is a compound represented by formula (II) or a pharmaceutically acceptable salt of a compound represented by formula (II), 2. A method for preparing a compound represented by formula (II), characterized in that: Include: subjecting the intermediate compound represented by formula (IV) to a thiol-ene reaction to obtain the compound represented by formula (II); wherein R2 is -SH, R3 is -CH=CH2, or R3 is -SH, R2 is -CH=CH2.

3. The method according to claim 2, characterized in that The intermediate compound represented by formula (IV) is obtained by solid phase synthesis.

4. The method according to claim 3, characterized in that The solid phase synthesis method is selected from the Fmoc solid phase synthesis method.

5. The method according to any one of claims 2 to 4, characterized in that The thiol-ene reaction comprises: The intermediate compound represented by formula (IV) is subjected to light treatment in the presence of guanidine hydrochloride and a photoinitiator.

6. The method according to claim 5, characterized in that The light treatment is carried out at a wavelength of 365 nm and a light intensity of (5-8)×10 5 μJ / cm 2 The experiment was carried out under the light conditions for 0.5h-2h.

7. The method according to claim 5, characterized in that The photoinitiator is selected from 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

8. The method according to claim 5, characterized in that The molar ratio of the photoinitiator to the intermediate compound represented by formula (IV) is (0.2-1):

1.

9. The method according to claim 5, characterized in that The final concentration of the intermediate compound represented by formula (IV) in the mixed solution of guanidine hydrochloride and the intermediate compound represented by formula (IV) is 3-5 mM.

10. The method according to claim 2, characterized in that Before the thiol-ene reaction is carried out, the intermediate compound represented by formula (IV) is purified in advance.

11. The method according to claim 10, characterized in that The purification process is carried out by high performance liquid chromatography.

12. The method according to claim 11, characterized in that The intermediate compound represented by formula (IV) obtained by the purification treatment is further freeze-dried.

13. A pharmaceutical composition, characterized in that Include: The compound according to claim 1 or the compound prepared by the method according to any one of claims 2 to 12.

14. The pharmaceutical composition according to claim 13, characterized in that The composition further comprises: Pharmaceutically acceptable excipients.

15. Use of the compound according to claim 1, the compound prepared according to the method according to any one of claims 2 to 12, or the pharmaceutical composition according to any one of claims 13 to 14 in the preparation of a medicament for preventing or treating induced labor and postpartum hemorrhage or promoting breast milk secretion.

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