CN115093461A

CN115093461A - Intermediate compound, preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN115093461A
Application number: CN202210488766.3A
Authority: CN
Inventors: 陈晨晨; 朱新旺
Original assignee: Hefei Jing Peptide Biological Technology Co ltd
Current assignee: Hefei Jing Peptide Biological Technology Co ltd
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2022-09-23

Abstract

The invention relates to an intermediate compound for synthesizing a novel oxytocin analogue, and a preparation method, a pharmaceutical composition and application thereof. The intermediate compound has a structure shown in a formula (IV); oxytocin compoundsThe analogue is a compound with a structure shown in a formula (I); the preparation method of the compound comprises the following steps: carrying out a thiol-ene reaction on an intermediate compound shown as a formula (IV) so as to obtain a compound shown as a formula (I); the pharmaceutical composition comprises a compound shown as a formula (I). The compound of the invention can effectively promote mammary gland milk secretion and prevent or treat induced labor bleeding and postpartum bleeding, and has longer half-life period and lower EC ₅₀ The value is obtained.

Description

Intermediate compound, preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to an intermediate compound, a preparation method of the intermediate compound, a pharmaceutical composition and application of the intermediate compound and the compound.

Background

Oxytocin is polypeptide hormone secreted by hypothalamus in mammals, and comprises nine amino acid residues and a pair of disulfide bonds. It is involved in many physiological processes such as neurotransmission in the central nervous system, contraction of smooth muscle in the breast and uterus, tumor growth in the breast, and autocrine and paracrine secretion of the ovary. The current oxytocin and analog products thereof have short existing half-life or EC ₅₀ Too large value and even no effect.

Therefore, there is a need for a novel oxytocin analog product.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems of the prior art at least to a certain extent. Therefore, the invention provides an intermediate compound for synthesizing a novel oxytocin analogue, a preparation method, a pharmaceutical composition and application thereof, wherein the oxytocin analogue has longer half-life period and lower EC ₅₀ The value is obtained.

The present invention has been completed based on the following findings of the inventors:

oxytocin is mainly applied to induction of labor and postpartum hemorrhage control in clinic, but has the defect of short half-life (2-5 minutes). Disulfide bonds play a crucial role in maintaining the rigidity and physiological activity of oxytocin structures, but disulfide bonds themselves are susceptible to reductive substances in the body (e.g. free sulfhydryl groups), thereby directly affecting the half-life of oxytocin. Thus, modification of disulfide modifications is one of the important modification directions for oxytocin. According to the literature, the disulfide bond of oxytocin is replaced by a C-C bond through olefin metathesis reaction, the modified compound (namely the oxytocin analogue) can greatly increase the half life (5-60 times), but the EC of the oxytocin analogue containing the C-C bond ₅₀ The value increases by three orders of magnitude with no effect. In addition, the S-S bond is transformed into the thioether bond which is a relatively popular disulfide bond transformation strategy, and the thioether bond is close to biochemical and geometric parameters such as dihedral angle, covalent radius and the like, so that the thioether bond hasHigher stability. The literature reports at present that one S atom of a disulfide bond of oxytocin is directly replaced by a C atom to obtain an oxytocin analogue containing a thioether bond, but the results show that the modification can increase the half-life period of the oxytocin analogue, but has an influence on the activity of the oxytocin analogue, so that the EC of the oxytocin analogue is greatly increased ₅₀ The value is obtained.

However, the present inventors have found through a large number of experiments that the side chain-SH of one of the cysteines in oxytocin is replaced by-R ₄ -CH＝CH ₂ (e.g. R) ₄ Is none or C ₁ -C ₅ Alkylene) which can be linked to the thiol group of another cysteine in a thiolane reaction to form a thioether bond and which can modify the-S-bond of the pro-oxytocin to-R ₁ -CH ₂ -S- (e.g. R) ₁ Is C ₁ -C ₆ Alkylene) and the modified compound (i.e., oxytocin analog) has a longer half-life while maintaining the original activity, the EC of the compound ₅₀ EC of value and oxytocin ₅₀ The values are comparable or even lower.

Based on this, in one aspect of the invention, the invention provides a compound which is a compound represented by formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt of the compound represented by formula (I),

wherein X is-S-CH ₂ -R ₁ -，R ₁ Is C ₁ -C ₆ An alkylene group.

The inventor obtains the compound as a novel oxytocin analogue through a great deal of experiments, and the oxytocin analogue has longer half-life period and lower EC ₅₀ The composition can effectively promote mammary gland milk excretion and prevent or treat induced labor and postpartum hemorrhage.

In yet another aspect of the invention, the invention provides an intermediate compound. According to an embodiment of the invention, the intermediate compound has a structure represented by formula (IV),

wherein R is ₂ is-SH, R ₃ is-R ₄ -CH＝CH ₂ Or R is ₃ is-SH, R ₂ is-R ₄ -CH＝CH ₂ ；R ₄ Is none or C ₁ -C ₅ An alkylene group.

The intermediate compound of the invention can be used for synthesizing the oxytocin analogue shown in the formula (I) in the specification.

In another aspect of the invention, the invention provides a process for preparing a compound of formula (I). According to an embodiment of the invention, the method comprises: carrying out a thiol-ene reaction on an intermediate compound shown as a formula (IV) so as to obtain a compound shown as a formula (I);

wherein X is-S-CH ₂ -R ₁ -；R ₁ Is C ₁ -C ₆ An alkylene group; r ₂ is-SH, R ₃ is-R ₄ -CH＝CH ₂ Or R is ₃ is-SH, R ₂ is-R ₄ -CH＝CH ₂ ；R ₄ Is none or C ₁ -C ₅ An alkylene group.

The inventor finds out through a large number of experiments that the oxytocin analogue shown in the formula (I) can be effectively synthesized by adopting the method, can effectively promote mammary gland lactation and prevent or treat induced labor and postpartum hemorrhage, and has a simple preparation method.

In another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the composition comprises: a compound as hereinbefore described or a compound prepared according to the process as hereinbefore described. The pharmaceutical composition of the invention has longer half-life and lower EC ₅₀ The composition can effectively promote mammary gland milk excretion and prevent or treat induced labor and postpartum hemorrhage.

In another aspect of the invention, the invention provides the use of a compound as described above, a compound prepared according to a process as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for use in promoting lactation of the breast and in the prevention or treatment of labour induction and postpartum haemorrhage.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic diagram of the steps for preparing oxytocin analogs in accordance with example 1 of the present invention;

FIG. 2 is an HPLC analysis chart of oxytocin analogue in example 1 of the present invention;

FIG. 3 is an ESI-MS mass spectrum of an oxytocin analogue in example 1 of the present invention;

FIG. 4 shows the EC of oxytocin analogs and oxytocin of example 2 of the present invention ₅₀ The value is obtained.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to encompass values close to these ranges or values. For numerical ranges, each range between the endpoints and the individual points, and each individual point can be combined with each other to give one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In this document, the terms "comprise" or "comprise" are open-ended expressions that include the meaning of the present invention, but do not exclude other aspects.

As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.

As used herein, the term "alkylene" refers to a straight or branched chain saturated divalent alkane group. Including but not limited to methylene (-CH) ₂ -) ethylene (-CH ₂ CH ₂ -) propylene (-CH) ₂ CH ₂ CH ₂ -) isopropylidene (-CH ₂ (CH ₃ )CH ₂ -)。

Herein, the term "C ₁ -C _n "refers to an alkyl group having a limited number of carbon atoms from 1 to n, for example 1, 2, 3, 4, 5 or 6 carbon atoms. It is further understood that the term "C ₁ -C ₆ "should be interpreted as including any sub-range therein, such as C ₁ -C ₆ 、C ₂ -C ₅ 、C ₃ -C ₄ 、C ₁ -C ₂ 、C ₁ -C ₃ 、C ₁ -C ₄ 、C ₁ -C ₅ (ii) a Especially C ₁ -C ₂ 、C ₁ -C ₃ 。

In this context, the compounds of the invention may be preferred compounds. The term "preferred compound" refers to a compound that results in a more desirable biological activity, e.g., a stereoisomer, tautomer, or pharmaceutically acceptable salt of the compound. Illustratively, isolated, purified, or partially purified stereoisomers of the compounds of the invention are also included within the scope of the invention. Purification and isolation of such materials can be accomplished by standard techniques known in the art.

It is to be noted that the compounds of the invention may also include all possible stereoisomers of the compounds of the invention, either as a single stereoisomer, or in any proportion of said stereoisomer, for example the (R) or (S) isomer, or any mixture of the (E) or (Z) isomers. Using any suitable art-described method, for example: chromatography, especially chiral chromatography, can effect the separation of individual stereoisomers of the compounds of the invention, for example: separation of a single enantiomer or a single diastereomer.

It is to be noted that the compounds of the invention may also include all possible tautomers of the compounds of the invention, as single tautomers, or any mixtures of said tautomers in any ratio.

As used herein, the term "pharmaceutically acceptable salt" refers to a compound or stereoisomer thereof, as well as acid and/or base salts formed with relatively non-toxic inorganic and/or organic acids and bases of the compound or stereoisomer thereof, and also includes zwitterionic (inner) salts, and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound or a stereoisomer thereof may be obtained by appropriately (e.g., equivalently) mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound. See, e.g., S.M.Berge et al, "Pharmaceutical Salts" J.pharm.Sci.1977, 66, 1-19.

The term "pharmaceutical composition" as used herein generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the adjuvants which constitute one or more accessory ingredients. In general, compositions are prepared by uniformly and sufficiently combining the active compound with liquid excipients, finely divided solid excipients, or both.

As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to those approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" can include any solvent, solid excipient, diluent, or other liquid excipient, and the like, suitable for the particular intended dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention.

Reference is made to the large literature on this subject for other pharmaceutically acceptable adjuvants mentioned herein and their processes, see in particular Handbook of Pharmaceutical Excipients, 3 rd edition, edited by Arthur h.kibbe, American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and Lexikon der Hilfsstuffe fur Pharmazie, Kosmetik and angrenzene Gebiete, edited by H.P. Fiedler, 4 th edition, Cantor, Aulentorf and earlier versions.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The fusion protein or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered by intravenous injection.

As used herein, the term "treating" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a compound described herein to an individual in need thereof.

The present invention provides an intermediate compound, a preparation method thereof, a pharmaceutical composition and a use thereof, which will be described in detail below.

Compound (I)

In one aspect of the invention, the invention provides a compound which is a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt of a compound of formula (I).

Wherein X is-S-CH ₂ -R ₁ -，R ₁ Is C ₁ -C ₆ An alkylene group.

Research shows that one S atom of disulfide bond of the original compound (oxytocin) is replaced by C atom, so that thioether bond is replaced by disulfide bond to form new compound (oxytocin analogue), and the new compound has long half life period and obviously reduced activityLow, greatly improves EC ₅₀ The value is obtained. Based on this, the inventors have found through experiments that the number of atoms at the thioether bond has an influence on the activity of the compound, and thus, the inventors have obtained the above compound through a large number of experiments, which has a long half-life and a low EC ₅₀ The composition can effectively promote mammary gland milk excretion and prevent or treat induced labor and postpartum hemorrhage.

According to an embodiment of the invention, R ₁ Is methylene, ethylene, propylene or isopropylene. The inventor finds through experiments that when the side chain-SH of cysteine is replaced by an alkenyl structure, the number of atoms of the alkenyl structure can influence the activity of the compound when R ₁ When the compound is a methylene group, an ethylene group, a propylene group or an isopropylene group, the activity of the resulting compound is good, and R is preferably R ₁ Is a methylene group.

According to an embodiment of the invention, the compound has a structure represented by formula (II) or formula (III),

the inventors have found that substitution of ethylene for one of the S atoms of oxytocin results in an oxytocin analogue as described above which has an increased half-life and which substantially retains the activity of oxytocin and which even further increases the activity of oxytocin.

Intermediate compound

wherein R is ₂ And R ₃ Each is independently selected from-R ₄ -CH＝CH ₂ or-SH, and R ₂ And R ₃ Must be one of-SH, R ₄ Is none or C ₁ -C ₅ An alkylene group.

Intermediates of the inventionThe compounds can be used for synthesizing the aforementioned compounds, so that the synthesized compounds have longer half-life and lower EC ₅₀ The composition can effectively promote mammary gland milk excretion and prevent or treat induced labor hemorrhage and postpartum hemorrhage.

According to an embodiment of the invention, R ₂ is-SH, R ₃ is-CH ═ CH ₂ Or R is ₃ is-SH, R ₂ is-CH ═ CH ₂ 。

Process for preparing compounds of formula (I)

In another aspect of the invention, the invention provides a process for preparing a compound of formula (I). According to an embodiment of the invention, the method comprises: carrying out a thiol alkene reaction on an intermediate compound shown in a formula (IV) so as to obtain a compound shown in a formula (I);

wherein X is-S-CH ₂ -R ₁ -，R ₁ Is C ₁ -C ₆ An alkylene group; r ₂ And R ₃ Each is independently selected from-R ₄ -CH＝CH ₂ or-SH, and R ₂ And R ₃ Must be-SH, R ₄ Is none or C ₁ -C ₅ An alkylene group.

The inventor finds that the compound shown in the formula (I) can be effectively synthesized by adopting the method through a large number of experiments, the compound can effectively promote mammary gland to discharge milk and prevent or treat induced labor and postpartum hemorrhage, and the preparation method is simple.

According to an embodiment of the invention, R ₁ Is methylene, ethylene, propylene or isopropylene, preferably methylene.

According to an embodiment of the present invention, the intermediate compound represented by formula (IV) is obtained by a solid phase synthesis method.

According to an embodiment of the present invention, the solid phase synthesis method is selected from Fmoc solid phase synthesis methods.

According to an embodiment of the invention, the thiol-ene reaction comprises: and (3) carrying out light treatment on the intermediate compound shown in the formula (IV) in the presence of guanidine hydrochloride and a photoinitiator.

According to an embodiment of the present invention, the light treatment is performed at a light wavelength of 365nm and a light intensity of (5-8). times.10 ⁵ μJ/cm ² Is carried out for 0.5h to 2h under the illumination condition. The present inventors have conducted extensive experiments to obtain the above-mentioned light treatment conditions, whereby the compound represented by the formula (I) can be efficiently synthesized.

According to an embodiment of the present invention, the photoinitiator is selected from 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (i.e., photoinitiator 2959). The inventors screened the above photoinitiator through a large number of experiments, and thus, the compound represented by formula (I) was efficiently synthesized. The inventors have found through experiments that when a compound represented by the formula (I) is synthesized using photoinitiators such as Dimethylaminopyridine (DMAP), an initiator VA-044, sodium anthraquinone sulfonate and a photoinitiator 819, it is found that the compound represented by the formula (I) cannot be obtained by using all the photoinitiators to form a dimer through a disulfide bond or directly without reaction.

According to an embodiment of the invention, the molar ratio of the photoinitiator to the intermediate compound of formula (IV) is (0.2-1): 1. the inventors have obtained the above-mentioned ratio through a large number of experiments, and thus, the compound represented by the formula (I) can be efficiently synthesized.

According to an embodiment of the present invention, the final concentration of the intermediate compound represented by formula (IV) in the mixture of guanidine hydrochloride and the intermediate compound represented by formula (IV) is 3-5 mM.

According to an embodiment of the present invention, the intermediate compound represented by formula (IV) is subjected to a purification treatment in advance before the thiol-ene reaction is performed.

According to an embodiment of the present invention, the purification treatment is performed using high performance liquid chromatography purification.

According to the embodiment of the invention, the intermediate compound shown in the formula (IV) obtained by the purification treatment is further subjected to freeze-drying treatment.

It will be appreciated by those skilled in the art that the features and advantages described above for the compounds of formula (I) and the intermediate compounds of formula (IV) apply equally to the process for the preparation of the compounds of formula (I) and will not be described in detail here.

Pharmaceutical composition

In another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: a compound as hereinbefore described or a compound prepared according to the process as hereinbefore described. The pharmaceutical composition can effectively promote mammary gland milk secretion and prevent or treat induced labor bleeding and postpartum bleeding, and has longer half-life period and lower EC ₅₀ The value is obtained.

According to an embodiment of the invention, the composition further comprises: pharmaceutically acceptable adjuvants. Including any solvent, solid excipient, diluent, or other liquid excipient, etc., as appropriate for the particular intended dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention. These pharmaceutical compositions can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including, but not limited to, liquid solutions (e.g., injection solutions and infusion solutions). Typical pharmaceutical compositions are injectable solutions. The pharmaceutical composition may be administered by intravenous injection.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the compounds of formula (I) and the process for preparing the compounds of formula (I) apply equally to the pharmaceutical composition and will not be described in detail here.

Method

In another aspect of the invention, a method of promoting breast milk production is provided. According to an embodiment of the invention, the method comprises: administering to the subject a pharmaceutically acceptable amount of a compound of formula (I) or a pharmaceutical composition of the foregoing. According to the embodiment of the invention, the method can effectively promote the mammary gland to discharge milk.

According to an embodiment of the invention, the route of administration of the method comprises intravenous injection.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the compounds of formula (I), the process for preparing the compounds of formula (I) and the pharmaceutical compositions are equally applicable to the method of promoting breast milk excretion and will not be described in detail herein.

In another aspect of the invention, the invention features a method of preventing or treating induced and postpartum hemorrhage. According to an embodiment of the invention, the method comprises: administering to the subject a pharmaceutically acceptable amount of a compound of formula (I) or a pharmaceutical composition of the foregoing. According to embodiments of the present invention, the method is effective for preventing or treating induced labor bleeding and postpartum bleeding.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the compounds of formula (I), the process for preparing the compounds of formula (I) and the pharmaceutical compositions are equally applicable to the method for preventing or treating induced and post-partum bleeding and will not be described in detail herein.

Use of

In another aspect of the invention, the invention provides the use of a compound of formula (I) as described above, a compound prepared according to the above method or a pharmaceutical composition as described above in the manufacture of a medicament for promoting mammary gland milk excretion and preventing or treating induced and postpartum hemorrhage.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the compounds of formula (I), the process for preparing the compounds of formula (I) and the pharmaceutical compositions are equally applicable to this use and will not be described in detail herein.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and manufacturers are not indicated.

Example 1: preparation of compounds (i.e. oxytocin analogues)

Fmoc solid phase synthesis of intermediate compounds

The amino acid sequence of the intermediate compound to be prepared is: allyl glycine-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-CONH ₂ 。

1300mg of an amino resin (Rink amide AM resin, available from Tianjin Nankai science and technology Co., Ltd.) having a degree of substitution of 0.31mmol/g (0.4mmol) was weighed into a polypeptide synthesis tube, 5mL of N, N-Dimethylformamide (DMF) and 5mL of Dichloromethane (DCM) were added, and the amino resin was allowed to swell at room temperature for 30 min. 10mL of 20% piperidine solution (20% piperidine solution is 100mL piperidine dissolved in 400mL DMF, and 2-oxime ethyl cyanoacetate (Oxyma) is additionally added to the final concentration of 0.1M) is added into a polypeptide synthesis tube to remove Fmoc protecting groups, and the reaction is carried out twice at 33 ℃, wherein the reaction time of the two times is 5min and 10min respectively. 475mg (1.6mmol) of Fmoc-Gly-OH (glycine) and 628mg (1.52mmol) of condensing agent HCTU (namely 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate) are weighed and put into a 50mL centrifuge tube, 10mL of DMF is dissolved, 528 mu L (3.2mmol) of N, N-Diisopropylethylamine (DIEA) is added, the obtained mixed solution is transferred into a polypeptide synthesis tube, and amino acid coupling reaction is carried out under the condition of oscillation at 33 ℃, and the reaction time is 1 h. The sequence of the rest amino acid sequence is Leu, Pro, Cys, Asn, Gln, Ile, Tyr and allyl glycine, and each amino acid is subjected to Fmoc deprotection and amino acid coupling processes in turn according to the sequence of the rest amino acid sequence. Coupling procedure 4equiv of Fmoc protected amino acid, 3.8equiv of condensing agent HCTU and 8equiv of DIEA were weighed as added amounts to the amino resin.

After completion of solid phase synthesis, 10mL of cleavage reagent (from 10mL of Trifluoroacetyl (TFA), 500. mu. L H) ₂ O, 500mg phenol and 250. mu.L Tips (triisopropylsilane, cas: 6485-79-6) were added to the aboveAfter the amino acid coupling reaction is completed, the reaction is carried out for 2.5h under the condition of 26 ℃ by shaking. The filtrate was filtered, and the resulting lysate was bubbled with nitrogen at room temperature, and the lysate was concentrated to 5mL or less. Adding a proper amount of glacial ethyl ether for precipitation, centrifuging and collecting the precipitate, and repeating the process of washing with glacial ethyl ether once to obtain the crude peptide. Semi-preparative separation of the crude peptide using HPLC followed by lyophilization gave 128mg of pure peptide in 32% overall yield.

Photoinitiated thiol-ene reaction of Compounds (oxytocin analogs)

50mg of pure peptide were weighed out and dissolved in 13mL of 6M guanidinium hydrochloride solution (final concentration of pure peptide 3.85mM) at pH 5. To the solution was added 5.6mg of photoinitiator 2959 (the amount of photoinitiator 2959 added was 0.5equiv of the pure peptide). Dissolving with ultrasound, placing into UVP CL-1000L ultraviolet crosslinking instrument, and irradiating at 365nm with light intensity of 7.2 × 10 ⁵ μJ/cm ² The light reaction was carried out for 1 h. Filtering the solution obtained after the light reaction by using a 0.22-micron biomembrane, performing semi-preparative separation by using High Performance Liquid Chromatography (HPLC), and freeze-drying to obtain 15mg of oxytocin analogue with the yield of 30%, and detecting the obtained oxytocin analogue by using HPLC and ESI-MS. The preparation steps of the oxytocin analogue are shown in figure 1, the detection results are shown in figures 2-3, wherein letters in circles in figure 1 are abbreviations of amino acids.

Example 2: calcium flux assay Activity assay for oxytocin analogs

1. Preparation of detection solution:

1)250mM probenecid (probenecid, sigma-Aldrich, Cat # P8761). Probenecid has the function of inhibiting cell pair Ca ²⁺ The efflux of the fluorescent dye ensures that the dye is retained in the cell.

2) Flex buffer (also known as Flex buffer) preparation: 10 × HBSS, final concentration l ×; 1M Hepes, final concentration 20 nM; 1M MgSO ₄ 1mM final concentration; 1M Na ₂ CO ₃ Final concentration 3.3 mM; 1M CaCl ₂ Final concentration 1.3 mM; 10% (w/v) BSA, final concentration 0.1%; 250mM probenecid with a final concentration of 2.5 mM; the pH was adjusted to 7.4 with 10M HCl (about 90. mu.L).

3) 20% (w/v) pluronic acid: each 20mg of Pluronic acid (trade name, Pluronic F-127, Sigma-Aldrich) was dissolved in 100. mu.L of dimethyl sulfoxide (DMSO) and stored at 4 ℃ until use. Pluronic acid is a surfactant that prevents the calcium ion fluorescent probe (Fluo-4 AM) dye from polymerizing in solution and helps it enter the cells through the cell membrane, and also reduces the damage to the cells caused by multiple changes of medium.

4)1 μ M Fluo-4AM solution (approximately 8mL per 96 well plate) was prepared as: to 8mL Flex buffer was added 8. mu.L of 1mM Fluo-4AM solution and 4. mu.L of 20% pluronic acid.

2. HEK293T cell preparation:

1) when HEK293T cells were grown to 60% to 70% confluence in the petri dish, transfection was performed to transfect 7 μ g of a plasmid expressing oxytocin receptor (OTR, purchased from kasei biotechnology).

2) After 24 hours of transfection, the cells were spread on a black 96-well plate by coating with 100. mu.L/well poly-lysine, then the cells were blown up evenly and counted in a medium, the cell density was adjusted with the medium and spread on a 96-well plate at 6 ten thousand cells/100. mu.L/well.

3. The experimental process comprises the following steps:

1) the cell culture medium was removed from the cells in HEK 293T-containing 96-well plates, 150. mu.L of Flex buffer was added to each well to wash the cells, 75. mu.L of Fluo-4AM solution was added to each well after aspiration, and the cells were incubated for 1 hour at 37 ℃ in a 5% carbon dioxide cell incubator.

2) The modified oxytocin analogue was dissolved using Flex buffer to obtain a drug solution with a final concentration of 0.2mM of oxytocin analogue. Then dispensed into a drug-loaded well plate, 50 μ Ι _ of drug solution per well cell, for a total of three wells. Meanwhile, prototype oxytocin (the amino acid sequence is Cys-Tyr-Ile-Gly-Asp-Cys-Pro-Leu-Gly, two Cys are connected through disulfide bonds) is added into three holes containing cells in the dosing orifice plate as a control after being treated in the same way.

3) After incubation of cells with Fluo-4AM was complete, the cells were washed with 150. mu.L Flex buffer, 50. mu.L Flex buffer was added after aspiration, and the cell plate, drug plate and tip were placed in a Flexstation (molecular devices) machine at a real time temperature of 37 ℃. After 20 seconds 50. mu.L of a double-concentrated drug solution was added by an autosampler. The dye fluorophore was excited to emit light by 485nm fluorescence and the emission at 525nm was recorded. Fluorescence values were recorded every 1.5s for 1 minute, see in particular FIG. 4.

After recording the experimental data, the half maximal Effect Concentrations (EC) of oxytocin analogue and oxytocin were obtained after GraphPad prism treatment ₅₀ ) 56nM and 68nM, respectively. The modified oxytocin is proved to have the biological activity similar to or slightly higher than that of the prototype.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A compound which is a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt of a compound of formula (I),

wherein X is-S-CH ₂ -R ₁ -，R ₁ Is C ₁ -C ₆ An alkylene group.

2. A compound of claim 1, wherein R is ₁ Is methylene, ethylene, propylene or isopropylene.

3. The compound of claim 1, wherein the compound has a structure represented by formula (II) or formula (III),

4. an intermediate compound characterized by having a structure represented by the formula (IV),

wherein R is ₂ is-SH, R ₃ is-R ₄ -CH＝CH ₂ Or R is ₃ is-SH, R ₂ is-R ₄ -CH＝CH ₂ ；

R ₄ Is absent or C ₁ -C ₅ An alkylene group.

5. A compound of claim 4, wherein R is ₂ is-SH, R ₃ is-CH ═ CH ₂ Or R is ₃ is-SH, R ₂ is-CH ═ CH ₂ 。

6. A process for preparing a compound of formula (I), comprising:

carrying out thiol-ene reaction on the intermediate compound shown in the formula (IV) so as to obtain the compound shown in the formula (I);

wherein X is-S-CH ₂ -R ₁ -；

R ₁ Is C ₁ -C ₆ An alkylene group;

R ₂ is-SH, R ₃ is-R ₄ -CH＝CH ₂ Or R is ₃ is-SH, R ₂ is-R ₄ -CH＝CH ₂ ；

R ₄ Is absent or C ₁ -C ₅ An alkylene group.

7. The method of claim 6, wherein R is ₁ Is methylene, ethylene, propylene or isopropylene;

optionally, R ₂ is-SH, R ₃ is-CH ═ CH ₂ Or R is ₃ is-SH, R ₂ is-CH ═ CH ₂ ；

Optionally, the intermediate compound shown in the formula (IV) is obtained by a solid-phase synthesis method;

optionally, the solid phase synthesis method is selected from Fmoc solid phase synthesis methods.

8. The method according to claim 6 or 7, wherein the thiol-ene reaction comprises:

subjecting the intermediate compound shown in the formula (IV) to light treatment in the presence of guanidine hydrochloride and a photoinitiator;

optionally, the light treatment is at a wavelength of 365nm and a light intensity of (5-8). times.10 ⁵ μJ/cm ² Under the illumination condition for 0.5h-2 h;

optionally, the photoinitiator is selected from 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone;

optionally, the molar ratio of the photoinitiator to the intermediate compound of formula (IV) is (0.2-1): 1;

optionally, the final concentration of the intermediate compound of formula (IV) in the mixture of guanidine hydrochloride and the intermediate compound of formula (IV) is 3-5 mM;

optionally, before the thiol-ene reaction, purifying the intermediate compound represented by the formula (IV) in advance;

optionally, the purification treatment is performed by high performance liquid chromatography purification;

optionally, the intermediate compound shown in the formula (IV) obtained by the purification treatment is further subjected to a freeze-drying treatment.

9. A pharmaceutical composition, comprising:

a compound according to any one of claims 1 to 3 or a compound prepared according to the process of any one of claims 6 to 8;

optionally, the composition further comprises:

pharmaceutically acceptable adjuvants.

10. Use of a compound according to any one of claims 1 to 3, a compound prepared by a process according to any one of claims 6 to 8 or a pharmaceutical composition according to claim 9 in the manufacture of a medicament for the prevention or treatment of induction of labour and post-partum bleeding or for the promotion of mammary gland milk production.