WO2022142534A1

WO2022142534A1 - Tetrazine compound, preparation method therefor, and application thereof

Info

Publication number: WO2022142534A1
Application number: PCT/CN2021/120831
Authority: WO
Inventors: 吴昊星; 毛梧宇; 孙洪宝; 贺欣羽; 杨豪杰
Original assignee: 四川大学华西医院
Priority date: 2020-12-31
Filing date: 2021-09-27
Publication date: 2022-07-07
Also published as: CN112812074B; CN112812074A

Abstract

Disclosed in the present invention are a tetrazine compound, a preparation method therefor, and an application thereof. The tetrazine compound provided by the present invention can be used for implementing selective tetrazine labeling of a mercapto-containing polypeptide, protein and biological material, and can be used for rapid, efficient and stable function derivatization of a substance having a tetrazine mark by using a bioorthogonal reaction. In addition, the tetrazine compound has high selectivity, when other functional groups are enriched in a body, the tetrazine compound can selectively bind to cysteine to generate a stable linked product, while no by-product is detected, and after formation of a linked product with mercapto, reaction power of dienophile and the linked product is rapid, which is significantly faster than a manner of using thiotetrazine as a tetrazine marker in the prior art, indicating that the tetrazine compound can be applied to in-vivo bioorthogonal pre-targeting and radiolabeling by a nanomolar concentration.

Description

Tetrazine compounds and preparation method and application thereof

technical field

The present invention relates to the field of tetrazine labeling, in particular to tetrazine compounds used for selective tetrazine labeling of thiol-containing amino acids, polypeptides, proteins and biological materials, as well as preparation methods and applications of the tetrazine compounds.

Background technique

Bioconjugation of proteins with cytotoxic drugs, imaging agents, and small molecule probes, bionanomaterials, etc., have become an attractive strategy for targeted chemotherapy, molecular imaging, and chemical biology. Because inappropriate protein modifications can have a major impact on protein tertiary structure and downstream activities, such as biodistribution, affinity, and drug release rate. To address this problem, several practical chemical and regioselective biological conjugation methods have been developed, which can achieve site-specific coupling and control the loading. In recent years, studies have reported that uniform and stable protein conjugates can be produced by methods such as enzymatic ligation, gene coding, and novel labeling reagents. If this strategy is combined with modular bioorthogonal chemistry, the protein can be further modified for more diverse biomedical applications.

Bioorthogonal probes based on tetrazine compounds are ideal bioconjugation labeling reagents because they react rapidly with dienophilic reagents and can be functionalized in various ways. At present, some studies have reported that tetrazine compounds are incorporated into E. coli proteins as an unnatural amino acid through tyrosyl-tRNA synthetase; other studies have reported that dichlorotetrazine is bound to the two proximal cysteine residues of the protein. Reversible protein labeling can be achieved through aromatic nucleophilic substitution reactions.

Due to the urgent need to selectively introduce reactive and functionalized bioorthogonal labels into proteins through simpler molecular biology techniques, tetrazine compounds have the potential to specifically label amino acids, polypeptides, and proteins. High research value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide tetrazine compounds, preparation methods and applications thereof, to realize selective tetrazine labeling of amino acids, polypeptides, proteins and biological materials containing sulfhydryl groups, and to use bioorthogonal reactions to label biological materials with tetrazine labels. Fast, efficient and stable functional derivatization application of functional molecules, so as to solve the selectivity of labeling reagents for labeling thiol-containing amino acids, polypeptides, proteins, and thiol-containing biological materials, drugs, and functional imaging molecules in the prior art Poor, difficult to functionalize biomolecules in the later stage, poor stability of coupling products, and great influence on the activity of biomolecules themselves.

The above purpose is achieved through the following technical solutions:

Tetrazine compound, its structural formula is shown in formula I:

In formula I, R is selected from hydrogen, alkyl, heteroatom-substituted alkyl, heterocycloalkyl, aldehyde, ketone, aryl, heteroaryl, alkenyl, ester, aminoacyl or polyethylene glycol group; R' is selected from substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl.

In this technical solution, the tetrazine compound is 1,2,4,5-tetrazine substituted at the 3-position and the 6-position, and has the general structural formula shown in formula I. Among them, there are many kinds of substituents that R group can choose, in addition to the aforementioned hydrogen, alkyl, heterocycloalkyl, aldehyde group, ketone group, aryl group, heteroaryl group, ester group, aminoacyl and polyethylene glycol In addition to the group, other substituents can also be selected.

In some embodiments, the R group is a substituted or unsubstituted chain alkyl, or a substituted or unsubstituted cyclic alkyl. The chain alkyl group may be either a straight chain alkyl group or a branched chain alkyl group, and the number of carbon atoms of the chain alkyl group is preferably C ₁ -C ₃₆ , more preferably C ₁ -C ₁₂ , still more preferably are C ₁ to C ₄ . In one or more embodiments, the chain alkyl is methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl. The number of carbon atoms of the cyclic alkyl group is preferably C ₄ to C ₃₆ , more preferably C ₄ to C ₁₂ , and still more preferably C ₄ to C ₆ . In one or more embodiments, the cyclic alkyl group is cyclobutyl, cyclopentyl, or cyclohexyl.

In some embodiments, the R group is a substituted or unsubstituted heterocycloalkyl, the heteroatom of the heterocycloalkyl can be sulfur, oxygen or nitrogen, and the number of carbon atoms of the heterocycloalkyl is preferably C ₄ - C ₁₂ , more preferably C ₄ to C ₅ . In one or more embodiments, the heterocycloalkyl group is pyrrolidinyl, azetidinyl, oxolane, or thiacyclopentyl.

In some embodiments, the R group is a substituted or unsubstituted aldehyde group, and the carbon number of the aldehyde group is preferably C ₁ -C ₃₆ , more preferably C ₁ -C ₂₄ , still more preferably C ₁ -C C ₄ . In one or more embodiments, the aldehyde group is formaldehyde, acetaldehyde, propionaldehyde, or butyraldehyde.

In some embodiments, the R group is a substituted or unsubstituted ketone group, and the carbon number of the ketone group is preferably C ₂ -C ₃₆ , more preferably C ₂ -C ₂₄ , still more preferably C ₂ -C C ₄ . In one or more embodiments, the ketone group is an ethanone group, an acetone group, or a butanone group.

In some embodiments, the R group is a substituted or unsubstituted aryl group, and the aryl group can be either a single-ring aryl group or a fused-ring aryl group. The number of carbon atoms of the aryl group is preferably C ₆ -C ₂₄ , more preferably C ₆ -C ₁₈ , and still more preferably C ₆ -C ₁₂ . In one or more embodiments, the aryl group is phenyl, tolyl, ethylphenyl, naphthyl, or biphenyl.

In some embodiments, the R group is a substituted or unsubstituted heteroaryl group, which can be a monocyclic or fused ring aryl group. The heteroatom of the heteroaryl group may be sulfur, oxygen or nitrogen, and the number of carbon atoms of the heteroaryl group is preferably C ₄ -C ₁₂ , more preferably C ₄ -C ₈ , and still more preferably C ₄ -C ₆ . In one or more embodiments, heteroaryl is pyridyl, methoxy, or amino-substituted pyridyl, furyl, thienyl, imidazolyl, quinolyl, pyrazolyl, or pteridyl.

In some embodiments, the R group is a substituted or unsubstituted ester group, the ester group can be a straight-chain or branched-chain ester group, and the carbon number of the ester group is preferably C ₂ -C ₂₄ , more preferably C ₂ ~ _C6 . In one or more embodiments, the ester group is carbomethoxy, ethyl ester, propyl ester, isopropyl ester, butyl ester, tert-butyl ester, or hexyl ester.

In some embodiments, the R group is a substituted or unsubstituted aminoacyl group, the aminoacyl group can be a straight-chain or branched-chain aminoacyl group, and the number of carbon atoms of the aminoacyl group is preferably C ₂ -C ₂₄ , more preferably C ₂ ~ _C6 . In one or more embodiments, the aminoacyl group is carbamoyl, ethylaminoyl, alanaminoyl, isopropylaminoyl, butylaminoyl, tert-butylaminoyl, or hexylaminoyl.

In some embodiments, the R group is a substituted or unsubstituted alkenyl group, and the carbon number of the alkenyl group is preferably C ₂ -C ₃₆ , more preferably C ₂ -C ₂₄ , still more preferably C ₂ -C C ₄ . In one or more embodiments, the alkenyl group is vinyl or propenyl.

In some embodiments, the R group is a polyethylene glycol group, and the polymerization degree of the polyethylene glycol group is preferably 1-400, more preferably 1-100.

Further, R is selected from the following groups:

The inventor found through a large number of experimental studies that the R group substituted at the 3-position has an influence on the tetrazine labeling and subsequent bioorthogonal reactions. Although tetrazine compounds used as tetrazine probes can quickly label thiol-containing polypeptides or proteins, the by-products generated by the Diels-Alder (IEDDA) reaction due to the counter-electron requirement during the reaction process will cause the tetrazine-labeled products to be collected. rate dropped significantly. In order to control the competing IEDDA side reaction, by changing the substituent on the 3-position of the tetrazine, namely the R group, to adjust the electronic and steric effects, the generation of by-products can be suppressed without reducing the reaction rate. After screening, preferably, the R group is selected from

More preferably, the R group is selected from

The R' group is selected from substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl. Among them, the alkenyl group may be a straight-chain or branched-chain alkenyl group, and the number of carbon atoms of the alkenyl group is preferably C ₂ -C ₁₈ , more preferably C ₂ -C ₄ . The alkynyl group may be a straight-chain or branched-chain alkenyl group, and the number of carbon atoms in the alkynyl group is preferably C ₂ to C ₁₈ , more preferably C ₂ to C ₄ .

In some embodiments, the R' group is a substituted or unsubstituted vinyl group, and the tetrazine compound has the general structural formula shown in formula II:

In formula II, the R group is selected from any one of the aforementioned technical solutions, R ₂ , R ₃ and R ₄ may be the same or different, and R ₂ , R ₃ and R ₄ are each independently selected from hydrogen, C ₁ -C ₃₆ chain alkyl group, C ₄ -C ₆ cyclic alkyl group, C ₄ -C ₅ heterocycloalkyl group whose heteroatom is sulfur, oxygen or nitrogen, C ₁ -C ₂₄ aldehyde group, C ₂ -C C ₂₄ keto group, C ₆ -C ₁₂ aryl group, C ₄ -C ₁₂ heteroaryl group whose heteroatom is sulfur, oxygen or nitrogen, C ₂ -C ₂₄ ester group, C ₂ -C ₂₄ aminoacyl group, C ₂ -C 24 C ₂₄ alkenyl or polyethylene glycol group.

In one or more embodiments, R ₂ , R ₃ and R ₄ are each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl or tert-butyl, isopropyl, butyl , tert-butyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, azetidinyl, oxolane, thiolanyl, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde , ethyl ketone, acetone, butanone, phenyl, tolyl, ethylphenyl, naphthyl, biphenyl, pyridyl, furanyl, thienyl, imidazolyl, quinolyl, pyrazolyl, pteroyl Acridyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, carbomethoxy, ethyl ester, propyl ester, isopropyl ester, butyl ester, tert-butylaminoyl, hexylesteryl, carbamoyl, ethylaminoyl, alanaminoyl, isopropylaminoyl, butylaminoyl, vinyl, propenyl, tert-butylaminoyl or hexylaminoyl.

Preferably, R ₂ , R ₃ and R ₄ are each independently selected from hydrogen or methyl. Further preferably, R ₂ , R ₃ and R ₄ are all hydrogen, ie, the R' group is an unsubstituted vinyl group. At this time, the tetrazine compound has the general structural formula shown in formula VIII:

Further preferably, R ₂ is methyl, and R ₃ and R ₄ are both hydrogen, ie, the R' group is methyl substituted vinyl. At this time, the tetrazine compound has the general structural formula shown in formula IX:

In some embodiments, the R' group is a substituted or unsubstituted ethynyl group, and the tetrazine compound has the general structural formula shown in formula III:

In formula III, the R group is selected from any of the foregoing technical solutions, and R ₅ is selected from hydrogen, C ₁ -C ₃₆ chain alkyl, C ₄ -C ₆ cyclic alkyl, heteroatom is sulfur, C ₄ -C ₆ heterocycloalkyl group of oxygen or nitrogen, C ₁ -C ₂₄ aldehyde group, C ₂ -C ₂₄ ketone group, C ₆ -C ₁₂ aryl group, C ₄ -C 6 -hetero atom of sulfur, oxygen or nitrogen C ₁₂ heteroaryl group, C ₂ -C ₂₄ ester group, C ₂ -C ₂₄ amine group, C ₂ -C ₂₄ alkenyl group or polyethylene glycol group.

_In one or more embodiments, R is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl or tert-butyl, isopropyl, butyl, tert-butyl, cyclobutyl, Cyclopentyl, cyclohexyl, pyrrolidinyl, azetidine, oxolane, thiacyclopentyl, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, ethanone, acetone, butanyl Keto, phenyl, tolyl, ethylphenyl, naphthyl, biphenyl, pyridyl, furyl, thienyl, imidazolyl, quinolinyl, pyrazolyl, pteridyl, methoxy, ethoxy group, propoxy group, isopropoxy group, butoxy group, tert-butoxy group, methyl ester group, ethyl ester group, propyl ester group, isopropyl ester group, butyl ester group, tert-butyl ester group, hexyl ester group, carbamoyl, ethylaminoyl, alanaminoyl, isopropylaminoyl, butylaminoyl, vinyl, propenyl, tert-butylaminoyl or hexaminoyl.

Preferably, _R5 is selected from hydrogen or methyl. Further preferably, _R5 is selected from hydrogen, ie the R' group is an unsubstituted ethynyl group. At this time, the tetrazine compound has the general structural formula shown in formula XI:

The present invention also provides a preparation method of the tetrazine compound, comprising the following steps:

Dissolving the compound shown in formula IV in the first reaction solvent, adding a sulfonic acid esterification reagent to obtain the compound shown in formula V, and performing elimination reaction of the compound shown in formula V under the action of a base to obtain vinyltetrazine compounds;

The compound shown in formula VI is dissolved in the second reaction solvent, trimethylsilane acetylene is added under the action of a catalyst to obtain the compound shown in formula VII, and the compound shown in formula VII is subjected to elimination reaction under the action of a base to obtain ethynyl tetrazine. compound; or

After dissolving the compound represented by the formula X, a methyl-substituted vinyltetrazine compound is generated by Stille coupling reaction;

In this technical solution, for vinyltetrazine compounds, the compound shown in formula IV can be used as a starting material, and the compound shown in formula V can be obtained by reacting with a sulfonic acid esterification reagent in the presence of the first reaction solvent, and then in The elimination reaction takes place under the action of the base to obtain the vinyltetrazine compound represented by the formula II or the formula VIII.

The synthesis technique of vinyltetrazine compound is as follows:

In some embodiments, the first reaction solvent is preferably dichloromethane, dichloroethane, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide or N,N-dimethylformamide. In some embodiments, the base is preferably triethylamine, diisopropylaminoethylamine, pyridine, sodium acetate, potassium carbonate, sodium carbonate or potassium tert-butoxide. In some embodiments, the sulfonic acid esterification reagent is methanesulfonyl chloride. In some embodiments, the reaction is carried out under the protection of an inert gas, preferably argon. In one or more embodiments, the reaction temperature is 0°C. In some embodiments, the substitute of the compound represented by the formula IV can be used to prepare the corresponding substitute of the vinyltetrazine compound represented by the formula II.

For the ethynyl tetrazine compounds, the compound represented by the formula VI can be used as the starting material, after dissolving in the second reaction solvent, the catalyst and trimethylsilane acetylene are added to the reaction system, and the formula is obtained after the reaction at room temperature. Compounds shown in VII. Next, the compound represented by the formula VII is subjected to an elimination reaction under the action of a base to obtain an ethynyl tetrazine compound.

The synthesis technique of ethynyl tetrazine compounds is as follows:

In some embodiments, the second reaction solvent is preferably ultra-dry toluene. In some embodiments, the base is preferably triethylamine, diisopropylaminoethylamine, pyridine, sodium acetate, potassium carbonate, sodium carbonate or potassium tert-butoxide. In some embodiments, the catalyst system is (triphenylphosphine) palladium dichloride, cuprous iodide, and the phosphine ligand is triphenylphosphorus. In some embodiments, the substitute of the compound represented by the formula VI can be used to prepare the corresponding substitute of the ethynyl tetrazine compound represented by the formula III.

For methyl-substituted vinyltetrazine compounds, a compound represented by formula X can be used as a starting material, and then a catalyst is added to generate methyl-substituted vinyltetrazine compounds through Stille coupling reaction. In some embodiments, the compound of formula X is dissolved in ultra-dry 1,4-dioxane, tetrakis(triphenylphosphine)palladium, copper thiophene-2-carboxylate, tributyl(1- After propen-2-yl)tin, react at 100°C to obtain the methyl-substituted vinyltetrazine compound represented by formula IX.

Further, the preparation method of the compound represented by the formula IV includes the following steps: using RCN and 3-hydroxypropionitrile as raw materials, adding a thiol compound and reacting with hydrazine hydrate, or adding a Lewis acid and reacting with hydrazine hydrate.

In some embodiments, RCN and 3-hydroxypropionitrile are used as raw materials, thiol compounds and hydrazine hydrate are added to react to prepare the compound shown in formula IV. In one or more embodiments, the thiol compound is 3-mercaptopropionic acid. Specifically, RCN, 3-hydroxypropionitrile, ethanol, 3-mercaptopropionic acid and hydrazine hydrate were added to the reaction vessel, and the reaction solution was stirred and reacted under the protection of argon. After the reaction is completed, the reaction solution is cooled, an ice-water solution of sodium nitrite is added to the reaction solution, and then HCl is slowly added in the ice bath until gas generation stops. At this time, the pH value of the reaction solution is 1 to 6, preferably, the pH value It is 1-4, More preferably, it is 2-3. Thereafter, the reaction solution is continuously stirred for a certain period of time, extracted, dried and concentrated, and purified by silica gel column chromatography to obtain the compound represented by formula IV. The specific synthetic route is as follows:

In some embodiments, RCN and 3-hydroxypropionitrile are used as raw materials, and Lewis acid and hydrazine hydrate are added for reaction. In one or more embodiments, the Lewis acid is Zn(OTf) ₂ . Specifically, RCN, 3-hydroxypropionitrile, Zn(OTf) ₂ and hydrazine hydrate were added to the reaction vessel. The reaction solution was heated and stirred under the protection of argon. After the reaction is completed, the reaction solution is cooled, an ice-water solution of sodium nitrite is added to the reaction solution, and then HCl is slowly added in the ice bath until gas generation stops. At this time, the pH value of the reaction solution is 1 to 6, preferably, pH value It is 1-4, More preferably, it is 2-3. Thereafter, the reaction solution is continuously stirred for a certain period of time, extracted, dried and concentrated, and purified by silica gel column chromatography to obtain the compound represented by formula IV. The specific synthetic route is as follows:

The present invention also provides the use of any of the aforementioned tetrazine compounds for selectively labeling amino acids having thiol groups and thiol groups of biomolecules or biological materials comprising the amino acids.

In this technical solution, alkenyl tetrazine compounds and alkynyl tetrazine compounds are used to selectively label sulfhydryl groups of polypeptides, proteins, biological materials, drugs, and functional imaging molecules containing cysteine or homocysteine , after the formation of the tetrazine label, the functionalization of the labeled biomolecules or biomaterials is achieved through bioorthogonal reactions. Those skilled in the art should understand that the alkenyl tetrazine compound or the alkynyl tetrazine compound can also label other sulfhydryl-containing substances.

The method for labeling tetrazine compounds is as follows: adding the tetrazine compounds into the buffer, adding the substance to be labeled after dissolving the tetrazine compounds, and obtaining a tetrazine-labeled product after the reaction. Bio-orthogonal reactions in vivo to achieve functional derivatization applications. In some embodiments, the dienophile is

In one or more embodiments, the dienophile can be a derivative of BCN or TCO, such as dyes, small molecule drugs, functional materials, etc., and the dienophile can also be cyclopropene, cyclobutene, isonitrile, etc. .

In some embodiments, the vinyl tetrazine compounds are used to selectively label cysteine-containing polypeptides and realize the process of polypeptide functionalization as follows:

Specifically, a vinyltetrazine compound is added to the buffer, and the cysteine-containing polypeptide is added after mixing. After stirring evenly, after reacting at 20-40° C. for a period of time, the reaction is detected by HPLC-MS, and the reaction solution can be purified by reverse-phase chromatography to obtain the product. After that, add the dienophile to the reaction solution containing the tetrazine-labeled polypeptide, stir and mix well, react at 20-40 °C for a period of time, and detect the reaction by HPLC-MS. The reaction solution can pass through a reversed-phase chromatographic column. The product was purified. In one or more embodiments, the buffer is a phosphate buffer, pH 6.0-8.5, and 5-30% dimethyl sulfoxide or tetrahydrofuran is a co-solvent.

In some embodiments, the vinyl tetrazine compounds are used to selectively label cysteine-containing proteins and realize the process of protein functionalization as follows:

Specifically, vinyltetrazine compounds are added to the buffer solution of the protein containing free sulfhydryl groups, and the tetrazine labeling of the protein is realized after a period of reaction at room temperature, and the reaction progress is monitored by HPLC. Subsequently, a dienophile-modified substance to be conjugated was added and reacted at room temperature. After purification, the conjugate was successfully introduced into the protein, thereby efficiently realizing the functionalization of the protein. The reaction solution was monitored by HPLC-MS.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The tetrazine compounds provided by the present invention can realize the selective tetrazine labeling of sulfhydryl-containing polypeptides, proteins and biological materials, and utilize the bioorthogonal reaction to rapidly, efficiently and stably function on the tetrazine-labeled substances Derivative applications;

2. The tetrazine compounds provided by the present invention have high selectivity. When the tetrazine compounds are rich in other functional groups in the body, the tetrazine compounds can selectively combine with cysteine to form a stable connection product, and no by-products can be detected;

3. After the tetrazine compound provided by the present invention forms a connection product with a sulfhydryl group, the reaction kinetics of the dienophile and the connection product is rapid, which is significantly faster than the method of using thiotetrazine as the tetrazine marker in the prior art, It shows that this tetrazine compound can be applied to nanomolar concentration in vivo bioorthogonal pretargeting and radiolabeling;

4. The tetrazine compounds provided by the present invention adjust the electronic and steric effects by changing the R group on the 3-position of the tetrazine, which can inhibit the generation of by-products without reducing the reaction rate and reduce the by-products generated by the IEDDA reaction. , which improves the yield of tetrazine-labeled products, so that even at millimolar labeling concentrations, no by-products of IEDDA reaction are observed;

5. The preparation process steps of the tetrazine compounds provided by the present invention are simple and the reaction conditions are mild, which can not only prepare a variety of alkenyl tetrazine compounds or alkynyl tetrazine compounds, but also can change the R group on the starting material. group, and modulate the electronic and steric effects of the R group at the 3-position of tetrazine compounds.

Description of drawings

The accompanying drawings described herein are used to provide further understanding of the embodiments of the present invention, and constitute a part of the present application, and do not constitute limitations to the embodiments of the present invention. In the attached image:

Fig. 1 shows the calculated value of the second order rate constant k ₂ of the tetrazine probe-labeled cysteine reaction in the specific embodiment of the present invention;

Fig. 2 shows the proportion of the ligation product of tetrazine probe and cysteine within a certain period of time in the specific embodiment of the present invention;

Figure 3 shows the competitive reaction of tetrazine probe and cysteine in the presence of N-Boc-lysine and arginine in a specific embodiment of the present invention;

Fig. 4 shows the HPLC trace of the reaction of bioorthogonal products and GSH in PB buffer according to the specific embodiment of the present invention;

Figure 5 shows the fluorescence imaging images of SKOV3 cells and 4T1 cells using Trastuzumab-Cy5 in a specific embodiment of the present invention, wherein (a)-(f) are the imaging of SKOV3 cells, (a) Hoechst 33342 , (b) Trastuzumab-Cy5, (c) a merge of images (a) and (b), (d-f) cells were preblocked with 100 nM trastuzumab, followed by trastuzumab-Cy5 ( 33 nM) treatment; (g)-(i) are images of 4T1 cells, (g) Hoechst 33342, (h) Trastuzumab-Cy5, (i) merge of pictures (g) and (h).

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. as a limitation of the present invention.

As used herein, "first", "second", etc. (eg, first reaction solvent, second reaction solvent, etc.) are used only to distinguish between corresponding reagents for descriptive clarity and are not intended to limit any order or emphasize importance Wait.

All the raw materials in the present invention are not particularly limited in their sources, and can be purchased in the market or prepared according to conventional methods well known to those skilled in the art.

All raw materials in the present invention are not particularly limited in their purity, and the present invention preferably adopts analytical purity or conventional purity requirements in the field of preparation of tetrazine compounds.

The present invention has no particular limitation on the expressions of the substituents, and expressions well-known to those skilled in the art are adopted. Those skilled in the art can correctly understand the meanings according to the expressions based on common sense.

All the raw materials of the present invention, its grades and abbreviations belong to the conventional grades and abbreviations in the field, and each grade and abbreviation are clear and definite in the field of its related use, and those skilled in the art can It can be purchased from the market or prepared by conventional methods.

1. The synthesis process of vinyltetrazine compounds is as follows:

The R group can be selected from a variety of substituents, for example, the R group can be selected from hydrogen, alkyl, heterocycloalkyl, aldehyde, ketone, aryl, heteroaryl, ester, aminoacyl or polyethylene diol group. The following embodiments are only used as preferred examples to illustrate the specific implementation process of the present invention.

[Example 1]

Synthesis of 3-phenyl-6-vinyl-1,2,4,5-tetrazine:

Benzonitrile (206 mg, 2 mmol), 3-hydroxypropionitrile (548 μL, 8 mmol), 3-mercaptopropionic acid (174 μL, 3 mmol), ethanol (0.6 mL) and hydrazine hydrate (1.2 mL, 24 mmol) were added to a mixture of Magnetic stir bar in a 10 mL reaction vessel. Under argon, the reaction solution was stirred at 40°C overnight. The reaction solution was then cooled to 0°C, and sodium nitrite (1.38 g, 20 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under the ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until gas generation stops, pH value is 1～6, preferably, pH value is 1～4, more preferably 2 to 3. The resulting mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 3), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 16 (247 mg, yield 61%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.69-8.42 (m, 2H), 7.73-7.53 (m, 3H), 4.31 (dd, J=10.9, 5.4 Hz, 2H), 3.63 (t, J=5.8 Hz,2H),2.69(t,J＝5.2Hz,1H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 168.29, 164.57, 132.79, 131.59, 129.32, 128.01, 60.08, 37.51.

The m/z of HRMS[C ₁₀ H ₁₁ N ₄ O] ⁺ [M+H] ⁺ is theoretically 203.0927, but actually 203.0929.

Intermediate 16 (181 mg, 0.8 mmol) and triethylamine (166 μL, 1.2 mmol) were dissolved in 4 mL of dichloromethane, and methanesulfonyl chloride (92 μL, 1.2 mmol) was slowly added dropwise at 0° C. under argon. The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 16 to mesylate intermediate 17 (about 1 hour). Triethylamine (166 μL, 1.2 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 1 hour. Then 20 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain the product 1 as a red solid (143.2 mg, two-step yield 97%), namely tetrazine probe 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.70-8.56 (m, 2H), 7.70-7.53 (m, 3H), 7.22 (dd, J=17.6, 10.9 Hz, 1H), 7.04 (dd, J=17.6 ,1.0Hz,1H),6.07(dd,J=10.8,1.0Hz,1H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 163.88, 163.78, 132.74, 131.77, 130.46, 129.30, 128.07, 127.42.

The m/z of HRMS[C ₁₀ H ₇ N ₄ ] ^- [MH] ^- is theoretically 183.0676, but actually 183.0671.

[Example 2]

Synthesis of 3-(2-methoxyethyl)-6-vinyl-1,2,4,5-tetrazine:

Combine 3-methoxypropionitrile (190 mg, 2 mmol), 3-hydroxypropionitrile (548 μL, 8 mmol), 3-mercaptopropionic acid (174 μL, 3 mmol), ethanol (0.6 mL) and hydrazine hydrate (1.2 mL, 24 mmol) Add to a 10 mL reaction vessel equipped with a magnetic stir bar. Under argon protection, the reaction solution was stirred at room temperature for 16 hours. The reaction solution was then cooled to 0°C, and sodium nitrite (1.38 g, 20 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under the ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until gas generation stops, pH value is 1～6, preferably, pH value is 1～4, more preferably 2 to 3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 6), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 18 (180.9 mg, yield) as a pink oil. rate 49%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.27 (t, J=5.8 Hz, 2H), 4.02 (t, J=6.3 Hz, 2H), 3.65-3.53 (m, 4H), 3.37 (s, 3H) ,2.56(s,1H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 168.66, 168.55, 69.73, 59.96, 58.79, 37.44, 35.27.

The m/z of HRMS [C ₇ H ₁₁ N ₄ O ₂ ] ⁺ [MH] ^- is theoretically 183.0887 and practically 183.0883.

Intermediate 18 (147 mg, 0.8 mmol) and triethylamine (166 μL, 1.2 mmol) were dissolved in 4 mL of dichloromethane, and methanesulfonyl chloride (92 μL, 1.2 mmol) was slowly added dropwise at 0°C under argon. The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 18 to mesylate intermediate 19. Triethylamine (166 μL, 1.2 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 2 hours. 20 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain a red oily product 2 (120.7 mg , two-step yield 91%), namely tetrazine probe 2.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.17 (dd, J=17.6, 10.8 Hz, 1H), 7.00 (dd, J=17.6, 1.1 Hz, 1H), 6.05 (dd, J=10.8, 1.1 Hz, 1H), 4.02(t, J＝6.3Hz, 2H), 3.59(t, J＝6.3Hz, 2H), 3.37(s, 3H).

¹³ C NMR (151MHz, CDCl ₃ ) δ 167.82, 164.14, 130.37, 127.46, 69.75, 58.74, 35.30.

The m/z of HRMS[C ₇ H ₉ N ₄ O] ^- [MH] ^- is theoretically 165.0782, but actually 165.0777.

[Example 3]

Synthesis of 3-isopropyl-6-vinyl-1,2,4,5-tetrazine:

2-Methylalanamide hydrochloride (292 mg, 2.6 mmol), 3-hydroxypropionitrile (712 μL, 10.4 mmol), 3-mercaptopropionic acid (226 μL, 2.6 mmol), ethanol (0.7 mL) and hydrazine hydrate were combined (1.5 mL, 30 mmol) was added to a 10 mL reaction vessel equipped with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred at room temperature overnight. The reaction solution was then cooled to 0°C, and sodium nitrite (1.8 g, 26 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under the ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until gas generation stops, pH value is 1～6, preferably, pH value is 1～4, more preferably 2 to 3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 5), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 20 (161.0 mg, yield) as a pink oil. rate 37%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.27 (t, J=5.5 Hz, 2H), 3.66 (dq, J=13.9, 7.0 Hz, 1H), 3.58 (t, J=5.8 Hz, 2H), 2.60 (s,1H),1.54(s,3H),1.52(s,3H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 174.20, 168.47, 59.99, 37.41, 34.27, 21.28.

The m/z of HRMS[C ₇ H ₁₁ N ₄ O] ^- [MH] ^- is theoretically 167.0938 and practically 167.0934.

Intermediate 20 (134.4 mg, 0.8 mmol) and triethylamine (166 μL, 1.2 mmol) were dissolved in 4 mL of dichloromethane, and methanesulfonyl chloride (92 μL, 1.2 mmol) was slowly added dropwise at 0° C. under argon. The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 20 to mesylate intermediate 21 (about 1 hour). Triethylamine (166 μL, 1.2 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 6 hours. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain a red oily product 3 (100.5 mg , two-step yield 83%), namely tetrazine probe 3.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.17 (dd, J=17.6, 10.8 Hz, 1H), 6.99 (dd, J=17.6, 1.1 Hz, 1H), 6.03 (dd, J=10.8, 1.1 Hz, 1H), 3.72–3.58(m, 1H), 1.54(s, 3H), 1.52(s, 3H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 173.56, 164.07, 130.50, 127.14, 34.29, 21.27.

The m/z of HRMS[C ₇ H ₉ N ₄ ] ^- [MH] ^- is theoretically 149.0833, but actually 149.0826.

[Example 4]

Synthesis of (R)-2-(6-vinyl-1,2,4,5-tetrazin-3-yl)pyrrolidine-1-carboxylate tert-butyl ester:

(R)-1-Boc-2-cyanopyrrolidine (294 mg, 1.5 mmol), 3-hydroxypropionitrile (410 μL, 6 mmol), 3-mercaptopropionic acid (130 μL, 1.5 mmol), ethanol (0.4 mL) and hydrazine hydrate (874 μL, 18 mmol) were added to a 10 mL reaction vessel equipped with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred at room temperature overnight. The reaction solution was then cooled to 0°C, and sodium nitrite (1.0 g, 15 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under the ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until gas generation stops, pH value is 1～6, preferably, pH value is 1～4, more preferably 2 to 3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 3), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 22 (308.3 mg, yield) as a purple oil. rate 70%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.41 (dd, J=8.1, 3.7 Hz, 1H), 5.31 (dd, J=8.0, 4.6 Hz, 1H), 4.26 (dd, J=11.5, 5.7 Hz, 4H), 3.85–3.49 (m, 8H), 2.71 (s, 1H), 2.59 (dt, J=11.3, 7.9Hz, 2H), 2.48 (s, 1H), 2.25–2.10 (m, 4H), 2.09 –1.99(m, 2H), 1.41(s, 9H), 1.14(s, 9H).

¹³ C NMR (101MHz, CDCl ₃ )δ171.90, 171.30, 168.97, 154.54, 153.39, 80.22, 80.05, 60.23, 60.11, 60.02, 59.94, 47.28, 47.08, 37.50, 33.99, 33.0, 2.18, 20,

The m/z of HRMS[C ₁₃ H ₂₁ N ₅ NaO ₃ ] ⁺ [M+Na] ⁺ is theoretically 318.1537 and practically 318.1540.

Both the above H NMR (25°C) and C NMR (25°C) spectra indicated an approximately 1:1 mixture of the two isomers.

Intermediate 22 (88.6 mg, 0.3 mmol) and triethylamine (62 μL, 0.45 mmol) were dissolved in 2 mL of anhydrous dichloromethane, and methanesulfonyl chloride (35 μL, 0.45 mmol) was slowly added dropwise at 0°C under argon protection. ). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 22 to mesylate intermediate 23 (about 2 hours). Triethylamine (62 μL, 0.45 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 15 hours. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain purple oily product 4 (72.1 mg , two-step yield 86%), namely tetrazine probe 4.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.18 (m, 10.8 Hz, 2H), 7.02 (d, J=13.8 Hz, 1H), 6.98 (d, J=13.6 Hz, 1H), 6.07 (d, J =10.8Hz,1H),6.04(d,J=10.7Hz,1H),5.42(dd,J=8.3,3.7Hz,1H),5.31(dd,J=8.2,4.4Hz,1H),3.82–3.58 (m, 4H), 2.67–2.50 (m, 2H), 2.24–2.10 (m, 4H), 2.08–1.98 (m, 2H), 1.42 (s, 9H), 1.15 (s, 9H).

¹³ C NMR(101MHz,CDCl ₃ )δ171.23,171.22,170.73,170.72,164.44,130.45,130.26,127.81,127.50,80.05,80.02,60.23,60.12,47.23,47.07,33.98,33.03,28.39,28.13,24.19,23.72 .

The m/z of HRMS[C ₁₃ H ₁₉ N ₅ NaO ₂ ] ⁺ [M+Na] ⁺ is theoretically 300.1431, but actually 300.1430.

[Example 5]

Synthesis of 3-tert-butyl-6-vinyl-1,2,4,5-tetrazine:

tert-Butylguanidine hydrochloride (680 mg, 5 mmol), 3-hydroxypropionitrile (1.37 mL, 20 mmol), 3-mercaptopropionic acid (436 μL, 5 mmol), ethanol (1.5 mL) and hydrazine hydrate (2.9 mL, 60 mmol) were combined ) was added to a 10 mL reaction vessel equipped with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred at room temperature overnight. The reaction solution was then cooled to 0°C, and sodium nitrite (3.5 g, 60 mmol) dissolved in ice water (80 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under the ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until gas generation stops, pH value is 1～6, preferably, pH value is 1～4, more preferably 2 to 3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 4), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 24 (388.4 mg, yield) as a purple oil. rate 43%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.27 (t, J=5.7 Hz, 2H), 3.58 (t, J=5.8 Hz, 2H), 2.60 (s, 1H), 1.59 (s, 9H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 176.08, 167.80, 59.96, 37.93, 37.35, 29.15.

The m/z of HRMS[C ₈ H ₁₃ N ₄ O] ^- [MH] ^- is theoretically 181.1095 and practically 181.1086.

Intermediate 24 (145.6 mg, 0.8 mmol) and triethylamine (166 μL, 1.2 mmol) were dissolved in 4 mL of anhydrous dichloromethane, and methanesulfonyl chloride (92 μL, 1.2 mmol) was slowly added dropwise at 0°C under argon protection. ). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 24 to mesylate intermediate 25 (about 1 hour). Triethylamine (166 μL, 1.2 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 12 hours. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain purple oily product 5 (119.1 mg, Two-step yield 91%), tetrazine probe 5.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.17 (dd, J=17.6, 10.8 Hz, 1H), 6.99 (dd, J=17.6, 1.2 Hz, 1H), 6.03 (dd, J=10.8, 1.2 Hz, 1H), 1.59(s, 9H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 175.45, 163.42, 130.50, 127.13, 37.95, 29.15.

The m/z of HRMS[C ₈ H ₁₂ N ₄ Na] ⁺ [M+Na] ⁺ is theoretically 187.0954 and practically 187.0961.

[Example 6]

Synthesis of 3-(pyridin-4-yl)-6-vinyl-1,2,4,5-tetrazine:

Intermediate 26 (19.2 mg, 0.06 mmol) was dissolved in 0.6 mL of anhydrous dichloromethane, followed by the addition of triethylamine (12 [mu]L, 0.072 mmol). The reaction solution was stirred at room temperature for 90 minutes, and purified by silica gel column chromatography to obtain product 6 (9.3 mg, yield 83%) as a purple solid, namely tetrazine probe 6.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.92 (d, J=6.0 Hz, 2H), 8.46 (d, J=6.0 Hz, 2H), 7.27 (dd, J=17.0, 11.2 Hz, 1H), 7.13 (d, J=17.5Hz, 1H), 6.18 (d, J=10.7Hz, 1H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 164.61, 162.68, 151.16, 139.10, 130.19, 129.11, 121.23.

The m/z of HRMS[C ₉ H ₈ N ₅ ] ⁺ [M+H] ⁺ is theoretically 186.0774, but actually 186.0776.

[Example 7]

Synthesis of 3-(3-methoxypyridin-4-yl)-6-vinyl-1,2,4,5-tetrazine:

Starting material 27 (134 mg, 1 mmol), 3-hydroxypropionitrile (410 μL, 6 mmol), Zn(OTf) ₂ (109 mg, 0.3 mmol) and hydrazine hydrate (600 mg, 12 mmol) were added to 10 mL equipped with a magnetic stir bar in the reaction vessel. Under the protection of argon, the reaction solution was stirred at 60°C overnight. The reaction solution was then cooled to 0°C, and sodium nitrite (690 mg, 10 mmol) dissolved in ice water (30 mL) was slowly added to the reaction solution. Then, 1M HCl was slowly added under an ice-water bath, during which the reaction solution turned bright red and gas was generated, and then continued to add HCl until gas generation stopped, and the pH was 1 to 6, preferably, the pH was 1 to 4. , more preferably 2 to 3. The mixture was further stirred for about 5 min, extracted with dichloromethane (30 mL x 4), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 28 (87.6 mg, yield) as a purple oil. rate 38%).

¹ H NMR (400MHz, CDCl ₃ ) δ 8.60 (s, 1H), 8.51 (d, J=4.8Hz, 1H), 7.86 (d, J=4.8Hz, 1H), 4.35 (t, J=5.8Hz) ,2H),4.04(s,3H),3.68(t,J＝5.8Hz,2H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 168.12, 164.89, 153.40, 142.93, 135.82, 128.47, 124.34, 59.90, 56.87, 37.71.

The m/z of HRMS[C ₁₀ H ₁₂ N ₅ O ₂ ] ⁺ [M+H] ⁺ is theoretically 234.0986, but actually 234.0989.

Intermediate 28 (69.9 mg, 0.3 mmol) and triethylamine (62 μL, 0.45 mmol) were dissolved in 3 mL of anhydrous dichloromethane, and methanesulfonyl chloride (35 μL, 0.45 mmol) was slowly added dropwise at 0°C under argon protection. ). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 28 to mesylate intermediate 29 (about 1 hour). Triethylamine (62 μL, 0.45 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 12 hours. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain purple oily product 7 (53.3 mg , two-step yield 83%), namely tetrazine probe 7.

¹ H NMR (400MHz, CDCl ₃ ) δ 8.60 (s, 1H), 8.51 (d, J=4.8Hz, 1H), 7.89 (d, J=4.8Hz, 1H), 7.25 (dd, J=17.6, 10.7Hz, 1H), 7.12 (dd, J=17.5, 1.0Hz, 1H), 6.15 (dd, J=10.7, 1.0Hz, 1H), 4.05 (s, 3H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.14, 163.22, 153.46, 142.97, 135.90, 130.26, 128.86, 128.56, 124.24, 56.89.

The m/z of HRMS[C ₁₀ H ₁₀ N ₅ O] ⁺ [M+H] ⁺ is theoretically 216.0880, but actually 216.0886.

[Example 8]

Synthesis of N,N-dimethyl-2-(6-vinyl-1,2,4,5-tetrazin-3-yl)pyridin-3-amine:

2-Cyano-3-fluoropyridine (2.44 g, 20 mmol) and 40% aqueous dimethylamine were stirred at room temperature for 2 hours. 60 mL of water was added to the reaction solution, and the reaction solution was extracted with ethyl acetate (60 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo, and purified by silica gel column chromatography to obtain compound 30 (2.69 g, yield 91%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.11 (dd, J=4.3, 1.2 Hz, 1H), 7.31 (dd, J=8.7, 4.3 Hz, 1H), 7.23 (dd, J=8.7, 1.2 Hz, 1H), 3.12(s, 6H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 151.70, 140.82, 126.83, 123.76, 121.14, 118.39, 42.46.

Compound 30 (294 mg, 2 mmol), 3-hydroxypropionitrile (546 μL, 8 mmol), Zn(OTf) ₂ (72 mg, 0.2 mmol) and hydrazine hydrate (594 μL, 12 mmol) were added to a 10 mL reaction vessel equipped with a magnetic stir bar middle. Under the protection of argon, the reaction solution was stirred at 60°C for 36 hours. After that, the reaction solution was cooled to room temperature with ice water, and sodium nitrite (1.38 g, 20 mmol) dissolved in ice water (40 mL) was slowly added to the reaction solution. Then, 1M HCl was slowly added under an ice-water bath, during which the reaction solution turned bright red and gas was generated, and then continued to add HCl until gas generation stopped, and the pH was 1 to 6, preferably, the pH was 1 to 4. , more preferably 2 to 3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (30 mL x 4), dried over anhydrous sodium sulfate and concentrated in vacuo, and purified by silica gel column chromatography to give the desired intermediate 31 as a brown oil (111 mg, yield twenty three%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.36 (dd, J=4.4, 1.3 Hz, 1H), 7.50 (dd, J=8.5, 1.2 Hz, 1H), 7.40 (dd, J=8.5, 4.4 Hz, 1H), 4.30(t, J＝5.8Hz, 2H), 3.66(t, J＝5.8Hz, 2H), 2.75(s, 6H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 167.98, 167.71, 149.00, 141.55, 140.56, 125.65, 125.38, 60.03, 43.48, 37.73.

The m/z of HRMS[C ₁₁ H ₁₅ N ₆ O] ⁺ [M+H] ⁺ is theoretically 247.1302, but actually 247.1302.

Intermediate 31 (9.8 mg, 0.04 mmol) and triethylamine (8.3 μL, 0.06 mmol) were dissolved in 0.4 mL of anhydrous dichloromethane, and methanesulfonyl chloride (4.6 μL was slowly added dropwise at 0°C under argon protection). , 0.06 mmol). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 31 to mesylate intermediate 32 (about 1.5 hours). Triethylamine (8.3 μL, 0.06 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 1 hour. After the reaction was completed, the product 8 (7.1 mg, two-step yield 78%) was obtained as a brown oily product, which was tetrazine probe 8, and was purified by silica gel column chromatography.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=3.1 Hz, 1H), 7.48 (d, J=8.5 Hz, 1H), 7.38 (dd, J=8.4, 4.4 Hz, 1H), 7.29 –7.18(m,1H),7.08(d,J=17.5Hz,1H),6.10(d,J=10.8Hz,1H),2.76(s,6H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 167.10, 163.15, 149.01, 141.71, 130.47, 128.08, 125.49, 125.11, 43.44.

The m/z of HRMS[C ₁₁ H ₁₃ N ₆ ] ⁺ [M+H] ⁺ is theoretically 229.1196, but actually 229.1200.

[Example 9]

Synthesis of (R)-1-(2-(6-vinyl-1,2,4,5-tetrazin-3-yl)pyrrolidin-1-yl)ethan-1one:

Intermediate 22 (118 mg, 0.4 mmol) was dissolved in 8 mL of dichloromethane, trifluoroacetic acid was added dropwise at 0°C, and stirred at room temperature for 1 hour. The solvent and trifluoroacetic acid were absolutely dried using a rotary evaporator and oil pump. To the residue in the round bottom flask were added acetate-N-succinimidyl ester (75.5 mg, 0.48 mmol), 4 mL of dichloromethane and trifluoroacetic acid (139 μL, 1 mmol) under argon. The mixture was stirred at room temperature for 3 h, concentrated in vacuo, and purified by silica gel column chromatography to give Intermediate 33 (68.2 mg, yield 72%) as a pink oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.58-5.48 (m, 2H), 4.27-4.17 (m, 4H), 3.94-3.78 (m, 2H), 36.78- 3.70 (m, 2H), 3.61-3.50 (m, 4H), 2.90 (s, 2H), 2.67–2.54 (m, 2H), 2.35–2.12 (m, 6H), 2.11 (s, 5.4H), 1.92 (s, 0.6H). The NMR Hydrogen spectrum (25°C) indicates a mixture of two isomers about 9:1.

¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.71, 169.67, 168.88, 59.98, 59.96, 48.43, 37.64, 32.44, 24.65, 22.38. The C NMR spectrum (25°C) indicated that the product had mainly two isomers.

The m/z of HRMS[C ₁₀ H ₁₅ N ₅ NaO ₂ ] ⁺ [M+Na] ⁺ is theoretically 260.1118 and practically 260.1124.

Intermediate 33 (52.1 mg, 0.22 mmol) and triethylamine (46 μL, 0.33 mmol) were dissolved in 2 mL of anhydrous dichloromethane, and methanesulfonyl chloride (26 μL, 0.33 mmol) was slowly added dropwise at 0°C under argon protection. ). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 33 to mesylate intermediate 34 (about 1 hour). Triethylamine (46 μL, 0.33 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 12 hours. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain purple oily product 9 (43 mg, 90% yield in two steps), namely tetrazine probe 9.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.24-7.10 (m, 2H), 7.06 (dd, J=17.6, 1.1 Hz, 0.2H), 6.98 (dd, J=17.6, 1.1 Hz, 1.8H), 6.13(dd,J=10.7,1.1Hz,0.2H),6.04(dd,J=10.8,1.1Hz,1.8H),5.57(dd,J=8.2,4.1Hz,1.8H),5.51(dd,J =8.3,2.3Hz,0.2H),3.95-3.79(m,2H),3.79-3.67(m,2H),2.64-2.52(m,2H),2.35-2.13(m,6H),2.11(s, 5.4H), 1.93(s, 0.6H). This H NMR spectrum (25°C) indicates a mixture of the two isomers about 9:1.

¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.22, 169.44, 164.34, 130.35, 127.62, 59.94, 48.37, 32.46, 24.67, 22.38. The C NMR spectrum (25°C) indicated the major isomer of the product.

The m/z of HRMS[C ₁₀ H ₁₃ N ₅ NaO] ⁺ [M+Na] ⁺ is theoretically 242.1012 and practically 242.1013.

[Example 10]

Synthesis of 3,6-divinyl-1,2,4,5-tetrazine:

Intermediate 35 (17.5 mg, 0.04 mmol) and triethylamine (43 μL, 0.32 mmol) were dissolved in 0.4 mL of anhydrous dichloromethane, and methanesulfonyl chloride (23.3 μL, 0.31 mmol). The reaction solution was stirred at room temperature and the progress of the reaction was monitored by TLC until complete conversion of intermediate 35 to mesylate intermediate 36 (about 1.5 hours). Triethylamine (43 μL, 0.32 mmol) was then added to the reaction solution, and the resulting mixture was stirred at room temperature for 1 hour. After the reaction, the product 10 (6.6 mg, two-step yield 49%) as a brown oily product was purified by silica gel column chromatography, namely tetrazine probe 10.

Second, the synthesis of methyl vinyl tetrazine compounds:

The synthesis process of ethynyl tetrazine compounds is based on

As a starting material, methyl-substituted vinyltetrazine compounds are generated by Stille coupling reaction.

The R group can be selected from a variety of substituents, for example, the R group can be selected from hydrogen, alkyl, aryl, heteroaryl, ester, aminoacyl or polyethylene glycol groups. The following embodiments are only used as preferred examples to illustrate the specific implementation process of the present invention.

[Example 11]

Synthesis of 3-phenyl-6-(1-propen-2-yl)-1,2,4,5-tetrazine:

In a glove box, phenyltetrazinethiomethyl (50 mg, 0.245 mmol) was dissolved in 50 mL of ultra-dry 1,4-dioxane, tetrakis(triphenylphosphine)palladium (43 mg, 0.037 mmol), Copper thiophene-2-carboxylate (94 mg, 0.49 mmol), tributyl(1-propen-2-yl)tin (162 μL, 0.49 mmol). The reaction was carried out at 100 °C for 30 min. 50 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a purple-red solid product 11 (20 mg, yield 41%), the tetrazine probe 11.

¹ H NMR (400MHz, Chloroform-d) δ8.73-8.50(m, 2H), 7.69-7.47(m, 3H), 6.84(s, 1H), 5.84(s, 1H), 2.42(s, 3H) .

¹³ C NMR (101MHz, Chloroform-d) δ164.80, 163.47, 137.58, 132.75, 131.90, 129.39, 128.09, 124.01, 19.16.

[Example 12]

Synthesis of 3-(6-cyano-phenyl)-6-(1-propen-2-yl)-1,2,4,5-tetrazine:

In a glove box, dissolve 3-(6-cyano-phenyl)tetraazinethiomethyl (50 mg, 0.218 mmol) in 50 mL of ultra-dry 1,4-dioxane, add tetrakis(triphenylphosphine) ) palladium (30 mg, 0.033 mmol), copper thiophene-2-carboxylate (83 mg, 0.436 mmol), tributyl(1-propen-2-yl)tin (143 μL, 0.436 mmol). The reaction was carried out at 100 °C for 30 min. 50 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a purple-red solid product 12 (18 mg, yield 38 %), namely the tetrazine probe 12.

¹ H NMR(400MHz, Chloroform-d)δ8.75(d,J=8.3Hz,2H),7.90(d,J=8.2Hz,2H),6.91(s,1H),5.92(s,1H), 2.44(s,3H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 165.11, 162.42, 137.41, 135.98, 133.14, 133.14, 128.44, 128.44, 125.47, 118.25, 116.17, 19.12.

[Example 13]

Synthesis of 3-isopropyl-6-(1-propen-2-yl)-1,2,4,5-tetrazine:

In a glove box, 3-isopropyltetrazinethiomethyl (50 mg, 0.294 mmol) was dissolved in 50 mL of ultra-dry 1,4-dioxane, and tetrakis(triphenylphosphine)palladium (50 mg, 0.044 mmol) was added. mmol), copper thiophene-2-carboxylate (112 mg, 0.588 mmol), tributyl(1-propen-2-yl)tin (195 μL, 0.588 mmol). The reaction was carried out at 100 °C for 30 min. 50 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a purple-red solid product 13 (18 mg, yield 37.3 %), namely the tetrazine probe 13.

¹ H NMR (400MHz, Chloroform-d) δ6.77(s,1H), 5.79(s,1H), 3.61-3.68(m,1H), 2.38(s,3H), 1.54(s,3H), 1.52 (s,3H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 173.25, 165.10, 137.67, 123.65, 34.33, 21.39, 19.17.

3. Synthesis of ethynyl tetrazine compounds:

The synthesis process of ethynyl tetrazine compounds is based on

As a starting material, trimethylsilane acetylene is added under the action of a catalyst to obtain

The elimination reaction is carried out under the action of a base to generate ethynyl tetrazine compounds.

[Example 14]

Synthesis of 3-methyl-6-ethynyl-1,2,4,5-tetrazine:

In a glove box, methyltetrazine bromide (50 mg, 0.286 mmol) was dissolved in 2 mL of ultra-dry toluene, bis(triphenylphosphine) palladium dichloride (10.1 mg, 0.014 mmol), cuprous iodide ( 5.5 mg, 0.029 mmol), triphenylphosphine (7.5 mg, 0.029 mmol), trimethylsilylacetylene (79 μL, 0.572 mmol), diisopropylamine (80.2 μL, 0.572 mmol). Overnight at room temperature. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a purple-red oily Intermediate 40 (47 mg, yield 85.9%).

¹ H NMR (400MHz, Chloroform-d) δ3.08(s, 3H), 0.35(s, 9H).

Intermediate 30 (351 mg, 1.83 mmol) was dissolved in 15 mL of MeOH, potassium carbonate (127 mg, 0.92 mmol) was added, and the reaction was completed in 5 min at room temperature. 20 mL of water was added, extracted with dichloromethane (20 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain purple oily product 14 (131.5 mg, yield 59.8 %), the tetrazine probe 14.

¹ H NMR (400MHz, Chloroform-d) δ3.69(s,1H), 3.11(s,3H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 166.33, 156.44, 85.62, 85.59, 21.71.

[Example 15]

Synthesis of 3-phenyl-6-ethynyl-1,2,4,5-tetrazine:

In a glove box, phenyltetrazine bromide (200 mg, 0.847 mmol) was dissolved in 8 mL of ultra-dry toluene, and bis(triphenylphosphine) palladium dichloride (30 mg, 0.042 mmol), cuprous iodide (16 mg) were added. , 0.085 mmol), triphenylphosphine (25.8 mg, 0.085 mmol), trimethylsilylacetylene (239 μL, 1.694 mmol), diisopropylamine (238 μL, 1.694 mmol). Overnight at room temperature. 50 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a purple-red oily intermediate 41 (380 mg, yield 89 %).

Intermediate 41 (300 mg, 1.18 mmol) was dissolved in 15 mL of MeOH, potassium carbonate (82 mg, 0.59 mmol) was added, and the reaction was completed in 5 min at room temperature. 50 mL of water was added, extracted with dichloromethane (50 mL x 3), the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain purple oily product 15 (133.8 mg, yield 62.3%) ), the tetrazine probe 15.

¹ H NMR (400MHz, Chloroform-d) δ8.65–8.60 (m, 2H), 7.68–7.59 (m, 3H), 3.76 (s, 1H).

¹³ C NMR (101MHz, Chloroform-d) δ162.19, 156.33, 133.57, 131.23, 129.57, 129.57, 128.78, 128.78, 86.06, 86.03.

4. Performance test of tetrazine compounds and bioorthogonal products

[Example 16]

Reaction Kinetics of Tetrazines and Cysteine

A cuvette was filled with 0.6 mL of 30% acetonitrile or 30% dimethyl sulfoxide in phosphate buffer and equilibrated at 22°C. Then, the tetrazine probes 1, 3, 8 and 9 prepared in the preceding examples were respectively added to the four groups of cuvettes. Cysteine was added to the cuvette and reacted with the tetrazine probe to generate a cysteine-ligated product. During the reaction process, the absorbance changes of tetrazine probes 1, 3, 8 and 9 at wavelengths of 260 to 350 nm were measured over time using a 6000+ (Quawell) UV-Vis spectrophotometer at 22°C to calculate the tetrazine Second order rate constant k ₂ for the reaction of the oxazine probe with cysteine.

As shown in FIGS. 1( a ) to ( h ), the second-order rate constant k ₂ of the reaction is calculated from the slope of (1/c-1/c ₀ ) with time. where c ₀ and c are the concentrations of tetrazine probes at the zero time and the specified time, respectively. Figure 1(a) and (b) show the calculated results of the _second order rate constant k for the reaction of tetrazine probe 1 and cysteine, where (a) the solvent is 30% acetonitrile and the wavelength is 300 nm, The solvent of (b) was 30% dimethyl sulfoxide and the wavelength was 305 nm. Figure 1(c) and (d) show the calculated results of the _second -order rate constant k for the reaction of the tetrazine probe 3 and cysteine, where (c) the solvent is 30% acetonitrile and the wavelength is 310 nm, The solvent of (d) was 30% dimethyl sulfoxide and the wavelength was 260 nm. Figure 1(e) and (f) show the calculated results of the _second order rate constant k for the reaction of tetrazine probe 8 and cysteine, where (e) the solvent is 30% acetonitrile and the wavelength is 260 nm, The solvent of (d) was 30% dimethyl sulfoxide and the wavelength was 260 nm. Figure 1 (g) and (h) show the calculated results of the _second order rate constant k for the reaction of the tetrazine probe 9 and cysteine, where (g) the solvent is 30% acetonitrile and the wavelength is 310 nm, The solvent of (d) was 30% dimethyl sulfoxide and the wavelength was 260 nm.

It can be seen from Fig. 1 that the second-order rate constants k ₂ of the above tetrazine probes are all between 3.6 and 202M ^-1 s ^-1 , indicating that thiol-containing polypeptides, proteins and other biomolecules can be treated by the tetrazine disclosed in the present invention. Compounds are rapidly and gently modified.

[Example 17]

Stability of tetrazine compounds

Tetrazine probes 3, 8 and 9 (20 mM, 2 μL) and cysteine (50 mM, 0.96 μL) were mixed in phosphate buffer solution (17.04 μL, 20 mM, pH 8.0, 30% MeCN) at room temperature . After the tetrazine probe was completely consumed, phosphate buffer solution (180 μL, 100 mM, pH 7.4) was added to the reaction solution again and kept at room temperature. The ligation product of the tetrazine probe and cysteine was analyzed by HPLC, and the stability of the ligation product was evaluated based on the peak area of the ligation product at 520 nm.

The analysis results are shown in Figure 2, and Figure 2(a) shows the stability of the ligation product of tetrazine probe 3 and cysteine in phosphate buffer solution. After 24 hours, the content of the ligation product was 98%; Figure 2(b) shows the stability of the ligation product of tetrazine probe 8 and cysteine in phosphate buffer solution. After 24 hours, the content of the ligation product is 94%; Figure 2(c) shows The stability of the ligation product of tetrazine probe 9 and cysteine in phosphate buffer solution was 97% after 24 hours. The analysis results show that the reaction product of the tetrazine compound and cysteine disclosed in the present invention is very stable, which is conducive to the bioorthogonal reaction between the labeled substance and the dienophile to realize the application of functional derivatization.

[Example 18]

Competitive Reaction of Tetrazines between Cysteine and Other Nucleophilic Amino Acids

Tetrazine probes at a final concentration of 200 μM in phosphate buffered solution (20 mM, 30% MeCN) were combined with 260 μM of cysteine and 2 mM of another amino acid (N-Boc-lysine or arginine). ) mixed solution reaction. After mixing and reacting for a period of time at room temperature, the peak area at 520 nm of the ligation product of the tetrazine probe and cysteine and the possible by-product of the tetrazine probe and another amino acid were detected by HPLC to evaluate the tetrazine compound selectivity.

Figure 3(a) and (b) show the competitive reaction of tetrazine probe 8 and cysteine in the presence of N-Boc-lysine and arginine, respectively, the results indicate that in the presence of other amino acids In this case, no competing by-products were detected within 5 min, and the reaction product of tetrazine probe 8 and cysteine accounted for 99%. Figure 3(c) and (d) show the competitive reaction of tetrazine probe 3 and cysteine in the presence of N-Boc-lysine and arginine, respectively, the results indicate that in the presence of other amino acids In this case, no competitive by-product was detected within 1 hour, and the reaction product of tetrazine probe 9 and cysteine accounted for more than 96%. Figure 3(e) and (f) show the competitive reaction of tetrazine probe 9 and cysteine in the presence of N-Boc-lysine and arginine, respectively, the results indicate that in the presence of other amino acids In this case, no competing by-products were detected within 1 hour, and the reaction product of tetrazine probe 8 and cysteine accounted for 98%.

It can be seen that the tetrazine compound disclosed in the present invention only reacts with cysteine, but not with another high-concentration amino acid containing amino group that exists at the same time, has high selectivity to amino acid containing thiol group, and can rapidly, Stable and accurate labeling of thiol-containing amino acids and biomolecules, biological materials and other substances containing such amino acids. The competitive reaction described above is as follows:

[Example 19]

Stability of Bioorthogonal Products

Tetrazine probe (20 mM, 2 μL) was mixed with cysteine (50 mM, 1.1 μL) in PB buffer (16.9 μL, 20 mM, 30% MeCN) at room temperature. BCN was added after the Cys was labeled with the tetrazine probe, and the mixture was left to react at room temperature until the tetrazine probe was completely consumed. The resulting bioorthogonal product was further diluted with PB buffer (180 μL, 100 mM, pH 7.4) to 0.2 mM, and then glutathione was added to a final concentration of 5 mM. Glutathione exchange was analyzed by monitoring HPLC peak areas of bioorthogonal products and GSH exchange products over time.

Figure 4(a) shows the HPLC trace (280 nm) of the reaction of the bioorthogonal product 42 with GSH in PB buffer (100 mM, pH 7.4) at room temperature; Figure 4(b) shows the bioorthogonal product at room temperature HPLC trace (280 nm) of the reaction of 43 with GSH in PB buffer (100 mM, pH 7.4). The analysis results showed that no glutathione exchange products were found at 40 or 68 hours, and only the impurity peaks produced by the reaction of GSH and BCN were observed.

V. Tetrazine Probe Labeling and Bioorthogonal Reaction

[Example 20]

To a reaction tube containing phosphate buffer (20 mM, pH 8.0, 5% DMSO) was added 110 [mu]M of tetrazine probe 8. After mixing, add cysteine-containing polypeptide ARI to the same reaction tube with a final concentration of 100 μM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 1 hour, and the formation of the target product was detected by HPLC-MS, and the yield was 96%.

Add dienophile BCN (cyclooctyne, 2mM) to the mixed solution after the reaction, pipette up and down 10 times with a pipette, react at room temperature for 2 hours, and detect the formation of the target polypeptide product by LC-MS. The step yield was 95%.

[Example 21]

To a reaction tube containing phosphate buffer (20 mM, pH 8.0, 5% DMSO) was added 110 [mu]M of tetrazine probe 8. After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 100 μM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 1 hour, and the formation of the target product was detected by HPLC-MS, and the yield was 97%.

After the labeling reaction with tetrazine probe 8, add dienophile BCN (cyclooctyne, 4mM) to the mixed solution, pipette up and down 10 times with a pipette, react at room temperature for 1 hour, and use LC-MS The generation of the target polypeptide product was detected, and the two-step yield was 95%.

After the labeling reaction with tetrazine probe 8, add dienophile aTCO (trans-cyclooctene, 1mM) to the mixed solution, pipette up and down 10 times, react at room temperature for 10 minutes, and use LC -MS detects the formation of the target polypeptide product with a two-step yield of 97%.

[Example 22]

To a reaction tube containing phosphate buffer (20 mM, pH 8.0, 5% DMSO) was added 110 [mu]M of tetrazine probe 9. After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 100 μM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 1.5 hours, and the target product was detected by HPLC-MS, and the yield was 93%.

[Example 23]

To a reaction tube containing phosphate buffer (20 mM, pH 8.0, 10% MeCN) was added 110 [mu]M of tetrazine probe 3. After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 100 μM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 2 hours, and the target product was detected by HPLC-MS with a yield of 97%.

After the labeling reaction with tetrazine probe 3, add dienophile aTCO (trans-cyclooctene, 1mM) to the mixed solution, mix well, react at room temperature for 10 minutes, and detect the target by LC-MS The yield of the polypeptide product was 97%.

[Example 24]

1 mM tetrazine probe 8 was added to a reaction tube containing phosphate buffer (20 mM, pH 8.0, 5% DMSO). After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 1 mM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 10 minutes, and the production of the target product was detected by HPLC-MS, and the yield was 98%.

[Example 25]

To a reaction tube containing phosphate buffer (20 mM, pH 8.0, 5% DMSO) was added 1 mM of tetraphilic probe 9. After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 1 mM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 20 minutes, and the production of the target product was detected by HPLC-MS, and the yield was 93%.

[Example 26]

1 mM tetrazine probe 3 was added to a reaction tube containing phosphate buffer (20 mM, pH 8.0, 10% MeCN). After mixing, add cysteine-containing polypeptide ASC to the same reaction tube with a final concentration of 1 mM and a total liquid volume of 200 μL. After mixing, the reaction was carried out at room temperature for 20 minutes, and the production of the target product was detected by HPLC-MS, and the yield was 96%.

[Example 27]

3 mM tetrazine probe 15 was added to a reaction tube containing phosphate buffer (20 mM, pH 8.0, 40% MeCN). After mixing, cysteine was added to the same reaction tube at a final concentration of 3.3 mM and the total liquid volume was 250 μL. After mixing, the reaction was carried out at room temperature for 1 hour, and the production of the target product was detected by HPLC-MS with a yield of 98%.

[Example 28]

Affibodies were labeled with vinyltetrazine probes and dye-labeled affibodies were obtained.

Tris buffer (60 μL, 200 mM, pH 8.0), TCEP (tris(2-carboxyethyl)phosphine, 100 mM, 2.3 μL, 10 equiv) were added to the PBS buffer with affibosome Z09591 (60 μL, 386 μM) dissolved , the reaction was carried out at 37°C for 1 hour. Reactions were monitored by HPLC-MS until the disulfide bonds of the affibodies were completely reduced. Tetrazine probe 8 (20 mM, 2.5 μL, 10 equiv.) was then added to PB buffer dissolved in reducing affine (50 μL, 100 μM) and reacted at room temperature with a yield of 96%. SulfoCy5-TCO (10 mM, 2 μL) was then added and allowed to stand at room temperature. The reaction was monitored by HPLC-MS and the two-step yield was 95%.

[Example 29]

Trastuzumab was labeled with a tetrazine probe and dye-labeled trastuzumab was obtained.

Tris buffer (60 μL, 213 mM, pH 8.0), TCEP (tris(2-carboxyethyl)phosphine, 50 mM, 1 μL, 10 equiv) were added to trastuzumab (49 μL, 102 μM) in PBS buffer , and reacted at 37°C for 2 hours. Reactions were monitored by HPLC-MS until the disulfide bonds of the affibodies were completely reduced. Tetrazine probe 8 (20 mM, 1 μL, 10 equiv) was then added to PB buffer dissolved in reduced trastuzumab (95 μL, 21 μM), and reacted at room temperature for 1 hour. SulfoCy5-TCO (10 mM, 3.2 μL) was then added to 80 μL of trastuzumab-8 and reacted at room temperature for 1 hour, and the reaction was monitored by HPLC-MS. LC-MS analysis showed that the labeling rate of tetrazine probe 8 to trastuzumab was 93%, and the tetrazine-modified trastuzumab could be fully functionalized by Cy5-modified dienophile (Cy-TCO). A two-step labeling yield of 93% was achieved, with a dye-to-antibody labeling ratio of 3.4 (an average of 3.4 Cy5 dyes labeled with one antibody). Surface plasmon resonance (SPR) experiments showed that tetrazine-modified trastuzumab (12pM) and unmodified trastuzumab (23pM) had similar affinity for HER2.

[Example 30]

SKOV3 cells (HER-2 positive) or 4T1 cells (HER-2 negative) were stained with Hoechst 33342 (250 nM) for 5 min to label the nuclei, washed with Trastuzumab-Cy5 (33 nM) for 30 min, after washing , imaged by confocal microscopy in DMEM medium containing 10% fetal bovine serum. SKOV3 was pre-incubated with 100 nM trastuzumab for 15 minutes, washed and then imaged. It can be seen that only HER-2-positive SHOV3 can be fluorescently labeled, while 4T1 cells that are pre-bound or negative for SKOV3 cannot be stained, indicating that the modified trastuzumab does not affect its selectivity for HER-2.

To sum up, the tetrazine compounds involved in the present invention can be used for polypeptides, proteins, drug molecules, molecular imaging probes, diagnostic reagents and various sulfhydryl-containing biological nanomaterials containing cysteine or homocysteine. Labeling and post-functionalization.

The specific embodiments described above further describe the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

Tetrazine compound, is characterized in that, its structural formula is shown in formula I:

In formula I, R is selected from hydrogen, alkyl, heterocycloalkyl, aryl, heteroaryl, alkenyl, polyethylene glycol group; R' is selected from substituted or unsubstituted alkenyl, or substituted or unsubstituted Substituted alkynyl.
tetrazine compound according to claim 1, is characterized in that, its structural formula is as shown in formula II:

In formula II, R, R 2 , R 3 and R 4 are each independently selected from hydrogen, C 1 -C 36 chain alkyl, C 4 -C 6 cyclic alkyl, heteroatom is sulfur, oxygen or nitrogen C 4 -C 5 heterocycloalkyl group, C 1 -C 24 aldehyde group, C 2 -C 24 ketone group, C 6 -C 12 aryl group, C 4 -C 12 heteroaryl group whose heteroatom is sulfur, oxygen or nitrogen group, C 2 -C 24 amide acyl, C 2 -C 24 alkenyl or polyethylene glycol group.
tetrazine compound according to claim 1, is characterized in that, its structural formula is as shown in formula III:

In formula III, R and R 5 are each independently selected from hydrogen, C 1 -C 36 chain alkyl, C 4 -C 6 cyclic alkyl, C 4 -C 5 heteroatom whose heteroatom is sulfur, oxygen or nitrogen Cycloalkyl, C 1 -C 24 aldehyde group, C 2 -C 24 ketone group, C 6 -C 12 aryl group, C 4 -C 12 heteroaryl group whose heteroatom is sulfur, oxygen or nitrogen, C 2 -C 24 ester group, C 2 -C 24 aminoacyl group, C 2 -C 24 alkenyl group or polyethylene glycol group.
The tetrazine compound according to claim 2 or 3, wherein the R is selected from the following groups:
A preparation method for preparing the tetrazine compound according to any one of claims 2 to 4, characterized in that, comprising the following steps:

Dissolving the compound shown in formula IV in the first reaction solvent, adding a sulfonic acid esterification reagent to obtain the compound shown in formula V, and performing elimination reaction of the compound shown in formula V under the action of a base to obtain vinyltetrazine compounds;

The compound shown in formula VI is dissolved in the second reaction solvent, trimethylsilane acetylene is added under the action of a catalyst to obtain the compound shown in formula VII, and the compound shown in formula VII is subjected to elimination reaction under the action of a base to obtain ethynyl tetrazine. compound; or

After dissolving the compound represented by the formula X, a methyl-substituted vinyltetrazine compound is generated by Stille coupling reaction;
The preparation method of tetrazine compound according to claim 5, is characterized in that, the preparation method of compound shown in described formula IV comprises the following steps:

Using RCN and 3-hydroxypropionitrile as raw materials, adding thiol compounds and reacting with hydrazine hydrate, or adding Lewis acid and reacting with hydrazine hydrate.
The method for preparing tetrazine compounds according to claim 5, wherein the first reaction solvent is dichloromethane, dichloroethane, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide or N,N - dimethylformamide, the second reaction solvent is ultra-dry toluene, and the base is triethylamine, diisopropylaminoethylamine, pyridine, sodium acetate, potassium carbonate, sodium carbonate or potassium tert-butoxide.
The method for preparing a tetrazine compound according to claim 5, wherein the sulfonic acid esterification reagent is methanesulfonyl chloride.
The use of the tetrazine compound according to any one of claims 1 to 4, wherein the tetrazine compound is used to selectively label an amino acid having a sulfhydryl group and a biomolecule or biological substance containing the amino acid The thiol group of the material.
The application of the tetrazine compound according to claim 9, wherein the labeling method is as follows: adding the tetrazine compound in a buffer solution, adding the substance to be labeled after the tetrazine compound is dissolved, and obtaining after the reaction Tetrazine-labeled products capable of functional derivatization applications via bioorthogonal reactions with dienophiles.