CN112010817A - Method for preparing tetrazine compound and application thereof - Google Patents

Abstract

The invention discloses a novel method for preparing tetrazine compounds, which uses nitrile and hydrazine hydrate, can carry out various substitutions in the presence of thiol catalysts to obtain tetrazine compounds with various functions, and facilitates the further modification of the tetrazine compounds, thereby obtaining more tetrazine derivatives with special functions. In addition, the method for preparing the tetrazine compound has the advantages of mild reaction conditions, easily obtained reagents, environmental friendliness, high product yield, gram-scale production and the like. The problems that in the prior art, the substitution is single, the variety of the obtained tetrazine compound is limited, the preparation condition is harsh, and the large-scale production cannot be realized when hydrazine hydrate is adopted to prepare the tetrazine compound are solved.

Description

A kind of method for preparing tetrazine compound and its application

technical field

The present invention relates to a method for preparing tetrazine compounds and its application.

Background technique

Tetrazines have promising applications in coordination chemistry, total synthesis, and in the design of high-energy-density materials. At the same time, with the development of science, tetrazine compounds and their derivatives have been found to have more physiological activities, such as anti-inflammatory, analgesic, antiviral, antibacterial and anticancer. In addition, with the uniqueness of tetrazine compounds in bioorthogonal chemistry, they have a wide range of applications in bioorthogonal fluorescent probes, targeted drug delivery of drugs, calibration of special proteins, and precursors of biological materials. value.

Although there are many methods for preparing tetrazine compounds at present, they all require a harsh reaction condition, such as heating and microwave; it requires the use of anhydrous hydrazine. In the classical two-step method for preparing tetrazine, the first Condensation to nitrile linkage in one step, followed by oxidation of 1,2-dihydrotetrazine in the second step; this method tolerates a narrow range of substrates. In 2012, a research group proposed a metal-catalyzed method to synthesize asymmetric tetrazine. Although a series of tetrazine derivatives were obtained, the controlled reagent anhydrous hydrazine and expensive metal catalyst were used in this method, and the reaction required under heating conditions. In addition, someone proposes to use the hydrazine hydrate solution of ethanol for the synthesis of tetrazine, but this method can only be used for the preparation of mono-substituted tetrazine, and the preparation conditions of this method are harsh, need to carry out under microwave conditions, and cannot be produced on a large scale, This greatly limits the application of this method. Therefore, due to the limitation of synthetic methods of tetrazine compounds, the development of tetrazine functions and their application in downstream fields are greatly restricted.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a method for preparing tetrazine compounds and its application.

The invention provides a method for preparing tetrazine compounds, which comprises the following steps:

Step 1: Dissolve the nitrile compound a, the nitrile compound b, the catalyst thiol compound, and the hydrazine hydrate in a solvent, and react to obtain a reaction solution;

Step 2: mixing the reaction solution with the oxidant-containing solution, slowly adding acid, adjusting the pH of the system to acidity, reacting, and purifying the reaction solution to obtain tetrazine compounds;

in,

R ₁ and R ₂ are independently selected from substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted 3- to 8-membered unsaturated cycloalkyl, substituted or unsubstituted 3- to 8-membered unsaturated heterocycle group, substituted or unsubstituted C ₁ -C ₁₀ alkoxy;

The substituents are substituted or unsubstituted C ₁ -C ₁₀ alkyl, halogen, cyano, nitro, carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₁₀ alkoxy, -NR ₄ R ₅ , azide Nitrogen, phosphate, tert-butoxycarbonyl;

R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₆ alkyl, and tert-butoxycarbonyl.

further,

取代或未取代的6元不饱和环烷基、取代或未取代5元不饱和杂环基；R ₁ and R ₂ are independently selected from unsubstituted C ₁ -C ₁₀ alkyl, -(CH ₂ ) _x R ₃ , -(CH ₂ ) _X N ₃ , -(CH ₂ CH ₂ O) _X N ₃ , -(CH ₂ ) _x (EtO) ₂ PO,

Substituted or unsubstituted 6-membered unsaturated cycloalkyl, substituted or unsubstituted 5-membered unsaturated heterocyclyl;

R ₃ is selected from cyano, nitro, carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₆ alkoxy, -NR ₄ R ₅ ;

R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₆ alkyl, and tert-butoxycarbonyl;

The substituents are substituted or unsubstituted C ₁ -C ₁₀ alkyl, halogen, substituted or unsubstituted C ₁ -C ₆ alkoxy, nitro, amino, cyano, hydroxyl, carboxyl, phosphate, tertiary Butoxycarbonyl, -NR ₄ R ₅ ;

x=1-10.

further,

-(CH₂)_XN₃、-(CH₂CH₂O)_XN₃、-(CH₂)_x(EtO)₂PO；R ₁ and R ₂ are independently selected from -(CH ₂ ) _x R ₃ , substituted or unsubstituted aryl, pyrrolyl, thienyl,

-(CH ₂ ) _X N ₃ , -(CH ₂ CH ₂ O) _X N ₃ , -(CH ₂ ) _x (EtO) ₂ PO;

R ₃ is selected from carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₂ alkoxy, -NR ₄ R ₅ ;

R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₄ alkyl, and tert-butoxycarbonyl;

The substituents are substituted or unsubstituted C ₁ -C ₈ alkyl, halogen, substituted or unsubstituted C ₁ -C ₂ alkoxy, nitro, amino, cyano, hydroxyl, carboxyl, phosphate, tertiary Butoxycarbonyl, -NR ₄ R ₅ ;

where x=1～10.

further,

In step 1, the molar ratio of the nitrile compound a, the nitrile compound b, the hydrazine hydrate, and the thiol compound is 1:1-30:6-32:0.2-1; the volume ratio of the hydrazine hydrate and the solvent is 2:0～4;

And/or, in step 2, the oxidant equivalents in the oxidant-containing solution are 15-20 times the total equivalents of the nitrile compounds a and the nitrile compounds b;

And/or, in step 2, an acid is added to adjust the pH of the system to 1-6.

further,

In step 1, the molar ratio of the nitrile compound a, the nitrile compound b, the hydrazine hydrate, and the thiol compound is 1:4 to 8:16:0.2 to 1; the volume ratio of the hydrazine hydrate to the solvent is 2: 1;

And/or, in step 2, the oxidant equivalent number in the oxidant-containing solution is 15 times the total equivalent number of the nitrile compound a and the nitrile compound b;

And/or, in step 2, acid is added to adjust the pH of the system to 3-4.

further,

In step 1, the solvent is selected from tetrahydrofuran, dimethyl sulfoxide, ethanol, dioxane, isopropanol, tert-butanol, n-propanol, n-butanol, ethylene glycol or hexafluoroisopropanol; preferably For ethanol or dimethyl sulfoxide.

further,

In step 1, the thiol compound is selected from cysteine, 1,3-propanedithiol, mercaptoethylamine, mercaptopropionic acid, thioglycolic acid, N-acetyl-L-cysteine, reduced glutathione Sathione, butanethiol or propanedithiol; preferably mercaptopropionic acid.

further,

In step 2, the oxidant in the oxidant-containing solution is sodium nitrite, hydrogen peroxide, iodobenzenediacetic acid, and oxygen; preferably sodium nitrite;

And/or, in step 2, the oxidant-containing solution is an oxidant-containing ice water solution, and the concentration of the oxidant-containing solution is 0.5-1.5 mmol/mL; preferably 1 mmol/mL.

further,

In step 2, the acid is hydrochloric acid, sulfuric acid, acetic acid, preferably hydrochloric acid; the concentration of the acid is 1M.

further,

In step 1, when the nitrile compounds a and b, the catalyst thiol compound, and the hydrazine hydrate are dissolved in the solvent, the hydrazine hydrate is added last, and the temperature during the addition is 0°C.

further,

In step 1, the reaction temperature is 0-60°C; the reaction time is 0.5-64h; preferably, the reaction temperature is 25°C or 40°C, and the reaction time is 6-18h.

further,

In step 2, the temperature at which the acid is added is 0°C.

further,

In step 2, the purification is to extract the reaction solution, and the organic phase is taken, washed, dried, filtered, concentrated under reduced pressure, and purified by silica gel chromatography.

further,

The extraction is 3 times of extraction with ethyl acetate; the washing is washing with saturated brine; the drying is drying with anhydrous sodium sulfate.

The present invention also provides the tetrazine compound prepared by the aforementioned method, and the structural formula of the tetrazine compound is as follows:

"Substituted" in the present invention means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

In the present invention, the minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix (C _a -C _b )alkyl indicates any alkyl group containing "a" to "b" carbon atoms . Thus, for example, C ₁ -C ₁₀ alkyl refers to a straight or branched chain alkyl group containing 1 to 10 carbon atoms; C ₁ -C ₁₀ alkoxy refers to an alkoxy group containing 1 to 10 carbon atoms .

In the present invention, a 3- to 8-membered unsaturated cycloalkyl group refers to a cycloalkyl group having one or more unsaturated bonds, and the cycloalkyl group is composed of 3-8 carbon atoms; a 3- to 8-membered unsaturated heterocyclic ring The group refers to a heterocyclic group having one or more unsaturated bonds, the heterocyclic group is composed of 3 to 8 atoms, at least one of the atoms is selected from O, S, or a substituted nitrogen atom or a silicon atom.

In the present invention, halogen is fluorine, chlorine, bromine or iodine.

In the present invention, the room temperature is 25°C±5°C.

In the prior art, anhydrous hydrazine is mainly used for the preparation of tetrazine compounds, but anhydrous hydrazine is a controlled reagent, which is very toxic and has very harsh reaction conditions. Although some people use hydrazine hydrate with relatively less toxicity to prepare tetrazine compounds, in the existing method, only mono-substituted tetrazine compounds can be prepared by using hydrazine hydrate, and the types of tetrazine compounds obtained are very limited, and when preparing It also requires very harsh reaction conditions, such as the need to carry out under microwave conditions, or the use of elemental sulfur, which is an explosive dangerous substance, which affects large-scale production and greatly limits the application of this method.

The invention discloses a novel method for preparing tetrazine compounds. The method uses nitrile and hydrazine hydrate, and can carry out various substitutions in the presence of thiol catalysts to obtain tetrazine compounds with various functions, which is convenient for tetrazine compounds. Further modification and transformation of the oxazine compound leads to more tetrazine derivatives with special functions. In addition, the method for preparing tetrazine compounds of the present invention has the advantages of mild reaction conditions, readily available reagents, environmental friendliness, high product yield, gram-scale production and the like. The method overcomes the problems in the prior art that the hydrazine hydrate is used to prepare the tetrazine compounds, the substitution is single, the types of the tetrazine compounds obtained are limited, the preparation conditions are harsh, and the large-scale production is impossible.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Detailed ways

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.

Embodiment 1, the preparation of tetrazine compound of the present invention

Add N-(tert-butylcarbonyl)-2-aminoacetonitrile (312 mg, 2 mmol), 3-mercaptopropionic acid (17.4 μL, 0.2 mmol), ethanol (389 μL) to a 10 mL reaction tube, and place the reaction tube in an ice bath Then, hydrazine hydrate (777 μL, 16 mmol) was added, and then stirred at room temperature for 18 h. The reaction was complete. The reaction solution was poured into an ice-water solution of sodium nitrite (1.03 g, 15 mmol), and hydrochloric acid (1 M) was slowly added at 0 °C. To the pH of the solution = 5, carry out the reaction, after the completion of the TLC detection, the obtained solution was extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, separated with a separatory funnel, combined the organic phases extracted 3 times, saturated common salt Wash with water (20ml) once, separate with a separatory funnel to obtain an organic phase, and then dry the organic phase with anhydrous sodium sulfate, filter with a glass sand funnel to obtain a filtrate, transfer the filtrate to a round-bottomed flask, and concentrate under reduced pressure to obtain a red color Crude oil. After purification by silica gel chromatography, a pink solid compound (262 mg) was obtained in a yield of 77%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.96 (d, J=1.0 Hz, 4H), 1.43 (s, 18H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 167.59, 155.78, 80.44, 43.50, 28.30.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₄ H ₂₄ N ₆ NaO ₄ ] ⁺ 363.1757, found 363.1763.

Embodiment 2, the preparation of tetrazine compound of the present invention

Add benzonitrile (102 μL, 1 mmol), 2-cyanoacetic acid (340 mg, 4 mmol), 3-mercaptopropionic acid (17.4 μL, 0.2 mmol) in a 10 mL reaction tube, place the reaction tube under an ice bath, add hydrazine hydrate ( 777μL, 16mmol), then stirred at 40°C for 18h, the reaction was complete, the reaction solution was poured into an ice-water solution of sodium nitrite (1.03g, 15mmol), and hydrochloric acid (1M) was slowly added at 0°C to pH=4 , carry out the reaction, and after the completion of the TLC detection, the obtained solution was extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, separated with a separatory funnel, combined the organic phases extracted 3 times, washed with saturated brine (20 ml) Once, separate the liquid with a separating funnel to obtain an organic phase, which is then dried over anhydrous sodium sulfate, filtered with a glass sand funnel to obtain a filtrate, which is transferred to a round-bottomed flask and concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel chromatography, a pink solid compound (145 mg) was obtained in a yield of 67.1%.

¹ H NMR (400 MHz, DMSO) δ 8.53 (dd, J=7.4, 1.7 Hz, 2H), 7.83–7.61 (m, 3H), 3.06 (s, 2H).

¹³ C NMR (101MHz, DMSO) δ171.86, 167.61, 163.74, 132.88, 132.35, 129.87, 127.86, 21.29.

HRMS[M+H] ⁺ m/z calcd.for[C ₁₀ H ₉ N ₄ O ₂ ] ⁺ 217.0726, found 217.0727.

Embodiment 3, the preparation of tetrazine compound of the present invention

Benzonitrile (102 μL, 1 mmol), 3-hydroxypropionitrile (272 μL, 4 mmol), 3-mercaptopropionic acid (87 μL, 1 mmol) were added to a 10 mL reaction tube, the reaction tube was placed in an ice bath, and hydrazine hydrate (777 μL) was added. , 16mmol), then, stirred at 40°C for 13h, the reaction was complete; the reaction solution was poured into an aqueous solution of sodium nitrite (1.03g, 15mmol), and hydrochloric acid (1M) was slowly added at 0°C until the solution pH=4, The reaction was carried out, and after the completion of the TLC detection, the obtained solution was extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, and the layers were separated with a separatory funnel. The organic phases of the 3 extractions were combined and washed once with saturated brine (20 ml). , the separatory funnel was used to separate the liquids to obtain the organic phase. The organic phase was then dried with anhydrous sodium sulfate and filtered with a glass sand funnel to obtain a filtrate. The filtrate was transferred to a round-bottomed flask and concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel chromatography, a pink solid compound (134 mg) was obtained in a yield of 66.3%.

¹ H NMR (400MHz, DMSO) δ8.53 (dt, J=8.2, 2.3Hz, 2H), 7.77-7.69 (m, 3H), 4.88 (s, 1H), 4.06 (d, J=5.8Hz, 2H) ),3.49(t,J=6.4Hz,2H).

¹³ C NMR (101MHz, DMSO) δ168.69, 164.02, 132.97, 132.33, 129.92, 127.91, 59.78, 38.62.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₀ H ₁₀ N ₄ NaO] ⁺ 225.0752, found 225.0751.

Embodiment 4, the preparation of tetrazine compound of the present invention

2-thiophenecarbonitrile (93 μL, 1 mmol), 3-hydroxypropionitrile (272 μL, 4 mmol), 3-mercaptopropionic acid (35 μL, 0.4 mmol) were added to a 10 ml reaction tube, dissolved in 383 μL of ethanol, and the reaction tube was placed in Under an ice bath, hydrazine hydrate (777 μL, 16 mmol) was added, and then stirred at 40 °C for 15 h, the reaction was complete; the reaction solution was poured into an aqueous solution of sodium nitrite (1.03 g, 15 mmol), and hydrochloric acid was slowly added at 0 °C (1M) to the solution pH=4, carry out the reaction, TLC detects that after the reaction is completed, the obtained solution is extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, the separatory funnel is used for separation, and the organic phases extracted for 3 times are combined. , washed once with saturated brine (20ml), separated with a separatory funnel to obtain an organic phase, which was then dried over anhydrous sodium sulfate, filtered with a glass sand funnel to obtain a filtrate, which was transferred to a round-bottomed flask and concentrated under reduced pressure , a red solid crude product was obtained. After purification by silica gel column chromatography, a pink solid compound (128 mg) was obtained, yield: 62%.

¹ H NMR (400MHz, DMSO) δ 8.24 (dt, J=2.7, 1.2Hz, 1H), 8.08-7.95 (m, 1H), 7.37 (td, J=4.3, 3.8, 1.1Hz, 1H), 4.92 (t,J=5.7Hz,1H),3.98(q,J=6.2Hz,2H),3.38(t,J=6.3Hz,2H).

¹³ C NMR (101MHz, DMSO) δ168.17, 161.99, 135.82, 133.68, 131.33, 129.83, 59.75, 38.52.

HRMS[M+Na] ⁺ m/z calcd.for[C ₈ H ₈ N ₄ NaOS] ⁺ 231.0317, found 231.0317.

Embodiment 5, the preparation of tetrazine compound of the present invention

Benzonitrile (102 μL, 1 mmol), 3-(dimethylamine)-propionitrile (452 μL, 4 mmol), and 3-mercaptopropionic acid (87 μL, 1 mmol) were added to a 10 ml reaction tube, which was placed in an ice bath , add hydrazine hydrate (777 μL, 16 mmol), then, stir at 40 ° C for 14 h, the reaction is complete; the reaction solution is poured into sodium nitrite (1.03 g, 15 mmol) ice-water solution, and hydrochloric acid (1 M) is slowly added at 0 ° C. To the pH of the solution = 4, carry out the reaction, TLC detection after the completion of the reaction, the obtained solution was extracted 3 times with ethyl acetate, 20 ml of ethyl acetate each time, separated with a separatory funnel, combined the organic phases extracted 3 times, saturated common salt Wash with water (20ml) once, separate with a separatory funnel to obtain an organic phase, and then dry the organic phase with anhydrous sodium sulfate, filter with a glass sand funnel to obtain a filtrate, transfer the filtrate to a round-bottomed flask, and concentrate under reduced pressure to obtain a red color Solid crude product. After purification by silica gel column chromatography, a pink solid compound (94 mg) was obtained, yield: 41%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.59 (dd, J=8.1, 1.6 Hz, 2H), 7.63-7.56 (m, 3H), 3.53 (t, J=7.1 Hz, 2H), 3.01 (t, J=7.1Hz, 2H), 2.33(s, 6H).

¹³ C NMR (101MHz, CDCl ₃ ) δ 168.94, 164.26, 132.53, 131.86, 129.21, 127.95, 57.45, 45.21, 33.03.

HRMS[M+H] ⁺ m/z calcd.for[C ₁₂ H ₁₆ N ₅ ] ⁺ 230.1406, found 230.1400.

Embodiment 6, the preparation of tetrazine compound of the present invention

4-Fluorobenzonitrile (121mg, 1mmol), 2-cyanoacetic acid (340mg, 4mmol), 3-mercaptopropionic acid (87μL, 1mmol) were added to a 10ml reaction tube, dissolved in 200μL of ethanol, and the reaction tube was placed on ice Under the bath, hydrazine hydrate (777 μL, 16 mmol) was added, and then stirred at room temperature for 15 h to complete the reaction; the reaction solution was poured into an aqueous solution of sodium nitrite (1.03 g, 15 mmol), and hydrochloric acid (1 M) was slowly added at 0° C. ) to the pH of the solution = 4, carry out the reaction, and after the completion of the TLC detection reaction, the obtained solution was extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, separated with a separatory funnel, combined the organic phases extracted 3 times, saturated Wash with salt water (20ml) once, and separate with a separatory funnel to obtain an organic phase. The organic phase is then dried with anhydrous sodium sulfate and filtered with a glass sand funnel to obtain a filtrate. The filtrate is transferred to a round-bottomed flask and concentrated under reduced pressure to obtain Red solid crude product. After purification by silica gel column chromatography, a pink solid compound (80 mg) was obtained, yield: 34%.

¹ H NMR (400 MHz, DMSO) δ8.61–8.39 (m, 2H), 7.51 (t, J=8.9 Hz, 2H), 3.00 (s, 2H).

¹³ C NMR (101MHz, DMSO) δ167.55, 165.28 (d, J=250.6Hz), 163.03, 130.56, 130.47,

128.96(d, J＝3.0Hz), 117.17, 116.95, 21.27.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₀ H ₇ FN ₄ NaO ₂ ] ⁺ 257.0451, found 257.0450.

Embodiment 7, the preparation of tetrazine compound of the present invention

4-Cyanophenylacetic acid (32.2 mg, 0.2 mmol), 3-hydroxypropionitrile (54.4 μL, 0.8 mmol), 3-mercaptopropionic acid (7.2 μL, 0.08 mmol) were added to a 10 ml reaction tube, and the reaction tube was placed Under an ice bath, hydrazine hydrate (156 μL, 3.2 mmol) was added, and then stirred at room temperature for 15 h to complete the reaction; the reaction solution was poured into an ice-water solution of sodium nitrite (206 mg, 3 mmol), and hydrochloric acid was slowly added at 0°C (1M) to the solution pH=4, carry out the reaction, TLC detects that after the reaction is completed, the obtained solution is extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, the separatory funnel is used for separation, and the organic phases extracted for 3 times are combined. , washed once with saturated brine (20ml), separated with a separatory funnel to obtain an organic phase, which was then dried over anhydrous sodium sulfate, filtered with a glass sand funnel to obtain a filtrate, which was transferred to a round-bottomed flask and concentrated under reduced pressure , a red solid crude product was obtained. After purification by silica gel column chromatography, compound 10 (36 mg) was obtained as a pink solid, yield: 69%.

¹ H NMR(400MHz,DMSO)δ8.40(d,J=8.3Hz,2H),7.55(d,J=8.3Hz,2H),4.01(t,J=6.3Hz,2H),3.64(s, 1H),3.43(t,J=6.3Hz,2H).There are four hydrogen atδ3.43in the H NMR spectra because it contains the water peakof DMSO.

¹³ C NMR (101MHz, DMSO) δ173.13, 168.53, 164.01, 141.83, 130.96, 130.21, 127.67, 59.76, 42.63, 38.57.

HRMS[M+H] ⁺ m/z calcd.for[C ₁₂ H ₁₃ N ₄ O ₃ ] ⁺ 261.0988, found 261.0688.

Example 8. Preparation of tetrazine compounds of the present invention

Add 4-cyanobenzoic acid (29.4 mg, 0.2 mmol), 3-hydroxypropionitrile (108.8 μL, 1.6 mmol), 3-mercaptopropionic acid (7.2 μL, 0.08 mmol) into a 10 ml reaction tube, dissolve with 78 μL ethanol , the reaction tube was placed in an ice bath, hydrazine hydrate (156 μL, 3.2 mmol) was added, and then, stirred at room temperature for 18 h, the reaction was complete; Slowly add hydrochloric acid (1M) at ℃ to pH=4 of the solution to carry out the reaction. After the completion of the TLC detection, the obtained solution was extracted with ethyl acetate 3 times, 20 ml of ethyl acetate each time, and the solution was separated with a separatory funnel. The organic phase of the first extraction was washed once with saturated brine (20ml), and the liquid was separated with a separatory funnel to obtain an organic phase. The organic phase was then dried with anhydrous sodium sulfate, filtered with a glass sand funnel to obtain a filtrate, and the filtrate was transferred to a round-bottomed flask. was concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel column chromatography, compound 11 (31 mg) was obtained as a pink solid, yield: 63%.

¹ H NMR(400MHz, DMSO)δ8.54(d,J=8.4Hz,2H),8.17(d,J=8.4Hz,2H),4.02(t,J=6.3Hz,2H),3.45(t, J=6.3Hz, 2H).

¹³ C NMR (101MHz, DMSO) δ168.76, 163.76, 163.64, 134.98, 130.59, 127.83, 59.74, 38.54.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₁ H ₁₀ N ₄ NaO ₃ ] ⁺ 269.0651, found 269.0658.

Embodiment 9, the preparation of tetrazine compound of the present invention

4-cyanobenzoic acid (29.4mg, 0.2mmol), N-Boc-2-cyanoethylamine (272mg, 1.6mmol), 3-mercaptopropionic acid (18μL, 0.2mmol) were added to a 10ml reaction tube, 78 μL of DMSO was dissolved, the reaction tube was placed in an ice bath, hydrazine hydrate (156 μL, 3.2 mmol) was added, and then stirred at room temperature for 18 h, the reaction was complete; the reaction solution was poured into sodium nitrite (206 mg, 3 mmol) ice-water solution , slowly add hydrochloric acid (1M) at 0°C to pH=4, carry out the reaction, after the TLC detection reaction is completed, the obtained solution is extracted 3 times with ethyl acetate, 20 ml of ethyl acetate each time, and the separatory funnel is used to separate the liquids. , combine the organic phases extracted 3 times, wash once with saturated brine (20ml), and separate the layers with a separatory funnel to obtain an organic phase. In a round-bottomed flask, concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel column chromatography, compound 12 (38 mg) was obtained as a pink solid, yield: 55%.

¹ H NMR (400MHz, CD ₃ OD) δ 8.65(d, J=8Hz, 2H), 8.25(d, J=8Hz, 2H), 3.64(t, J=6Hz, 2H), 3.50(t, J =6Hz,2H),1.31(s,9H).

¹³ C NMR (400MHz, DMSO) δ168.59, 167.34, 163.41, 156.07, 135.57 (d, J=188Hz), 130.68, 127.96, 78.25, 39.25, 36.00, 28.57.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₆ H ₁₉ N ₅ NaO ₄ ] ⁺ 368.1335, found 368.1331.

Example 10. Preparation of tetrazine compounds of the present invention

(R)-(+)-1-Boc-2-cyanopyrrolidine (196 mg, 1 mmol), acetonitrile (167 μL, 4 mmol), 3-mercaptopropionic acid (35 μL, 0.4 mmol) were added to a 10 ml reaction tube, with 200 μL of DMSO was dissolved, the reaction tube was placed in an ice bath, hydrazine hydrate (777 μL, 16 mmol) was added, and then stirred at room temperature for 40 h, the reaction was complete; the reaction solution was poured into sodium nitrite (1.03 g, 15 mmol) in an ice-water solution , slowly add hydrochloric acid (1M) at 0°C to pH=4, carry out the reaction, after the TLC detection reaction is completed, the obtained solution is extracted 3 times with ethyl acetate, 20 ml of ethyl acetate each time, and the separatory funnel is used to separate the liquids. , combine the organic phases extracted 3 times, wash once with saturated brine (20ml), and separate the layers with a separatory funnel to obtain an organic phase. In a round-bottomed flask, concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel column chromatography, compound 13 (198 mg) was obtained as a pink solid, yield: 75%.

¹ H NMR(400MHz, DMSO)δ5.24(m,2H),3.66-3.45(m,4H),2.98(d,J=9.7Hz,6H),2.53-2.47(m,2H),2.12-1.82 (m, 6H), 1.35(s, 8H), 1.04(s, 10H). This ¹ HNMR spectra (25°C) represents a mixture of two isomers of 4:5, as shown in Figure S1.

¹³ C NMR (101MHz, DMSO) δ171.00, 170.52, 168.34, 154.01, 153.07, 79.52, 79.20, 60.19, 60.12, 47.46, 47.32, 33.55, 32.59, 28.50, 28.13, 24.07, 23.48, 21.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₂ H ₁₉ N ₅ NaO ₂ ] ⁺ 288.1436, found 288.1432.

Example 11. Preparation of tetrazine compounds of the present invention

(R)-(+)-1-Boc-2-cyanopyrrolidine (39.2 mg, 0.2 mmol), 3-(2-hydroxyethoxy)propionitrile (92 mg, 0.8 mmol), 3-mercaptopropane Acid (7.2 μL, 0.08 mmol) was added to a 10 ml reaction tube, dissolved in 78 μL of ethanol, the reaction tube was placed in an ice bath, hydrazine hydrate (155 μL, 3.2 mmol) was added, and then stirred at room temperature for 15 h, the reaction was complete; The reaction solution was poured into an ice-water solution of sodium nitrite (206 mg, 3 mmol), and hydrochloric acid (1 M) was slowly added at 0° C. until the pH of the solution was 4, and the reaction was carried out. After the completion of the reaction was detected by TLC, the obtained solution was extracted with ethyl acetate 3 times, 20ml of ethyl acetate each time, separate the layers with a separatory funnel, combine the organic phases extracted 3 times, wash once with saturated brine (20ml), and separate with a separatory funnel to obtain an organic phase, which is then washed with anhydrous sulfuric acid Dry over sodium, filter with a glass sand funnel to obtain a filtrate, transfer the filtrate to a round-bottomed flask, and concentrate under reduced pressure to obtain a red solid crude product. After purification by silica gel column chromatography, compound 14 (47.4 mg) was obtained as a pink solid, yield: 69%.

¹ H NMR (400MHz, CDCl ₃ ) δ 5.34 (m, 2H), 4.13 (m, 4H), 3.80–3.57 (m, 16H), 2.58 (m, 2H), 2.23–2.11 (m, 4H), 2.07–2.00(m, 2H), 1.72(s, 2H), 1.41(s, 9H), 1.13(s, 9H).

This ¹ HNMR spectra (25°C) represents a mixture of two isomers of 1:1, as shown in Figure S2. ¹³ C NMR (400MHz, DMSO) δ171.62,169.22,153.01,79.25,72.54,68.41,65.73,60.28,47.47, 35.41, 33.69, 28.05, 23.62.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₅ H ₂₅ N ₅ NaO ₄ ] ⁺ 362.1804, found 362.1809.

Example 12. Preparation of tetrazine compounds of the present invention

Boc-L-4-cyanophenylalanine (29.4mg, 0.1mmol), 3-hydroxypropionitrile (54.4ul, 0.8mmol), 3-mercaptopropionic acid (9μL, 0.1mmol) were added to a 10ml reaction tube , the reaction tube was placed in an ice bath, hydrazine hydrate (78 μL, 0.16 mmol) was added, and then, stirred at 40 ° C for 15 h, the reaction was complete; the reaction solution was poured into sodium nitrite (103 mg, 1.5 mmol) ice water solution, Slowly add hydrochloric acid (1M) at 0°C to pH=4 to carry out the reaction. After the completion of the TLC detection, the obtained solution was extracted with ethyl acetate for 3 times, 20 ml of ethyl acetate each time, and the solution was separated with a separatory funnel. The organic phases extracted three times were combined, washed once with saturated brine (20 ml), and the organic phase was separated with a separatory funnel to obtain an organic phase. In the bottom flask, concentrated under reduced pressure to obtain a red solid crude product. After purification by silica gel column chromatography, compound 15 (27.5 mg) was obtained as a pink solid, yield: 71%.

¹ H NMR (400MHz, DMSO) δ 8.44–8.32 (m, 2H), 7.53 (q, J=7.0, 5.2Hz, 2H), 4.11 (dd, J=8.2, 3.9Hz, 1H), 4.02 (d , J=6.4Hz, 2H), 3.63–3.50 (m, 1H), 3.20–3.04 (m, 1H), 2.86 (dd, J=13.7, 8.6Hz, 1H), 2.09 (s, 2H), 1.11 ( s, 9H).

¹³ C NMR (101MHz, DMSO) δ173.91, 168.53, 164.03, 155.70, 143.99, 130.80, 130.27, 127.64, 78.38, 59.75, 55.66, 38.59, 37.26, 28.62.

HRMS[M+Na] ⁺ m/z calcd.for[C ₁₈ H ₂₃ N ₅ NaO ₅ ] ⁺ 412.1597, found 412.2283.

Example 13. Preparation of tetrazine compounds of the present invention

Diethyl cyanomethyl phosphate (177 mg, 1 mmol), acetonitrile (833 μL, 20 mmol), 3-mercaptopropionic acid (87 μL, 1 mmol) were added to a 10 mL reaction tube, dissolved in 389 μL of ethanol, and the reaction tube was placed in an ice bath , hydrazine hydrate (78 μL, 0.16 mmol) was added, and then, stirred at room temperature for 18 h, the reaction was complete; the reaction solution was poured into an ice-water solution of sodium nitrite (1.03 g, 15 mmol), and hydrochloric acid (1 M) was slowly added at 0 °C To the pH of the solution = 4, carry out the reaction, TLC detection after the completion of the reaction, the obtained solution was extracted 3 times with ethyl acetate, 20 ml of ethyl acetate each time, separated with a separatory funnel, combined the organic phases extracted 3 times, saturated common salt Wash with water (20ml) once, separate with a separatory funnel to obtain an organic phase, and then dry the organic phase with anhydrous sodium sulfate, filter with a glass sand funnel to obtain a filtrate, transfer the filtrate to a round-bottomed flask, and concentrate under reduced pressure to obtain a red color Crude oil. After purification by silica gel column chromatography, a pink oil (120.8 mg) was obtained with a yield of 49%.

¹ H NMR (400MHz, CDCl3) δ4.35-4.00 (m, 4H), 3.93 (d, J=22.1Hz, 2H), 3.07 (p, J=0.9Hz, 3H), 1.38-1.29 (m, 6H) ).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 167.57 (d, J=3.0 Hz), 164.22 (d, J=10.0 Hz), 62.98, 62.92, 34.23 (d, J=34.0 Hz), 21.11, 16.31, 16.25 .

HRMS[M+Na] ⁺ m/z calcd.for[C ₈ H ₁₅ N ₄ NaO ₃ P] ⁺ 269.0779, found 269.0779.

To sum up, the present invention discloses a novel method for preparing tetrazine compounds. The method uses nitrile and hydrazine hydrate, and can carry out various substitutions in the presence of thiol catalysts to obtain tetrazine compounds with various functions. , to facilitate the further modification and transformation of tetrazine compounds, so as to obtain more tetrazine derivatives with special functions. In addition, the method for preparing tetrazine compounds of the present invention has the advantages of mild reaction conditions, readily available reagents, environmental friendliness, high product yield, gram-scale production and the like. The method overcomes the problems in the prior art that the hydrazine hydrate is used to prepare the tetrazine compounds, the substitution is single, the types of the tetrazine compounds obtained are limited, the preparation conditions are harsh, and the large-scale production is impossible.

Claims (15)

1. a method for preparing tetrazine compounds, is characterized in that: it comprises the steps:

Step 1: Dissolve the nitrile compound a, the nitrile compound b, the catalyst thiol compound, and the hydrazine hydrate in a solvent, and react to obtain a reaction solution; Step 2: mixing the reaction solution with the oxidant-containing solution, slowly adding acid, adjusting the pH of the system to acidity, reacting, and purifying the reaction solution to obtain tetrazine compounds; in, R ₁ and R ₂ are independently selected from substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted 3- to 8-membered unsaturated cycloalkyl, substituted or unsubstituted 3- to 8-membered unsaturated heterocycle group, substituted or unsubstituted C ₁ -C ₁₀ alkoxy; The substituents are substituted or unsubstituted C ₁ -C ₁₀ alkyl, halogen, cyano, nitro, carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₁₀ alkoxy, -NR ₄ R ₅ , azide Nitrogen, phosphate, tert-butoxycarbonyl; R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₆ alkyl, and tert-butoxycarbonyl. 2. method according to claim 1, is characterized in that: R ₁ and R ₂ are independently selected from unsubstituted C ₁ -C ₁₀ alkyl, -(CH ₂ ) _x R ₃ , -(CH ₂ ) _X N ₃ , -(CH ₂ CH ₂ O) _X N ₃ , -(CH ₂ ) _x (EtO) ₂ PO,

Substituted or unsubstituted 6-membered unsaturated cycloalkyl, substituted or unsubstituted 5-membered unsaturated heterocyclyl; R ₃ is selected from cyano, nitro, carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₆ alkoxy, -NR ₄ R ₅ ; R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₆ alkyl, and tert-butoxycarbonyl; The substituents are substituted or unsubstituted C ₁ -C ₁₀ alkyl, halogen, substituted or unsubstituted C ₁ -C ₆ alkoxy, nitro, amino, cyano, hydroxyl, carboxyl, phosphate, tertiary Butoxycarbonyl, -NR ₄ R ₅ ; x=1-10. 3. method according to claim 2, is characterized in that: R ₁ and R ₂ are independently selected from -(CH ₂ ) _x R ₃ , substituted or unsubstituted aryl, pyrrolyl, thienyl,

-(CH ₂ ) _X N ₃ , -(CH ₂ CH ₂ O) _X N ₃ , -(CH ₂ ) _x (EtO) ₂ PO; R ₃ is selected from carboxyl, hydroxyl, substituted or unsubstituted C ₁ -C ₂ alkoxy, -NR ₄ R ₅ ; R ₄ and R ₅ are independently selected from hydrogen, C ₁ -C ₄ alkyl, and tert-butoxycarbonyl; The substituents are substituted or unsubstituted C ₁ -C ₈ alkyl, halogen, substituted or unsubstituted C ₁ -C ₂ alkoxy, nitro, amino, cyano, hydroxyl, carboxyl, phosphate, tertiary Butoxycarbonyl, -NR ₄ R ₅ ; where x=1～10. 4. method according to claim 1, is characterized in that: In step 1, the molar ratio of the nitrile compound a, the nitrile compound b, the hydrazine hydrate, and the thiol compound is 1:1-30:6-32:0.2-1; the volume ratio of the hydrazine hydrate and the solvent is 2:0～4; And/or, in step 2, the oxidant equivalents in the oxidant-containing solution are 15-20 times the total equivalents of the nitrile compounds a and the nitrile compounds b; And/or, in step 2, an acid is added to adjust the pH of the system to 1-6. 5. method according to claim 4, is characterized in that: In step 1, the molar ratio of the nitrile compound a, the nitrile compound b, the hydrazine hydrate, and the thiol compound is 1:4 to 8:16:0.2 to 1; the volume ratio of the hydrazine hydrate to the solvent is 2: 1; And/or, in step 2, the oxidant equivalent number in the oxidant-containing solution is 15 times the total equivalent number of the nitrile compound a and the nitrile compound b; And/or, in step 2, acid is added to adjust the pH of the system to 3-4. 6. The method according to any one of claims 1 or 4 to 5, wherein in step 1, the solvent is selected from the group consisting of tetrahydrofuran, dimethyl sulfoxide, ethanol, dioxane, isopropanol, tertiary Butanol, n-propanol, n-butanol, ethylene glycol or hexafluoroisopropanol; preferably ethanol or dimethyl sulfoxide. 7. The method according to any one of claims 1 or 4 to 5, wherein in step 1, the thiol compound is selected from cysteine, 1,3-propanedithiol, mercaptoethylamine , mercaptopropionic acid, thioglycolic acid, N-acetyl-L-cysteine, reduced glutathione, butanethiol or propanedithiol; preferably mercaptopropionic acid. 8. The method according to any one of claims 1 or 4 to 5, wherein in step 2, the oxidant in the oxidant-containing solution is sodium nitrite, hydrogen peroxide, iodobenzenediacetic acid, and oxygen; preferably sodium nitrite; And/or, in step 2, the oxidant-containing solution is an oxidant-containing ice water solution, and the concentration of the oxidant-containing solution is 0.5-1.5 mmol/mL; preferably 1 mmol/mL. 9. The method according to any one of claims 1 or 4 to 5, wherein in step 2, the acid is hydrochloric acid, sulfuric acid, acetic acid, preferably hydrochloric acid; the acid concentration is 1M. 10. The method according to claim 1, wherein in step 1, when the nitrile compounds a and b, the catalyst thiol compound and the hydrazine hydrate are dissolved in the solvent, the hydrazine hydrate is added last, and the temperature during the addition is is 0°C. 11. The method according to claim 1, characterized in that: in step 1, the temperature of the reaction is 0～60°C; the reaction time is 0.5～64h; preferably, the reaction temperature is 25°C or 40°C, and the reaction The time is 6 to 18h. 12. The method according to claim 1, wherein in step 2, the temperature at which the acid is added is 0°C. 13. The method according to claim 1, characterized in that: in step 2, the purification is an extraction reaction solution, and the organic phase is taken, washed, dried, filtered, concentrated under reduced pressure, and purified by silica gel chromatography. . 14. The method according to claim 13, characterized in that: the extraction is three times extraction with ethyl acetate; the washing is washing with saturated brine; and the drying is drying with anhydrous sodium sulfate. 15. The tetrazine compound prepared by the method of any one of claims 1 to 14, wherein the structural formula of the tetrazine compound is as follows: