A monosubstituted 1,2,4,5-tetrazine compound based on a five-membered heterocycle and a preparation method thereof

Abstract

The application discloses a kind of single-substituted 1,2,4,5-tetrazine compounds based on five-membered heterocyclic ring and preparation method thereof.The single-substituted 1,2,4,5-tetrazine compounds based on five-membered heterocyclic ring keep high reaction rate (10 4 ~10 5 M ‑1 s ‑1 ), improve the LUMO+1 orbital energy of single-substituted tetrazine compound to improve its stability, or improve the reaction ability of tetrazine compound under the same or similar LUMO+1 orbital energy, so that the single-substituted 1,2,4,5-tetrazine compounds based on five-membered heterocyclic ring have higher stability when the reaction rate is certain, have higher reaction rate when the stability is certain, provide a new idea for breaking the limitation of orbital energy and reaction ability with trans-cyclooctene, can promote the retro Diels-Alder reaction of 1,2,4,5-tetrazine compound and trans-cyclooctene in vivo, and is of great significance to the field of medicine.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle and its preparation method. Background Technology

[0002] Bioorthogonal reactions refer to chemical reactions that can occur in living cells or tissues without interfering with the organism's own biochemical reactions; they are generally addition reactions (e.g., A + B → C). Utilizing the principle of addition reactions, and combined with other techniques, bioorthogonal reactions are widely used in fields such as the coupling modification of proteins with small molecules (or macromolecules), intracellular protein labeling, tumor pre-targeting imaging, and cell surface super-resolution imaging. The types of bioorthogonal reactions mainly focus on cycloaddition reactions that are not easily found in biological systems, especially the electron-demanding Diels-Alder reaction and the ring-strain-promoted [3+2] cycloaddition reaction. Bioorthogonal reactions require high reaction rates to ensure that considerable yields can be achieved in a short time at low concentrations; otherwise, even if the criteria for bioorthogonal reactions are met, they have no value for in vivo application. Although there are some non-cycloaddition bioorthogonal reactions such as the Staudinger linkage reaction, their in vivo application is greatly limited due to insufficient orthogonality and reactivity (too slow reaction rate) or the use of toxic catalysts (such as copper ions and palladium metal).

[0003] Currently, the two most widely used bioorthogonal reactions are the ring-strained [3+2] cycloaddition reaction between an azide group and a ring-strained alkyne, and the electron-demanding Diels-Alder reaction between a 1,2,4,5-tetraazine compound and trans-cyclooctene. The latter is favored due to its higher reaction rate (second-order reaction rate constant is around 10). 1 ~10 7 M -1 s -1 The orthogonal reaction between powers has garnered significant attention and has become the most valuable and effective biological orthogonal reaction for in vivo applications. One medical product utilizing this reaction has even entered Phase I clinical trials.

[0004] However, due to their electron-deficient nature, 1,2,4,5-tetraazines are easily destroyed by nucleophiles in biological systems. Although many different 1,2,4,5-tetraazine compounds have been developed, those that react with trans-cyclooctene tend to be more susceptible to destruction by other substances in biological systems (i.e., lower stability) the faster the reaction rate. The balance between the stability and reactivity of 1,2,4,5-tetraazine compounds (hereinafter referred to as "tetraazine compounds") has always been the "ceiling" for this reaction. When using more stable tetraazine compounds, their reactivity is lower (reaction rate is around 10). 1~10 3 M -1 s -1 However, when using more reactive tetrazine compounds, their stability is low, which greatly limits the further application of this reaction in living organisms.

[0005] Therefore, the research and development of a new tetrazine compound with both high reactivity and high stability is crucial for the reverse electron-demanding Diels-Alder reaction between 1,2,4,5-tetrazine compounds and trans-cyclooctene, and is of great significance for the further application of bioorthogonal reactions in vivo. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle and a method for its preparation. The monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle exhibits both high stability and high reaction rate.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle, having the structure shown in Formula I:

[0009]

[0010] Where X is NR 1 , O or S;

[0011] R 1 R 2 R 3 Independently selected from hydrogen, amino, hydroxyl, halogen, azide, substituted or unsubstituted C1-C7 alkoxy groups, substituted or unsubstituted straight-chain or branched C1-C groups. 10 One or more of alkyl, substituted or unsubstituted C2-C7 alkynyl, substituted or unsubstituted phenyl, and substituted or unsubstituted acyl;

[0012] The C1-C7 alkoxy group is preferably a C1-C4 alkoxy group, specifically including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, etc.

[0013] The C1-C 10 The alkyl group is preferably a C1-C6 alkyl group, specifically including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, etc.

[0014] The C2-C7 alkynyl group is preferably a C2-C4 alkynyl group, more preferably an ethynyl, propynyl, or butynyl group.

[0015] Preferably, the C1-C7 alkoxy group, C1-C 10 The alkyl and C2-C7 alkynyl substituents are independently selected from one or more of the halogen atom, amino group, and azide group.

[0016] The halogen atom of the substituent is preferably a fluorine atom, a chlorine atom, or a bromine atom.

[0017] The substituent amino group is preferably an amino group containing 1 to 5 carbon atoms, specifically including but not limited to one or more of methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino, sec-butylamino, n-pentanamino, and neopentanamino.

[0018] Preferably, the substituents of the phenyl group are selected from one or more of amino, hydroxyl, halogen, azide, alkoxy, alkyl, alkynyl, and acyl groups.

[0019] Preferably, the substituent of the acyl group is selected from one or more of amino, halogen, azide, and alkynyl groups.

[0020] The alkynyl substituent is preferably a C2-C5 alkynyl group, specifically including but not limited to one or more of ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, tert-butynyl, sec-butynyl, n-pentynyl, and neopentynyl.

[0021] Preferably, the monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle has any one of the following structures: Formulas 1 to 3

[0022]

[0023] Formulas 1, 2, and 3 above are respectively tetrazine compounds Oxazole-H, imi-penta, or Thiazole-H.

[0024] This invention also provides a method for preparing a monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle, comprising the following steps:

[0025] 1) A nitrile compound containing a substituted or unsubstituted five-membered heterocycle, a thiol compound, and hydrazine hydrate are mixed and reacted to obtain reaction system S1;

[0026] 2) Add formamidin salt to the above reaction system S1, and the reaction results in reaction system S2;

[0027] 3) After mixing the above reaction system S2 with the oxidant, adjust the pH of the reaction system to neutral or acidic, and carry out the reaction to prepare a monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle.

[0028] The five-membered heterocycle is selected from imidazole rings, thiazole rings, or oxazole rings.

[0029] Preferably, in step 1), the substituents of the five-membered heterocycle are selected from hydrogen, amino, hydroxyl, halogen, azide, substituted or unsubstituted C1-C7 alkoxy groups, substituted or unsubstituted straight-chain or branched C1-C7 groups. 10 One or more of alkyl, substituted or unsubstituted C2-C7 alkynyl, substituted or unsubstituted phenyl, and substituted or unsubstituted acyl.

[0030] The C1-C7 alkoxy groups, C1-C 10 The preferred and more preferred ranges for alkyl, C2-C7 alkynyl, and aryl groups are the same as above, and will not be repeated here.

[0031] Preferably, in step 1), the molar ratio of the nitrile compound containing a substituted or unsubstituted five-membered heterocycle, the thiol compound, and the hydrazine hydrate is 1:(0.1-2):(16-100); more preferably, it is 1:(0.2-1.5):(16-50); and even more preferably, it is 1:0.2:16 or 1:1:16.

[0032] In this invention, the reaction system S1 can proceed with or without the addition of a reaction solvent.

[0033] When a reaction solvent is added to reaction system S1, the volume ratio of the hydrazine hydrate to the solvent in reaction system S1 is (1-40):4; more preferably (4-12):4; in some specific embodiments of the present invention, the volume ratio of the hydrazine hydrate to the solvent in reaction system S1 is (10-14):4 or 2.4:1.

[0034] In step 1) of the above preparation method, the solvent for reaction system S1 is preferably one or more of tetrahydrofuran, ethanol, dioxane, N,N-dimethylformamide, isopropanol, tert-butanol, n-butanol, ethylene glycol, and pyridine. More preferably, it is N,N-dimethylformamide or ethanol.

[0035] Preferably, the thiol compound in step 1) of this invention is selected from one or more of cysteine, mercaptopropionic acid, mercaptoethanol, mercaptoacetic acid, 1,3-propanedithiol, N-acetyl-L-cysteine, reduced glutathione, butanethiol, or mercaptopropane. More preferably, it is one or more of cysteine, mercaptopropionic acid, mercaptoethanol, and mercaptoacetic acid; even more preferably, it is mercaptopropionic acid.

[0036] Preferably, in step 1), after mixing the nitrile compound, the thiol compound, and hydrazine hydrate, the reaction temperature is first raised to 25°C to 60°C and the reaction is carried out for 0 to 10 minutes, more preferably 50°C for 5 minutes.

[0037] Preferably, the molar ratio of formamidinium salt to nitrile compound containing substituted or unsubstituted five-membered heterocycles in step 2) is (1-8):1; more preferably (3-6):1; in some specific embodiments of the present invention, the molar ratio of formamidinium salt to nitrile compound containing substituted or unsubstituted five-membered heterocycles is 4:1.

[0038] Preferably, the molar ratio of oxidant to hydrazine hydrate in step 3) is (1-2):1; more preferably (1.5-2):1; and even more preferably 2:1.

[0039] Preferably, the oxidant is selected from one or more of sodium nitrite, hydrogen peroxide, iodophenylacetic acid, and oxygen; more preferably, it is sodium nitrite or hydrogen peroxide.

[0040] In step 2), after adding methyl methyl salt, the reaction temperature is controlled at 30℃~50℃ and the reaction time is 2~12h, more preferably 2h at 35℃.

[0041] In step 3) described above, an acidic substance is used to adjust the pH value of the reaction system.

[0042] The acidic substance is preferably one or more of acetic acid, sulfuric acid, hydrochloric acid, and hydrobromic acid;

[0043] The concentration of the acidic substance has an effect of 0.5M to 4M, more preferably 1M.

[0044] The pH value is preferably 0 to 7; more preferably pH = 6.

[0045] In the above preparation method, after the reaction in step 3) is completed, purification post-treatment is also included.

[0046] The present invention does not specifically limit the above purification method, and can use purification methods known to those skilled in the art such as extraction, filtration, and column chromatography.

[0047] The present invention preferably employs a combination of extraction, filtration, and column chromatography for purification to prepare the monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle as described in the present invention.

[0048] The extraction is preferably performed using dichloromethane or ethyl acetate as the extraction solvent.

[0049] After extraction, the organic phase is preferably washed with saturated brine.

[0050] Compared with the prior art, the monosubstituted 1,2,4,5-tetraazine compound based on a five-membered heterocycle provided by the present invention maintains an extremely high reaction rate (10 4 ~10 5 M -1 s -1 This method increases the LUMO+1 orbital energy of monosubstituted tetrazine compounds, thereby improving their stability, or enhances the reactivity of tetrazine compounds at the same or similar LUMO+1 orbital energies. This allows the monosubstituted 1,2,4,5-tetrazine compounds based on five-membered heterocycles to exhibit higher stability at a given reaction rate, and higher reaction rates at a given stability. This provides a new approach to breaking the limitations of orbital energy and reactivity with trans-cyclooctene, and is beneficial for promoting the reverse electron-demanding Diels-Alder reaction between 1,2,4,5-tetrazine compounds and trans-cyclooctene in vivo, which is of great significance to the pharmaceutical field. Attached Figure Description

[0051] Figure 1 This is a comparison chart of the stability and reaction rates of the tetrazine compounds in Table 1;

[0052] Figure 2 The image shows the 1H NMR spectrum of the tetrazine compound Oxazole-H.

[0053] Figure 3 The image shows the carbon NMR spectrum of the tetrazine compound Oxazole-H.

[0054] Figure 4 The image shows the 1H NMR spectrum of the tetrazine compound imi-penta.

[0055] Figure 5 The image shows the carbon NMR spectrum of the tetrazine compound imi-penta.

[0056] Figure 6 The image shows the 1H NMR spectrum of the tetrazine compound Thiazole-H.

[0057] Figure 7 This is the carbon NMR spectrum of the tetrazine compound Thiazole-H. Detailed Implementation

[0058] To further illustrate the present invention, the following detailed description of the monosubstituted 1,2,4,5-tetraazine compounds based on five-membered heterocycles and their preparation methods, with reference to the embodiments, is provided by the present invention.

[0059] Example 1

[0060]

[0061] 0.25 g of 4-cyanoxazole (2.7 mmol) was mixed with 2.1 mL of hydrazine hydrate (42.5 mmol) in a 25 mL round-bottom flask to prepare a solution. 0.23 mL of 3-mercaptopropionic acid (2.7 mmol) was slowly added dropwise under ice bath conditions. The mixture was then moved to 50 °C and reacted for 5 minutes. 1.1 g of formamidin acetate (10.8 mmol) was added to the solution, and the mixture was reacted at 40 °C under an argon atmosphere for 3 hours. At this point, the system turned brownish-yellow.

[0062] The system was cooled to room temperature, diluted with 50 mL of water, and 5 g of sodium nitrite (72.5 mmol) was added. 1 M hydrochloric acid solution was added dropwise under ice bath until the pH reached 5–6. Stirring was continued for 30 minutes, followed by extraction with ethyl acetate (3 × 50 mL). The collected organic layer was washed with saturated brine (2 × 50 mL) and dried overnight with anhydrous sodium sulfate. The filtrate was filtered and collected. Concentration of the filtrate yielded the crude product (pink solid). Purification by silica gel column chromatography yielded 0.13 g of the monosubstituted tetrazine compound Oxazole-H, a pink solid, in a yield of 33%.

[0063] The proton and carbon NMR spectra of the monosubstituted tetrazine compound Oxazole-H prepared above are shown below. Figure 2 , Figure 3 As shown.

[0064] 1 H NMR (400MHz, Chloroform-d) δ10.26 (s, 1H), 8.79 (d, J = 0.9Hz, 1H), 8.22-–8.14 (m, 1H).

[0065] 13 C NMR(101MHz,Chloroform-d)δ162.91,158.19,152.98,143.01,135.31.

[0066] Example 2

[0067]

[0068] 0.261 g of 1-n-pentyl-4-cyanoimidazole (1.6 mmol) and 1.21 mL of hydrazine hydrate (25.6 mmol) were mixed in a 5 mL round-bottom flask. 0.5 mL of N,N-dimethylformamide was added to prepare a solution. 0.14 mL of 3-mercaptopropionic acid (1.6 mmol) was slowly added dropwise under ice bath conditions. After reacting at 50 °C for 5 minutes, 0.65 g of formamidin acetate (6.4 mmol) was added to the solution, and the mixture was reacted at 40 °C for 3 hours under an argon atmosphere. At this point, the system turned brownish-yellow.

[0069] The system was cooled to room temperature, diluted with 50 mL of water, and 5 g of sodium nitrite (72.5 mmol) was added. 1 M hydrochloric acid solution was added dropwise under ice bath until the pH reached 5–6. Stirring was continued for 30 minutes, followed by extraction with ethyl acetate (3 × 50 mL). The collected organic layer was washed with saturated brine (2 × 50 mL) and dried overnight with anhydrous sodium sulfate. The filtrate was filtered and collected. Concentration of the filtrate yielded the crude product (pink solid). Purification by silica gel column chromatography yielded the monosubstituted tetrazine compound imi-penta, 0.12 g of a pink solid, in a yield of 32%.

[0070] The proton and carbon NMR spectra of the monosubstituted tetrazine compound imi-penta prepared above are shown below. Figure 4 , Figure 5 As shown.

[0071] 1 H NMR(400MHz,DMSO-d6)δ10.38(s,1H),8.43--8.37(m,1H),8.03--7.97(m,1H),4. 11(t,J=7.1Hz,2H),1.89--1.69(m,2H),1.36--1.18(m,4H),0.93--0.80(m,3H).

[0072] 13 C NMR (101MHz, DMSO-d6) δ157.38,140.28,125.14,46.60,29.94,27.97,21.53,13.78.

[0073] Example 3

[0074]

[0075] 0.24 g of 4-cyanothiazole (2.2 mmol) and 1.7 mL of hydrazine hydrate (35.2 mmol) were mixed in a 10 mL round-bottom flask. 0.5 mL of N,N-dimethylformamide was added to prepare a solution. 0.19 mL of 3-mercaptopropionic acid (2.2 mmol) was slowly added dropwise. The mixture was moved to 50 °C and reacted for 3 minutes. Then, 0.91 g of formamidin acetate (8.8 mmol) was added to the solution, and the mixture was reacted at 40 °C for 2 hours under an argon atmosphere. At this point, the system turned brownish-yellow.

[0076] The system was cooled to room temperature, diluted with 50 mL of water, and 5 g of sodium nitrite (72.5 mmol) was added. 1 M hydrochloric acid solution was added dropwise under ice bath until the pH reached 5–6. Stirring was continued for 30 minutes, followed by extraction with ethyl acetate (3 × 50 mL). The collected organic layer was washed with saturated brine (2 × 50 mL) and dried overnight with anhydrous sodium sulfate. The filtrate was filtered and collected. Concentration of the filtrate yielded the crude product (pink solid). Purification by silica gel column chromatography yielded 0.13 g of the monosubstituted tetrazine compound Thiazole-H, a bright red solid, in a yield of 36%.

[0077] The proton and carbon NMR spectra of the monosubstituted tetrazine compound Thiazole-H prepared above are shown below. Figure 6 , Figure 7 As shown.

[0078] 1 H NMR (400MHz, Chloroform-d) δ10.27 (s, 1H), 9.11 (d, J = 2.0Hz, 1H), 8.78 (d, J = 2.1Hz, 1H).

[0079] 13 C NMR (101MHz, Chloroform-d) δ163.35,157.91,155.13,149.35,125.60.

[0080] Specific implementation plan for determining the rate of tetrazine compounds:

[0081] Trans-cyclooctene was dissolved in phosphate buffer (pH = 7.4) to prepare a 10 mM solution. Various tetrazine compounds for which the rate constants needed to be determined were dissolved in phosphate buffer (pH = 7.4) to prepare 1 mM solutions. The reaction rates were measured using a residence time spectrometer (manufacturer: Applied; instrument model: Π*-180). The prepared solutions were first injected into the sample loading chamber of the residence time spectrometer. Since 520 nm is the characteristic absorption peak of the 1,2,4,5-tetraazine heterocycle, absorbance data were recorded at 520 nm. Since the measurements were taken at room temperature, no temperature setting was required. The program was then started to begin the measurement. The reaction rate of each tetrazine compound with trans-cyclooctene was measured in parallel four times. Finally, the absorbance curve as a function of time was obtained (the absorbance at 520 nm gradually decreases according to the first-order reaction curve). The data was exported and processed using Prism6 (Graphpad) software. The data was plotted with time on the x-axis and absorbance on the y-axis to fit the built-in decay exponential function in the software. Finally, the pseudo-first-order reaction rate constant was obtained. Dividing this constant by the concentration of trans-cyclooctene in the final mixed solution (5 mM) yielded the second-order reaction rate constant between the tetrazine compound and trans-cyclooctene.

[0082] Stability testing methods for tetrazine compounds in biological environments:

[0083] 1. Mother liquor and solvent

[0084] The stability of tetrazines was assessed in cell growth medium (DMEM + 10% fetal bovine serum). The tetrazine compounds to be tested were first dissolved in cell culture grade DMSO to a final concentration of 42.5 mM, and then further diluted in the medium to a final concentration of 500 μM (DMSO content (v / v): 2.5%).

[0085] 2. Stability Test

[0086]

[0087] All tetrazine solutions to be tested were placed in 1.5 mL centrifuge tubes and incubated at 37°C.

[0088] Three parallel treatments were performed. After 24 hours, the absorbance of the tetrazine solution was measured at 520 nm using a Synergy H1 microplate reader. The measured absorbance was compared with the initial absorbance at 0 hours to calculate the remaining percentage of intact tetrazine compound in the culture medium (24 hours remaining amount). Table 1 shows the implementation data.

[0089] Table 1. Second-order reaction rates of tetrazine compounds and their stability in biological environments.

[0090] name <![CDATA[Second-order reaction rate k (M -1 s -1 )]]> 24-hour remaining amount 1 19448 27.20％ Thiazole-H 41360 15.60％ Oxazole-H 31380 27.90％ 8 17330 18.10％ 9 16824 35.50％ 11 23300 19.54％ 12 25300 19.45％ dipy-Tz 12024 10.00％ imi-penta 21820 60.40％

[0091] Note: Monosubstituted tetrazine compounds 1, 8, 9, 11, and 12, as well as polysubstituted tetrazine compound dipy-Tz, are comparative examples in the table.

[0092] Table 1 shows that, compared with existing tetrazine compounds, the monosubstituted tetrazine compounds Oxazole-H and Thiazole-H based on five-membered heterocycles prepared in Examples 1-3 of this invention have higher reaction rates, reaching 10. 4 M -1 s -1 imi-penta exhibits good stability, with a remaining amount of 60.40% after 24 hours.

[0093] Figure 1 This is a comparison chart of the stability and reaction rate of the tetrazine compounds in Table 1. Figure 1Plotting the aforementioned stability against the second-order reaction rate constant, with the horizontal axis representing the remaining percentage of the tetrazine compound in the biological environment after 24 hours, and the vertical axis representing the second-order reaction rate constant with trans-cyclooctene, the dashed line in the graph represents the "ceiling" (i.e., the balance between the stability and reactivity of the tetrazine compound) in the reaction with trans-cyclooctene. Currently, all existing monosubstituted tetrazine compounds are below this "ceiling." Among them, monosubstituted tetrazine compounds 11 and 12 are commonly used monosubstituted tetrazine compounds. Our designed tetrazine compounds, Thiazole-H, Oxazole-H, and imi-penta, are all above this "ceiling," meaning that the five-membered heterocyclic tetrazine compounds described in this invention exhibit higher stability at a given reaction rate; and at a given stability, they exhibit a higher reaction rate, reaching 10. 4 ~10 5 M -1 s -1 This indicates that the Diels-Alder reaction, which involves the reverse electron requirement of a five-membered heterocyclic tetrazine compound and trans-cyclooctene, described in this invention, is more efficient, which is of great significance for the further application of bioorthogonal reactions in vivo.

[0094] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A monosubstituted 1,2,4,5-tetrazine compound based on a five-membered heterocycle, characterized in that, Any one of the following formula 1~2 Formula 1 Formula 2.

2. A process for the preparation of a monosubstituted 1,2,4,5-tetrazine compound based on a five-membered heterocycle according to claim 1, characterized in that Comprising the following steps: 1) mixing and reacting a nitrile compound containing a five-membered heterocyclic ring, a mercaptan compound and hydrazine hydrate to obtain a reaction system S1; 2) adding formamidine salt to the above reaction system S1, and obtaining a reaction system S2 after reaction; 3) mixing the above reaction system S2 with an oxidizing agent, adjusting the pH value of the reaction system to neutral or acidic, and reacting to prepare a monosubstituted 1,2,4,5-tetrazine compound based on a five-membered heterocyclic ring; The nitrile compound containing a five-membered heterocyclic ring is 4-cyano oxazole or 1-n-pentyl-4-cyano imidazole; The mercaptan compound is 3-mercapto propionic acid; The oxidizing agent is sodium nitrite.

3. The method of claim 2, wherein, The molar ratio of the nitrile compound containing a five-membered heterocyclic ring, the mercaptan compound and hydrazine hydrate in step 1) is 1:(0.1~2):(16~100); The volume ratio of hydrazine hydrate to reaction system S1 solvent is (1~40):

4.

4. The preparation method according to claim 2, characterized in that, The molar ratio of formamidine salt to nitrile compound containing a five-membered heterocyclic ring in step 2) is (1~8):

1.

5. The preparation method according to claim 2, characterized in that, The molar ratio of oxidizing agent to hydrazine hydrate in step 3) is (1~2):1.

Images