A 1,2,3-triazole feed additive and its preparation method and application

Abstract

The present invention discloses a 1,2,3-triazole feed additive and its preparation method and application, which belongs to the field of functional feed additive synthesis technology. The technical solution of the present invention is as follows: the 1,2,3-triazole feed additive molecule has a structure wherein n is 1 or 2; R is 4-methylphenyl, 4-fluorophenyl or zidovudine. The present invention uses 2-Boc aminobenzoic acid as a starting material, first reacts to obtain 2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline-1,3-diketone, then introduces terminal alkynes, and finally reacts with an azide compound to obtain a target molecule with a novel structure through a click reaction, which has a certain inhibitory effect on urease and can be used as a potential feed additive.

Description

Technical Field

[0001] The invention belongs to the technical field of synthesis of feed additives, and particularly relates to a 1,2,3-triazole feed additive and a preparation method and application thereof. Background Art

[0002] The 1,2,3-triazole structure is a very important class of nitrogen-containing heterocyclic compounds. It is a five-membered heterocyclic ring constructed by three nitrogen atoms and two carbon atoms, with a molecular formula of C2N3H3. 1,2,3-triazole has a special planar rigid structure, which gives it a strong ability to embed into DNA. It also has a large dipole moment and can form various non-covalent interactions with different biological targets, such as hydrophobic, hydrogen bonds, van der Waals forces, and dipole-dipole bonds. In addition, the structural characteristics of 1,2,3-triazole allow it to be an electronic equivalent substitute for amides, esters, carboxylic acids, olefin rigid analogs, etc., and therefore has a broad spectrum of biological activity. It is often used as an important molecular building block in the synthesis of active compounds, such as the preparation of antibacterial, antimalarial, antifungal, antiviral, antituberculosis and anticancer active compounds. A variety of clinical drugs and active molecules have enhanced their original biological activities or acquired new biological activities after being modified with 1,2,3-triazole. For example, the terminal alkyne was introduced into the core structure of the HIV integrase inhibitor drug dolutegravir, and reacted with 2-trifluoromethylphenyl azide to obtain the compound DTHP, which has good inhibitory activity against various lung cancer cells, especially H1975 cells, causing cell apoptosis and enhancing ROS levels. The tumor inhibition effect in mouse experiments is also very significant.

[0003]

[0004] Pomalidomide, chemically known as (RS)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, is an oral small molecule derivative and a newly launched third-generation immunomodulator. Developed and manufactured by Celgene Corporation in the United States, it is a modified version of the first-generation IMiD thalidomide. It can enhance immune responses mediated by T cells and natural killer cells, inhibit the production of pro-inflammatory cytokines by monocytes, and induce tumor cell apoptosis. It has garnered widespread attention in the treatment of various malignancies and immune diseases. It is a widely used thalidomide derivative. Compared to thalidomide, the amino group attached to the benzene ring of pomalidomide makes it more chemically stable and exhibits stronger immunomodulatory effects than thalidomide. Furthermore, in clinical practice, pomalidomide offers improved safety and fewer adverse reactions than thalidomide. It is virtually teratogenic and neurotoxic, and is effective against a variety of hematologic malignancies and solid tumors.

[0005]

[0006] Given the significant effect of introducing phenyl-1,2,3-triazole into the drug molecule group, in order to find a newer and more effective anti-tumor drug, we modified thalidomide and pomalidomide and introduced 1,2,3-triazole groups into their structures. Because triazole and the two amines contain nitrogen atoms, they can be used as nitrogen sources in feed additives. Our company has developed a variety of feed additives containing 1,2,3-triazole, which have good inhibitory effects on urease activity. Therefore, after cells modify these two amines, they can also be used as feed additives. Summary of the Invention

[0007] The technical problem solved by the present invention is to provide a preparation method of a novel pomalidomide derivative.

[0008] The present invention adopts the following technical solution to solve the above technical problems, a method for preparing a pomalidomide derivative, characterized by the following specific steps:

[0009] 1. Add a certain amount of 2-N-Boc aminobenzoic acid to dichlorothionyl, stir for a period of time under nitrogen protection, and then vacuum concentrate the reaction system. Dissolve the concentrate with dichloromethane, and then slowly add a dichloromethane solution containing 2-aminoglutaric acid dimethyl ester hydrochloride under stirring. After the addition is complete, continue stirring for a period of time and then concentrate the reaction system. Add N,N-dimethylformyl, and after complete dissolution, add a certain amount of tert-butyl lithium, barium hydroxide, cesium carbonate and palladium acetate under nitrogen protection. Maintain a nitrogen atmosphere and transfer to a high-pressure reactor. The autoclave is fully protected with nitrogen, and then stirred at room temperature for a period of time to replace the gas in the autoclave with carbon dioxide, and then introduce carbon dioxide to make the pressure in the autoclave reach a certain level. value, slowly heated to a certain temperature, after a period of reaction, vacuum removed carbon dioxide, cooled to a certain temperature, then introduced ammonia to make the pressure in the autoclave reach a certain value, maintained the temperature and pressure and stirred for a period of time, cooled to room temperature, and then added water and dichloromethane to the reaction system, stirred and filtered the reaction liquid, separated the organic phase, the aqueous phase was washed with dichloromethane for many times, and then the organic phase was separated, 2 mol / L dilute hydrochloric acid was added to the organic phase, heated to 30 ° C and stirred for a period of time, and then the reaction system was adjusted to neutral with a saturated solution of potassium carbonate, and the organic phase was separated again, concentrated and purified by silica gel column chromatography to obtain 2-(2,6-dioxopiperidine-3-yl)-4-aminoisoindoline-1,3-dione.

[0010] 2. Add a certain amount of benzoic acid to thionyl chloride, stir under nitrogen protection, and vacuum concentrate the reaction system. Dissolve the concentrate with dichloromethane, and then slowly add a dichloromethane solution containing 2-aminoglutaric acid dimethyl ester hydrochloride at 0°C. After the addition is complete, heat the temperature to room temperature and stir for a period of time. Concentrate the reaction system and add N,N-dimethylformamide. After complete dissolution, add tert-butyl lithium, barium hydroxide, cesium carbonate and palladium acetate under nitrogen protection. Maintain nitrogen atmosphere and transfer to a high-pressure reactor. Nitrogen protection is maintained in the autoclave throughout the process. Then stir at room temperature for 30 minutes and replace the carbon dioxide in the autoclave. Gas, then carbon dioxide is introduced to make the pressure in the autoclave reach 0.2MPa, and it is slowly heated to 120℃. After a period of reaction, carbon dioxide is removed in vacuo, and the temperature is lowered to 80℃. Then ammonia is introduced to make the pressure in the autoclave reach 0.12MPa. The temperature and pressure are maintained and stirred for a period of time, and then the reaction is cooled to room temperature. Water and dichloromethane are then added to the reaction system. After stirring, the reaction liquid is filtered to separate the organic phase. The aqueous phase is washed with dichloromethane several times, and then the organic phase is separated. After concentration, it is separated and purified by silica gel column chromatography to obtain 2-(2,6-dioxopiperidine-3-yl)-isoindoline-1,3-dione.

[0011] 3. Add a certain amount of methyl benzoate and calcium carbonate to a dichloroethane solution, stir, and then slowly dropwise add a dichloroethane solution containing N-iodosuccinimide. After the addition is complete, stir at room temperature for a period of time, then add 3-aminopiperidine-2,6-dione hydrochloride, triethylamine, and DCC reagent. Stir and react at room temperature for a period of time. After concentration, wash the concentrate with ether several times, then add it to a mixed solution of tert-butyl alcohol and methyl tert-butyl ether, place it in a high-pressure reactor, and then add triphenylphosphine rhodium chloride and silver carbonate. and potassium dihydrogen phosphate, stir evenly and react for a period of time, then add palladium acetate and triethylamine, remove the air in the reactor by vacuum, then introduce carbon monoxide to make the pressure in the reactor reach 0.5-0.8 MPa, slowly heat to 100°C, keep the pressure in the reactor unchanged, filter the reaction liquid after the reaction, and then adjust the reaction system to neutral with acetic acid, add water, stir, separate the organic phase, concentrate and separate and purify by silica gel column chromatography to obtain 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione.

[0012] 4. A certain amount of methyl benzoate and calcium carbonate were added to a dichloroethane solution, stirred, and then a dichloroethane solution containing N-iodosuccinimide was slowly added dropwise. After the addition was completed, the mixture was stirred at room temperature for a period of time, and then 3-aminopiperidine-2,6-dione hydrochloride, triethylamine, and DCC reagent were added. The mixture was stirred at room temperature for a period of time. After concentration, the concentrate was washed with ether several times. The concentrate was then added to toluene, and triphenylphosphine and iodine were added. The mixture was heated to reflux, and water in the reaction system was removed through a water separator. Then, palladium acetate and triethylamine were added. After stirring under nitrogen protection, anhydrous formic acid was added. The nitrogen atmosphere was maintained, and the temperature of the reaction system was adjusted to 80°C. After the reaction was completed, the reaction solution was filtered while hot, water was added, and the mixture was extracted with dichloromethane several times after stirring. The organic phases were combined, concentrated, and separated by silica gel column chromatography to obtain 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione.

[0013] 5. Add pomalidomide or thalidomide and potassium carbonate to dichloromethane, stir, then add 3-bromopropyne or 4-bromobutyne, stir and heat to reflux and continue stirring for a while, add saturated sodium chloride solution to the reaction system, stir and separate the organic phase, extract the aqueous phase with dichloromethane several times, combine the organic phases, and concentrate to obtain the terminal acetylene compound.

[0014] 6. Add the terminal acetylene compound and the azide compound to a mixed solvent of distilled water, tetrahydrofuran and tert-butanol, then add cuprous iodide, protect with nitrogen, heat to 80°C, reflux for a period of time, extract with dichloromethane several times, combine the organic phases, concentrate the organic phases, and separate by silica gel column chromatography to obtain the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the H NMR spectrum of the target compound prepared in Example 1.

[0016] Figure 2 This is the H NMR spectrum of the target compound prepared in Example 2.

[0017] Figure 3 This is the H NMR spectrum of the target compound prepared in Example 5.

[0018] Figure 4 This is the H NMR spectrum of the target compound prepared in Example 6.

[0019] Figure 5 This is the H NMR spectrum of the target compound prepared in Example 8.

[0020] Figure 6 This is the H NMR spectrum of the target compound prepared in Example 12. DETAILED DESCRIPTION

[0021] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0022] Example 1

[0023]

[0024] In a reaction flask with a stirring device, 2.4 g of 2-N-Boc aminobenzoic acid was added to 50 mL of dichlorothionyl, stirred under nitrogen protection for 1 hour, and then the reaction system was concentrated in vacuo. The concentrate was stirred and dissolved with 100 mL of dichloromethane, and then 70 mL of a dichloromethane solution containing 2.3 g of 2-amino-1,5-pentanedioic acid dimethyl ester hydrochloride was slowly added dropwise at 0 ° C. After the addition was complete, the mixture was heated to room temperature and stirred for 5 hours. The reaction system was concentrated in vacuo, and then added to 200 mL of N, N-dimethylformamide and stirred. After complete dissolution, 1.28 g of tert-butyl lithium, 3.4 g of barium hydroxide, 6.5 g of cesium carbonate and 0.45 g of palladium acetate were added under nitrogen protection. The nitrogen atmosphere was maintained and the reaction was transferred to a high-pressure reactor. The autoclave was fully protected with nitrogen, and then stirred at room temperature for 10 minutes. The gas in the autoclave was replaced with carbon dioxide. Then, carbon dioxide was introduced to make the pressure in the autoclave reach 0.2MPa, and the mixture was slowly heated to 100°C. After reacting for 20h, the carbon dioxide was removed by vacuum replacement and the temperature was lowered to 70°C. Then, ammonia was introduced to make the pressure in the autoclave reach 0.1MPa. The temperature and pressure were maintained and stirred for 15h, and then the mixture was cooled to room temperature. 200mL of water and 200mL of dichloromethane were added to the reaction system. After stirring, the reaction liquid was filtered and the organic phase was separated. The aqueous phase was washed four times with 100mL of dichloromethane, and then the organic phase was separated. 50mL of 2mol / L dilute hydrochloric acid was added to the organic phase, and the mixture was heated to 30°C and stirred for 2h. The reaction system was then adjusted to neutral with a saturated solution of potassium carbonate, and the organic phase was separated. After concentration, 2.31g of 2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline-1,3-dione was obtained by separation and purification by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.10(s,1H),7.48(s,1H),7.02(s,2H),6.53(s,2H),5.06(s,1H),3.11-2.58(m,3H),2.04(s,1H).

[0025] Example 2

[0026]

[0027] In a reaction flask with a stirring device, 1.25 g of benzoic acid was added to 50 mL of dichlorothionyl, stirred under nitrogen protection for 1 hour, and then the reaction system was concentrated in vacuo. The concentrate was dissolved with 100 mL of dichloromethane, and then 70 mL of a dichloromethane solution containing 2.3 g of 2-amino-1,5-pentanedioic acid dimethyl ester hydrochloride was slowly added dropwise at 0°C. After the addition was complete, the temperature was raised to room temperature and stirred for 2 hours. The reaction system was concentrated in vacuo and then added to 180 mL of N,N-dimethylformamide. After complete dissolution, 1.28 g of tert-butyl lithium, 3.4 g of barium hydroxide, 6.5 g of cesium carbonate and 0.45 g of palladium acetate were added under nitrogen protection. The nitrogen atmosphere was maintained and the reaction was transferred to a high-pressure reactor. The autoclave was protected by nitrogen throughout the process. , then stirred at room temperature for 30 minutes, replaced the gas in the autoclave with carbon dioxide, and then introduced carbon dioxide to make the pressure in the autoclave reach 0.2MPa, slowly heated to 120°C, reacted for 10 hours, and then vacuum-excluded carbon dioxide. At the same time, the temperature was lowered to 80°C, and then ammonia was introduced to make the pressure in the autoclave reach 0.12MPa. The temperature and pressure were stirred and reacted for 8 hours, then cooled to room temperature, and 100mL of water and 150mL of dichloromethane were added to the reaction system. After stirring, the reaction liquid was filtered and the organic phase was separated. The aqueous phase was washed several times with 50mL of dichloromethane, and then the organic phase was separated. After vacuum concentration, it was separated and purified by silica gel column chromatography to obtain 2.27g of 2-(2,6-dioxopiperidine-3-yl)-isoindoline-1,3-dione; 1 HNMR (400MHz, DMSO-d6) δ11.14(s,1H),7.95-7.89(m,4H),5.20-5.15(m,1H),2.95-2.86(m,2H),2.73-2.53(m,2H),2.10-2.07(m,1H).

[0028] Example 3

[0029]

[0030] In a reaction flask with a stirring device, 1.4 g of methyl benzoate and 3 g of calcium carbonate were added to a 100 mL dichloroethane solution, and after stirring, 100 mL of a dichloroethane solution containing 2.5 g of N-iodosuccinimide was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 1.5 h. Then, 1.7 g of 3-aminopiperidine-2,6-dione hydrochloride, 1 g of triethylamine, and 2 g of DCC reagent were added. The mixture was stirred at room temperature for 15 h. After vacuum concentration, the concentrate was washed several times with 40 mL of ether and 20 mL of petroleum ether. The concentrate was then added to a mixed solution of 100 mL of tert-butyl alcohol and 100 mL of methyl tert-butyl ether, placed in a high-pressure reactor, and 0.14 g of triphenylphosphine rhodium chloride and carbonate were added. 0.55 g of silver and 1.7 g of potassium dihydrogen phosphate were stirred and reacted evenly for 1 h, and then 0.22 g of palladium acetate and 1 g of triethylamine were added, and 50 mL of anhydrous toluene was added. The air in the reactor was removed by vacuum, and then carbon monoxide was introduced to make the pressure in the reactor reach 0.8 MPa. The reaction was slowly heated to 100 ° C., and the pressure in the reactor was kept constant. The reaction was reacted for 5 h, the reaction was stopped, and the reaction liquid was filtered after cooling. The reaction system was then adjusted to neutral with acetic acid, and partially dissolved was removed by vacuum concentration. Then 150 mL of water and 150 mL of dichloromethane were added, and the organic phase was separated after stirring. The organic phase was concentrated and purified by silica gel column chromatography to obtain 2.09 g of 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione.

[0031] Example 4

[0032]

[0033] In a reaction flask equipped with a stirring device and a water separator, 1.4 g of methyl benzoate and 3 g of calcium carbonate were added to a 100 mL dichloroethane solution. After stirring, 100 mL of a dichloroethane solution containing 2.5 g of N-iodosuccinimide was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 1.5 h. Then, 1.7 g of 3-aminopiperidine-2,6-dione hydrochloride, 1 g of triethylamine, and 2 g of DCC reagent were added. The mixture was stirred at room temperature for 15 h. After concentration, the concentrate was washed several times with 40 mL of ether and 20 mL of petroleum ether. The concentrate was then dissolved in 150 mL of toluene and 2.6 g of triphenylphosphine was added. g and 3 g of elemental iodine, heated to reflux, removed water from the reaction system through a water separator, then added 0.23 g of palladium acetate and 1 g of triethylamine, added 50 mL of anhydrous toluene, stirred for 30 min under nitrogen protection, then added 1.5 g of anhydrous formic acid, maintained in a nitrogen atmosphere, adjusted the temperature of the reaction system to 80 ° C, reacted for 13 h, stopped the reaction, filtered the reaction solution while hot, added 250 mL of water, stirred, and extracted several times with 100 mL of dichloromethane, combined the organic phases, concentrated, and separated by silica gel column chromatography to obtain 2.15 g of 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione.

[0034] Example 5

[0035]

[0036] In a stirred reaction flask, 2.75 g of 2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline-1,3-dione and 1.4 g of potassium carbonate were added to 150 mL of dichloromethane. After stirring, 1.43 g of 3-bromopropyne was added, stirred for 30 minutes, heated to reflux and continued to stir for 4 hours, 100 mL of saturated sodium chloride solution was added to the reaction system, stirred, and the organic phase was separated. The aqueous phase was extracted several times with 100 mL of dichloromethane, and the organic phases were combined and concentrated to obtain 2.64 g of N-propynyl pomalidomide. 1 H NMR (400 MHz, DMSO) 1 H NMR (400MHz, DMSO) δ7.48 (t, J1=4.0Hz, J2=4.0Hz, 1H), 7.02 (t, J1=4.0Hz, J2=8.0Hz, 2H), 6.54 (s, 2H), 5.20 (t, J1=4.0Hz, J2= 8.0Hz,1H),4.41-4.35(m,2H),3.11-3.10(m,1H),3.06-3.00(m,1H),2.82-2.78(m,1H),2.60-2.53(m,1H),2.06-1.99(m,1H).

[0037] Example 6

[0038]

[0039] In a stirred reaction flask, 2.6 g of 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione and 1.4 g of potassium carbonate were added to 150 mL of dichloromethane. After stirring, 1.43 g of 3-bromopropyne was added, stirred for 30 minutes, heated to reflux and stirred for 4 hours, 100 mL of saturated sodium chloride solution was added to the reaction system, stirred, and the organic phase was separated. The aqueous phase was extracted several times with 100 mL of dichloromethane. The organic phases were combined, concentrated, and separated and purified by silica gel column chromatography to obtain 2.52 g of N-propynyl thalidomide. 1 H NMR (400MHz, DMSO-d6) δ7.96-7.90 (m, 4H), 5.32 (dd, J1=4.0Hz, J2=4.0Hz, 1H), 4.39 (s, 2H) ,3.11(s,1H),3.07-3.01(m,1H),2.84-2.80(m,1H),2.62-2.55(m,1H),2.13-2.09(m,1H).

[0040] Example 7

[0041]

[0042] In a stirred reaction flask, 2.6 g of 2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione and 1.4 g of potassium carbonate were added to 150 mL of dichloromethane, stirred, and then 1.6 g of 4-bromobutyne was added. The mixture was stirred for 30 min, heated to reflux, and stirred for 6.5 h. 100 mL of saturated sodium chloride solution was added to the reaction system. After stirring, the organic phase was separated, and the aqueous phase was extracted several times with 100 mL of dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, and then concentrated in vacuo to obtain 2.77 g of N-butynyl thalidomide. MS (ESI + )m / z:311[M+H] + .

[0043] Example 8

[0044]

[0045] In a reaction flask with a stirring device, 3.1 g of N-propynyl pomalidomide and 1.65 g of 4-fluorophenyl azide were added to 120 mL of a mixed solvent of distilled water, tetrahydrofuran, and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 9 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 50 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography to obtain 3.77 g of the product. 1 H NMR (400 MHz, DMSO-d6) 1 HNMR (400MHz, DMSO) δ8.50(s,1H),7.93-7.90(m,2H),7.49-7.43(m,3H),7.02(t,J1=8.0Hz,J2=8.0Hz,2H),6.51(s,2H),5.23(d d, J1=8.0Hz, J2=4.0Hz,1H),5.06-4.96(m,2H),3.09-3.00(m,1H),2.83(d,J=16.0Hz,1H),2.69-2.59(m,1H),2.12-2.08(m,1H).

[0046] Example 9

[0047]

[0048] In a reaction flask with a stirring device, 3.1 g of N-propynyl pomalidomide and 3.2 g of zidovudine were added to 150 mL of a mixed solvent of distilled water, tetrahydrofuran and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 9 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 70 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography (PE:EA=12:1) to obtain 4.27 g of the product.

[0049] Example 10

[0050]

[0051] In a reaction flask with a stirring device, 3.1 g of N-propynyl pomalidomide and 1.6 g of 4-methylphenyl azide were added to 120 mL of a mixed solvent of distilled water, tetrahydrofuran, and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 10 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 50 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography to obtain 3.58 g of the product. 1 H NMR (600MHz, DMSO-d6) δ8.51 (s, 1H), 7.79 (d, J = 6.0Hz, 2H), 7.53-7.43 (m, 3H), 7.06 (t, J1 = 6.0Hz, J2 = 6.0Hz, 2H), 6.57 (s, 2H), 5.29 (dd, J1 = 6.0Hz, J2=6.0Hz,1H),5.06(dd,J1=12.0Hz,J2=18.0Hz,2H),3.12-3.06(m,1H),2 .88(d,J=18.0Hz,1H),2.72-2.64(s,1H),2.42(s,3H),2.15-2.13(m,1H).

[0052] Example 11

[0053]

[0054] In a reaction flask with a stirring device, 3.1 g of N-butynylthalidomide and 3.2 g of zidovudine were added to 150 mL of a mixed solvent of distilled water, tetrahydrofuran and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 5 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 70 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography (PE:EA=12:1) to obtain 2.94 g of the product.

[0055] Example 12

[0056]

[0057] In a reaction flask with a stirring device, 3.1 g of N-butynyl thalidomide and 1.6 g of 4-methylphenyl azide were added to 120 mL of a mixed solvent of distilled water, tetrahydrofuran and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 11 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 50 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography to obtain 3.36 g of the product. 1 H NMR (400MHz, CDCl3) δ7.96 (s, 1H), 7.89 (dd, J1 = 4.0Hz, J2 = 4.0Hz, 2H), 7.77 (dd, J1 = 4.0Hz, J2 = 4.0Hz, 2H), 7.61 (d, J = 8.0Hz, 2H), 7 .30(d,J=8.0Hz,2H),5.28-5.17(m,2H),2.84-2.75(m,2H),2.42(s,3H),2.18-2.11(m,1H),1.72-1.62(m,3H),1.00-0.94(m,1H).

[0058] Example 13

[0059]

[0060] In a reaction flask with a stirring device, 3.0 g of N-propynyl thalidomide and 1.65 g of 4-fluorophenyl azide were added to 120 mL of a mixed solvent of distilled water, tetrahydrofuran and tert-butanol (the volume ratio of the three was 1:1:1), and then 0.38 g of cuprous iodide was added. Under nitrogen protection, the mixture was heated to 80°C and refluxed for 9 hours. After that, the reaction solution was filtered and the filtrate was extracted several times with 50 mL of dichloromethane. The organic phases were combined, concentrated, and separated by silica gel column chromatography (PE:EA=10:1) to obtain 3.08 g of the product. MS (ESI + )m / z:434[M+H] + .

[0061] Example 14

[0062] We conducted an in vitro urease inhibition test on the six target compounds obtained: yellow cattle of about 12 months old were selected. After feeding for 1 hour, 400 mL of rumen fluid was collected through an artificial rumen fistula using a special rumen fluid collector. The fluid was filtered through 4 layers of gauze and set aside. After adding the corresponding reagents in the amount shown in the table below to each culture tube, 4 drops of liquid paraffin were added dropwise, and the tubes were placed in a (39.0±0.5)°C constant temperature water bath shaker and gently shaken. After incubation for 8 hours, some culture tubes were removed from each group, and 4 drops of saturated mercuric chloride solution were immediately added and shaken to terminate the reaction. The ammoniacal nitrogen content of each tube was determined by the Kjeldahl semi-micro-saturated magnesium oxide distillation method. We can find that the two products obtained in Example 8 and Example 11 have increasingly significant inhibitory activity against urease over time, showing a very significant inhibitory effect.

[0063]

[0064]

[0065] Inhibition rate (%) = (ammonia content of control group - ammonia content of test group) ÷ ammonia content of control group × 100%

[0066] serial number Time (h) Inhibition rate of control group Inhibition rate of test group 1 Inhibition rate of test group 2 Example 8 product 8 0％ 33.71％ 56.09％ Example 9 product 8 0％ 31.88％ 47.79％ Example 10 Product 8 0％ 14.05％ 21.70％ Example 11 Product 8 0％ 35.94％ 60.18％ Example 12 Product 8 0％ 18.78％ 25.13％ Example 13 Product 8 0％ 28.06％ 40.92％

[0067] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A 1,2,3-triazole feed additive, characterized in that The structure of the 1,2,3-triazole feed additive is ; wherein n is 1 or 2; R is 4-methylphenyl, 4-fluorophenyl or ; X is hydrogen or NH2; and, When n is 1 and X is NH2, R is 4-fluorophenyl, 4-methylphenyl or When n is 2 and X is H, R is 4-methylphenyl or .

2. A method for preparing the 1,2,3-triazole feed additive according to claim 1, characterized in that: A certain amount of 2-N-Boc aminobenzoic acid was added to dichlorothionyl, and the reaction system was concentrated in vacuo after stirring for a period of time under nitrogen protection. The concentrate was dissolved with dichloromethane, and then a dichloromethane solution containing 2-aminoglutaric acid dimethyl ester hydrochloride was slowly added dropwise under stirring. After the addition was complete, the stirring reaction was continued for a period of time, and then the reaction system was concentrated. N,N-dimethylformamide was added, and after complete dissolution, a certain amount of tert-butyl lithium, barium hydroxide, cesium carbonate and palladium acetate were added under nitrogen protection. The nitrogen atmosphere was maintained and the reaction was transferred to a high-pressure reactor. The autoclave was fully protected with nitrogen, and then stirred at room temperature for a period of time to replace the gas in the autoclave with carbon dioxide, and then carbon dioxide was introduced to make the pressure in the autoclave reach 0.15~0.2MPa. The reaction was slowly heated to 100~110℃, and the carbon dioxide was discharged in vacuo after a period of reaction, and the temperature was lowered at the same time. to 60-70°C, then introducing ammonia gas to make the pressure in the autoclave reach 0.1-0.12 MPa, maintaining the temperature and pressure with stirring for a period of time, cooling to room temperature, adding water and dichloromethane to the reaction system, stirring and filtering the reaction solution, separating the organic phase, washing the aqueous phase with dichloromethane several times, and then separating the organic phase, adding 2 mol / L dilute hydrochloric acid to the organic phase, heating to 30°C and stirring for a period of time, and then adjusting the reaction system to neutral with a saturated potassium carbonate solution, separating the organic phase again, concentrating and separating and purifying by silica gel column chromatography to obtain thalidomide; the molar ratio of the 2-N-Boc aminobenzoic acid to 2-aminoglutaric acid dimethyl ester hydrochloride is 1:1-1.1; the molar ratio of the 2-N-Boc aminobenzoic acid to tert-butyl lithium to barium hydroxide to cesium carbonate and palladium acetate is 1:2:2:2:0.2; Thalidomide and potassium carbonate are added to dichloromethane, and 3-bromopropyne or 4-bromobutyne is added after stirring. The mixture is heated to reflux with stirring and continued to stir for a period of time. A saturated sodium chloride solution is added to the reaction system, and the organic phase is separated after stirring. The aqueous phase is extracted with dichloromethane several times, and the organic phases are combined and concentrated to obtain a thalidomide terminal alkyne compound. Thalidomide terminal alkyne compound and azide compound are added to a mixed solvent of distilled water, tetrahydrofuran and tert-butanol, and then cuprous iodide is added, nitrogen protection is applied, and the mixture is heated to 80°C. After reflux reaction for a period of time, the mixture is extracted with dichloromethane several times, and the organic phases are combined, concentrated, and separated by silica gel column chromatography to obtain the product.

3. A method for preparing the 1,2,3-triazole feed additive according to claim 1, characterized in that: A certain amount of benzoic acid was added to thionyl chloride, and the reaction system was concentrated in vacuo under nitrogen protection after stirring. The concentrate was dissolved with dichloromethane, and then a dichloromethane solution containing dimethyl 2-aminoglutarate hydrochloride was slowly added dropwise at 0°C. After the addition was complete, the temperature was raised to room temperature and stirred for a period of time. The reaction system was concentrated, and N,N-dimethylformamide was added. After complete dissolution, tert-butyl lithium, barium hydroxide, cesium carbonate and palladium acetate were added under nitrogen protection. The nitrogen atmosphere was maintained and the reaction system was transferred to a high-pressure reactor. The high-pressure reactor was fully protected with nitrogen. After stirring at room temperature, the gas in the reactor was replaced with carbon dioxide, and then carbon dioxide was introduced to make the pressure in the reactor reach 0.2MPa. The reaction system was slowly heated to 1 20°C, after reacting for a period of time, the carbon dioxide is discharged in vacuo, and the temperature is lowered to 80°C, and then ammonia is introduced to make the pressure in the autoclave reach 0.12MPa, the temperature and pressure are maintained and stirred for a period of time, and then the temperature is lowered to room temperature, water and dichloromethane are added to the reaction system, and the reaction liquid is filtered after stirring to separate the organic phase, and the aqueous phase is washed with dichloromethane for multiple times, and then the organic phase is separated, concentrated, and separated and purified by silica gel column chromatography to obtain pomalidomide; the molar ratio of the benzoic acid to 2-aminoglutarate dimethyl ester hydrochloride is 1:1~1.1; the molar ratio of the benzoic acid to tert-butyl lithium to barium hydroxide to cesium carbonate and palladium acetate is 1:2:1.5:2:0.2; Pomalidomide and potassium carbonate are added to dichloromethane, and 3-bromopropyne or 4-bromobutyne is added after stirring. The mixture is heated to reflux with stirring and continued to stir for a period of time. A saturated sodium chloride solution is added to the reaction system, and the organic phase is separated after stirring. The aqueous phase is extracted with dichloromethane several times, and the organic phases are combined and concentrated to obtain a pomalidomide terminal alkyne compound. The terminal alkyne compound of pomalidomide and the azide compound are added to a mixed solvent of distilled water, tetrahydrofuran and tert-butanol, and then cuprous iodide is added. Under nitrogen protection, the mixture is heated to 80°C, and refluxed for a period of time. The mixture is extracted with dichloromethane several times, and the organic phases are combined, concentrated, and separated by silica gel column chromatography to obtain the product.

4. A method for preparing the 1,2,3-triazole feed additive according to claim 1, characterized in that: A certain amount of methyl benzoate and calcium carbonate are added to a dichloroethane solution, stirred, and then a dichloroethane solution containing N-iodosuccinimide is slowly added dropwise. After the addition is completed, the mixture is stirred at room temperature for a period of time, and then 3-aminopiperidine-2,6-dione hydrochloride, triethylamine, and DCC reagent are added. The mixture is stirred and reacted at room temperature for a period of time. After concentration, the concentrate is washed with ether several times, and then added to a mixed solution of tert-butyl alcohol and methyl tert-butyl ether. The mixture is placed in a high-pressure reactor, and triphenylphosphine rhodium chloride, silver carbonate, and potassium dihydrogen phosphate are added. The mixture is stirred and reacted for a period of time, and then palladium acetate and triethylamine are added. The air in the reactor is evacuated by vacuum, and then carbon monoxide is introduced to make the pressure in the reactor reach 0.5~0.8MP. a. slowly heating to 80-100° C., maintaining the pressure in the reactor constant, filtering the reaction solution after the reaction is completed, adjusting the reaction system to neutral with acetic acid, adding water, stirring, separating the organic phase, concentrating, and separating and purifying by silica gel column chromatography to obtain pomalidomide; the molar ratio of the methyl benzoate to calcium carbonate and N-iodosuccinimide is 1:3:1.1; the molar ratio of the methyl benzoate to 3-aminopiperidine-2,6-dione hydrochloride to triethylamine and DCC is 1:1:1:1; the mass ratio of the methyl benzoate to triphenylphosphine rhodium chloride is 1:0.1; the molar ratio of the methyl benzoate to silver carbonate and potassium dihydrogen phosphate is 1:0.2:1.2; Pomalidomide and potassium carbonate are added to dichloromethane, and 3-bromopropyne or 4-bromobutyne is added after stirring. The mixture is heated to reflux with stirring and continued to stir for a period of time. A saturated sodium chloride solution is added to the reaction system, and the organic phase is separated after stirring. The aqueous phase is extracted with dichloromethane several times, and the organic phases are combined and concentrated to obtain a pomalidomide terminal alkyne compound. The terminal alkyne compound of pomalidomide and the azide compound are added to a mixed solvent of distilled water, tetrahydrofuran and tert-butanol, and then cuprous iodide is added. Under nitrogen protection, the mixture is heated to 80°C, and refluxed for a period of time. The mixture is extracted with dichloromethane several times, and the organic phases are combined, concentrated, and separated by silica gel column chromatography to obtain the product.

5. A method for preparing the 1,2,3-triazole feed additive according to claim 1, characterized in that: A certain amount of methyl benzoate and calcium carbonate were added to a dichloroethane solution, stirred, and then a dichloroethane solution containing N-iodosuccinimide was slowly added dropwise. After the addition was completed, the mixture was stirred at room temperature for a period of time, and then 3-aminopiperidine-2,6-dione hydrochloride, triethylamine, and DCC reagent were added. The mixture was stirred at room temperature for a period of time, concentrated, and washed with ether several times. The concentrate was then added to toluene, and triphenylphosphine and iodine were added. The mixture was heated to reflux, and water in the reaction system was removed through a water separator. Then, palladium acetate and triethylamine were added. After stirring under nitrogen protection, anhydrous formic acid, maintaining a nitrogen atmosphere, adjusting the temperature of the reaction system to 80°C, filtering the reaction solution while hot after the reaction is completed, adding water, stirring, extracting with dichloromethane for multiple times, combining the organic phases, concentrating, and separating by silica gel column chromatography to obtain pomalidomide; the molar ratio of the feeding amount of methyl benzoate to calcium carbonate and N-iodosuccinimide is 1:3:1.1; the molar ratio of the feeding amount of methyl benzoate to 3-aminopiperidine-2,6-dione hydrochloride to triethylamine and DCC is 1:1:1:1; the molar ratio of the feeding amount of methyl benzoate to triphenylphosphine and iodine element is 1:1.2:1.2; Pomalidomide and potassium carbonate are added to dichloromethane, and 3-bromopropyne or 4-bromobutyne is added after stirring. The mixture is heated to reflux with stirring and continued to stir for a period of time. A saturated sodium chloride solution is added to the reaction system, and the organic phase is separated after stirring. The aqueous phase is extracted with dichloromethane several times, and the organic phases are combined and concentrated to obtain a pomalidomide terminal alkyne compound. The terminal alkyne compound of pomalidomide and the azide compound are added to a mixed solvent of distilled water, tetrahydrofuran and tert-butanol, and then cuprous iodide is added. Under nitrogen protection, the mixture is heated to 80°C, and refluxed for a period of time. The mixture is extracted with dichloromethane several times, and the organic phases are combined, concentrated, and separated by silica gel column chromatography to obtain the product.

6. Use of the 1,2,3-triazole feed additive according to claim 1 in the preparation of a product for inhibiting urease activity.

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