CN109232646B - Triazole urease inhibitor type feed additive and preparation method thereof - Google Patents
Triazole urease inhibitor type feed additive and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a triazole urease inhibitor type feed additive and a preparation method thereof, belonging to the technical field of feed additive synthesis. The technical scheme provided by the invention has the key points that: a triazole urease inhibitor type feed additive has a structural formula as follows:
Description
Technical Field
The invention belongs to the technical field of feed additive synthesis, and particularly relates to a triazole urease inhibitor type feed additive and a preparation method thereof.
Background
Urease is also called urea hydrolase, is a nickel-containing oligomeric enzyme, has absolute specificity, can specifically catalyze urea to hydrolyze into ammonia and carbon dioxide, and the hydrolysate ammonia can provide a nitrogen source for the metabolism of a plurality of microorganisms and plants and plays a key role in regulating the pH value of the surrounding environment; at the same time, this also causes many negative effects in nature. For example, urease can excessively catalyze and degrade volatile ammonia and carbon dioxide produced by urea, so that the loss of the fertilizer effect of the nitrogen fertilizer is caused, the concentration of ammonia ions in soil is increased, the pH value is increased, and the normal growth of plants is influenced; urease can also be used as a pathogen toxin to cause diseases such as gastritis, gastric ulcer and gastric cancer. For example, helicobacter pylori uses a large amount of urease produced by helicobacter pylori itself to catalyze the hydrolysis of urea, resulting in an increase in the surrounding pH, which counteracts the killing action of gastric acid. Therefore, it is necessary to find a suitable urease inhibitor to scientifically regulate and control the activity of urease and eliminate the negative effects generated by the urease inhibitor, and the currently reported urease inhibitors mainly comprise two types, namely transition metal ion type urease inhibitors and organic compound type urease inhibitors, wherein the transition metal ion type urease inhibitors and the organic compound type urease inhibitors are limited in application due to the fact that heavy metal ions have high toxicity; the latter has limitations in application due to short effective inhibition time, low efficiency and toxic and side effects. If the organic small molecules and the heavy metal ions form the complex, the organic ligand and the metal ions are both fixed in the complex framework, so that the toxicity is greatly reduced, the acting time is prolonged, and the potential is good.
Urease, which decomposes urea to NH, is widely present in the stomach of ruminants3At a rate of not catalytically decomposing 1014Double, the use of NH by rumen microorganisms3The rate of the nitrogen is 4 times that of the nitrogen, the utilization rate of the nitrogen is greatly reduced, and is usually only 30% -40%, so that not only is the serious waste of agricultural and animal husbandry resources caused and the production cost increased, but also the growth of animals and plants and the ecological environment and the like are seriously damaged, and serious water source pollution, water eutrophication and the like can be caused. Therefore, how to solve the problems of economy, environment and health caused by urease is a challenging subject to be solved urgently in the sustainable development of economy, agriculture and animal husbandry in the world today.
Triazole and derivatives thereof serving as a five-membered azacyclo-your compound have the advantages of multiple coordination sites, strong coordination capacity and small volume, and show excellent biological activity, such as antibacterial activity, anti-tumor activity, anti-inflammatory activity and the like, and a large amount of triazole and complexes thereof are used for research on the aspects of biological activity and the like at present. Copper is an indispensable trace element for the growth and development of piglets, and copper is a naturally-existing trace element in feed raw materials. In animals, copper is present in enzymes and collagen, and not only participates in the synthesis of hemoglobin and erythrocytes, but also has an effect on reproductive and cardiac functions. In addition, researches show that the copper element can inhibit harmful microbial flora in the intestinal tract, promote the growth of beneficial microbial flora and change the microbial structure of the intestinal tract, thereby promoting the growth of organisms.
Therefore, the triazole compound is modified to be subjected to coordination and complexation with copper ions, so that the azole compound with a novel structure is synthesized, can be used as non-protein nitrogen, has a certain inhibition effect on urease, has the effects of sterilization and ulcer resistance, and can be used as an excellent animal feed additive.
Disclosure of Invention
The invention aims to provide a triazole urease inhibitor type feed additive which has the advantages of simple synthesis method, low raw material price and novel structure and a preparation method thereof.
The invention adopts the following technical scheme for solving the technical problems, and the structure of the triazole urease inhibitor type feed additive is as follows:
the invention adopts the following technical scheme for solving the technical problems, and the preparation method of the azole urease inhibitor type feed additive is characterized by comprising the following specific steps:
(1) respectively adding a carrier and sodium nitrate into a reaction bottle, slowly pouring concentrated sulfuric acid, uniformly stirring, adding perchloric acid, heating to 40 ℃, stirring for a period of time, adding deionized water, heating to 90 ℃, stirring for a period of time, cooling to room temperature, adding a proper amount of deionized water and hydrogen peroxide, standing for precipitation, washing, centrifuging for multiple times, continuously performing ultrasonic treatment, and performing freeze drying to obtain an oxidized carrier; dissolving the prepared oxidation carrier in an N, N-dimethylformamide solution, performing ultrasonic dispersion, weighing bismuth nitrate pentahydrate and scandium nitrate hexahydrate to dissolve in N, N-dimethylformamide, weighing sodium trifluoromethanesulfonate to dissolve in ethylene glycol, mixing the solutions, stirring for a period of time, pouring into a polytetrafluoroethylene high-pressure hydrothermal kettle, heating to 140 ℃ for reaction for a period of time, naturally cooling to room temperature, alternately washing with ethanol and deionized water, centrifuging, and drying the precipitate in a 80 ℃ blast drying box to obtain the composite catalyst; the carrier is graphite powder; the feeding amount molar ratio of the bismuth nitrate pentahydrate to the scandium nitrate trihydrate is 1: 0.5-1.0.
(2) Adding 1, 3-dichlorobenzene into a mixed solution of trifluoroacetic anhydride, formic acid and dimethyl sulfoxide under the condition of an internal temperature of 5-10 ℃ in a high-pressure reaction kettle under the protection of argon, keeping the temperature unchanged, adding a certain amount of composite catalyst, slowly heating to 90 ℃, stirring for a period of time, introducing ammonia gas into the reaction kettle to ensure that the pressure in the kettle reaches 0.1MPa, keeping the pressure unchanged, continuously heating to 140 ℃ for reaction until TLC monitors that raw materials are completely reacted, cooling to room temperature, filtering reaction liquid, adding water into filtrate, extracting with dichloromethane for multiple times, combining organic phases, drying with anhydrous magnesium sulfate, and concentrating to obtain 3, 5-dichlorobenzamide; the feeding amount molar ratio of the 1, 3-dichlorobenzene to the trifluoroacetic anhydride to the formic acid is 1:2: 2; the mass ratio of the 1, 3-dichlorobenzene to the composite catalyst is 100: 5.
(3) Adding 3, 5-dichlorobenzamide and benzylamine into N, N-dimethylformamide, heating to 100 ℃, reacting until the raw materials disappear, cooling the reaction liquid to room temperature, pouring into water, extracting the reaction liquid for multiple times by using ethyl acetate, combining organic phases, drying by using anhydrous magnesium sulfate, and concentrating to obtain 3, 5-dibenzylamino benzamide; the feeding amount molar ratio of the 3, 5-dichlorobenzamide to the benzylamine is 1: 2.5.
(4) Adding 3, 5-dibenzylamine benzamide and palladium carbon into methanol, introducing hydrogen into an autoclave, reacting until the pressure reaches 0.2MPa and the reaction temperature is 40 ℃ until the raw materials completely react, filtering the reaction solution, and concentrating the filtrate to obtain the 3, 5-aminobenzamide.
(5) Adding phosphorus oxychloride into dichloromethane, adding a dichloromethane mixed solution dissolved with 3, 5-aminobenzamide into a constant-pressure dropping funnel, dropwise adding the mixed solution at room temperature under the protection of nitrogen, continuously stirring until the raw materials completely react, introducing ammonia gas into a reaction bottle, observing by a thermometer inserted into the reaction solution, rapidly increasing the reaction temperature, then rapidly decreasing the reaction temperature, when the reaction temperature is decreased to room temperature, finishing the reaction, filtering the reaction solution, drying an organic phase by anhydrous magnesium sulfate, and concentrating to obtain the 3, 5-dimethylureidobenzamide.
(6) Dissolving 3, 5-dimethylureidobenzamide in N, N-dimethylformamide to prepare a solution A; dissolving propargyl bromide and triethylamine in N, N-dimethylformamide to prepare a solution B; feeding the solution A and the solution B into a silicon carbide reactor with the reaction temperature of 40 ℃ through a tetrafluoro pump, carrying out substitution reaction on the materials in a microreactor within a certain residence time, collecting the reacted materials, pouring all obtained reaction liquid into water, extracting the reaction liquid for multiple times by using chloroform, combining organic phases, and concentrating to obtain N-propynyl-3, 5-dimethylureidobenzamide; the retention time is 3-6 min.
(7) Adding N-propynyl-3, 5-dimethylureidobenzamide and cuprous iodide into a mixed solution of N, N-dimethylformamide and methanol in a reaction bottle, stirring under the protection of nitrogen, adding azidotrimethylsilane, heating to 95 ℃ to react until the raw materials react completely, washing with water, extracting with ethyl acetate, drying with anhydrous sodium sulfate, filtering, evaporating the solvent to dryness, and separating by silica gel column chromatography to obtain the N- ((1H-1,2, 3-triazole-4-yl) -3, 5-dimethylureidobenzamide.
(8) Dissolving copper nitrate in acetonitrile, dissolving N- ((1H-1,2, 3-triazole-4-yl) -3, 5-dimethyl ureido benzamide in ethanol, mixing the solutions, heating to a certain temperature, stirring for a period of time, cooling to room temperature, adding anhydrous ether, standing, slowly volatilizing until crystals are separated out, and filtering the reaction solution to obtain a complex
The invention adopts the following technical scheme for solving the technical problems, and the preparation method of the azole urease inhibitor type feed additive is characterized by comprising the following specific steps:
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Example 1
Respectively adding 10g of graphite and 10g of sodium nitrate into a reaction bottle, slowly adding 150mL of concentrated sulfuric acid at 0 ℃, mechanically stirring for 10min, adding 50g of perchloric acid, heating to 50 ℃, stirring for 1h, adding 500mL of deionized water, heating to 80 ℃, stirring for 50min, taking out the reaction bottle, adding 200mL of deionized water and 100mL of hydrogen peroxide at room temperature, standing, precipitating, cooling to room temperature, washing, centrifuging for several times, continuously performing ultrasonic treatment for 8h, and freeze-drying for 10h to obtain graphite oxide; and adding all the obtained graphite oxide into 60mL of N, N-dimethylformamide, performing ultrasonic dispersion, weighing 4.8g of bismuth nitrate pentahydrate and 2.3g of scandium nitrate hexahydrate, dissolving in 40mL of N, N-dimethylformamide, weighing 10g of sodium trifluoromethanesulfonate, dissolving in 40mL of ethylene glycol, stirring for 1.5h, mixing the above solutions, and stopping stirring for 1 h. Pouring the mixed solution into a polytetrafluoroethylene high-pressure hydrothermal kettle, carrying out hydrothermal reaction at 140 ℃ for 24h, naturally cooling to room temperature after the reaction is stopped, alternately washing with ethanol and deionized water, and centrifuging. And (3) drying the precipitate in an air drying oven at the temperature of 80 ℃ for 12 hours to obtain 14.9g of the graphite-supported bismuth/scandium catalyst.
Example 2
Respectively adding 10g of graphite and 10g of sodium nitrate into a reaction bottle, slowly adding 150mL of concentrated sulfuric acid at 0 ℃, mechanically stirring for 10min, adding 50g of perchloric acid, heating to 50 ℃, stirring for 1h, adding 500mL of deionized water, heating to 80 ℃, stirring for 50min, taking out the reaction bottle, adding 200mL of deionized water and 100mL of hydrogen peroxide at room temperature, standing, precipitating, cooling to room temperature, washing, centrifuging for several times, continuously performing ultrasonic treatment for 8h, and freeze-drying for 10h to obtain graphite oxide; and adding all the obtained graphite oxide into 60mL of N, N-dimethylformamide, performing ultrasonic dispersion, weighing 4.8g of bismuth nitrate pentahydrate and 1.2g of scandium nitrate hexahydrate, dissolving in 40mL of N, N-dimethylformamide, weighing 10g of sodium trifluoromethanesulfonate, dissolving in 40mL of ethylene glycol, stirring for 1.5h, mixing the above solutions, and stopping stirring for 1 h. Pouring the mixed solution into a polytetrafluoroethylene high-pressure hydrothermal kettle, carrying out hydrothermal reaction at 140 ℃ for 24h, naturally cooling to room temperature after the reaction is stopped, alternately washing with ethanol and deionized water, and centrifuging. The precipitate was dried in an air-blast drying oven at 80 ℃ for 12 hours to obtain 11.6g of a graphite-supported bismuth/scandium catalyst B11.
Example 3
In a high-pressure reaction kettle, under the protection of argon, starting cooling, adding 15g of 1, 3-dichlorobenzene into a mixed solution of 42g of trifluoroacetic anhydride, 9.2g of formic acid and 150mL of dimethyl sulfoxide at the internal temperature of 5-10 ℃, keeping the temperature unchanged, and adding 0.7g of a graphite-loaded bismuth/scandium catalyst; slowly heating to 90 ℃, stirring for 50min, and introducing ammonia gas into the reaction kettle to enable the pressure in the kettle to reach 0.1 MPa; keeping the pressure unchanged, continuously raising the temperature to 140 ℃, reacting for 2 hours, then monitoring the reaction completion of the raw materials by TLC (thin layer chromatography), cooling to room temperature, filtering the reaction solution, adding 100mL of water into the filtrate, extracting for multiple times by using 100mL of dichloromethane, combining organic phases, drying by using anhydrous magnesium sulfate, and concentrating to obtain 13g of 3, 5-dichlorobenzamide; call for C7H5NCl2O:C,44.24；H,2.65；N,7.37.Found:C,44.51；H,2.69；N,7.32。
Example 4
In a high-pressure reaction kettle, under the protection of argon, starting cooling, adding 15g of 1, 3-dichlorobenzene into a mixed solution of 42g of trifluoroacetic anhydride, 9.2g of formic acid and 150mL of dimethyl sulfoxide at the internal temperature of 5-10 ℃, keeping the temperature unchanged, and adding 0.7g of a graphite-loaded bismuth/scandium catalyst; slowly heating to 90 ℃, stirring for 50min, and introducing ammonia gas into the reaction kettle to enable the pressure in the kettle to reach 0.1 MPa; keeping the pressure unchanged, continuously raising the temperature to 140 ℃, reacting for 2 hours, then monitoring the reaction completion of the raw materials by TLC (thin layer chromatography), cooling to room temperature, filtering the reaction solution, adding 100mL of water into the filtrate, extracting for multiple times by using 100mL of dichloromethane, combining organic phases, drying by using anhydrous magnesium sulfate, and concentrating to obtain 9g of 3, 5-dichlorobenzamide; call for C7H5NCl2O:C,44.24；H,2.65；N,7.37.Found:C,44.51；H,2.69；N,7.32。
Example 5
Adding 19g of 3, 5-dichlorobenzamide and 25g of benzylamine into 130mL of N, N-dimethylformamide in a reaction bottle, heating to 100 ℃, reacting for 1h, then monitoring by TLC to show that the raw materials disappear, cooling the reaction liquid to room temperature, and pouring the cooled reaction liquid into 100mL of water; extracting the reaction solution for three times by using 100mL of ethyl acetate, combining organic phases, drying the organic phases by using anhydrous magnesium sulfate, and concentrating the organic phases to obtain 31g of 3, 5-dibenzylamino benzamide;1H NMR(400M,CDCl3):7.98(s,2H),7.74(d,J＝2.0Hz,2H),7.49(d,J＝2.0Hz,4H),7.33-7.32(m,2H),7.27(s,2H),6.89(s,2H),6.77(s,1H),4.38(s,4H)；MS(ESI)m/z:332.4[M+H]+.Anal.Calcd for C21H21N3O:C,76.11；H,6.39；N,12.68.Found:C,76.35；H,6.32；N,12.57。
example 6
Adding 33g of 3, 5-dibenzylamine benzamide and 1.6g of palladium carbon (the content is 5%) into 200mL of methanol in a 500mL reaction bottle, introducing hydrogen into an autoclave, leading the pressure to reach 0.2MPa, leading the reaction temperature to be 40 ℃, monitoring the complete reaction of raw materials by TLC after reacting for 12 hours, filtering the reaction solution, and concentrating the filtrate to obtain 12g of pure 3, 5-aminobenzamide; call7H9N3O:C,55.62；H,6.00；N,27.80.Found:C,55.83；H,6.13；N,27.54。
Example 7
Adding 30g of phosphorus oxychloride into 300mL of dichloromethane in a reaction bottle, adding 100mL of dichloromethane dissolved with 15g of 3, 5-aminobenzamide into a constant-pressure dropping funnel, dropwise adding the mixed solution at room temperature under the protection of nitrogen, continuing stirring for 1h after dropwise adding, monitoring the complete reaction of the raw materials by TLC (thin layer chromatography), introducing ammonia gas into the reaction bottle, and insertingThe thermometer added into the reaction liquid can observe that the reaction temperature rises sharply and then drops rapidly, when the reaction temperature drops to room temperature, the reaction is finished, the reaction liquid is filtered, and the organic phase is dried by anhydrous magnesium sulfate and then concentrated to obtain 29g of 3, 5-dimethylureido benzamide; call7H15N7O2P2:C,27.37；H,4.92；N,31.92.Found:C,27.14H,4.81；N,31.76。
Example 8
Dissolving 31g of 3, 5-dimethylureidobenzamide into 200mL of N, N-dimethylformamide to prepare a solution A; dissolving 12g of bromopropyne and 12g of triethylamine in 100mL of N, N-dimethylformamide to prepare a solution B; feeding the solution A and the solution B into a silicon carbide reactor with the reaction temperature of 40 ℃ through a tetrafluoropump at the flow rate of 10mL/min and 5mL/min respectively, carrying out substitution reaction on materials in a microreactor, keeping the materials for 3min, collecting the reacted materials, pouring all obtained reaction liquid into water, extracting the reaction liquid for 4 times by using 200mL of chloroform, combining organic phases, and concentrating to obtain 32g of N-propynyl-3, 5-dimethylureido benzamide;1H NMR(400M,DMSO-d6):8.41(s,1H),7.22(s,2H),6.97(s,1H),4.55(d,J＝8.0Hz,2H),4.16(s,2H),3.25(s,1H),2.13-2.11(m,8H)；MS(ESI)m/z:346.2[M+H]+.Anal.Calcd for C10H17N7O3P2:C,34.79；H,4.96；N,28.40.Found:C,34.61H,4.92；N,28.55。
example 9
Dissolving 31g of 3, 5-dimethylureidobenzamide into 200mL of N, N-dimethylformamide to prepare a solution A; dissolving 12g of bromopropyne and 12g of triethylamine in 100mL of N, N-dimethylformamide to prepare a solution B; feeding the solution A and the solution B into silicon carbide reaction at a reaction temperature of 40 ℃ through a tetrafluoropump at flow rates of 10mL/min and 5mL/min respectivelyIn the reactor, the materials are subjected to substitution reaction in a microreactor, the residence time is 6min, the materials after the reaction are collected, all obtained reaction liquid is poured into water, 200mL of chloroform is used for extracting the reaction liquid for 4 times, organic phases are combined, and 27g of N-propynyl-3, 5-dimethylureido benzamide is obtained after concentration;1H NMR(400M,DMSO-d6):8.41(s,1H),7.22(s,2H),6.97(s,1H),4.55(d,J＝8.0Hz,2H),4.16(s,2H),3.25(s,1H),2.13-2.11(m,8H)；MS(ESI)m/z:346.2[M+H]+.Anal.Calcd for C10H17N7O3P2:C,34.79；H,4.96；N,28.40.Found:C,34.61H,4.92；N,28.55。
example 10
3.5g of N-propynyl-3, 5-dimethylureidobenzamide, 0.15g of cuprous iodide and 30mL (V) of a mixture of N, N-dimethylformamide and methanol were added to a reaction flaskDMF：VCH3OH2:1) under nitrogen protection, stirring, adding 1.5g of azidotrimethylsilane, heating to 95 ℃, stirring for 24h, cooling, adding 20mL of water and 30mL of ethyl acetate, extracting for three times, drying with anhydrous sodium sulfate, filtering, evaporating the solvent to dryness, and performing silica gel column chromatography (V)CH3OH：VDCM1:15) to obtain 3.6g of N- ((1H-1,2, 3-triazole-4-yl) -3, 5-dimethylureidobenzamide;1H NMR(400M,DMSO-d6):11.17(s,1H),8.09(d,J＝8.0Hz,1H),7.66(s,1H),7.04-7.02(m,1H),6.83-6.82(m,2H),4.68(d,J＝8.0Hz,2H)，4.15(s,2H),2.17-2.15(m,8H)；13C NMR(100M,DMSO-d6):166.5,147.1,136.2,129.4,128.8,107.9,102.3,56.5；MS(ESI)m/z:389.1[M+H]+.Anal.Calcd for C10H18N10O3P2:C,30.93；H,4.67；N,36.08.Found:C,30.84；H,4.75；N,36.27。
example 11
Dissolving 19g of copper nitrate in 300mL of acetonitrile,
dissolving 39g of the above solution in 600mL of ethanol, mixing the above solutions, heating to 70 ℃, stirring for 30min, cooling to room temperature, adding 1000mL of anhydrous ether, standing, slowly volatilizing, filtering the reaction solution after two days to obtain a complex
35g。
Elemental analysis
The contents of carbon, hydrogen and nitrogen were measured by an elemental analyzer, the content of copper ions was measured by a copper-ammonia complex method, and the results of elemental analysis are shown in table 1.
TABLE 1 elemental analysis results of the complexes
And (4) conclusion: the elemental analysis results of the samples were in agreement with the theoretical values.
Example 12
In vitro urease inhibition assay
After feeding the cattle for 1h, collecting 400mL of rumen fluid by a special rumen fluid collector through an artificial rumen fistula, and filtering the rumen fluid by 4 layers of gauze for later use. After adding the corresponding reagents in the amount shown in Table 1 into each culture tube, 4 drops of paraffin were added dropwise and the mixture was gently shaken on a constant temperature water bath shaker at 39.0. + -. 0.5 ℃. At 1,2,4,6 and 8h of culture, part of the culture tubes were taken out from each group, and 4 drops of saturated mercuric chloride solution were immediately added and shaken to terminate the reaction. The ammonia nitrogen content of each tube is measured by a Kjeldahl half-trace-saturated magnesium oxide distillation method.
Percent inhibition (%) (control ammonia content-test ammonia content) ÷ control ammonia content × 100%
| Time (h) | Inhibition rate of control group | Test 1 group inhibition | Test 2 group inhibition | Test 3 groups inhibition |
| 1 | 0％ | 16.3％ | 21.9％ | 39.6％ |
| 2 | 0％ | 26.8％ | 29.4％ | 49.1％ |
| 4 | 0％ | 43.4％ | 48.2％ | 60.4％ |
| 6 | 0％ | 52.7％ | 61.8％ | 71.9％ |
| 8 | 0％ | 59.3％ | 69.5％ | 80.5％ |
Example 13
Biological activity assay
Coli (gram-negative brevibacterium) and staphylococcus aureus (gram-positive bacterium) were selected as the test subjects for antibacterial activity. Firstly, preparing a liquid culture medium (1 g of peptone, 0.5g of yeast extract, 1g of sodium chloride and 100mL of distilled water are placed in a 250mL conical flask and are placed on an electric furnace to be heated while stirring, when the peptone, the yeast extract, the sodium chloride and the distilled water are mixed and clarified uniformly, the heating is stopped, and the bottleneck is sequentially sealed by gauze and kraft paper for standby) and a solid culture medium (1 g of peptone, 0.5g of yeast extract, 1g of sodium chloride, 2g of agar and 100mL of distilled water are placed in a 250mL conical flask and are placed on an electric furnace to be heated while stirring, when the peptone, the yeast extract, the agar and the distilled water are mixed and clarified uniformly, the heating is stopped, and the bottleneck is sequentially sealed by gauze and; then the culture medium is sterilized by an autoclave. Secondly, preparing bacterial liquid, namely activating escherichia coli and staphylococcus aureus strains, transferring 100 mu L of activated bacterial liquid by using a liquid transfer gun, and putting the activated bacterial liquid into sterilized 100mL of distilled water for uniformly mixing. And finally, sterilizing the flat plate by an ultraviolet lamp, quickly pouring the culture medium into the flat plate while the culture medium is hot, uniformly paving the flat plate with the thickness of about 0.15cm, standing the flat plate, slowly solidifying the flat plate, and putting the solidified flat plate into an incubator at 37 ℃ for culturing for one day for carrying out non-impurity bacteria detection.
The target compound and the control compound solutions are prepared respectively with DMF and placed in volumetric flasks for later use. Punching a hole on the filter paper by using a puncher, wherein the aperture is 5mm, and then sterilizing the filter paper sheet and soaking the filter paper sheet in a sample solution with the concentration of 1mg/mL for later use.
On a clean bench, an alcohol lamp was lit, and 10. mu.L of diluted culture solution was applied to the surface of the solid medium with a pipette and spread uniformly. The soaked round filter paper sheet was spread on the surface of the medium with sterile forceps. 4 plates were placed on each plate and 3 replicates were run, one of which was blank controlled. The plate with the tablets was incubated at 37 ℃ for 24h and observed for signs. The transparent circular rings-bacteriostatic rings with different sizes are respectively arranged on the agar culture medium, and the bacteriostatic activity of each sample can be seen by measuring the diameters of the bacteriostatic rings.
Example 14
The novel feed additive is subjected to oral acute toxicity tests according to the technical requirements of general toxicity tests of novel veterinary drugs and GB 15193.3-2003: on the basis of a pre-test, 20 mice and 20 rats are respectively taken by oral drenching at intervals of 4h, the administration dose is 16.0g/kg, the administration volume is 0.2mL/10g of body weight, the result shows that the animals normally eat, drink water, excrement and act within 14d, no toxic reaction is seen, and the novel feed additive orally takes LD (laser diode) for the mice and the rats50Are all more than 16.0 g/kg.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
Claims (7)
1. A triazole urease inhibitor type feed additive is characterized in that the molecular structure of the feed additive is as follows:
2. a preparation method of the triazole urease inhibitor type feed additive of claim 1 is characterized by comprising the following specific steps:
(1) respectively adding a carrier and sodium nitrate into a reaction bottle, slowly pouring concentrated sulfuric acid, uniformly stirring, adding perchloric acid, heating to 40 ℃, stirring for a period of time, adding deionized water, heating to 90 ℃, stirring for a period of time, cooling to room temperature, adding a proper amount of deionized water and hydrogen peroxide, standing for precipitation, washing and centrifuging for multiple times, continuously performing ultrasonic treatment, and performing freeze drying to obtain an oxidized carrier; dissolving the prepared oxidation carrier in an N, N-dimethylformamide solution, performing ultrasonic dispersion, weighing bismuth nitrate pentahydrate and scandium nitrate hexahydrate to dissolve in N, N-dimethylformamide, weighing sodium trifluoromethanesulfonate to dissolve in ethylene glycol, mixing the solutions, stirring for a period of time, pouring into a polytetrafluoroethylene high-pressure hydrothermal kettle, heating to 140 ℃ for reaction for a period of time, naturally cooling to room temperature, alternately washing with ethanol and deionized water, centrifuging, and drying the precipitate in a 80 ℃ blast drying box to obtain the composite catalyst;
(2) adding 1, 3-dichlorobenzene into a mixed solution of trifluoroacetic anhydride, formic acid and dimethyl sulfoxide under the condition of an internal temperature of 5-10 ℃ in a high-pressure reaction kettle under the protection of argon, keeping the temperature unchanged, adding a certain amount of composite catalyst, slowly heating to 90 ℃, stirring for a period of time, introducing ammonia gas into the reaction kettle to ensure that the pressure in the kettle reaches 0.1MPa, keeping the pressure unchanged, continuously heating to 140 ℃, reacting until TLC monitors that raw materials are completely reacted, cooling to room temperature, filtering reaction liquid, adding water into filtrate, extracting with dichloromethane for multiple times, combining organic phases, drying with anhydrous magnesium sulfate, and concentrating to obtain 3, 5-dichlorobenzamide;
(3) adding 3, 5-dichlorobenzamide and benzylamine into N, N-dimethylformamide, heating to 100 ℃, reacting until the raw materials disappear, cooling the reaction liquid to room temperature, pouring into water, extracting the reaction liquid for multiple times by using ethyl acetate, combining organic phases, drying by using anhydrous magnesium sulfate, and concentrating to obtain 3, 5-dibenzylamino benzamide;
(4) adding 3, 5-dibenzylamine benzamide and palladium carbon into methanol, introducing hydrogen into an autoclave, reacting until the pressure reaches 0.2MPa and the reaction temperature is 40 ℃ until the raw materials completely react, filtering the reaction solution, and concentrating the filtrate to obtain 3, 5-aminobenzamide;
(5) adding phosphorus oxychloride into dichloromethane, adding a dichloromethane mixed solution dissolved with 3, 5-aminobenzamide into a constant-pressure dropping funnel, dropwise adding the mixed solution at room temperature under the protection of nitrogen, continuously stirring until the raw materials completely react, introducing ammonia gas into a reaction bottle, observing by a thermometer inserted into the reaction solution, rapidly increasing the reaction temperature, then rapidly decreasing the reaction temperature, when the reaction temperature is decreased to room temperature, finishing the reaction, filtering the reaction solution, drying an organic phase by anhydrous magnesium sulfate, and concentrating to obtain the 3, 5-dimethylureidobenzamide;
(6) dissolving 3, 5-dimethylureidobenzamide in N, N-dimethylformamide to prepare a solution A; dissolving propargyl bromide and triethylamine in N, N-dimethylformamide to prepare a solution B; feeding the solution A and the solution B into a silicon carbide reactor with the reaction temperature of 40 ℃ through a tetrafluoro pump, carrying out substitution reaction on the materials in a microreactor within a certain residence time, collecting the reacted materials, pouring all obtained reaction liquid into water, extracting the reaction liquid for multiple times by using chloroform, combining organic phases, and concentrating to obtain N-propynyl-3, 5-dimethylureidobenzamide;
(7) adding N-propynyl-3, 5-dimethylureidobenzamide and cuprous iodide into a mixed solution of N, N-dimethylformamide and methanol in a reaction bottle, stirring under the protection of nitrogen, adding azidotrimethylsilane, heating to 95 ℃ to react until the raw materials react completely, washing with water, extracting with ethyl acetate, drying with anhydrous sodium sulfate, filtering, evaporating the solvent to dryness, and separating by silica gel column chromatography to obtain N- ((1H-1,2, 3-triazole-4-yl) -3, 5-dimethylureidobenzamide;
(8) dissolving copper nitrate in acetonitrile, dissolving N- ((1H-1,2, 3-triazole-4-yl) -3, 5-dimethyl ureido benzamide in ethanol, mixing the solutions, heating to a certain temperature, stirring for a period of time, cooling to room temperature, adding anhydrous ether, standing, slowly volatilizing until crystals are separated out, and filtering the reaction solution to obtain a complex
3. The method for producing a triazole urease inhibitor type feed additive according to claim 2, wherein: the carrier in the step (1) is graphite powder; the feeding amount molar ratio of the bismuth nitrate pentahydrate to the scandium nitrate trihydrate is 1: 0.5-1.0.
4. The method for producing a triazole urease inhibitor type feed additive according to claim 2, wherein: the feeding amount molar ratio of the 1, 3-dichlorobenzene, the trifluoroacetic anhydride and the formic acid in the step (2) is 1:2: 2; the mass ratio of the 1, 3-dichlorobenzene to the composite catalyst is 100: 5.
5. The method for producing a triazole urease inhibitor type feed additive according to claim 2, wherein: the feeding amount molar ratio of the 3, 5-dichlorobenzamide to the benzylamine in the step (3) is 1: 2.5.
6. The method for producing a triazole urease inhibitor type feed additive according to claim 2, wherein: the retention time in the step (6) is 3-6 min.
7. The method for producing a triazole urease inhibitor type feed additive according to claim 2, wherein: the reaction temperature in the step (8) was 70 ℃.
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