Use of nitrothiazole derivatives and their hydrochloride and sulfate salts for the preparation of a medicament for the treatment of intestinal infections in poultry and livestock

Abstract

The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of nitrothiazole derivatives and hydrochloride and sulfate thereof in preparation of medicines for treating intestinal infection of poultry and livestock. The nitrothiazole derivatives synthesized by the present application have high biological activity at the cell level, and have good biological activity on bacterial enteritis of poultry and livestock, especially chicken, pig, sheep and deer, and have high application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to the application of nitrothiazole derivatives and their hydrochlorides and sulfates in the preparation of drugs for treating intestinal infections in poultry and livestock. Background Technology

[0002] The livestock industry frequently faces a complex and diverse range of bacterial infections, with those caused by Escherichia coli, Salmonella, Pasteurella, Clostridium perfringens, Haemophilus parasuis, Staphylococcus aureus, and Streptococcus being the most serious. Complex bacterial infections, especially intestinal bacterial infections, occur frequently in livestock farming. For example, necrotizing enteritis in chickens is a bacterial infectious disease caused by Clostridium perfringens, but there are reports of Salmonella pullorum and Escherichia coli also occurring concurrently during this disease process. Clinically, a single drug is often insufficient to effectively control intestinal infections in animals; therefore, developing highly effective antibacterial drugs to control intestinal infections in livestock and poultry is of paramount importance.

[0003] Thiazoles and their derivatives are important functional groups in many drugs. Current research indicates that nitrothiazole derivatives can improve cardiac function and alleviate ischemia-reperfusion injury. In 2004, Dharmara Sriram et al. reported that compounds with a nitrothiazole core can act on the ATPase domain of mycobacterial gyrase, thereby inhibiting bacterial growth. Heterocyclic nitro compounds such as nitrofurans and metronidazole are commonly used to treat intestinal infections. Tinidazole, ornidazole, and other nitroimidazole compounds are approved for the treatment of parasitic infections. Megazol is used to treat protozoan infections. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides the application of nitrothiazole derivatives and their hydrochloride and sulfate salts in the preparation of drugs for treating intestinal infections in poultry and livestock. The nitrothiazole derivatives synthesized by this invention have high biological activity at the cellular level and exhibit good biological activity against intestinal infections in poultry and livestock, especially bacterial enteritis in chickens, pigs, sheep, and deer. They have significant therapeutic effects, high application value, and solve the problems existing in the prior art.

[0005] This invention is achieved through the following technical solution:

[0006] Application of nitrothiazole derivatives and their hydrochloride and sulfate salts in the preparation of drugs for treating intestinal infections in poultry and livestock.

[0007] Furthermore, the intestinal infections of poultry and livestock include infections caused by Escherichia coli and / or Salmonella, ileitis caused by Lawsonia, or infections caused by drug-resistant Clostridium perfringens.

[0008] Furthermore, the intestinal infections of poultry and livestock include chicken infections caused by Escherichia coli and / or Salmonella, swine ileitis caused by Lawsonia, or infections caused by drug-resistant Clostridium perfringens in deer.

[0009] The application of nitrothiazole derivatives and their hydrochloride and sulfate salts in the preparation of drugs for treating peritonitis in poultry and livestock, wherein the poultry and livestock peritonitis is peritonitis caused by Haemophilus parasuis infection.

[0010] The application of nitrothiazole derivatives and their hydrochloride and sulfate salts in the preparation of drugs for treating lung infections in poultry and livestock, wherein the lung infection in poultry and livestock is caused by Streptococcus suis infection.

[0011] Furthermore, the dosage of the nitrothiazole derivative and its hydrochloride and sulfate is 1 mg / kg for large and medium-sized livestock and 3 mg / kg for small poultry.

[0012] Furthermore, the structural formula of the nitrothiazole derivative is as follows: Its synthetic route is as follows:

[0013]

[0014] Furthermore, the synthesis of the nitrothiazole derivative includes the following steps:

[0015] Starting with 2-aminothiazole, compound 2 (2-amino-5-nitrothiazole) is generated by nitration with sulfuric acid and nitric acid.

[0016] b) The compound 3 (2-bromo-5-nitrothiazole) is generated by diazotization with sodium nitrite and Sanger reaction under CuSO4 catalysis. Both steps a and b are carried out in water.

[0017] c) Compound 4 is obtained by reacting 2-mercapto-5-amino-1,3,4-thiadiazole with compound 3 obtained in step b under alkaline conditions via a nucleophilic substitution reaction.

[0018] Further, the base used in step c, the alkaline condition, includes one or a mixture of several of triethylamine, N,N-diisopropylethylamine, sodium hydroxide, potassium hydroxide, potassium carbonate, piperidine, N-methylmorpholine, and diaminopyridine; the amount of base used is 1-4 times that of the compound 2-mercapto-5-amino-1,3,5-thiadiazole; the solvent used in the alkaline condition includes one or a mixture of several of methanol, ethanol, tetrahydrofuran, and acetone.

[0019] Furthermore, the reaction temperature for the synthesis of compound 4 in step c is 0-30℃.

[0020] A pharmaceutical composition comprising the above-mentioned nitrothiazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] The beneficial effects of this invention:

[0022] The 5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazole compound synthesized by the process of this invention is simple to operate and significantly improves the product yield. The obtained derivatives, after application studies, exhibit high bioactivity at the cellular level and show significant efficacy in the preparation of treatments for intestinal infections in poultry and livestock, specifically treating infections caused by *Escherichia coli*, *Lactosomalemia*-induced ileitis, or infections caused by drug-resistant *Clostridium perfringens*. Attached Figure Description

[0023] Figure 1 This is a culture medium diagram for identifying pathogenic bacteria in the cecal contents of a dead sika deer, as shown in Example 7 of the present invention.

[0024] Figure 2 This is the growth curve of Escherichia coli ATCC-25922 at different concentrations of compound 4 in Example 8 of the present invention;

[0025] Figure 3 This is the growth curve of Staphylococcus aureus ATCC-29213 at different concentrations of compound 4 in Example 8 of the present invention;

[0026] Figure 4 These are photos of the feces of pigs in the experimental group of Example 10 of this invention before medication;

[0027] Figure 5 This is a photograph of the feces of the pigs in the experimental group of Example 10 of this invention on the second day after medication.

[0028] Figure 6 This is a photograph of the feces of the pigs in the experimental group of Example 10 of this invention on the third day after medication.

[0029] Figure 7 This is a photograph of the feces of the pigs in the experimental group of Example 10 of the present invention on the second day after drug withdrawal;

[0030] Figure 8 This is a photograph of the pig herd in the experimental group of Example 10 of the present invention on the 9th day after treatment and withdrawal;

[0031] Figure 9 This is a photograph of the deer feces in the experimental group of Example 11 of the present invention before drug administration;

[0032] Figure 10 This is a photograph of the feces of deer in the experimental group of Example 11 of the present invention on the second day after drug administration;

[0033] Figure 11 These are photos of deer feces on the 4th day after drug withdrawal in the 11 experimental groups of this invention.

[0034] Figure 12 The results of RAP detection of Clostridium perfringens in the intestine before treatment in this invention;

[0035] Figure 13 The results of RAP detection of Clostridium perfringens in the intestine after 11 treatments according to this invention;

[0036] Figure 14 The results of fecal microbiota detection in 11 young deer treated at different times during the present invention;

[0037] Figure 15 The results of fecal microbiota testing of adult deer at different treatment times in this invention;

[0038] Figure 16 These are photographs of the anus of the diseased sheep on the first, second, and third days of medication in Example 12 of this invention.

[0039] in, Figure 1 Image A shows the results of streaking the chromogenic medium for Clostridium perfringens from the French company, Colmarga. Figure 1 Figure B shows the results of streaking on tryptone-sulfite-cyclic serine agar medium. Detailed Implementation

[0040] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0041] Example 1

[0042] The synthesis of nitrothiazole derivatives for treating intestinal infections in poultry and livestock includes the following steps:

[0043] 1. Synthesis of compound 2:

[0044] 20 g (0.2 mol) of compound 1:2-aminothiazole was added to a 250 mL flask. 30 mL of concentrated sulfuric acid and 10 mL of 40% nitric acid were added dropwise under ice bath conditions, and the mixture was stirred overnight at 15 °C. The pH was adjusted to 8 using 1 M NaOH, the precipitate was filtered, and the precipitate was washed thoroughly with plenty of water. The precipitate was purified by silica gel column chromatography using petroleum ether:ethyl acetate (5:1) as the mobile phase to give 17.1 g of compound 2 (59% yield). 1 H NMR (500MHz, d6-DMSO) δ8.81 (s, 1H), 8.25 (s, 1H).

[0045] 2. Synthesis of compound 3:

[0046] Add 2.88 g of compound 2 to a 100 mL flask, add 14 mL of water, and slowly add 18 mL of 40% HBr dropwise under ice bath conditions. Stir the mixture for 30 min and cool to -15 °C. Slowly add 10 mL of a mixture of NaNO2 and copper sulfate (containing 3 g of NaNO2 and 3 g of copper sulfate pentahydrate) dropwise to the reaction solution. After the addition is complete, stir the reaction at 0 °C for 12 h. Raise the temperature to 25 °C and continue stirring for 2 h. Stop stirring and filter. Wash the residue three times with 10 mL of water and collect the residue. Add 3 mL of ethanol, stir at 50 °C for 3 h, and filter again. Wash the residue with 3 mL of cold ethanol. Filter and dry. The sample is dried to obtain 2.7 g of product (yield 65%).

[0047] 3. Synthesis of compound 4:

[0048] Weigh 2.67 g of 2-mercapto-5-amino-1,3,4-thiadiazole, add 2.72 g of solid K₂CO₃ and 15 mL of ethanol, purge with nitrogen, and stir at room temperature for 30 min. Add 3.31 g of the previously prepared compound 3, and stir overnight at room temperature. Concentrate the reaction solution to 8 mL, filter, and wash three times with 20 mL of water, collecting the residue. Add 15 mL of a 6:1 solution of petroleum ether:ethyl acetate and slurry for 2 h, repeating twice. Filter and wash with petroleum ether. Obtain 41.92 g of compound (73% yield).

[0049] Example 2

[0050] The synthesis of nitrothiazole derivatives for treating intestinal infections in poultry and livestock includes the following steps:

[0051] 1.1. The synthesis method of compound 2 is the same as that of compound 2 in Example 1 above.

[0052] 2. Synthesis of compound 3:

[0053] Add 2.88 g of compound 2 to a 100 mL flask, along with 14 mL of water and 2 g of NaBr. Slowly add 9 mL of concentrated sulfuric acid dropwise under ice bath conditions, and stir the mixture for 30 min. Slowly add 10 mL of NaNO2 solution (containing 3 g of NaNO2) dropwise to the reaction mixture, and stir at 0 °C for 12 h. Increase the temperature to 25 °C and continue stirring for 2 h. Stop stirring and filter, washing three times with 10 mL of water and collecting the residue. Add 3 mL of ethanol, slurry at 50 °C for 3 h, and filter again, washing the residue with 3 mL of cold ethanol. Filter and dry, and the sample is dried to obtain 2.40 g of product (yield 58%).

[0054] 3. Synthesis of compound 4:

[0055] Weigh 2.67 g of 2-mercapto-5-amino-1,3,4-thiadiazole, add 0.8 g of solid NaOH and 15 mL of ethanol, and stir at room temperature for 30 min under nitrogen protection. Add 3.31 g of compound 3 and stir overnight at room temperature. Concentrate the reaction solution to 8 mL, filter, and wash three times with 20 mL of water, collecting the residue. Add 15 mL of a 6:1 solution of petroleum ether and ethyl acetate and stir for 2 h, repeating twice. Filter and wash with petroleum ether to obtain 1.76 g of compound 4 (68% yield). 1 HNMR(500MHz,d6-DMSO)δ8.74(s,1H),7.94(s,2H).

[0056] Example 3

[0057] The synthesis of nitrothiazole derivatives for treating intestinal infections in poultry and livestock includes the following steps:

[0058] 1. The synthesis method of compound 2 is the same as that of compound 2 in Example 1 above.

[0059] 2. Synthesis of compound 3:

[0060] Add 2.88 g of compound 2 to a 100 mL flask, add 14 mL of water, and slowly add 18 mL of 40% hydrobromic acid dropwise under ice bath conditions. Stir the mixture for 30 min. Slowly add 10 mL of NaNO2 solution (containing 3 g of NaNO2) to the reaction mixture and stir at 0 °C for 12 h. Increase the temperature to 25 °C and continue stirring for 2 h. Stop stirring and filter. Wash three times with 10 mL of water and collect the residue. Add 3 mL of ethanol, stir at 50 °C for 3 h, and filter again. Wash the residue with 3 mL of cold ethanol. Filter and dry. The sample is dried to obtain 3.1 g of product (yield 75%). 1 H NMR (500MHz, CDCl3) δ8.34 (s, 1H).

[0061] 3. Synthesis of compound 4:

[0062] Weigh 2.67 g of 2-mercapto-5-amino-1,3,4-thiadiazole, add 2.5 mL of triethylamine and 35 mL of ethanol, and stir at room temperature for 30 min under nitrogen protection. Add 3.31 g of compound 3 and stir overnight at room temperature. Concentrate the reaction solution to 15 mL, filter, and wash three times with 20 mL of water, collecting the residue. Add 15 mL of a 6:1 solution of petroleum ether and ethyl acetate and stir for 2 h, repeating twice. Filter and wash with petroleum ether to obtain 1.24 g of compound 4 (yield 48%).

[0063] Example 4

[0064] The synthesis of nitrothiazole derivatives for treating intestinal infections in poultry and livestock includes the following steps:

[0065] 1. The synthesis method of compound 2 is the same as that of compound 2 in Example 1 above.

[0066] 2. One-pot synthesis of compound 3:

[0067] Add 20 g (0.2 mol) of compound 2 to a 250 mL flask. Add 30 mL of concentrated sulfuric acid and 10 mL of nitric acid (40%) dropwise under ice bath conditions, and stir overnight at 15 °C. Cool the reaction solution to 0 °C and add 30 mL of saturated NaBr solution. Add 60 mL of a mixture of NaNO2 and copper sulfate (containing 18 g of NaNO2 and 18 g of copper sulfate pentahydrate), and stir at 0 °C for 12 h after the addition is complete. Raise the temperature to 25 °C and continue stirring for 2 h. Stop stirring and filter. Wash three times with 10 mL of water and collect the residue. Add 3 mL of ethanol, slurry at 50 °C for 3 h, and filter again. Wash the residue with 3 mL of cold ethanol. Filter and dry. The sample yielded 17.3 g of product (yield 42%).

[0068] 3. Synthesis of compound 4:

[0069] Weigh 2.67 g of 2-mercapto-5-amino-1,3,4-thiadiazole, add 2.72 g of solid K₂CO₃ and 15 mL of ethanol, and stir at room temperature for 30 min under nitrogen protection. Add 3.31 g of compound 3 and stir overnight at room temperature. Concentrate the reaction solution to 8 mL, filter, and wash three times with 20 mL of water, collecting the residue. Add 15 mL of a 6:1 solution of petroleum ether and ethyl acetate and stir for 2 h, repeating twice. Filter and wash with petroleum ether to obtain 1.92 g of compound 4 (73% yield).

[0070] Example 5

[0071] The synthesis of nitrothiazole derivatives for treating intestinal infections in poultry and livestock includes the following steps:

[0072] 1. The synthesis method of compound 2 is the same as that of compound 2 in Example 1 above;

[0073] 2. Effect of changes in synthesis conditions on the yield of compound 3

[0074] 2.88 g (0.2 mol) of compound 2 was added to a 100 mL flask. Different amounts of 40% hydrobromic acid were added at 0 °C, followed by dropwise addition of different amounts of n-pentanol and 3 g of NaNO2 at different temperatures. After reacting for 4 h, the mixture was stirred overnight at 15 L. The temperature was raised to 25 °C and stirring was continued for 2 h. Stirring was stopped, and the mixture was filtered, washed three times with 50 mL of water, and the residue was collected. 10 mL of ethanol was added, and the mixture was recrystallized at 80 L. The residue was washed with 10 mL of ethanol. The mixture was then filtered and dried to obtain compound 3. The results are shown in Table 1.

[0075] 3. Compound 4 can be synthesized using any of the synthesis methods described in the foregoing examples.

[0076] Table 1

[0077]

[0078] In the synthesis of compound 3 in this route, copper sulfate was removed and replaced with n-pentanol. Table 1 shows that different amounts of hydrobromic acid, different temperatures, and different amounts of n-pentanol significantly affect the yield. Since the yield of compound 3 has the most significant impact on the yield of compound 4 in this route, the synthesis conditions indicated by number 3 were selected for this route.

[0079] Compound 4, synthesized in Example 1 of this invention, was selected for subsequent determination and application experiments.

[0080] Example 6: Determination of the minimum inhibitory concentration of compound 4

[0081] Escherichia coli, Staphylococcus aureus, Salmonella paratyphi, Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), Enterococcus faecalis, vancomycin-resistant Enterococcus faecium (VRE), Haemophilus parasuis, and Streptococcus suis were resuscitated using blood agar plates. After resuscitation, the bacteria were inoculated into nutrient broth for 12-24 hours, diluted to a concentration of MH broth. 5 CFU / mL. The minimum inhibitory concentration (MIC) for each type of bacteria was determined using the broth dilution method.

[0082] First, compound 4 was dissolved in DMSO and filtered using a sterile organic filter membrane. Different volumes were added to the bacterial broth to prepare solutions of different concentrations. After culturing for 16-24 hours, the turbidity of the broth was observed to determine the results. The results are shown in Table 2.

[0083] Table 2 Antibacterial activity of different bacteria

[0084]

[0085] Note: a TSB medium supplemented with 1% NAD chocolate agar.

[0086] Table 2 shows the minimum inhibitory concentration (MIC) of compound 4 of the present invention against the aforementioned pathogenic bacteria. The MIC determinations for different bacteria indicate that compound 4 is more effective against Gram-positive bacteria, less effective against Gram-negative bacteria, and less effective against Bacillus subtilis and Pseudomonas aeruginosa. Considering all factors, compound 4 can be used as an intestinal flora regulator to inhibit partial intestinal flora imbalance.

[0087] Example 7: Isolation of Clostridium perfringens and Determination of the Minimum Inhibitory Concentration of Compound 4

[0088] The contents of the cecum of a dead sika deer were streaked onto Clostridium perfringens chromogenic medium from the French Colmargasse (French). Figure 1 A) and tryptone-sulfite-cycloserine agar medium (TSC) ( Figure 1 B), after anaerobic incubation at 37℃ for 48 hours, distinct orange-red colonies were observed, and it was identified as Clostridium perfringens (B). Figure 1 ).

[0089] Culture media containing Compound 4 at concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL were prepared using the agar dilution method. Clostridium perfringens from different sources were plated and anaerobically incubated at 37°C for 48 h. The sources of Clostridium perfringens included cattle, sheep, pigs, chickens, seafood, and vultures. All Clostridium perfringens strains were clinically isolated and identified using selective culture media.

[0090] Table 3 Antibacterial activity of different bacteria

[0091]

[0092] Note: a. This strain was isolated from a deer farm infected with Clostridium perfringens, and drug resistance testing revealed that it was resistant to dimetridazole.

[0093] As shown in Tables 2 and 3, compound 4 exhibits good efficacy against Clostridium perfringens from different sources, indicating its ability to inhibit Clostridium perfringens infection in different animals. Compound 4 showed good efficacy against Clostridium perfringens from both large and medium-sized animals, with a MIC of 4 μg / mL. It showed the best efficacy against Clostridium perfringens from rabbits and vultures at 0.5 μg / mL. However, it was less effective against Clostridium perfringens from chickens, at 16 μg / mL.

[0094] The combined results of Examples 6 and 7 indicate that Compound 4 exhibits good antibacterial effects against common aerobic, anaerobic, and facultative anaerobic bacteria. Compound 4 has therapeutic value against common intestinal infections in livestock. Considering the clinical use of Compound 4 as an intestinal flora regulator, a dose not exceeding 1 mg / kg is recommended for large and medium-sized livestock, and 3 mg / kg for small animals.

[0095] Example 8: Time-killing curves of compound 4 against Staphylococcus aureus and Escherichia coli.

[0096] Escherichia coli ATCC25922 (MIC 8 μg / mL) and Staphylococcus aureus ATCC29213 (MIC 0.5 μg / mL) were resuscitated using blood agar plates and inoculated into LB broth for 14 hours. The inoculated broth was then diluted with MH broth to the corresponding concentrations (10 μg / mL). 6(Approximately), add the corresponding chemicals to make the final concentrations 0, 1*MIC, 2*MIC, 4*MIC, and 8*MIC. Take samples at 0h, 2h, 4h, 8h, 12h, and 24h, dilute and plate them, and record the number of single colonies.

[0097] The results indicate that compound 4 has a significant inhibitory effect on bacterial growth.

[0098] Example 9: Application of Compound 4 in the preparation of a drug for treating intestinal infections in poultry and livestock

[0099] To investigate the therapeutic effect of compound 4 on diarrhea caused by mixed infections of multiple bacteria, compound 4, prepared according to Example 1 of the synthetic route of this invention, was used in a therapeutic and protective study on naturally infected broiler chickens. Submission for testing confirmed that the infection was caused by a combination of Salmonella and Escherichia coli.

[0100] Compound 4, prepared in Example 1, along with metronidazole and amoxicillin, were used in different doses for treatment. The treatment effects are shown in Table 4 below.

[0101] Table 4. In vivo antibacterial activity of compound 4, metronidazole and amoxicillin against mixed bacterial diarrhea.

[0102]

[0103] As shown in Table 4, at low doses, the in vivo antibacterial activity of compound 4 of the present invention against mixed interfering diarrhea caused by multiple bacteria was significantly higher than that of metronidazole and amoxicillin. At a dose of 0.75 mg / kg, the number of broilers surviving after treatment with compound 4 in Example 1 of the present invention was 30, while no broilers survived after treatment with the same dose of metronidazole and amoxicillin. Furthermore, as the dose of compound 4 increased, its antibacterial activity gradually increased, demonstrating a significant increase in the number of surviving broilers compared to metronidazole and amoxicillin at the same increased dose. At a dose of 1.5 mg / kg, the number of surviving broilers after treatment with compound 4 in Example 1 of the present invention reached 100, while the same dose of metronidazole and amoxicillin remained ineffective against mixed infections, with the number of surviving broilers remaining at 0.

[0104] Example 10: Application of Compound 4 in the preparation of a drug for treating porcine ileitis caused by Lawsonia.

[0105] To investigate the therapeutic effect of compound 4 on porcine ileitis caused by Lawsonia, compound 4 from Example 1 of this invention was selected for a therapeutic and protective study on naturally occurring porcines.

[0106] In early June 2021, 854 38-day-old pigs were initially stocked in Unit 1 of a fattening farm in Tai'an City, Shandong Province. The pigs were primarily weak and diarrhea-prone pigs selected from Units 4, 5, and 6. After being transferred to Unit 1, some pigs experienced persistent diarrhea for several days, accounting for approximately 20% of the total stock. The feces were mostly porridge-like or watery, gray or cement-like in color, with a small amount being greenish / yellowish-green. Severely ill pigs had feces on their buttocks, red and swollen anus, and rough, disheveled coats; some pigs had pale skin. The majority of the pigs were in good spirits and showed no other abnormalities. The group had been given medications such as amoxicillin, colistin, doxycycline, and florfenicol powder in their drinking water, but the effects were unsatisfactory, and new cases of diarrhea continued to appear daily. Individual pigs were treated with a combination of medications including dysentery-relieving agents, enrofloxacin, florfenicol, and lincomycin injections, which showed some effectiveness in treating some of the diarrhea-prone pigs. A small number of pigs that recovered experienced a recurrence of diarrhea after several days or weeks. Subsequent fecal sampling and testing revealed the presence of PPE and PED pathogens in the fecal samples, leading to a diagnosis of Lawsonia solani ileitis.

[0107] Treatment method: Thirty pigs with severe diarrhea were selected from the herd and placed in a test pen. Anal swabs were randomly collected from three pigs with diarrhea and marked accordingly. Samples were sent for testing before medication. The test pigs were fasted for half a day. Approximately 8 kg of feed was mixed with the test drug using a stepwise artificial dilution method before feeding. To ensure that all pigs had equal access to feed, three additional feed trays were added to the pen in conjunction with the existing feed troughs. To ensure the objectivity of the experiment, no drugs were added to the drinking water during the period. Each pig was given 60 mg of compound 4 daily for three consecutive days, and anal swabs were collected from the sick pigs daily for testing. The results are shown in Table 5.

[0108] Table 5. Follow-up effects of drug treatment on pigs with diarrhea

[0109]

[0110] Combined with appendix Figures 4-7 , Figure 4 Feces of pigs in the experimental group before medication; feces on day 2 of medication; feces on day 3 of medication; and feces on day 2 after medication withdrawal. Most pigs with diarrhea recovered after medication intervention, with their feces becoming formed or normal. The redness and swelling around the anus in some pigs subsided. Analysis of the data collected in Table 2 shows that the drug had a significant effect. The three pigs that were not cured still had diarrhea, which is speculated to be related to insufficient drug intake, differences in individual physical condition / recovery time—their intestinal mucosal inflammation was more severe, and intestinal repair may have taken longer than other pigs. Further daily clinical observation and follow-up revealed that all pigs' feces were normal on day 9 after medication withdrawal.

[0111] Before treatment, the 30 selected pigs exhibited severe diarrhea, rough and disheveled coats, and most showed signs of "exposed backs." Combined with... Figure 8It is evident that the pigs showed significantly improved health and uniformity after treatment. This result indicates that compound 4, compared to commonly used treatments for porcine ileitis such as florfenicol and enrofloxacin, has the advantages of rapid onset of action and quick recovery.

[0112] Example 11: Application of Compound 4 in the preparation of a drug for treating Clostridium perfringens-induced infections in deer.

[0113] This experiment was conducted at a breeding farm in Dongying, Shandong Province. The breeding farm introduced 100 breeding deer. During the feeding process, bacterial infection occurred in the breeding deer herd due to feed contamination. Veterinary identification confirmed it as Clostridium perfringens infection. After infection, the deer were found to have bloody stools and eventually died from dehydration. Sporadic deer deaths continued despite long-term use of dimetridazole.

[0114] In the early stages, deer exhibited bloody stools, and later ceased rumination. They also showed eyelid hemorrhage and tearing. Post-mortem examination upon death revealed peritoneal hemorrhage, intestinal hemorrhage and necrosis, and extensive kidney necrosis and edema. PCR amplification using Clostridium perfringens-specific primers showed clear amplification bands and detected Clostridium perfringens α-toxin.

[0115] Before this experiment, the deer farm had 21 surviving deer. Four deer were randomly selected as the control group; three of these died three days after discontinuation of dimetridazole, and one died five days later. Sixteen deer were selected as the experimental group. The drug was encapsulated based on the deer's average weight, and the capsules were wrapped in grass to entice them to eat. Each deer was given 4140 mg of the compound daily for three consecutive days, and the observation period was 11 days. The results are shown in Table 6 below.

[0116] Table 6. Statistical table of treatment for deer infected with Clostridium perfringens.

[0117]

[0118] As shown in Table 6, when compound 4 from Example 1 of this invention was administered at a low dose for 2 days, all deer exhibited improved diarrhea, with no deaths observed during the treatment period. All deer were cured by the third day of treatment, demonstrating significant efficacy. This treatment is specifically effective against infections caused by drug-resistant Clostridium perfringens in deer.

[0119] Combined with appendix Figure 9-11 It was found that the feces of deer infected with drug-resistant Clostridium perfringens before medication, on the second day after medication, and on the fourth day after medication was stopped, showed that the disease was completely cured with no recurrence after 3-4 days of stopping medication, and the average feed intake of the deer herd increased by 30%. Figure 12-13 The results show the results of Clostridium perfringens RAP testing before and after treatment. Clostridium perfringens infection was observed before treatment, but it could not be detected after treatment.

[0120] Fecal samples were collected from sick fawns on the first day of medication (Z1), the third day of medication (Z2), the first day after medication was stopped (Z3), and 20 days after medication was stopped (Z4) for intestinal flora testing. Figure 14 The results showed that the number of Clostridium species was effectively reduced during treatment, while the number of other bacterial species increased.

[0121] Microbial community analysis was performed on fecal samples taken from asymptomatic fawns on the first day of treatment (Z5), the first day after treatment (Z6), and the 20th day after treatment (Z7). Figure 15 The results showed that compound 4 had little effect on Bacteroides, but a greater effect on Firmicutes and Proteobacteria.

[0122] Example 12: Application of Compound 4 in the treatment of sheep dysentery caused by Clostridium perfringens

[0123] To investigate the effect of compound 4 on sheep dysentery caused by Clostridium perfringens, compound 4 from Example 1 of this invention was selected for a therapeutic and protective study on naturally infected sheep.

[0124] Sheep infected with Clostridium perfringens from a farm in Yantai City, Shandong Province were selected for the experiment. Before the experiment, the sheep had been treated with ceftiofur for 10 days but the disease did not improve. The veterinarian diagnosed the sheep with dysentery caused by Clostridium perfringens infection.

[0125] Treatment method: Four affected sheep were selected. These sheep were fasted for half a day. Approximately 2 kg of feed was mixed with the experimental drug using a stepwise artificial dilution method before feeding. To ensure the objectivity of the experiment, no drug was added to the drinking water during this period. The drug was administered for three consecutive days, with each sheep receiving 60 mg of compound 4 daily. The feces of the flock were observed after three days of treatment. Figure 16 ; Figure 16 The three pictures show the sheep's diarrhea at the anus on the first, second, and third days after taking the medication.

[0126] The experimental results showed that the sheep stopped having diarrhea after 3 days, the anus was clean, and the feces were granular. After 14 days of continuous observation, there was no recurrence of diarrhea.

[0127] Example 13: Application of Compound 4 in the treatment of swine septicemia caused by Streptococcus suis

[0128] To investigate the effect of compound 4 on swine septicemia caused by Streptococcus suis, compound 4 synthesized in this invention was selected for therapeutic and protective studies on naturally occurring swine.

[0129] Treatment method: Ten sick pigs were selected and divided into two groups. The experimental pigs were fasted for half a day. Approximately 1 kg of feed was mixed with the experimental drug using a stepwise artificial dilution method before feeding. To ensure the objectivity of the experiment, no drugs were added to the drinking water during this period. Each pig was given 20 mg of compound 4 for 3 consecutive days. The survival of the pigs was observed after three days of treatment, as shown in Table 7.

[0130] Table 7

[0131]

[0132] Three days after medication, the pigs were observed for another 10 days. Two pigs in the treatment group survived, while all pigs in the control group died. The results indicate that compound 4 has a certain therapeutic effect on Streptococcus suis infection.

[0133] The above specific embodiments should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0134] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. Use of nitrothiazole derivatives and their hydrochloride and sulfate salts for the preparation of a medicament for the treatment of intestinal infections in livestock, characterized in that: The enteric infection of the livestock is ileitis caused by Lawsonia intracellularis; The structural formula of the nitrothiazole derivative is: 。

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