Use of a nitrothiazole derivative for the preparation of an antibacterial agent for inhibiting helicobacter pylori

Abstract

The application provides a use of a nitrothiazole derivative in preparation of an antibacterial agent for inhibiting helicobacter pylori, and belongs to the field of pharmacy.The structure of the amino nitrothiazole derivative is shown as formula I.The experimental results show that the compound can effectively inhibit the growth of helicobacter pylori, and the inhibiting effect is better than that of control compounds nitazoxanide and lauric acid; the compound can effectively treat mice infected with helicobacter pylori, clear helicobacter pylori in the stomach and reduce the inflammatory response of the stomach tissue, and the treatment effect is better than that of control compounds nitazoxanide and lauric acid.The compound provided by the application can be used for preparing the antibacterial agent for inhibiting helicobacter pylori, and can be used for preparing a medicine for preventing and / or treating helicobacter pylori infection and related diseases caused by the helicobacter pylori, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and particularly relates to a use of a nitrothiazole derivative in preparation of an antibacterial agent for inhibiting Helicobacter pylori. BACKGROUND

[0002] Helicobacter pylori is a spiral-shaped, micro-anaerobic, and very demanding bacteria in growth conditions. It was first isolated successfully from gastric mucosa biopsy tissues of patients with chronic active gastritis in 1983, and is the only microbial species known to survive in human stomach. On October 27, 2017, the International Agency for Research on Cancer of the World Health Organization announced the preliminary sorting reference of the list of carcinogens, and Helicobacter pylori (infection) is in the list of class I carcinogens.

[0003] Helicobacter pylori exists in the human stomach antrum, and is one of the most common bacterial pathogens. More than half of the world's population has been infected with Helicobacter pylori, and almost 90% of people in some countries have been infected with the bacteria. People are usually infected in childhood, and 50% of children under 5 years old are infected. The bacterial infection first causes chronic gastritis, and leads to gastric ulcer and gastric atrophy, and in severe cases, develops into gastric cancer.

[0004]

[0005] Nitazoxanide is a derivative of nitazoxanide, and its actual mechanism of action has not been clarified, but it is believed to be related to inhibiting the enzyme-dependent electron transfer reaction of pyruvate, iron oxidoreductase, which is important for anaerobic energy metabolism. Studies have found that in addition to Cryptosporidium and intestinal Giardia, nitazoxanide also has activity on many intestinal parasites, such as Besnoitia, amoeba, human roundworm, hookworm, Trichuris trichiura, Taenia saginata, Hymenolepis nana, and Fasciola hepatica. However, a study (Foreign Medicine Antibiotic Supplement, July 2004, Vol. 25, No. 4, pp. 191-192) reported that nitazoxanide as a single therapeutic agent cannot eradicate Helicobacter pylori.

[0006] In order to effectively prevent and treat Helicobacter pylori infection and related diseases caused thereby, it is urgent to develop an antibacterial agent with better inhibitory effect on Helicobacter pylori. SUMMARY

[0007] The purpose of the present application is to provide a use of a nitrothiazole derivative in preparation of an antibacterial agent for inhibiting Helicobacter pylori, and a medicine for preventing and / or treating Helicobacter pylori infection and related diseases caused thereby.

[0008] The present application provides a use of a compound represented by formula (I), or a crystal form thereof, or a salt thereof in preparation of an antibacterial agent for inhibiting Helicobacter pylori:

[0009] The present application provides a use of a compound represented by formula (I), or a crystal form thereof, or a salt thereof in preparation of an antibacterial agent for inhibiting Helicobacter pylori:

[0010] wherein R is selected from C3, C4 linear alkanes, C5-C 15 linear or branched alkanes.

[0011] Further, the compound is one of the following compounds:

[0012]

[0013] Further, the bacteriostatic agent is capable of inhibiting the growth of H. pylori.

[0014] Further, the bacteriostatic agent is a drug for preventing and / or treating H. pylori infection.

[0015] Further, the bacteriostatic agent is a drug for preventing and / or treating diseases caused by H. pylori.

[0016] Further, the disease caused by H. pylori is gastritis, gastric ulcer, duodenal ulcer, gastric cancer.

[0017] Further, the bacteriostatic agent is a preparation prepared by adding a pharmaceutically acceptable excipient or auxiliary ingredient to the compound, or a crystal form thereof, or a salt thereof as an active ingredient.

[0018] Further, the excipient or auxiliary ingredient is selected from one or more of diluents, fillers, colorants, glidants, lubricants, binders, stabilizers, suspending agents, and buffers.

[0019] The compound of the present application can effectively inhibit the growth of H. pylori, and the inhibitory activity of compounds I-1 to I-4 is much stronger than that of the control compound lauric acid, and the inhibitory activity of compounds I-1, I-3, and I-4 is much stronger than that of the control compound nitazoxanide.

[0020] The compound of the present application can effectively treat H. pylori infected mice, clear H. pylori infection in the stomach and reduce gastric tissue inflammatory response, and the treatment effect is comparable to that of the positive control clarithromycin, and the treatment effect is obviously better than that of the control compounds nitazoxanide and lauric acid at the same molar dose.

[0021] The compound provided by the present application can be used for preparing a bacteriostatic agent for inhibiting H. pylori, and can be used for preparing a drug for preventing and / or treating H. pylori infection and related diseases caused thereby, and has a wide application prospect.

[0022] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0023] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0024] Figures in the specification

[0025] Figure 1 These are the gastric tissue pathology scoring results of each group of mice in Example 2.

[0026] Figure 2 These are the OD values ​​of Helicobacter pylori urease detection results for each group of mice in Example 2. DETAILED DESCRIPTION

[0027] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0028] Preparation of test compounds I-1 to I-4:

[0029] The test compounds I-1 to I-4 were prepared according to the preparation method disclosed in CN114044761A. The specific operation is as follows:

[0030]

[0031] 2.0 g of tizoxanide (7.5 mmol) was placed in a 50 ml single-necked flask, 20 ml of ethyl acetate and 0.9 g of triethylamine (8.8 mmol) were added, and 0.9 g of n-butyryl chloride (8.4 mmol) was added dropwise with stirring. After the addition was complete, the mixture was heated to reflux in an oil bath and allowed to react for 2 hours. TLC confirmed the complete reaction. The mixture was cooled to room temperature and filtered to remove the solid. The filtrate was diluted with 20 ml of ethyl acetate and washed twice with 10 ml of 0.1 M dilute hydrochloric acid, once with 10 ml of 10% sodium bicarbonate solution, and finally with 10 ml of saturated brine until neutral. The ethyl acetate layer was dried over 2.5 g of anhydrous sodium sulfate for 30 minutes; the sodium sulfate was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain compound I-1.

[0032] Referring to the above method, n-butyryl chloride was replaced by octanoyl chloride, lauroyl chloride, and tetradecanoyl chloride, respectively, to prepare compounds I-2, I-3, and I-4, respectively:

[0033]

[0034]

[0035] Example 1. In vitro inhibitory activity of the test compounds against Helicobacter pylori

[0036] (1) Experimental methods

[0037] Weigh 3.9g of Columbia blood agar base medium, heat and dissolve in 100ml distilled water, and sterilize in a triangular flask at 121℃ for 15min. When cooled to about 50℃, add 5% sterile defibrillated sheep blood, mix well, and keep in a 50℃ water bath to ensure that the medium does not solidify.

[0038] DMSO was used to dissolve the test compounds I-1 to I-4, the control compound nitazoxanide and lauric acid. In a 12-well culture plate, 10ul of the diluted test sample solution was added to each well, followed by 1ml of 50℃ melted Columbia blood agar medium, and the test compound concentration was 0-200ug / ml, with a two-fold concentration dilution, a total of 10 concentration gradients.

[0039] The -80℃ frozen Helicobacter pylori strain ATCC26695 was recovered and inoculated on a Columbia blood plate, and microaerophilic 37℃ culture was performed for 48h. The growth state of the colonies was scraped using a pipette tip, and resuspended in a Brucella medium containing 5% peptide bovine serum, and the OD value of the bacterial solution was adjusted to 0.2. 2ul of the bacterial solution was aspirated using a micropipette, inoculated on the Columbia blood agar containing the test sample in the 12-well plate, 3 bacterial spots were inoculated in each well, and after the bacterial solution was dried. Put into the microaerophilic environment and culture at 37℃ for 48h.

[0040] After 48h of culture in the 12-well plate, the colony growth was photographed and recorded, and the minimum inhibitory concentration (MIC) of the test compound on Helicobacter pylori was determined. The DMSO-dissolved control group had well-grown colonies in the plate wells, forming obvious colonies. The minimum concentration at which the colonies did not grow was the minimum inhibitory concentration of the test sample on Helicobacter pylori.

[0041] The results are shown in Table 1 below.

[0042] (2) Experimental results

[0043] Table 1. MIC of each compound on Helicobacter pylori

[0044] Compound MIC (μg / ml) Compound MIC (μg / ml) I-1 12.5 I-2 25 I-3 3.125 I-4 12.5 Nitazoxanide 25 Laurylic acid 100

[0045] The experimental results show that the test compounds of the present application can effectively inhibit the growth of Helicobacter pylori, and the inhibitory activity of compounds I-1 to I-4 is much stronger than that of the control compound lauric acid, and the inhibitory activity of compounds I-1, I-3 and I-4 is much stronger than that of the control compound nitazoxanide.

[0046] The above experimental results show that the test compounds of the present application can be used to prepare bacteriostatic agents for inhibiting Helicobacter pylori, and can be used for the prevention and / or treatment of Helicobacter pylori infection and related diseases caused thereby.

[0047] Example 2, therapeutic effect of the test compound on a mouse H. pylori infection model

[0048] (1) Experimental method

[0049] H. pylori SS1 strain preparation. The bacteria were resuscitated using a Columbia blood plate, and after 48 hours of culture, colonies were selected and inoculated in a Brucella broth medium containing 5% fetal bovine serum and a selective additive, and after 48 hours of culture at 37°C under microaerophilic conditions, the bacteria were removed and centrifuged to adjust the concentration to 5 x 10 9 CFU for standby.

[0050] C57BL / 6 mice, female, 6-8 weeks old, a total of 42, were randomly divided into 6 groups after adaptive feeding for one week, namely the blank group, the model group, the positive control clarithromycin group, the compound I-3 group, the control compound nitazoxanide group and the control compound lauric acid group, 7 in each group. Except for the blank group, the rest of the mice were given 200 μl of the above bacterial resuspension system, i.e. 1 x 10 9 CFU per mouse. Before gavage of bacteria, the mice were fasted for 12 h and deprived of water for 4 h; one hour before gavage of bacteria, 250 μL of 0.2 mol / L sodium bicarbonate was gavaged to neutralize stomach acid; two hours after gavage of bacteria, the mice were allowed to eat and drink freely; the above process was repeated after 1 day, a total of 6 times. Two weeks after the last gavage, the corresponding drugs were given to each group daily, the clarithromycin was given at a dose of 50 mg / kg, the compound I-3 was given at a dose of 100 mg / kg, the nitazoxanide was given at a dose of 68 mg / kg, the lauric acid was given at a dose of 45 mg / kg, and the blank group was given an equal volume of normal saline; the mice were sacrificed after two weeks of drug administration, and the samples were analyzed.

[0051] The stomachs of the mice in each group were cut from the cardiac to the pylorus and placed in a sterile dish, the greater curvature was cut open, part of which was used for histopathological examination, the gastric contents were gently removed with a sterile cotton swab, and after rinsing with normal saline, the samples were fixed with 10% neutral formaldehyde. After HE staining, the inflammatory changes in the stomach of the mice after live bacteria challenge were observed, scored and recorded, and the scoring criteria were as follows:

[0052] 0 points: occasional inflammatory cell infiltration in the lamina propria; 1 point: scattered inflammatory cell infiltration in the lamina propria; 2 points: moderate inflammatory cell infiltration in the lamina propria; 3 points: massive inflammatory cell infiltration in the lamina propria, and individual lymphoid follicles formed under the gastric mucosa.

[0053] The same weight of gastric tissue was taken from each group of mice, and a high-throughput homogenizer was used for homogenization. The rapid urease kit was prepared, 100 μL of enzyme reaction solution was added to each well, after the drug film was completely dissolved, the tissue homogenate was added, and incubated at room temperature for 5 minutes for color development, the OD value of each well was tested and the results were recorded.

[0054] (2) Experimental results

[0055] The pathological score of each group of mice stomach tissue is recorded as shown in Table 2, wherein * represents P<0.5 compared with the model group; the H. pylori urease detection result of the stomach tissue homogenate of each group of mice is shown in Table 2, wherein ** represents P<0.05 compared with the model group; and # represents P<0.5 of the I-3 group compared with the nitazoxanide group. Figure 1 Figure 2 The pathological score of each group of mice stomach tissue is recorded as shown in Table 2, wherein * represents P<0.5 compared with the model group; the H. pylori urease detection result of the stomach tissue homogenate of each group of mice is shown in Table 2, wherein ** represents P<0.05 compared with the model group; and # represents P<0.5 of the I-3 group compared with the nitazoxanide group.

[0056] Table 2. Pathological score of stomach tissue of H. pylori infected model mice

[0057] Group Score Blank group 0.3 Model group 2.4 Clarithromycin group 1.3 * ]]> I-3 group 1.1 * ]]> Nitazoxanide group 1.9 Laurylic acid group 2.1

[0058] The experimental results show that in the pathological score of the stomach tissue of the mice, the I-3 group and the clarithromycin group significantly reduce the inflammatory response of the mice after the live bacteria attack, and there is a statistical difference compared with the model group; the nitazoxanide group and the lauric acid group slightly reduce the inflammatory response, but there is no statistical difference compared with the model group. In the H. pylori urease detection result of the stomach tissue of the mice, the I-3 group, the clarithromycin group and the nitazoxanide group significantly reduce the expression of urease, indicating that the three groups of compounds significantly inhibit the growth of H. pylori, and there is a significant difference compared with the model group; at the same time, the urease expression result of the I-3 group is obviously lower than that of the nitazoxanide group and the lauric acid group, indicating that the compound I-3 has a significantly better effect on inhibiting the growth of H. pylori than the nitazoxanide and lauric acid, and there is a statistical difference compared with the nitazoxanide group. The above results show that the test compound of the present application can effectively treat H. pylori infected mice, clear the stomach infected H. pylori and reduce the inflammatory response of the stomach tissue, and the treatment effect is equivalent to the positive control clarithromycin, and the treatment effect is obviously better than the control compounds nitazoxanide and lauric acid under the same molar dose.

[0059] The above experimental results show that the test compound of the present application can effectively inhibit H. pylori infection, and can be used for preventing and / or treating H. pylori infection and related diseases caused thereby.

[0060] In summary, the present application provides the use of the nitrothiazole derivative shown in formula I in the preparation of an antibacterial agent for inhibiting H. pylori. The experimental results show that the compound can effectively inhibit the growth of H. pylori, and the inhibitory effect is better than the control compounds nitazoxanide and lauric acid; the compound can effectively treat H. pylori infected mice, clear the stomach infected H. pylori and reduce the inflammatory response of the stomach tissue, and the treatment effect is better than the control compounds nitazoxanide and lauric acid. The compound provided by the present application can be used for preparing an antibacterial agent for inhibiting H. pylori, and can be used for preparing a medicine for preventing and / or treating H. pylori infection and related diseases caused thereby, and has a wide application prospect.​

Claims

1. Use of a compound or a salt thereof in the manufacture of an antibacterial agent for inhibiting Helicobacter pylori, the compound being one of the following compounds:

2. Use according to claim 1, characterized in that: The antibacterial agent is capable of inhibiting the growth of Helicobacter pylori.

3. Use according to claim 1, characterized in that: The antibacterial agent is a medicament for preventing and / or treating Helicobacter pylori infection.

4. Use according to claim 1, characterized in that: The antibacterial agent is a medicament for preventing and / or treating a disease caused by Helicobacter pylori.

5. Use according to claim 4, characterized in that: The disease caused by Helicobacter pylori is gastritis, gastric ulcer, duodenal ulcer, or gastric cancer.

6. Use according to any one of claims 1 to 5, characterized in that: The antibacterial agent is a preparation prepared by adding a pharmaceutically acceptable adjuvant or auxiliary ingredient to the compound or a salt thereof as an active ingredient.

7. Use according to claim 6, characterized in that: The adjuvant or auxiliary ingredient is selected from one or two or more of a diluent, a filler, a coloring agent, a glidant, a lubricant, a binder, a stabilizer, a suspending agent, and a buffer.

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