WO2023198095A1 - Use of nitrothiazole derivative in preparing bacteriostat for inhibiting helicobacter pylori - Google Patents
Use of nitrothiazole derivative in preparing bacteriostat for inhibiting helicobacter pylori Download PDFInfo
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- WO2023198095A1 WO2023198095A1 PCT/CN2023/087744 CN2023087744W WO2023198095A1 WO 2023198095 A1 WO2023198095 A1 WO 2023198095A1 CN 2023087744 W CN2023087744 W CN 2023087744W WO 2023198095 A1 WO2023198095 A1 WO 2023198095A1
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- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 42
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 42
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 20
- ALNUOSXDMYFQNQ-UHFFFAOYSA-N 2-nitro-1,3-thiazole Chemical class [O-][N+](=O)C1=NC=CS1 ALNUOSXDMYFQNQ-UHFFFAOYSA-N 0.000 title abstract description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 47
- 206010019375 Helicobacter infections Diseases 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 14
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 239000000022 bacteriostatic agent Substances 0.000 claims description 17
- 229940079593 drug Drugs 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 208000007882 Gastritis Diseases 0.000 claims description 4
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 4
- 239000013078 crystal Substances 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 3
- 208000007107 Stomach Ulcer Diseases 0.000 claims description 3
- 206010017758 gastric cancer Diseases 0.000 claims description 3
- 201000011549 stomach cancer Diseases 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 239000003086 colorant Substances 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 208000000718 duodenal ulcer Diseases 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 201000005917 gastric ulcer Diseases 0.000 claims description 2
- -1 glidants Substances 0.000 claims description 2
- 239000000314 lubricant Substances 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 239000000375 suspending agent Substances 0.000 claims description 2
- 239000006172 buffering agent Substances 0.000 claims 1
- 230000001568 sexual effect Effects 0.000 claims 1
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 abstract description 26
- YQNQNVDNTFHQSW-UHFFFAOYSA-N acetic acid [2-[[(5-nitro-2-thiazolyl)amino]-oxomethyl]phenyl] ester Chemical compound CC(=O)OC1=CC=CC=C1C(=O)NC1=NC=C([N+]([O-])=O)S1 YQNQNVDNTFHQSW-UHFFFAOYSA-N 0.000 abstract description 19
- 241000699670 Mus sp. Species 0.000 abstract description 16
- 229960002480 nitazoxanide Drugs 0.000 abstract description 13
- 239000005639 Lauric acid Substances 0.000 abstract description 12
- 230000002496 gastric effect Effects 0.000 abstract description 11
- 210000002784 stomach Anatomy 0.000 abstract description 10
- 230000001225 therapeutic effect Effects 0.000 abstract description 7
- 230000028709 inflammatory response Effects 0.000 abstract description 4
- SHVHFDTVXHMMNP-UHFFFAOYSA-N 2-nitro-1,3-thiazol-4-amine Chemical class NC1=CSC([N+]([O-])=O)=N1 SHVHFDTVXHMMNP-UHFFFAOYSA-N 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 108010046334 Urease Proteins 0.000 description 6
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
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- 230000008595 infiltration Effects 0.000 description 4
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- 238000000034 method Methods 0.000 description 4
- 210000004400 mucous membrane Anatomy 0.000 description 4
- DQXKOHDUMJLXKH-PHEQNACWSA-N (e)-n-[2-[2-[[(e)-oct-2-enoyl]amino]ethyldisulfanyl]ethyl]oct-2-enamide Chemical group CCCCC\C=C\C(=O)NCCSSCCNC(=O)\C=C\CCCCC DQXKOHDUMJLXKH-PHEQNACWSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 229960002626 clarithromycin Drugs 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000589562 Brucella Species 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- DVECBJCOGJRVPX-UHFFFAOYSA-N butyryl chloride Chemical compound CCCC(Cl)=O DVECBJCOGJRVPX-UHFFFAOYSA-N 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
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- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- ANPAWAXFULSFEF-UHFFFAOYSA-N 5-bromo-3-[4-(prop-2-enoylamino)piperidin-1-yl]sulfonyl-1H-indole-2-carboxamide Chemical group C(C=C)(=O)NC1CCN(CC1)S(=O)(=O)C1=C(NC2=CC=C(C=C12)Br)C(=O)N ANPAWAXFULSFEF-UHFFFAOYSA-N 0.000 description 1
- 241000224489 Amoeba Species 0.000 description 1
- 241001465677 Ancylostomatoidea Species 0.000 description 1
- 208000004300 Atrophic Gastritis Diseases 0.000 description 1
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- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000242722 Cestoda Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000223935 Cryptosporidium Species 0.000 description 1
- FDTZUTSGGSRHQF-UHFFFAOYSA-N Desacetyl-nitazoxanide Chemical compound OC1=CC=CC=C1C(=O)NC1=NC=C([N+]([O-])=O)S1 FDTZUTSGGSRHQF-UHFFFAOYSA-N 0.000 description 1
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- 241000242711 Fasciola hepatica Species 0.000 description 1
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- 241000224467 Giardia intestinalis Species 0.000 description 1
- 241001473947 Helicobacter pylori SS1 Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- SKZKKFZAGNVIMN-UHFFFAOYSA-N Salicilamide Chemical compound NC(=O)C1=CC=CC=C1O SKZKKFZAGNVIMN-UHFFFAOYSA-N 0.000 description 1
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- 241001489151 Trichuris Species 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- NQGIJDNPUZEBRU-UHFFFAOYSA-N dodecanoyl chloride Chemical group CCCCCCCCCCCC(Cl)=O NQGIJDNPUZEBRU-UHFFFAOYSA-N 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
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- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 244000000053 intestinal parasite Species 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 238000010172 mouse model Methods 0.000 description 1
- REEZZSHJLXOIHL-UHFFFAOYSA-N octanoyl chloride Chemical group CCCCCCCC(Cl)=O REEZZSHJLXOIHL-UHFFFAOYSA-N 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
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- 229960000581 salicylamide Drugs 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/426—1,3-Thiazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D277/58—Nitro radicals
Definitions
- the invention belongs to the field of pharmaceuticals, and specifically relates to the use of a nitrothiazole derivative in preparing a bacteriostatic agent for inhibiting Helicobacter pylori.
- Helicobacter pylori is a spiral-shaped, slightly anaerobic bacterium that has very demanding growth conditions. It was first successfully isolated from the gastric mucosal biopsy tissue of a patient with chronic active gastritis in 1983. It is currently the only microbial species known to be able to survive in the human stomach. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer released a preliminary reference list of carcinogens, and Helicobacter pylori (infection) was included in the list of carcinogens.
- Helicobacter pylori lives in the pylorus of the human stomach and is one of the most common bacterial pathogens. More than half of the world's population is infected with Helicobacter pylori, and in some countries almost 90% of the population is infected with this bacterium. People are usually infected at an early age, with 50% of cases under 5 years old. This bacterial infection first causes chronic gastritis, and leads to gastric ulcers and gastric atrophy. In severe cases, it may develop into gastric cancer.
- Nitazoxanide is a derivative of nitethyl salicylic acid amide. Although its actual mechanism of action has not yet been clarified, it is believed to be related to the inhibition of the enzyme-dependent electron transfer reaction of pyruvate and ferredoxin oxidoreductase. , the latter is crucial for anaerobic energy metabolism. Studies have found that in addition to Cryptosporidium and Giardia intestinalis, nitazoxanide is also effective against many intestinal parasites, such as Isospora burnetii, amoeba, human roundworms, hookworms, Trichuris trichuris, Beef tapeworm, brevis tapeworm and Fasciola hepatica are all active. However, there are research reports (Foreign Medicine and Antibiotics Volume, July 2004, Volume 25, Issue 4, Pages 191-192) that nitazoxanide cannot eradicate Helicobacter pylori as a single therapeutic agent.
- the object of the present invention is to provide the use of a nitrothiazole derivative in the preparation of bacteriostatic agents for inhibiting Helicobacter pylori and medicaments for preventing and/or treating Helicobacter pylori infection and related diseases caused by it.
- the present invention provides the use of the compound represented by formula (I), or its crystal form, or its salt in the preparation of a bacteriostatic agent for inhibiting Helicobacter pylori:
- R is selected from C 3 and C 4 linear alkanes, C 5 -C 15 linear or branched alkanes.
- the compound is one of the following compounds:
- the bacteriostatic agent can inhibit the growth of Helicobacter pylori.
- the bacteriostatic agent is a drug for preventing and/or treating Helicobacter pylori infection.
- the bacteriostatic agent is a drug for preventing and/or treating diseases caused by Helicobacter pylori.
- the diseases caused by Helicobacter pylori are gastritis, gastric ulcer, duodenal ulcer, and gastric cancer.
- the bacteriostatic agent is a preparation made of the compound, or its crystal form, or its salt as an active ingredient, and adding pharmaceutically acceptable excipients or auxiliary ingredients.
- auxiliary materials or auxiliary ingredients are selected from one or more of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and buffers.
- the compounds of the present invention can effectively inhibit the growth of Helicobacter pylori, and the inhibitory activities of compounds I-1 to I-4 are much stronger than the control compound lauric acid, and the inhibitory activities of compounds I-1, I-3, and I-4 are much stronger than the control.
- the compound nitazoxanide is much stronger than the control compound lauric acid, and the inhibitory activities of compounds I-1, I-3, and I-4 are much stronger than the control.
- the compound of the present invention can effectively treat mice infected with Helicobacter pylori, eliminate Helicobacter pylori infection in the stomach and reduce the inflammatory reaction of gastric tissue. Its therapeutic effect is equivalent to that of the positive control clarithromycin, and the therapeutic effect is obviously superior at equimolar doses. to the control compounds nitazoxanide and lauric acid.
- the compounds provided by the invention can be used to prepare bacteriostatic agents that inhibit Helicobacter pylori, and can be used to prepare drugs for preventing and/or treating Helicobacter pylori infection and related diseases caused by it.
- the scenery is vast.
- Figure 1 shows the gastric histopathological scoring results of mice in each group in Example 2.
- Figure 2 shows the results of OD value detection of Helicobacter pylori urease in each group of mice in Example 2.
- the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
- Test compounds I-1 to I-4 were prepared with reference to the preparation method disclosed in CN114044761A. The specific operations are as follows:
- the filtrate is diluted with 20 ml of ethyl acetate, washed twice with 10 ml of 0.1M dilute hydrochloric acid, once with 10 ml of 10% sodium bicarbonate solution, and finally with Wash with 10 ml saturated saline until neutral.
- the ethyl acetate layer was dried with 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 crude product was purified by silica gel column chromatography to obtain compound I-1.
- n-butyryl chloride was replaced with octanoyl chloride, lauroyl chloride, and myristanoyl chloride to prepare compounds I-2, I-3, and I-4 respectively:
- Example 1 In vitro inhibitory activity of test compounds against Helicobacter pylori
- DMSO DMSO was used to dissolve the test compounds I-1 to I-4, the control compounds nitazoxanide and lauric acid.
- DMSO DMSO was used to dissolve the test compounds I-1 to I-4, the control compounds nitazoxanide and lauric acid.
- a 12-well culture plate add 10 ⁇ l of diluted test sample solution to each well, then add 1 ml of Columbia blood agar culture medium melted at 50°C, shake and mix until the concentration of the test compound is 0-200 ⁇ g/ml, twice Concentration dilution, a total of 10 concentration gradients.
- Helicobacter pylori strain ATCC26695 frozen at -80°C was resuscitated and inoculated on Columbia blood agar plates, and cultured microaerobically at 37°C for 48 hours.
- Use a pipette tip to scrape well-growing colonies, resuspend them in Brucella's medium containing 5% peptide bovine serum, and adjust the OD value of the bacterial solution to 0.2.
- Use a micropipette to absorb 2 ⁇ l of the bacterial solution and inoculate it onto the Columbia blood agar containing the test sample in a 12-well plate. Inoculate 3 bacterial spots in each well and wait until the bacterial solution dries. Place in a microaerophilic environment and incubate at 37°C for 48 hours.
- the minimum inhibitory concentration (MIC) of the test compound against Helicobacter pylori After culturing the 12-well culture plate for 48 hours, take photos to record the colony growth, and determine the minimum inhibitory concentration (MIC) of the test compound against Helicobacter pylori. DMSO dissolved the control group, and the colonies in the plate wells grew well and formed obvious colonies. For the test sample group, the minimum concentration at which bacterial colonies do not grow is the minimum inhibitory concentration of the test sample against Helicobacter pylori.
- test compounds of the present invention can effectively inhibit the growth of Helicobacter pylori, and the inhibitory activities of compounds I-1 to I-4 are much stronger than the control compound lauric acid.
- the inhibitory activities of compounds I-1, I-3, and I-4 are The inhibitory activity is much stronger than that of the control compound nitazoxanide.
- test compound of the present invention can be used to prepare a bacteriostatic agent that inhibits Helicobacter pylori, and can be used to prevent and/or treat Helicobacter pylori infection and related diseases caused by it.
- Example 2 Therapeutic effect of test compounds on mouse Helicobacter pylori infection model
- Helicobacter pylori SS1 strain preparation Use Columbia blood plate to resuscitate bacteria. After 48 hours of culture, select bacterial colonies and inoculate them into Brucella broth medium containing 5% fetal calf serum and selective additives. After culturing for 48 hours under microaerophilic conditions at 37°C, take them out. Centrifuge and adjust the bacterial concentration to 5 ⁇ 10 9 CFU for later use.
- mice Female, 6-8 weeks old, were randomly divided into 6 groups after adaptive feeding for one week, namely blank group, model group, positive control clarithromycin group, and compound I-3 group. , control compound nitazoxanide group and control compound lauric acid group, 7 animals in each group. Except for the blank group, the other mice were orally administered 200 ⁇ l of the above bacterial resuspension system, that is, 1 ⁇ 10 9 CFU per mouse.
- the dosage of clarithromycin was 50 mg/kg, the dosage of compound I-3 was 100 mg/kg, and the dosage of nitazoxanide was The dosage of lauric acid was 68 mg/kg, the dosage of lauric acid was 45 mg/kg, and the blank group was given an equal volume of physiological saline; the mice were killed two weeks after the administration, and the materials were collected for analysis.
- the stomachs of mice in each group were cut off from the cardia to the pylorus, placed in a sterile dish, and cut open along the greater curvature of the stomach. Parts were taken for histopathological examination.
- the stomach contents were gently removed with sterile cotton swabs, and the stomach contents were removed with a physiological After rinsing with saline, they were fixed with 10% neutral formaldehyde.
- HE staining observe the inflammatory changes in the stomach of mice after live bacteria challenge, score and record the results.
- the scoring standards are as follows:
- stomach tissues of equal weight from each group of mice were taken and homogenized using a high-throughput homogenizer for later use.
- Prepare a rapid urease kit add 100 ⁇ L of enzymatic reaction solution to each well, add tissue homogenate after the drug film is completely dissolved, incubate at room temperature for 5 minutes to develop color, test the OD value of the test solution in each well and record the results.
- mice showed that in the gastric histopathological scores of mice, treatment in the I-3 group and the clarithromycin group significantly reduced the inflammatory response of mice after live bacteria challenge, with statistical differences compared with the model group; Treatment in the nitazoxanide group and the lauric acid group slightly reduced the inflammatory response, but there was no statistical difference compared with the model group.
- test compound of the present invention can effectively treat mice infected with Helicobacter pylori, clear the Helicobacter pylori infection in the stomach and reduce the inflammatory reaction of gastric tissue. Its therapeutic effect is equivalent to that of the positive control clarithromycin, and in et al. The therapeutic effect at molar dosage was significantly better than that of the control compounds nitazoxanide and lauric acid.
- test compound of the present invention can effectively inhibit Helicobacter pylori infection and can be used to prevent and/or treat Helicobacter pylori infection and related diseases caused by it.
- the present invention provides the use of nitrothiazole derivatives represented by formula I in the preparation of bacteriostatic agents for inhibiting Helicobacter pylori.
- Experimental results show that the compound can effectively inhibit the growth of Helicobacter pylori, and the inhibitory effect is better than the control compounds nitazoxanide and lauric acid; the compound can effectively treat mice infected with Helicobacter pylori, clear the Helicobacter pylori infection in the stomach and Reduce the inflammatory response of gastric tissue, and its therapeutic effect is better than that of the control compounds nitazoxanide and lauric acid.
- the compounds provided by the invention can be used to prepare bacteriostatic agents that inhibit Helicobacter pylori, and can be used to prepare drugs for preventing and/or treating Helicobacter pylori infection and related diseases caused by it, and have broad application prospects.
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Abstract
The present invention relates to the field of pharmaceuticals, and particularly provides use of a nitrothiazole derivative in preparing a bacteriostat for inhibiting Helicobacter pylori. The amino nitrothiazole derivative has a structure of formula I. Experimental results show that the compound can effectively inhibit the growth of Helicobacter pylori, with an inhibitory effect superior to that of reference compounds nitazoxanide and lauric acid; the compound can effectively treat mice infected with Helicobacter pylori, remove Helicobacter pylori affecting the stomach and ameliorate the inflammatory response of the gastric tissue, with a therapeutic effect superior to that of the reference compounds nitazoxanide and lauric acid. The compound can be used for preparing a bacteriostat for inhibiting Helicobacter pylori, and can be used for preparing a medicament for preventing and/or treating Helicobacter pylori infection and a related disease caused by Helicobacter pylori infection, featuring broad application prospects.
Description
本发明属于制药领域，具体涉及一种硝基噻唑衍生物在制备抑制幽门螺杆菌的抑菌剂中的用途。The invention belongs to the field of pharmaceuticals, and specifically relates to the use of a nitrothiazole derivative in preparing a bacteriostatic agent for inhibiting Helicobacter pylori.
幽门螺杆菌是一种螺旋形、微厌氧、对生长条件要求十分苛刻的细菌。1983年首次从慢性活动性胃炎患者的胃黏膜活检组织中分离成功，是目前所知能够在人胃中生存的唯一微生物种类。2017年10月27日，世界卫生组织国际癌症研究机构公布的致癌物清单初步整理参考，幽门螺杆菌(感染)在一类致癌物清单中。Helicobacter pylori is a spiral-shaped, slightly anaerobic bacterium that has very demanding growth conditions. It was first successfully isolated from the gastric mucosal biopsy tissue of a patient with chronic active gastritis in 1983. It is currently the only microbial species known to be able to survive in the human stomach. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer released a preliminary reference list of carcinogens, and Helicobacter pylori (infection) was included in the list of carcinogens.
幽门螺杆菌生存于人体胃幽门部位，是最常见的细菌病原体之一。世界有多半人口受到过幽门螺杆菌的感染，而在有些国家几乎90％的人都感染过这种细菌。人们通常是在幼年时就受到感染，5岁以下达到50％。这种细菌感染首先引起慢性胃炎，并导致胃溃疡和胃萎缩，严重者则发展为胃癌。
Helicobacter pylori lives in the pylorus of the human stomach and is one of the most common bacterial pathogens. More than half of the world's population is infected with Helicobacter pylori, and in some countries almost 90% of the population is infected with this bacterium. People are usually infected at an early age, with 50% of cases under 5 years old. This bacterial infection first causes chronic gastritis, and leads to gastric ulcers and gastric atrophy. In severe cases, it may develop into gastric cancer.
Helicobacter pylori lives in the pylorus of the human stomach and is one of the most common bacterial pathogens. More than half of the world's population is infected with Helicobacter pylori, and in some countries almost 90% of the population is infected with this bacterium. People are usually infected at an early age, with 50% of cases under 5 years old. This bacterial infection first causes chronic gastritis, and leads to gastric ulcers and gastric atrophy. In severe cases, it may develop into gastric cancer.
硝唑尼特是一种硝噻柳酸酰胺的衍生物，其实际的作用机制虽尚未弄清，但被认为与抑制丙酮酸盐，铁氧化还原蛋白氧化还原酶的酶依赖性电子转移反应有关，后者对厌氧能量代谢至为重要。研究发现，除了对隐孢子虫和肠贾第鞭毛虫之外，硝唑尼特还对许多肠寄生虫，如贝氏等孢子虫、阿米巴原虫、人蛔虫、钩虫、毛首鞭虫、牛肉绦虫、短膜壳绦虫和肝片吸虫均有活性。但是，有研究报道(国外医药抗生素分册，2004年7月，第25卷，第4期，第191-192页)硝唑尼特作为单一治疗剂不能根除幽门螺杆菌。Nitazoxanide is a derivative of nitethyl salicylic acid amide. Although its actual mechanism of action has not yet been clarified, it is believed to be related to the inhibition of the enzyme-dependent electron transfer reaction of pyruvate and ferredoxin oxidoreductase. , the latter is crucial for anaerobic energy metabolism. Studies have found that in addition to Cryptosporidium and Giardia intestinalis, nitazoxanide is also effective against many intestinal parasites, such as Isospora burnetii, amoeba, human roundworms, hookworms, Trichuris trichuris, Beef tapeworm, brevis tapeworm and Fasciola hepatica are all active. However, there are research reports (Foreign Medicine and Antibiotics Volume, July 2004, Volume 25, Issue 4, Pages 191-192) that nitazoxanide cannot eradicate Helicobacter pylori as a single therapeutic agent.
为了有效预防和治疗幽门螺杆菌感染及其引起的相关疾病，亟需开发出对幽门螺杆菌的抑制效果更佳的抑菌剂。In order to effectively prevent and treat Helicobacter pylori infection and related diseases caused by it, there is an urgent need to develop bacteriostatic agents with better inhibitory effects on Helicobacter pylori.
发明内容Contents of the invention
本发明的目的在于提供一种硝基噻唑衍生物在制备抑制幽门螺杆菌的抑菌剂，预防和/或治疗幽门螺杆菌感染及其引起的相关疾病的药物中的用途。The object of the present invention is to provide the use of a nitrothiazole derivative in the preparation of bacteriostatic agents for inhibiting Helicobacter pylori and medicaments for preventing and/or treating Helicobacter pylori infection and related diseases caused by it.
本发明提供了式(I)所示的化合物、或其晶型、或其盐在制备抑制幽门螺杆菌的抑菌剂中的用途：
The present invention provides the use of the compound represented by formula (I), or its crystal form, or its salt in the preparation of a bacteriostatic agent for inhibiting Helicobacter pylori:
The present invention provides the use of the compound represented by formula (I), or its crystal form, or its salt in the preparation of a bacteriostatic agent for inhibiting Helicobacter pylori:
其中，R选自C3、C4的直链烷烃，C5-C15的直链或支链烷烃。Wherein, R is selected from C 3 and C 4 linear alkanes, C 5 -C 15 linear or branched alkanes.
进一步地，所述化合物为如下化合物之一：
Further, the compound is one of the following compounds:
Further, the compound is one of the following compounds:
进一步地，所述抑菌剂能够抑制幽门螺杆菌生长。Further, the bacteriostatic agent can inhibit the growth of Helicobacter pylori.
进一步地，所述抑菌剂为预防和/或治疗幽门螺杆菌感染的药物。Further, the bacteriostatic agent is a drug for preventing and/or treating Helicobacter pylori infection.
进一步地，所述抑菌剂为预防和/或治疗幽门螺杆菌引起的疾病的药物。Further, the bacteriostatic agent is a drug for preventing and/or treating diseases caused by Helicobacter pylori.
进一步地，所述幽门螺杆菌引起的疾病为胃炎、胃溃疡、十二指肠溃疡、胃癌。Further, the diseases caused by Helicobacter pylori are gastritis, gastric ulcer, duodenal ulcer, and gastric cancer.
进一步地，所述抑菌剂是以所述化合物、或其晶型、或其盐为活性成分，加入药学上可接受的辅料或辅助性成分制成的制剂。Further, the bacteriostatic agent is a preparation made of the compound, or its crystal form, or its salt as an active ingredient, and adding pharmaceutically acceptable excipients or auxiliary ingredients.
进一步地，所述辅料或辅助性成分选自稀释剂、填充剂、着色剂、助流剂、润滑剂、粘合剂、稳定剂、助悬剂和缓冲剂中的一种或两种以上。Further, the auxiliary materials or auxiliary ingredients are selected from one or more of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and buffers.
本发明化合物能够有效抑制幽门螺杆菌生长，并且化合物I-1～I-4的抑制活性远强于对照化合物月桂酸，化合物I-1、I-3、I-4的抑制活性远强于对照化合物硝唑尼特。The compounds of the present invention can effectively inhibit the growth of Helicobacter pylori, and the inhibitory activities of compounds I-1 to I-4 are much stronger than the control compound lauric acid, and the inhibitory activities of compounds I-1, I-3, and I-4 are much stronger than the control. The compound nitazoxanide.
本发明化合物能够有效治疗幽门螺杆菌感染的小鼠，清除胃部感染的幽门螺杆菌并减少胃组织炎症反应，其治疗效果与阳性对照克拉霉素相当，且在等摩尔剂量下治疗效果明显优于对照化合物硝唑尼特和月桂酸。The compound of the present invention can effectively treat mice infected with Helicobacter pylori, eliminate Helicobacter pylori infection in the stomach and reduce the inflammatory reaction of gastric tissue. Its therapeutic effect is equivalent to that of the positive control clarithromycin, and the therapeutic effect is obviously superior at equimolar doses. to the control compounds nitazoxanide and lauric acid.
本发明提供的化合物可以用于制备抑制幽门螺旋杆菌的抑菌剂，可以用于制备预防和/或治疗幽门螺旋杆菌感染及其引起的相关疾病的药物，应用前
景广阔。The compounds provided by the invention can be used to prepare bacteriostatic agents that inhibit Helicobacter pylori, and can be used to prepare drugs for preventing and/or treating Helicobacter pylori infection and related diseases caused by it. The scenery is vast.
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.
说明书附图Instructions with pictures
图1为实施例2中各组小鼠胃组织病理学评分结果。Figure 1 shows the gastric histopathological scoring results of mice in each group in Example 2.
图2为实施例2中各组小鼠幽门螺杆菌尿素酶检测OD值结果。Figure 2 shows the results of OD value detection of Helicobacter pylori urease in each group of mice in Example 2.
本发明所用原料与设备均为已知产品，通过购买市售产品所得。The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
制备受试化合物I-1～I-4：Preparation of test compounds I-1 to I-4:
参照CN114044761A公开的制备方法，制备受试化合物I-1～I-4。具体操作如下：
Test compounds I-1 to I-4 were prepared with reference to the preparation method disclosed in CN114044761A. The specific operations are as follows:
Test compounds I-1 to I-4 were prepared with reference to the preparation method disclosed in CN114044761A. The specific operations are as follows:
取2.0g替唑尼特(7.5mmol)于50毫升单口瓶中，加入20毫升乙酸乙酯和0.9g三乙胺(8.8mmol)，搅拌下逐滴加入0.9g正丁酰氯(8.4mmol)，滴加完后油浴加热至回流，反应2小时。TLC检测原料反应完全，降至室温，过滤除去固体，滤液用20毫升乙酸乙酯稀释，再分别用10毫升0.1M稀盐酸洗2次，10毫升10％碳酸氢钠溶液洗1次，最后用10毫升饱和食盐水洗至中性。乙酸乙酯层用2.5克无水硫酸钠干燥30分钟；过滤除去硫酸钠，滤液减压浓缩至干，所得粗品经硅胶柱层析精制，得化合物I-1。Take 2.0g tizoxanide (7.5mmol) in a 50ml single-mouth bottle, add 20ml of ethyl acetate and 0.9g triethylamine (8.8mmol), add 0.9g n-butyryl chloride (8.4mmol) dropwise under stirring, After the dropwise addition, the oil bath was heated to reflux and the reaction was carried out for 2 hours. TLC detects that the reaction of the raw materials is complete, cools down to room temperature, and removes the solid by filtration. The filtrate is diluted with 20 ml of ethyl acetate, washed twice with 10 ml of 0.1M dilute hydrochloric acid, once with 10 ml of 10% sodium bicarbonate solution, and finally with Wash with 10 ml saturated saline until neutral. The ethyl acetate layer was dried with 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 crude product was purified by silica gel column chromatography to obtain compound I-1.
参照上述方法，分别将正丁酰氯替换为辛酰氯、月桂酰氯、十四烷酰氯，分别制得化合物I-2、I-3、I-4：
Referring to the above method, n-butyryl chloride was replaced with octanoyl chloride, lauroyl chloride, and myristanoyl chloride to prepare compounds I-2, I-3, and I-4 respectively:
Referring to the above method, n-butyryl chloride was replaced with octanoyl chloride, lauroyl chloride, and myristanoyl chloride to prepare compounds I-2, I-3, and I-4 respectively:
实施例1、受试化合物对幽门螺杆菌的体外抑制活性Example 1. In vitro inhibitory activity of test compounds against Helicobacter pylori
(1)实验方法(1) Experimental methods
称取哥伦比亚血琼脂基础培养基3.9g，加热溶解于100ml蒸馏水中，分装三角瓶121℃高压灭菌15min，冷至50℃左右时，加入5％无菌脱纤维羊血，混匀，50℃水浴保温，确保培养基不凝固。Weigh 3.9g of Columbia blood agar basic culture medium, heat and dissolve it in 100ml of distilled water, place it in a triangular flask and sterilize it under high pressure at 121°C for 15 minutes. When cooled to about 50°C, add 5% sterile defibrinated sheep blood, mix well, and 50 ℃ water bath insulation to ensure that the culture medium does not solidify.
使用DMSO溶解受试化合物I-1～I-4、对照化合物硝唑尼特和月桂酸。在12孔培养板中，每孔加入10μl的稀释好的测试样品溶液，再加入1ml 50℃融化的哥伦比亚血琼脂培养基，振荡混匀，使受试化合物浓度在0-200μg/ml，两倍浓度稀释，共10个浓度梯度。DMSO was used to dissolve the test compounds I-1 to I-4, the control compounds nitazoxanide and lauric acid. In a 12-well culture plate, add 10 μl of diluted test sample solution to each well, then add 1 ml of Columbia blood agar culture medium melted at 50°C, shake and mix until the concentration of the test compound is 0-200 μg/ml, twice Concentration dilution, a total of 10 concentration gradients.
将-80℃冻存的幽门螺旋杆菌菌株ATCC26695复苏接种在哥伦比亚血平板上，微需氧37℃培养48h。使用移液器吸头刮取生长状态良好的菌落，并在含5％肽牛血清的布氏培养基中重悬，调整菌液OD值为0.2。使用微量移液器吸取2μl菌液，接种到12孔板含测试样品的哥伦比亚血琼脂上，每孔接种3个菌点，待菌液干后。放入微需氧环境中37℃培养48h。Helicobacter pylori strain ATCC26695 frozen at -80°C was resuscitated and inoculated on Columbia blood agar plates, and cultured microaerobically at 37°C for 48 hours. Use a pipette tip to scrape well-growing colonies, resuspend them in Brucella's medium containing 5% peptide bovine serum, and adjust the OD value of the bacterial solution to 0.2. Use a micropipette to absorb 2 μl of the bacterial solution and inoculate it onto the Columbia blood agar containing the test sample in a 12-well plate. Inoculate 3 bacterial spots in each well and wait until the bacterial solution dries. Place in a microaerophilic environment and incubate at 37°C for 48 hours.
12孔培养板培养48h后，拍照记录菌落生长情况，判定受试化合物对幽门螺旋杆菌的最小抑菌浓度(MIC)。DMSO溶解对照组，板孔内的菌落生长良好，形成明显的菌落。受试样品组，菌落不生长的最小浓度，则为该受试样品对幽门螺旋杆菌的最小抑菌浓度。After culturing the 12-well culture plate for 48 hours, take photos to record the colony growth, and determine the minimum inhibitory concentration (MIC) of the test compound against Helicobacter pylori. DMSO dissolved the control group, and the colonies in the plate wells grew well and formed obvious colonies. For the test sample group, the minimum concentration at which bacterial colonies do not grow is the minimum inhibitory concentration of the test sample against Helicobacter pylori.
结果如下表1所示。The results are shown in Table 1 below.
(2)实验结果(2)Experimental results
表1.各化合物对幽门螺旋杆菌的MIC
Table 1. MIC of each compound against Helicobacter pylori
Table 1. MIC of each compound against Helicobacter pylori
实验结果表明，本发明受试化合物能够有效抑制幽门螺杆菌生长，并且化合物I-1～I-4的抑制活性远强于对照化合物月桂酸，化合物I-1、I-3、I-4的抑制活性远强于对照化合物硝唑尼特。Experimental results show that the test compounds of the present invention can effectively inhibit the growth of Helicobacter pylori, and the inhibitory activities of compounds I-1 to I-4 are much stronger than the control compound lauric acid. The inhibitory activities of compounds I-1, I-3, and I-4 are The inhibitory activity is much stronger than that of the control compound nitazoxanide.
上述实验结果表明，本发明受试化合物可以用于制备抑制幽门螺旋杆菌的抑菌剂，可以用于预防和/或治疗幽门螺旋杆菌感染及其引起的相关疾病。The above experimental results show that the test compound of the present invention can be used to prepare a bacteriostatic agent that inhibits Helicobacter pylori, and can be used to prevent and/or treat Helicobacter pylori infection and related diseases caused by it.
实施例2、受试化合物对小鼠幽门螺杆菌感染模型的治疗效果Example 2. Therapeutic effect of test compounds on mouse Helicobacter pylori infection model
(1)实验方法
(1) Experimental methods
幽门螺杆菌H.pylori SS1菌株准备。使用哥伦比亚血平板复苏细菌，培养48小时后，挑选菌落接种在含5％胎牛血清和选择性添加剂的布氏肉汤培养基中，在37℃的微需氧条件下培养48小时后，取出离心调整细菌浓度为5×109CFU备用。Helicobacter pylori SS1 strain preparation. Use Columbia blood plate to resuscitate bacteria. After 48 hours of culture, select bacterial colonies and inoculate them into Brucella broth medium containing 5% fetal calf serum and selective additives. After culturing for 48 hours under microaerophilic conditions at 37°C, take them out. Centrifuge and adjust the bacterial concentration to 5×10 9 CFU for later use.
取C57BL/6小鼠，雌性，6-8周龄，共计42只，适应性喂养一周后随机分为6组，分别为空白组、模型组、阳性对照克拉霉素组、化合物I-3组、对照化合物硝唑尼特组和对照化合物月桂酸组，每组7只。除空白组外，其余小鼠灌胃上述细菌重悬体系200μl即1×109CFU每只小鼠。灌胃细菌前需禁食12h，断水4h；灌胃细菌前1小时，需灌胃250μL0.2mol/L碳酸氢钠中和胃酸；小鼠灌胃细菌后两小时恢复自由进食和饮水；间隔1天后重复上述过程，共计6次。末次灌胃两周后，各给药组每日灌胃给予对应药物，克拉霉素给药剂量为50mg/kg，化合物I-3给药剂量为100mg/kg，硝唑尼特给药剂量为68mg/kg，月桂酸给药剂量为45mg/kg，空白组给予等体积生理盐水；给药两周后处死小鼠，取材分析。A total of 42 C57BL/6 mice, female, 6-8 weeks old, were randomly divided into 6 groups after adaptive feeding for one week, namely blank group, model group, positive control clarithromycin group, and compound I-3 group. , control compound nitazoxanide group and control compound lauric acid group, 7 animals in each group. Except for the blank group, the other mice were orally administered 200 μl of the above bacterial resuspension system, that is, 1×10 9 CFU per mouse. It is necessary to fast for 12 hours and deprive water for 4 hours before gavage bacteria; 1 hour before gavage bacteria, 250 μL 0.2mol/L sodium bicarbonate needs to be gavaged to neutralize gastric acid; mice can resume eating and drinking freely two hours after gavage bacteria; interval 1 Day after day, repeat the above process for a total of 6 times. Two weeks after the last intragastric administration, each administration group was given corresponding drugs by intragastric administration daily. The dosage of clarithromycin was 50 mg/kg, the dosage of compound I-3 was 100 mg/kg, and the dosage of nitazoxanide was The dosage of lauric acid was 68 mg/kg, the dosage of lauric acid was 45 mg/kg, and the blank group was given an equal volume of physiological saline; the mice were killed two weeks after the administration, and the materials were collected for analysis.
各组小鼠从贲门到幽门处剪取胃，置无菌平皿内，沿胃大弯剖开，取部分用于组织病理学检测，用无菌棉拭子轻轻去除胃内容物，用生理盐水漂洗后，用10％中性甲醛进行固定。通过HE染色，观察小鼠胃部在活菌攻击后的炎症改变，评分并记录结果，评分标准如下：The stomachs of mice in each group were cut off from the cardia to the pylorus, placed in a sterile dish, and cut open along the greater curvature of the stomach. Parts were taken for histopathological examination. The stomach contents were gently removed with sterile cotton swabs, and the stomach contents were removed with a physiological After rinsing with saline, they were fixed with 10% neutral formaldehyde. Through HE staining, observe the inflammatory changes in the stomach of mice after live bacteria challenge, score and record the results. The scoring standards are as follows:
0分：固有层偶见炎症细胞浸润；1分：固有层散在炎症细胞浸润；2分：固有层有中等量炎症细胞浸润；3分：固有层大量炎症细胞浸润，个别在胃粘膜下有淋巴滤泡形成。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: Large amounts of inflammatory cell infiltration in the lamina propria, with some lymph nodes under the gastric mucosa. Follicle formation.
另取各组小鼠等重胃部组织，使用高通量匀浆器匀浆备用。准备快速尿素酶试剂盒，每孔加入酶促反应液100μL，待药膜完全溶解后加入组织匀浆液，在室温下孵育5分钟显色，测试每孔测试液OD值并记录结果。In addition, stomach tissues of equal weight from each group of mice were taken and homogenized using a high-throughput homogenizer for later use. Prepare a rapid urease kit, add 100 μL of enzymatic reaction solution to each well, add tissue homogenate after the drug film is completely dissolved, incubate at room temperature for 5 minutes to develop color, test the OD value of the test solution in each well and record the results.
(2)实验结果(2)Experimental results
各组小鼠胃组织病理学评分记录如图1和表2所示，其中，*代表与模型组相比P<0.5；各组小鼠胃组织匀浆幽门螺杆菌尿素酶检测结果如图2所示，其中，**代表与模型组相比，P<0.05；#代表I-3组与硝唑尼特组相比，P<0.5。The gastric histopathological score records of mice in each group are shown in Figure 1 and Table 2, where * represents P<0.5 compared with the model group; the results of Helicobacter pylori urease detection in gastric tissue homogenates of mice in each group are shown in Figure 2 As shown, ** represents P<0.05 compared with the model group; # represents P<0.5 compared with the nitazoxanide group in the I-3 group.
表2.幽门螺杆菌感染模型小鼠胃组织病理学评分
Table 2. Gastric histopathological scores of Helicobacter pylori infection model mice
Table 2. Gastric histopathological scores of Helicobacter pylori infection model mice
实验结果表明，在小鼠胃组织病理学评分中，I-3组和克拉霉素组治疗显著的降低了小鼠在活菌攻击后的炎症反应，与模型组相比具有统计学差异；
硝唑尼特组与月桂酸组治疗略微降低炎症反应，但与模型组相比没有统计学差异。在小鼠胃组织幽门螺杆菌尿素酶检测结果中，I-3组、克拉霉素组和硝唑尼特组治疗显著降低了尿素酶表达，说明三组化合物显著抑制了幽门螺杆菌生长，且与模型组相比具有显著性差异；同时I-3组尿素酶表达结果明显低于硝唑尼特组和月桂酸组，说明化合物I-3抑制幽门螺杆菌生长的效果显著优于硝唑尼特和月桂酸，且与硝唑尼特组相比有统计学差异。综合以上结果表明，本发明受试化合物能够有效治疗幽门螺杆菌感染的小鼠，清除胃部感染的幽门螺杆菌并减少胃组织炎症反应，其治疗效果与阳性对照克拉霉素相当，且在等摩尔剂量下治疗效果明显优于对照化合物硝唑尼特和月桂酸。The experimental results showed that in the gastric histopathological scores of mice, treatment in the I-3 group and the clarithromycin group significantly reduced the inflammatory response of mice after live bacteria challenge, with statistical differences compared with the model group; Treatment in the nitazoxanide group and the lauric acid group slightly reduced the inflammatory response, but there was no statistical difference compared with the model group. In the test results of Helicobacter pylori urease in mouse gastric tissue, treatment in the I-3 group, clarithromycin group and nitazoxanide group significantly reduced the expression of urease, indicating that the three groups of compounds significantly inhibited the growth of Helicobacter pylori, and There is a significant difference compared with the model group; at the same time, the urease expression result of group I-3 is significantly lower than that of nitazoxanide group and lauric acid group, indicating that the effect of compound I-3 on inhibiting the growth of Helicobacter pylori is significantly better than that of nitazoxanide group and lauric acid, and there was a statistical difference compared with the nitazoxanide group. Based on the above results, it is shown that the test compound of the present invention can effectively treat mice infected with Helicobacter pylori, clear the Helicobacter pylori infection in the stomach and reduce the inflammatory reaction of gastric tissue. Its therapeutic effect is equivalent to that of the positive control clarithromycin, and in et al. The therapeutic effect at molar dosage was significantly better than that of the control compounds nitazoxanide and lauric acid.
上述实验结果表明，本发明受试化合物能够有效抑制幽门螺杆菌感染，可以用于预防和/或治疗幽门螺旋杆菌感染及其引起的相关疾病。The above experimental results show that the test compound of the present invention can effectively inhibit Helicobacter pylori infection and can be used to prevent and/or treat Helicobacter pylori infection and related diseases caused by it.
综上，本发明提供了式I所示的硝基噻唑衍生在制备抑制幽门螺旋杆菌的抑菌剂中的用途。实验结果表明，该化合物能够有效抑制幽门螺杆菌生长，抑制效果优于对照化合物硝唑尼特和月桂酸；该化合物能够有效治疗幽门螺杆菌感染的小鼠，清除胃部感染的幽门螺杆菌并减少胃组织炎症反应，其治疗效果优于对照化合物硝唑尼特和月桂酸。本发明提供的化合物可以用于制备抑制幽门螺旋杆菌的抑菌剂，可以用于制备预防和/或治疗幽门螺旋杆菌感染及其引起的相关疾病的药物，应用前景广阔。
In summary, the present invention provides the use of nitrothiazole derivatives represented by formula I in the preparation of bacteriostatic agents for inhibiting Helicobacter pylori. Experimental results show that the compound can effectively inhibit the growth of Helicobacter pylori, and the inhibitory effect is better than the control compounds nitazoxanide and lauric acid; the compound can effectively treat mice infected with Helicobacter pylori, clear the Helicobacter pylori infection in the stomach and Reduce the inflammatory response of gastric tissue, and its therapeutic effect is better than that of the control compounds nitazoxanide and lauric acid. The compounds provided by the invention can be used to prepare bacteriostatic agents that inhibit Helicobacter pylori, and can be used to prepare drugs for preventing and/or treating Helicobacter pylori infection and related diseases caused by it, and have broad application prospects.
Claims (8)
- 式(I)所示的化合物、或其晶型、或其盐在制备抑制幽门螺杆菌的抑菌剂中的用途：
Use of the compound represented by formula (I), or its crystal form, or its salt in the preparation of a bacteriostatic agent for inhibiting Helicobacter pylori:
其中，R选自C3、C4的直链烷烃，C5-C15的直链或支链烷烃。Wherein, R is selected from C 3 and C 4 linear alkanes, C 5 -C 15 linear or branched alkanes. - 根据权利要求1所述的用途，其特征在于：所述化合物为如下化合物之一：
The use according to claim 1, characterized in that: the compound is one of the following compounds:
- 根据权利要求1或2所述的用途，其特征在于：所述抑菌剂能够抑制幽门螺杆菌生长。The use according to claim 1 or 2, characterized in that the bacteriostatic agent can inhibit the growth of Helicobacter pylori.
- 根据权利要求1或2所述的用途，其特征在于：所述抑菌剂为预防和/或治疗幽门螺杆菌感染的药物。The use according to claim 1 or 2, characterized in that the bacteriostatic agent is a drug for preventing and/or treating Helicobacter pylori infection.
- 根据权利要求1或2所述的用途，其特征在于：所述抑菌剂为预防和/或治疗幽门螺杆菌引起的疾病的药物。The use according to claim 1 or 2, characterized in that the bacteriostatic agent is a drug for preventing and/or treating diseases caused by Helicobacter pylori.
- 根据权利要求5所述的用途，其特征在于：所述幽门螺杆菌引起的疾病为胃炎、胃溃疡、十二指肠溃疡、胃癌。The use according to claim 5, characterized in that: the diseases caused by Helicobacter pylori are gastritis, gastric ulcer, duodenal ulcer, and gastric cancer.
- 根据权利要求1-6任一项所述的用途，其特征在于：所述抑菌剂是以所述化合物、或其晶型、或其盐为活性成分，加入药学上可接受的辅料或辅助性成分制成的制剂。The use according to any one of claims 1 to 6, characterized in that: the bacteriostatic agent uses the compound, or its crystal form, or its salt as an active ingredient, and adds pharmaceutically acceptable auxiliary materials or auxiliaries. Preparations made from sexual ingredients.
- 按照权利要求7所述的用途，其特征在于：所述辅料或辅助性成分选自稀释剂、填充剂、着色剂、助流剂、润滑剂、粘合剂、稳定剂、助悬剂和 缓冲剂中的一种或两种以上。 The use according to claim 7, characterized in that: the auxiliary materials or auxiliary ingredients are selected from the group consisting of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and One or more than two buffering agents.
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